See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/355037287

Text Book on Marine Microbiology

Book · October 2020

CITATIONS READS

0 20

2 authors, including:

Rajeswari Anburaj

27 PUBLICATIONS   49 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

tannin-rich compounds View project

All content following this page was uploaded by Rajeswari Anburaj on 03 October 2021.

The user has requested enhancement of the downloaded file.

 A Textbook
on
Marine Microbiology
By

Dr. P.F.Steffi
and
Mrs. R. Rajeswari Anburaj

1
 Author: Dr. P.F.Steffi and Mrs. R. Rajeswari Anburaj

ISBN:
E-ISBN:

Publisher: Ryan Publishers

B-3, Lakshmi Pride, 80 feet road, 10th Cross
West, Thillainagar, Trichy, 620018,
Tamil Nadu, India, Ph- + 91 6374561101

Copyright © 2020 by Ryan Publishers

No part of this book shall be reproduced or transmitted in any
form or by any means (electronic or mechanical including
photocopy, recording or any information storage or retrieval
system) without the permission in writing of the publisher.

2
 Table of Contents
1 Unit-I: Marine Microbial and Diversity…………………6
1.1 Physical and Chemical Properties of the marine
environment……………………………………………….6
1.2 Ecology of Coastal microorganism…………………...8
1.3 Ecology of Shallow Microorganisms…………………10
1.4 Deep-sea microorganism……………………………..13
1.5 Significance of marine microflora……………………16
1.6 Diversity of Archaebacteria…………………………..20
1.7 Actinobacteria………………………………………..23
1.8 Cyanobacteria………………………………………...26
1.9 Algae…………………………………………………29
1.10 Fungi in Anoxic Marine Environments……………..36
1.11 Viruses………………………………………………39
1.12 Protozoa……………………………………………..43
1.13 Epiphytes – Coral Microbial association……………47
1.14 Sponge Microbial association……………………….51

2 Unit-II: Cultivation of Marine microbes and Nutrient

cycling……………………………………………………56
2.1 Methods of studying marine microbes……………….56
2.2 Physiological and Biochemical properties……………59
2.3 Preservation of microbes……………………………..62
2.4 Role of microbes in carbon cycle…………………….65
2.5 Role of microbes in Nitrogen Cycle………………….68
2.6 Role of microbes in Phosphorus cycle……………….72
2.7 Role of microbes in sulphur cycle……………………76
3
3 Unit-III: Marine Extremophiles and Bioremediation…..81
3.1 Thermophilic microorganism………………………...81
3.2 Haloalkaliphiles………………………………………85
3.3 Osmophiles…………………………………………...86
3.4 Barophilic microbe…………………………………...88
3.5 Psychrophiles………………………………………...91
3.6 Hyperthermophiles…………………………………...93
3.7 Halophiles……………………………………………97
3.8 Bioremediation of heavy metals…………………….100
3.9 Bioremediation of Oils……………………………...104
3.10 Biofouling and their control……………………….106

4 Unit –IV:Sea food Microbiology……………………..112

4.1 Distribution of pathogenic microorganisms…………112
4.2 Indicator Organisms in Marine Environment……….115
4.3 Prevention and control of marine pollution……….…117
4.4 Rapid diagnosis of contamination in sea foods……..124

5 Unit – V: Marine Microbial Products………………….129

5.1 Properties of carrageenan…………………………...129
5.2 Agar Agar…………………………………………...132
5.3 Seaweed fertiliser…………………………………...136
5.4 Astaxanthin…………………………………………139
5.5 Beta carotene………………………………………..142
5.6 Enzymes…………………………………………….145
5.7 Antitumour agents derived from marine sources……152
4
5.8 Polysaccharides derived from marine microbes.........155
5.9 Biosurfactants derived from marine microbes………157
5.10 Pigments produced by marine microbes…………...161
5.11 Preservation methods of Sea foods………………...163
5.12 Quality control for microbial quality of fishes……..166
5.13 Marine resources used as food and drugs…………..169

6 References…………………………………………….174

5
1 Unit-I: Marine Microbial and Diversity

1.1 Physical and Chemical Properties of the

marine environment
Seawater is a combination of a variety of salts and water.
Most waters in the ocean basins originated from
the condensation of water found in the early atmosphere as the
Earth cooled after its formation. This water was released from
the lithosphere as the Earth's crust solidified. Further water has
also been added to the oceans over geologic time from
periodic volcanic action. Some scientists have speculated that
comets entering the Earth's atmosphere may be another vital
source of water for the oceans.
The majority of the dissolved chemical constituents or salts
found in seawater have a continental origin. These chemicals were
released from continental rocks through weathering and then
passed to the oceans by stream runoff. Over time, the
concentration of these chemicals amplified until it met an
equilibrium. This equilibrium takes place when the ocean's water
could not dissolve any more material in the solution. The
comparison between fossilized sea life and living today specifies
that seawater composition stopped changing considerably about
600 million years ago.
Only six elements and compounds consist of about 99% of
sea salts: chlorine (Cl-), sodium (Na+), sulfur (SO4-2),
magnesium (Mg+2), calcium (Ca+2), and potassium (K+)
(Figure-1). The relative quantity of the major salts in seawater is
constant regardless of the ocean. Only the mixture's water quantity
varies because of the variation between ocean basins because of
regional differences in freshwater loss (evaporation) and gain
(runoff and precipitation).
The chlorine ion makes up 55% of the salt in seawater.
Estimation of seawater salinity is completed of the parts per 1000
of the chlorine ion existing in one kilogram of seawater. Typically,
seawater has a salinity of 35 parts per thousand.
6
 Figure: 1 Various Proportion of salts in seawater

Water is one of the few materials accessible on the Earth's

surface in all three forms of matter. At zero degrees Celsius, liquid
water turns into ice and has a density of around 917 kilograms per
cubic meter. Liquid water at the equivalent temperature has a
density of nearly 1,000 kilograms per cubic meter. The density of
seawater generally boosts with decreasing temperature, increasing
salinity, and increasing depth in the ocean. The density of
seawater at the surface of the ocean varies from 1,020 to 1,029
kilograms per cubic meter. Maximum densities are attained with
depth because of the overlying weight of water. In the deepest
parts of the oceans, seawater densities can be as high as 1,050
kilograms per cubic meter.
Seawater freezes at a temperature that is, to some extent,
colder than freshwater (0.0° Celsius). The freezing temperature of
seawater also differs from the concentration of salts—the more
salt, the minor the initial freezing temperature. At a salinity of 35
parts per thousand, seawater freezes at a temperature of -1.9°
Celsius.
Sea ice usually contains significantly less salt than seawater.
Most of the salts found in liquid seawater are forced out when
freezing occurs. The cause for the exclusion is because the
molecules of the various salts do not fit well in the extremely
7
orderly molecular structure of frozen water. Due to the density
difference between ice and seawater, ice floats on the surface of
the ocean.
Seawater also contains minute amounts of dissolved gases.
Many of these gases are added to seawater from the atmosphere
during the stable stirring of the sea surface by wind and waves.
The concentration of gases that can be dissolved into seawater
from the atmosphere is determined by the temperature and salinity
of the water. Rising the temperature or salinity reduces the amount
of gas that ocean water can dissolve. Some of the important
atmospheric gases found in seawater consist of nitrogen, oxygen,
carbon dioxide (in the form of bicarbonate HCO3), argon, helium,
and neon. In contrast to the other atmospheric gases, the amount
of carbon dioxide dissolved in saturated seawater is unusually
large.
Some gases originated within seawater are also concerned in
oceanic organic and inorganic processes that are indirectly related
to the atmosphere. For example, oxygen and carbon dioxide may
be temporally generated or depleted by such processes to varying
concentrations at specific locations within the ocean.
1.2 Ecology of Coastal Microorganism
Opening the microbial black box involved in hydrocarbon
degradation needs to take into concern the whole ecosystem. In
situ studies allow reaching the entire microbial communities in
their real context, considering (biotic and abiotic) ecological
interactions. Over the past few years, field studies have been
achieved in marine sediments addressing the impact of oil on
microbial communities by subsequently following their
organization and/or by characterizing their composition. In most
cases, this has been advanced by spatial assessment of contrasting
contaminated and uncontaminated sites; or sites with different oil
contents. In this way, following the bacterial diversity of oil-
polluted retention basin sediments from the Berre lagoon (France)
through nine stations, it is confirmed that bacterial community
structure was connected with the gradient of oil contamination.
Nevertheless, the adaptation of the bacterial community to oil
8
contamination was not described by the dominance of oil-
degrading bacteria, but a prevalence of bacterial populations
related to the sulfur cycle was observed. Other in situ studies have
highlighted the existence or the role of sulfur cycle
microorganisms in oil-polluted coastal marine sediments, with a
focus on sulfate-reducing bacteria. Researchers suggested that
sulfate-reducers comprise a large fraction of the bacteria present
in oil-contaminated mangrove sediment. They also confirmed that
the abundance of sulfate-reducers reduced with depth and showed
greater bacterial abundance and diversity in top layers (0–5 cm)
than in deeper layers (below 15 cm). Similarly, applying culture-
dependent and molecular techniques to describe the bacterial
populations after the prestige oil spill at two sampling times (2004
and 2007) reported the dominance of sulfate-reducing bacteria in
oil-polluted sediments. Sulfate reduction was the main type of
respiration connected to hydrocarbon oxidative capacities, and
sulfate-reducing bacteria comprise the prevalent populations
(being maximal at 12–15 cm depth). These studies emphasized the
ecological importance of sulfate-reducers in oil-polluted marine
sediments. Sulfate-reducing microorganisms are known to play a
key role in coastal marine ecosystems by recycling the organic
matter, even after an oil spill. Acosta-González also confirmed
that community structure was initially dominated by Gamma and
Deltaproteobacteria (2004), while three years late, in 2007, the
phylum Bacteroidetes was a major component of the community.
These results, showing the great plasticity of bacterial community
structures and suggesting that they were constantly adapting to the
changing environmental factors, highlight the significance to
consider the impact of environmental parameters on
microorganisms when studying oil degradation in coastal marine
sediments. In this way, considering the spatial-temporal
difference in oil-contaminated beach ecosystems, showed the
selective response of bacterial communities to oil from the DH oil
spill. Microbial communities were dominated by members of the
Gammaproteobacteria and Alphaproteobacteria, recommended as
key players in oil-degradation. These groups are comprised of
hydrocarbon-degrading members, the former contributing to the
early stages of oil hydrocarbon degradation (oxidizing more
reactive components such as n-alkanes), and the latter leads to the
9
later stages of degradation (oxidizing more recalcitrant
compounds such as PAHs). In the same way, investigating the
microbial response to the DH oil spill in coastal sediments, Horel
(2012) showed no seasonal differences in the abundances of total
hydrocarbon and alkane degraders in marsh ecosystem with high
physical-chemical parameters variations. Different molecular
approaches, mainly concerning 16S rRNAs gene analyses such as
DGGE or T-RFLP, clone libraries, and phyllo/geochip, were
employed to describe the microbial communities established in
oil-polluted coastal environments. More recently, NGS and
“omics” approaches have been applied, allowing an in-depth
description of microbial communities. These studies allowed us to
evaluate the complexity of autochthonous microbial communities
related to oil pollution, enlightening in situ changes in microbial
diversity, and their selective response to the presence of oil.
Nevertheless, in situ approaches have many drawbacks since
information access on the ecophysiology of oil-degrading
microorganisms, their movement, and degradation pathways
remain still limited.
1.3 Ecology of Shallow Microorganisms
Shallow water marine environment refers to the area between
the shore and deeper water, such as a reef wall or a shelf break.
This environment is characterized by oceanic, geological, and
biological conditions, as described. The water in this environment
is shallow and clear, allowing the arrangement of different
sedimentary structures, carbonate rocks, coral reefs, and allowing
certain organisms to survive and become fossils.
Sediments
The sediment itself is often comprised of limestone, which
forms readily in shallow, warm, calm waters. The shallow
marine environments are not exclusively composed
of siliciclastic or carbonaceous sediments. While they cannot
always coexist, it is possible to have a shallow marine
environment comprised solely of carbonaceous sediment or one
that is composed wholly of siliciclastic sediment. Shallow water
marine sediment is made up of larger grain sizes because smaller
10
grains have been washed out to deeper water. Within sedimentary
rocks consist of carbonaceous sediment, there may also
be evaporite minerals. The most common evaporite minerals
found within modern and ancient deposits are gypsum, anhydrite,
and halite; they can occur as crystalline layers, isolated crystals,
or clusters of crystals.
In terms of geologic time, it is said that the majority of
phanerozoic sedimentary rock was deposited in shallow marine
environments as about 75% of the sedimentary carapace is made
up of shallow marine sediments; it is then understood that
Precambrian sedimentary rocks were too, deposited in shallow
marine waters, unless it is specifically identified otherwise. This
trend is seen in the North American and Caribbean region. Also,
as a consequence of supercontinent breakup and other shifting
tectonic plate processes, shallow marine sediment exhibits a huge
difference in terms of quantity in the geologic time.
Water Composition
The water in this environment is mostly clear and shallow. It
is said that if shallow marine environments can be distinct by their
distributional patterns of marine organisms in terms of
temperature, then deductions can be made from that of the past
model in terms of paleolithic zones. Today, there are three major
defining criteria used in defining shallow marine environments;
these are the faunal provinces, the faunal elements, and the degree
of latitude. However, the restrictions of different present-day
shallow marine environments in terms of climatic zones are
seldom agreed on.
Also, many shallow marine environments are often related to
carbonate factory zones. In these zones, the method that removes
the CO₂ from water to cause the bicarbonate ions to change into
carbonate ions are vital and support lime precipitation. Increasing
temperature, intense evaporation, and mixing water that is high in
CO₃ and low in calcium cations with seawater are some instances
of processes that change bicarbonate ions to carbonate ions.
Carbon dioxide is removed from the atmosphere by being
dissolved in water and is turned into carbonic acid. The carbonic
acid then weathers rocks, creating bicarbonate and other ions.
11
Then the calcium carbonate is a precipitate from calcium and the
bicarbonate ions that are formed through organisms like coral, and
then the carbon is stored in layers of limestone on the seafloor. In
terms of geologic time, the composition of limestone has changed
from calcite rich limestone to aragonite rich limestone. The
presence of magnesium ions at certain concentrations reduces the
ability to precipitate calcite. Aragonite, however, has the same
chemical formula as calcite, but it is in a different crystal system
that is much less prone to the magnesium to avoid the precipitation
of this mineral, which would stop it from forming carbonate rocks.
At times in geologic history where the Mg and Ca ratio were
different, and the seas were more abundant in calcite, and this was
as a consequence of the high rates of seafloor spreading because
of tectonic plate movement and action. The more spreading, the
more the magnesium gets removed, so more calcite is precipitated,
and calcite will be more plentiful than aragonite.
Some organisms in this environment, specifically those in the
intertidal zone, are sea stars, sea anemones, sponges, worms,
clams, mussels, predatory crustaceans, barnacles, and small
fish. Hydrozoa, or hydroids, also exist in shallow marine
ecosystems and eat surrounding algae and zooplankton. Some
species of isopods and amphipods are originated in the intertidal
zones and create diverse burrows and surface tracks in the
sediment. Brittle stars have been seen buried in sediment with
their arms showing through the sediment; this behavior has been
noted in numerous shallow marine areas.
As well, carbonate reefs can be established in the
depositional environment that shallow marine areas; they are host
to reefs and organisms that exist in reefs. Recent estimates
concerning the information of species on coral reefs range from
1–9 million. There are three main types of reef formations:
fringing reefs, these reefs are attached to the shore, barrier reefs,
which are separated from the mainland by a lagoon, and atoll
reefs. Organisms that live in this environment consist of red algae,
green algae, bivalves, and echinoderms. Many of these organisms
contribute to the formation of reefs. Also,
Unicellular dinoflagellates exist in the tissues of corals and have
a mutualistic relationship in which the dinoflagellates supply the
12
corals with organic molecules.
Fossils
The majority of the fossil record has been found after the
shallow water marine environment has been lithified. A lot of
these fossils were deposited at times when much of Earth was
covered with shallow seas, supporting an extensive diversity of
organisms.
Several fossils can be found/formed in this environment.
Some examples are:
 Skolithos ichnofacies are trace fossils that are vertical,
cylindrical, or u-shaped burrows formed by organisms for
protection.
 Glossifungites ichnofacies are trace fossils that are vertical,
cylindrical, u or tear-shaped borings or burrows produced by
organisms like shrimp, crabs, worms, and bivalves.
 Stromatolites are fossils that are laminated sedimentary
structures that are produced when cyanobacteria form
microbial mats, which then trap clay and/or silt sediment and
organic materials to form the fossil.
1.4 Deep-sea microorganism
Despite the well-recognized significance of microorganisms
in the biogeochemistry of hydrothermal plumes, few studies have
characterized microbial communities that inhabit them. Thus the
physical source, taxonomic composition, and ecological nature of
these organisms remain poorly understood. Possible sources of
microbes for deep-sea hydrothermal plumes can be divided into
three categories: (i) seafloor (or sub-seafloor) communities, (ii)
background deep seawater communities, or (iii) growth within the
plume. The traditional view is that plume microbes are likely
sourced from extremely productive biological communities that
inhabit vent chimneys and surrounding areas. Such sources could
also include bottom water that is heavily inclined by low-
temperature diffuse flow, which may be accountable for
geochemical flux comparable to focused hydrothermal venting
and contains microbes from the subsurface biosphere. Indeed,
13
tracer studies specify that diffuse flow and larvae of vent fauna
can be entrained into plumes. Alternatively, plume microbes could
be resultant primarily from ambient background seawater, which
seems feasible given that plumes are a mix of >99% seawater and
∼0.01% hydrothermal fluid and that benthic and pelagic habitats
vary greatly. Regardless of the original cause of microbes, it is
likely that plume communities are dynamic in time and space.
Shifts in microbial community structure are to be projected as the
geochemical environment of hydrothermal fluids evolves with
plume age (i.e., become more dilute and oxidized). Consistent
with this notion are observations of progressive removal of
electron donors and morphological proof of changes in microbial
communities with plumes age. However, only recently has plume
microbial diversity been sampled and investigated with molecular
tools in a spatially resolved manner. Many studies have reported
elevated microbial biomass and activity in plumes relative to
background, signifying that plume microbes are discrete from
those in the ambient water column. In one of the first applications
of molecular tools to deep-sea hydrothermal plumes, Researchers
revealed that the Suiyo Seamount hydrothermal plume is
dominated by just two phylotypes, one group of
Gammaproteobacteria and one group of Epsilonproteobacteria.
Interestingly, both of these phylotypes were most closely
associated (at the time) to symbionts of hydrothermal vent animals
– the Gammaproteobacteria to bivalve gill symbionts and the
Epsilonproteobacteria to ectosymbionts of the tubeworm Riftia
pachyptila and shrimp Rimicaris exoculata. The
Gammaproteobacteria, designated “SUP05,” were also found to
dominate low-temperature diffuse flow emanating from a bivalve-
colonized mound (99% of cells), raising the opportunity that
microbial communities in the subsurface or animal symbioses are
sources of plume microbes. Indeed, SUP05-like sequences have
also been retrieve from diffuse hydrothermal fluids on the seafloor
at the Juan de Fuca ridge. However, recent rRNA gene surveys
show that SUP05 are also widely dispersed in pelagic
environments, raising the question of whether the connection
between plumes and subsurface is physical (i.e., transport only) or
ecological (i.e., actively operating in similar niches in both
environments) in nature. An extreme model of the physical
14
transport of seafloor and/or subsurface material to the water
column is “snowblower” vents that discharge elemental sulfur and
bacterial filaments. Thermophilic microbes resultant from the
subsurface have also been experimented in eruptive event plumes.
Epsilonproteobacteria are also commonly encountered in vent
seafloor environments, again highlighting potential connections
between the seafloor and plumes. Clear signals of
Epsilonproteobacteria and other seafloor hydrothermal microbes
have been observed in plumes at the MidCayman Rise, in the
Iheya hydrothermal field, the Logatchev hydrothermal plume, and
in descending particles from plumes at the East Pacific Rise,
which are genetically distinct from surrounding seawater. Recent
investigation employing fine-scale phylogenetic approaches and
coupled DNA and RNA approaches hold great assure for
elucidating the niche space and allocation of sulfur-oxidizing
Gammaproteobacteria and Epsilonproteobacteria in subsurface,
seafloor, plume, and background environments. Researchers
noted partitioning of distinct clades of sulfur-oxidizing
Gammaproteobacteria in vent environments (SUP05 in plumes
and animal symbioses) versus others [Arctic96BD-19 in
background and oxygen minimum zones (OMZs)] and recognized
that sulfide concentration likely controls the balance of
Gammaproteobacteria versus Epsilonproteobacteria. Consistent
with that view, coupled RNA and DNA analyses revealed that
Epsilonproteobacteria are more energetic in the reducing
environment of the subseafloor, whereas Gammaproteobacteria
are more active at the seafloor where integration with seawater is
more prevalent. In difference with the hypothesis that seafloor
environments are the major cause of plume biota, microbial
communities in hydrothermal plumes at the Guaymas Basin in the
Gulf of California are distinct from those of the underlying
seafloor habitats such as hydrothermal sediments or chimneys.
Rather, Guaymas Basin plume communities closely look like
those from background seawater samples taken just above the
plume or in the neighboring Carmen Basin, which is 100 km away
and does not host hydrothermal activity. Metagenomic and
metatranscriptomic data additionally reinforces the plume-water
column connection, showing that the metabolically active
microbes in Guaymas plumes are pelagic rather than benthic in
15
nature, and signifying an ecological boundary between seafloor
and plume. These studies understand that the above-plume and
Carmen Basin samples as true background communities, and this
is held up by the absence of detectable physical and chemical
tracers of hydrothermal activity in those samples. However,
another possibility is that this deep seawater surrounding
Guaymas Basin is impacted by microbes that are exported from
highly productive chemoautotrophic plumes. Methods that could
assist export include ascending and descending particles and
migratory zooplankton, buoyant transparent exopolymeric
substances, and large scale advection such as mesoscale eddies.
Regional manipulation of vents on deepsea microbial
communities may be particularly significant in the Gulf of
California, where restricted basins could limit dispersal of plumes
and mix with true non-hydrothermally-impacted seawater.
1.5 Significance of marine microflora
Marine microorganisms as an antimicrobial and antiviral
source:
On earlier studies, marine microorganisms are found to be
able of producing antimicrobial metabolites. The amides of D-
allo- and L-isoleucine derivatives were isolated from the culture
of a marine strain of Acremonium furcatum. Structural elucidation
of these compounds was then achieved by examination of
spectroscopic data and confirmed by synthesis. From the
experiment, the sample exhibited moderate antimicrobial
activity against Bacillus subtilis, Staphylococcus
aureus, Escherichia coli , and Botrytis cynerea and a very
simple pattern by Thin Layer Chromatography (TLC).
Experimentation that has been carried out for its purification
and partial characterization of marinocine, a new broad-
spectrum antibacterial protein produced by Marinomonas
mediterranea. Marinomonas mediterranea is a melanogenic
marine bacterium isolated from the Mediterranean Sea at the
Murcia coast. From the study, M. mediterranea synthesizes a
macromolecular compound with an antibacterial activity that has
been called marinocine. The interest of marinocine was reinforced
16
by its activity on three nosocomial strains of hard
treatment with commercial antibiotics (Pseudomonas sp.,
ceftazidime, Pseudomonas sp. imipenem, Staphylococcus
aureus methicillin).
Besides, an enzyme such as lysozyme that is extensively
distributed in animals, plants, and microorganisms also possessed
antimicrobial and antiviral properties. A study
by researchers revealed that lysozyme isolated Bacillus sp. strain
S-12-86 from marine able to disrupt Candida albicans cell wall
seriously and reason for asymmetry of the cytoplasm. The
investigation also shows that this lysozyme has a extensive
antimicrobial activity against numerous standard strains including
gram-positive bacteria, gram-negative bacteria, and fungi.
Other antimicrobial compound isolate from marine
microorganisms is sisomicin. Sisomicin show antibacterial
activity against Bacillus cereus and Escherichia coli.
Studies by investigation productively recognize sisomicin from
marine Streptomyces sp.
Marine microorganisms as biosurfactant and bioremediation:
Marine biosurfactants formed by some marine
microorganisms have been paid more attention by researchers,
mainly for the bioremediation of the sea polluted by crude oil.
Biosurfactants are the surface-active molecules synthesized by
microorganisms. With the advantage of environmental
compatibility, the demand for biosurfactants has been steadily
rising and may ultimately substitute their chemically synthesized
counterparts. They are significant predominantly in the
bioremediation of the sea polluted by crude oil. Species that can
generate polymeric biosurfactant is Pseudomonas nautical. The
major constituents were proteins, carbohydrates and lipid at the
ratio of 35:63:2, respectively. Investigators also reported
that Yarrowia lipolytica, a tropical marine strain formed the
emulsifier in the presence of alkanes or crude oil. In the study also
conclude that biosurfactant as the promising agents for
bioremediation of hydrocarbons, chiefly oil polluted in marine
environment.
Researchers reported that Alcaligenes sp., is able to generate
17
glucose lipid. The lipophilic constituent consisting of four β-
hydroxydecanoic acids linked together by ester bonds is coupled
glycosidically with C-1 of glucose. Alcanivorax borkumensis also
produces an anionic glucose lipid with a tetrameric oxyacyl side
chain. According to investigators, Myroides sp. is able to form
bile acids, cholic acid, deoxycholic acid and their glycine
conjugate when cultivated in marine broth. Besides that, there are
marine microorganisms that are able to oxidize heavy metal
ion. Lee and Tebo (1994) has reported that Bacillus sp., strain
SG-1 able to oxidize Manganese (Mn) (II) and Cobalt (Co).
Marine microorganisms as enzyme producer:
It is predicted that global market for industrial enzyme grows
by 10-15% yearly with the current value of USD 4.1 billion. With
the unique capacity of marine microorganisms, that are able of
catalyzing reactions at temperature near the freezing point of
water, it have raise significant interest for both industrial use and
fundamental studies as alternative of common mesophilic enzyme
since cold-adapted enzymes would contribute towards energy
saving strategies. Besides, marine enzyme biotechnology can
suggest novel biocatalysts with properties like high salt tolerance,
hyperthermostability, barophilicity, cold adaptivity and ease in
large scale cultivation.
Lipase producer: Lipase are among such valuable products.
Therefore, scientists began to isolate and screen a variety of
microorganisms, mainly bacteria, in the search to discover better
lipases. Lipase is extensively distributed among animals, plants
and also microorganisms. Lipase-producing microorganisms have
been found in diverse habitats such as industrial wastes, vegetable
oil processing factories, dairies, soil contaminated with oil,
oilseeds and decaying food, compost heaps, coal tips and hot
springs.
Few bacterial genera have been isolated and characterized
which include Aeromonas sp; Pseudoalteromonas sp; and
Psychrobacter sp; Photobacterium lipolyticum; Pseudomonas sp.
strain B11-1, Pseudomonas sp. 7323 and Bacillus sp. S14. Some
of the lipase producer not only cold-adapted, it also have
another useful characteristics such as lipases produced
18
by Pseudomonas sp. 7323 that shows optimum activity at alkaline
condition (pH 9.0) which can profit industrial field.
Chitinase producer: Chitinase is another enzyme that is
significant for industrial application. Chitin, a (1>4)-β-linked
homopolymer of N-acetyl-d-glucosamine, is an abundant
structural polysaccharide formed by many marine organisms.
Through study of chitinase genes we can recognize the ecology of
chitin-degrading bacteria and chitin degradation in aquatic
systems. Other marine microorganism that known to have
interaction with chitinase substrate that is chitin is Vibrio cholera.
Since chitin is an abundant source of carbon, nitrogen and also as
energy source in the aquatic environment have made V.
cholerae as an autochthonous member of diverse aquatic
ecosystems around the globe. These bacteria have an immense
potential to be exploited even though it know to be pathogenic
microorganisms.
Protease producer: Protease is another chief industrial enzyme
employed in many industrial applications such as detergents,
leather processing, food processing, feeds and chemical
industries, as well as waste treatment. Proteases catalyzed
conversion of proteins to amino acids and peptides.
Psychrophiles that form protease has been identified.
Study by Yu et al. (2009), manage to detect 222 isolates
positively producing protease that derived
from Bacillus sp., Marinomonas sp., Pseudoaltermonas sp.
and Sulfitobacter sp. Besides that Pseudomonas strains 145-2 are
also capable of generating extracellular protease at low
temperature (20- 30°C) with presence of nitrogen and carbon
source in a concentration 0.5-1.0%.
Agarase producer: Agarase catalyze the hydrolysis of agar are
categorized into α-agarase (E.C. 3.2.1.158) and β-agarase (E.C.
3.2.1.81). Agarase have extensive applications in food industry,
cosmetics and medical fields. It been employed as enzyme in
biology, physiological and cytological studies. Marine
microorganisms that producing agarase have been isolated from
seawater and marine sediments. Agarases have been isolated
from Alteromonas sp., Pseudomonas sp., Vibrio sp.
19
Cytophaga sp., Agarivorans sp., Thalassomonas sp., Pseudoalter
omonas sp., Bacillus sp. and Acinetobacter sp. Most isolates
formed extracellular agarases than intracellularly. Agarases have
been employed in production of agar derived oligosaccharides and
degradation of the cell wall of red algae for extraction of labile
material with biological actions such as unsaturated fatty acids,
vitamins, carotenoids from algae.
1.6 Diversity of Archaebacteria
Archaea were documented to be very diverse from bacteria
and were categorized as a separate domain in 1990. The archaea
were first assumed to exist only in extreme environments, such as
hot springs, brines and deep hydrothermal vents, but researchers
was capable to show that they had a broad distribution, they
reported the survival of novel, non-extreme archaea in marine
waters. Similar kind of archaea were shortly thereafter reported
from soils and freshwater lakes and this marked the starting point
of an completely novel branch of microbial ecological studies.
Two main archaeal phyla, Euryarchaeota and Crenarchaeota, are
presently accepted. Recent phylogenetic studies propose that the
mesophilic Crenarchaeota form a third phylum, Thaumarchaeota,
and certain 16S rRNA gene sequences from uncultured archaea
from hot springs are diverse enough to potentially be assigned to
yet a separate phylum, the Korarchaeota. Members of both
Euryarchaeota and Crenarchaeota have been found in freshwater
environments. In these, the most frequently found Euryarchaeota
belong to many different methanogenic groups, but also members
of the Thermoplasmatales and numerous other still uncultured
euryarchaeotal groups have been identified by molecular means.
The Crenarchaeota existing in freshwater environments generally
affiliate with Groups I.1a, I.1b, I.2 and I.3 (also known as Marine
Benthic Group C) of the non-thermophilic Crenarchaeota, but no
members of these freshwater Crenarchaeota have yet been isolated
in culture. The relative abundance of archaea in diverse
environments is normally much lower than that of the bacteria,
and they habitually constitute less than 10% of the microbial
population in freshwater habitats. Though, particularly in the
water column, the archaea can reach abundances of up to 37%
20
(4.1·105 cells ml-1) of the microbial cells. The microbial
phylogenetic groupings based on the 16S ribosomal RNA (rRNA)
gene, are changing as more sequences become accessible. This is
particularly true when it comes to archaea, of which only a few
groups with cultured representatives exist. Most archaeal
phylogenetic groups consist only of 16S rRNA gene sequences
that have been acquired directly from the environment and have
no cultured members. These sequences, which typically cover
only a part of the 16S rRNA gene, are deposited in the gene banks
in exponentially growing numbers. The sequence data allow for
the formation of distinct phylogenetic groups, but the functions
and physiology of these organisms are mostly unknown.
However, metagenomic studies have newly given some sign of
the function of some of the uncultured archaea.
Euryarchaeota
The phylum Euryarchaeota contains identified methanogenic
archaea as well as extreme halophiles and cell wall lacking
thermoplasma. The anaerobic methanogenic archaea exist in and
have been isolated from very diverse habitats, such as the rumen,
bioreactors and lake sediments where the common factors are low
redox and absence of oxygen. Extreme halophiles are readily
isolated from brines and salt lakes, such as the Dead Sea, and
many of these micro organisms have been characterized. Due to
this fact, the euryarchaeotal phylogeny appears more
comprehensible than that of the Crenarchaeota. Methanogens
were also the first microorganosms whos 16S rRNA genes
sequences demonstrates that archaea were not bacteria.
Methanosarcinales and Methanomicrobiales
Alexandro Volta confirmed that swamps emit a combustible
gas, methane. Methane is produced in anaerobic conditions by
methanogenic archaea, The vertical division profile of
methanogens differs between lakes. Chan et al. found, using T-
RFLP analysis, that in Lake Dagow, Methanosaeta dominated in
the upper sediment layers, which were directly influenced by the
sedimentation of organic matter, and Methanomicrobiales
affiliating with Methanospirillum and cluster WCHD (according
to the phylogenetic analyses of this review, Table 1 were dominant
21
in deeper, acetate depleted sediments. Zepp-Falz et al., on the
other hand, found by fluorescent in situ hybridization that in the
profundal sediment of Swiss Lake Rotsee, Methanospirillum-like
Methanomicrobiales archaea (sequences of which in this review
affiliate with cluster WCHD, Table 1, were existing only in the
uppermost layer whereas Methanosaeta could be found
throughout the 50 cm length of the studied sediment cores. Waters
receiving excessive organic input, such as leaves and other plant
materials from the surroundings have also been shown to host
genera of methanogens has the capacity to utilize C1-compounds,
such as methanol, and methylamines, which are formed during
hydrolysis of complex organic matter. A general characteristic of
microorganisms in lake sediments is that the number of microbial
cells decreases with depth in lake sediments, but the relative
abundance of archaea increases. Methanogenic archaea have been
identified in deep subsurface groundwater. In the granitic rock
groundwater of Äspö Hard Rock Laboratory (Sweden), vaarious
types of methanogens were found in surprisingly high numbers at
depths of 446 m bsl and the methane concentrations in the water
at this depth measured up to 100 µM. The methanogens found in
this habitat belonged to the genera Methanosarcina and
Methanobacterium Table 1. Interestingly, the acetoclastic
Methanosarcina were the most abundant archaea in the
groundwater at depths down to 112 m, while the autotrophic
hydrogenotrophic Methanobacterium existed in the deeper parts
of the boreholes down to 446 m. An alkalophilic and halotolerant
hydrogenotrophic methanogen, Methanobacterium subterraneum
strain A8p, was isolated from the granitic groundwater at a depth
of 420 m. Methanosaeta and Methanospirillum 16S rRNA gene
sequences were also acquired from the groundwater of deep gold
mines in South Africa at depths down to 3.8 km below ground.

22
Table: 1 Euryarchaeotal 16S rRNA gene sequences found in
freshwater environments and affiliate with Methanosarcinales
and Methanomicrobiales from freshwaterenvironments.

1.7 Actinobacteria
Actinobacteria Associated with Marine Flora:
Mangroves, Seagrasses, and Seaweeds
Mangroves are main intertidal bionetworks of the tropical
and subtropical zones of the world. The actinobacterial genera
such as Agromyces, Gordonia, Microbacterium, Mycobacterium,
Rhodococcus, and Streptomyces were isolated from mangrove
roots in the Iriomote Island and were tested against diverse
pathogenic bacteria and fungi. Researchers have accounted that a
huge diversity of actinobacterial populations live in the mangrove
23
phyllosphere. Several novel species named Agromyces brachium,
Agromyces rhizospherae, Agromyces luteolus, Gordonia
rhizosphera, Lysinimicrobium mangrovi, and Micromonospora
avicenniae were accounted from mangroves with a variety of
bioactivities. Seagrasses are the marine angiosperms, dwell in the
tidal and subtidal marine ecosystem. Cultivable and non-
cultivable molecular approaches revealed that seagrasses harbor
wealthy bacterial diversity. Thalassia hemprichii is related with
ten genera of actinobacteria. Totally, 43 16S rRNA gene base
pairs were selected from the gene data bank to recognize the
diversity of mangrove endophytic actinobacteria, and the
investigation of RDP assignment tools revealed that all the
actinobacteria belong to the order Actinomycetales including 11
families and 14 genera. Based on the 16S rRNA data in the
GenBank, a assortment of seagrass-associated actinobacteria is
moderately higher, including 15 families of three subclasses, viz.,
Acidimicrobidae, Actinobacteridae, and Nitriliruptoridae. The
symbiont from T. hemprichii displays the NRPS and PKS genes,
which recommend that the actinobacteria might function as an
antibacterial agent. Researchers have reported that Streptomyces
is the leading genus in seagrasses, AU1 which shows efficient
microbicidal activity against multiple drug-resistant pathogens.
Seaweeds harbor a diverse group of bacteria, depending on the
season, species, and thallus structure. Beneficial interactions
between seaweeds and bacteria are exchange of nutrients and
secondary molecules. Researchers have analyzed the 16S rRNA
sequences recovered from the GenBank, among them 22 genera
within two orders of Acidimicrobiales and Actinomycetales were
found to be related with seaweeds.
Marine Fauna:
Ascidians and Mollusks
Ascidians, a class of tunicates (phylum: Chordata), marine
sessile filter feeders, are present in solitary and colonial forms.
Actinobacteria, namely, Arthrobacter, Brevibacterium, Gordonia,
Micrococcus, and Nocardia, were reported from Didemnum
ligulum, and Didemnum sp. might play an significant role in
ascidians as well as the isolates were recorded for its activity
24
against a variety of human pathogens. Mollusks are a group of
invertebrates that comprise more than 100,000 species.
Biodiversity of mollusk-associated 109 actinobacteria was
preliminarily revealed by researchers. Strains were isolated that
belong to ten genera of actinobacteria along with two unidentified
isolates from Donax trunculus. Actinobacteria were leading in the
cone snails such as Conus pulicarius, C. rolani, and C. tribblei
and included 16 diverse genera of 11 families with four common
genera (Brevibacterium, Microbacterium, and Streptomyces).
Based on the 16S rRNA data acquired from the GenBank, it is
predicted that 34 genera within 16 families of the order
Actinomycetales are related with the ascidians. Among them,
Streptomyces (93 sequences), Micromonospora (37 sequences),
and Nocardia (13 sequences) were abundant in the ascidians.
Phylogenetic analysis of the mollusk-associated actinobacterial
sequences from the GenBank specify that 28 actinobacterial
genera within the order Actinomycetales were associated with the
mollusks.
Corals, Sponges, and Fish
Coral reefs support mainly biodiverse and biologically
productive important communities in the tropical and subtropical
marine ecosystem. Primarily, culture-dependent investigation of
coral-associated marine bacteria discovered the 126 occurrence of
actinobacteria in corals. Sponges harbor microbes up to 40% of
their total biomass by providing with favorable environmental
circumstances. The novel actinobacteria, Janibacter alkaliphilus
and Janibacter corallicola, were isolated 130 from the corals,
Acropora gemmifera and Anthogorgia sp., respectively, which has
been revealed for active compounds. Actinobacteria acquired
from these healthy corals have strong microbicidal activity,
signifying that these bacteria can protect the hosts from the
pathogens. Researchers reported that coral associated
actinobacterial community comprise about 523 sequences
in GenBank, which include 62 genera of 32 families within
five orders, viz., Acidimicrobiales, Nitriliruptorales,
Solirubrobacterales and Thermoleophilales. Among the Porifera-
associated bacteria, members of Actinomycetales are frequently
sponge specific and a leading producer of bioactive natural
25
products. There are various bioactives isolated and screened for a
variety of pharmacological activities. Many of them verified to be
good drug molecules with considerable results for preclinical and
clinical studies. Studies using culture-dependent and culture-
independent molecular methods established that new, abundant
actinobacterial accumulations are related with marine sponges.
According to literature survey, 44 genera of the sponge-derived
actinobacteria have been recognized based on culture-dependent
methods. Phylogenetic classification using RDP classifier signify
that the sponge-associated actinobacteria consisted of three
orders, namely, Acidimicrobiales, Actinomycetales, and
Rubrobacteriales, which together encompassed 112 genera in 39
families. Eighty-seven unique Streptomyces were acquired from
homogenized gut materials of marine ornamental fish, and these
Streptomyces species effectively formed bioactive metabolites
against target pathogens. Subsequently, Sanchez(2012) have
reported three suborders of actinobacteria (Corynebacterineae,
Micromonosporineae, and Micrococcineae) from the fish
microbiome.
1.8 Cyanobacteria
Cyanobacteria are oxy-photosynthetic bacteria. One of the
distinctiveness of cyanobacteria is their thylakoids, the seats of
photosynthesis, respiration, and in some species, molecular
nitrogen fixation. One of the initial signs of life on Earth was the
formation of stromatolite reefs, which survive now as fossil
structures in the oldest rocks known. This cyanobacterial fossil
record is among the oldest of any group of organism, possibly
reaching back to 3500 million years (myr) ago. Throughout the
succeeding 3000 myr, many shallow reefs arose and afford a
habitat for cyanobacteria.
Benthic Cyanobacteria:
Microbialites
Microbialites are organosedimentary deposits of trapped
benthic microbes and detrital sediment and/or mineral
precipitation. Thus, microbialites may display various degrees of
mineral induration. Based on their internal structure, Burne and
26
Moore divided microbialites into stromatolites described as
sedimentary structures containing lithified laminae, thrombolites
(clotted texture), cryptic microbialites (vague, mottled or patchy
texture), oncolites (concentric lamination), and spherulitic
microbialites (spherular aggregates). Microbialites may
characterize a major structural component of the reef.
Microbialites consist entirely of millimetre- to centimetre-thick
thrombolite crusts. In the barrier reef-edge of Tahiti, they may
form 80% of the rock by volume and reflect at least 13,500 years
of continuous reef formation. However, the growth of
microbialites in the cryptic niches of the reef framework ceased
about 6000 years ago when the sea level approached at its present
level. Soft, biscuit-shaped, internally finely laminated
stromatolitic structures, with substantial quantities of fine grain
(micritic) carbonate, have been discovered in a lagoon on Tikehau
atoll (Tuamotu Archipelago, French Polynesia) at depths of 15–
23 m. These modern stromatolites cover large areas of the lagoon
floor and are particularly numerous around patch reefs. They
consist of filamentous, sheathed, non-heterocystous cyanobacteria
predicted as two new species of Phormidium. The constructional
elements of carbonate precipitates fall into two categories
characterized by distinctive forms and size ranges:
micrometresized (0.5–2.0 µm) mineral fibres, rounded (0.1–0.2
µm) bodies, and grape-like clusters. The growth of modern marine
stromatolites correspond to a dynamic balance between
sedimentation and intermittent lithification of cyanobacterial
mats.
Endolithic Cyanobacteria
Carbonate skeletons of hermatypic corals harbour diverse
populations of microboring organisms. Skeletons of live colonies
are bored from the inside outward by Chlorophyta, while dead and
denuded parts of coral skeletons are colonized at the surface and
bored inward by a succession of euendoliths, starting with
Chlorophyta and followed by cyanobacteria, to establish a stable
Chlorophyta-dominated endolith community within 2 years. The
distribution of boring cyanobacteria generally depends on light
level and depth; however some other features may also influence
their distribution. In Jamaica, in clear water, the boring
27
cyanobacteria community structure changes below 20–30 m.
Boring cyanobacteria can also infest shells. In French Polynesia,
infestations of cyanobacteria identified as Hyella, Mastigocoleus,
and Plectonema destroy the commercially valuable shells of the
black oyster Pinctada margaritifera. In the carbonate cycle,
cyanobacteria play a significant and decisive role. Cycling of
carbon and carbonate is linked to biological processes: some build
up specific carbonate structures, some demolish carbonate
substrates, and others do both simultaneously. The photosynthetic
activity of cyanobacteria, their extracellular polymeric substances,
and possibly also their adherent heterotrophic bacteria are
responsible for the production of various carbonate structures and
the ability to penetrate carbonate material. The boring activity of
euendoliths results in biological corrosion and disintegration of
carbonate surfaces. Grazing organisms on carbonate surfaces
colonized by epiand endolithic cyanobacteria form specific
biokarst forms and specific grains that can contribute to near-shore
sedimentation. Biological corrosion and abrasion together
constitute bioerosion. Endolithic phototrophs (cyanobacteria and
Chlorophytes) are one of the major primary producers in dead
coral substrates in a wide range of coral reef environments. In an
investigation of the photosynthetic activity and N2 fixation rates
of coral rubble endoliths in fringing reefs at La Reunion Island
(France) and Sesoko Island (Okinawa, Japan), the main endolith
flora was composed of the cyanobacteria Hyella (cf.) caespitosa,
Plectonema (cf.) terabrans, Mastigocoelus testarumin, and
Scytonema (cf.) conchophyllum (the last two species with
heterocysts). Their primary production rate varied seasonally
between 1.6 and 4.8 µg C µg chl−1 day−1 and were comparable
to those of scleractinian corals [16].
Symbiotic Cyanobacteria
Marine sponges can host a mixture of cyanobacterial and
bacterial symbionts. For example, the filamentous
cyanobacterium Oscillatoria spongeliae is found in the sponge
Dysidea on the Great Barrier Reef (Australia) and also in three
species of Dysidea found around Guam. In the Western Central
Pacific reefs from Taiwan to the Ryukyu Archipelago, the
encrusting sponge Terpios hoshinata is connected with unicellular
28
cyanobacteria first illustrated as Aphanocapsa raspaigellae and
later reclassified using molecular tools as closely related to
Prochoron sp. [20]. In the shallow waters of the Caribbean Sea,
the encrusting sponges Terpios manglaris and T. belindae are
related with the cyanobacterium Hypheothrix sp. (Oscillatoriales,
Schizotrichaceae). The sponge Terpios sp. aggressively
participate for space by killing and overgrowing live corals and is
accountable for devastating extensive areas of coral reef.
Phylogenetic analyses of 16S rRNA sequences of sponge-
associated cyanobacteria have shown them to be polyphyletic.
Many sequences are affiliated with Synechococcus and
Prochlorococcus species. Cyanobacteria fill the cortical region of
the sponge and penetrate inward into the choanosomal region.
Microbial symbionts may generate many of the pharmaceutically
active compounds isolated from marine sponges. These
compounds can serve a mixture of ecological functions, from
predator and competitor deterrence and resistance to malignant
microbial infections. Because cyanobacterial symbionts can also
overgrow and kill their host sponge, it is not known whether
sponges can energetically normalize their symbiont populations.
1.9 Algae
Green algae (Chlorophyta) are clearly signifcant
photosynthetic contributors in coastal waters as demonstrated by
the Ocean Sampling Day dataset where they constituted the
second major photosynthetic group (dinofagellates excluded) afer
Ochrophyta (mostly diatoms) both in terms of read contribution
and number of Operational Taxonomic Units. The importance of
Chlorophyta had previously been previously highlighted in
several specifc environments. In European coastal waters, the
contribution of Chlorophyta to photosynthetic 18S rRNA clones
was found to be 42%. In the OSD dataset, the percentage of
Chlorophyta was maximum in tropical waters with oceanic
characteristics (94% OSD7 of Moorea,). Such high Chlorophyta
involvement in oceanic waters have been also been experimented
in clone library studies as well as in the Tara Ocean dataset10. In
contrast, low Chlorophyta contribution (less than 1% Chlorophyta
reads) was observed at very few stations in the North Atlantic
29
Ocean (e.g. of Norway OSD155 and 157) and in the Arctic
(OSD128). Such low contribution of Chlorophyta does not mean
that their quantity is always low in these waters since sampling
was restricted to a single day and Chlorophyta have been isolated
earlier in these environments, e.g. in Norwegian coastal waters. In
the OSD dataset, Mamiellophyceae (especially Micromonas,
Ostreococcus and Bathycoccus) was the major Chlorophyta class
in coastal waters under a broad range of environmental conditions,
as previously experimented by several studies in coastal and
nutrient-rich environments from the Arctic Ocean to the
Mediterranean Sea through the Pacifc and Indian Oceans. Not et
al. 48 found Micromonas to be the most widespread genus in the
world ocean coastal waters and at a more local scale, Micromonas
dominates coastal picoplankton in the Western English Channe.
Researchers found that Mamiellophyceae (Micromonas,
Ostreococcus and Bathycoccus mostly) were dominant in the
upwelling-infuenced coastal waters of Chile. Using quantitative
PCR, Marie et al. 44 found Bathycoccus to be leading in a transect
through the Mediterranean Sea.
In contrast, the contribution of Mamiellophyceae was low at
oceanic OSD stations, which confrms data from the oceanic Tara
Ocean dataset, where only 17% of the Chlorophyta reads belonged
to Mamiellophyceae10. Nutrient used up environments have been
reported earlier to host Chloropicophyceae10 and clade IX11.
These two groups however appear to be diferentially dispersed in
the OSD dataset with prasinophytes clade IX in more oligotrophic
areas than Chloropicophyceae, as experimented previously in the
South China Sea or the Pacifc gyre. Picocystophyceae (formerly
prasinophytes clade VIIC7 ) were completely absent from the
OSD dataset, validates that this class is absent from marine waters.
Pyramimonadales were recovered everywhere in OSD and were
the second most rich Chlorophyta class as found in the Tara
Oceans dataset12 and ofen co-occurred with Mamiellophyceae.
They were particularly widespread in the Mediterranean Sea and
the North Atlantic Ocean, where microplankton microscopy
inventories earlierly recorded the presence of the genera
Halosphaera and Pterosperma. In the OSD dataset,
Pyramimonadales did not show any environmental preferendum
30
supporting the observation made by Viprey et al. that
Pyramimonadales were found in almost all metadata ranges they
sampled in the Mediterranean Sea. Pyramimonadales strains have
been isolated from a huge range of environments including polar
Mediterranean and different coastal waters. Surprisingly,
Pyramimonadales were not recovered from coastal waters of
Japan (OSD124), while numerous strain or natural samples
sequences from GenBank initiate from this area and South Africa,
where a wide diversity of Pyramimonas have been isolated.
In the OSD dataset, Chlorodendrophyceae replaced
Mamiellophyceae at some stations in particular in the
Mediterranean Sea and contribute to Chlorophyta of the US coast
and in the Indian Ocean. In contrast they were mostly absent from
boreal waters. This group has been somewhat overlooked in 18S
rRNA surveys, most of which focused on the picophytoplankton
size fraction, while Chlorodendrophyceae species, such as those
from the genus Tetraselmis, are rather nanoplanktonic. Some 18S
rRNA sequences have been recovered from the Mediterranean Sea
from surface, low nutrients samples, which corroborate the pattern
observed in the OSD data. The major Chlorodendrophyceae genus
Tetraselmis has been accounted in several microscopic
inventories in the Mediterranean Sea and North Atlantic Ocean
and strains have been isolated in a wide variety of environment.
Interestingly, Tetraselmis strains are used for biotechnology
applications and can grow heterotrophically, which may enlighten
their presence in low nutrient environments.
Relative abundance and diversity of the different Chlorophyta
classes in coastal waters
Overall, Mamiellophyceae dominated Chlorophyta in terms
of mean contribution (55%) and number of OTUs. They were
subsequently followed by Pyramimonadales (12%),
Chlorodendrophyceae (12%), and the UTC clade (Ulvophyceae,
Trebouxiophyceae and Chlorophyceae: 3.5%, 7.5% and 3.2%
respectively. The distribution of OTUs among the diverse classes
was somewhat related to the mean contribution of each class.
However, although Pyramimonadales and Chlorodendrophyceae
had similar contribution, the former class had three time more
31
OTUs than the latter. Chlorodendrophyceae were dominated by
OTUs with a large number of reads (29,899 reads for the larger
one), while Pyramimonadales OTUs had a smaller number of
reads, the larger one with 5,089 reads. Ulvophyceae and
Chlorophyceae had more OTUs (respectively 95 and 94 OTUs)
than expected from their relative contribution (respectively 3.5
and 3.2%). Several classes with low overall contributions had a
quite large number of OTUs. For example, the
Palmophyllophyceae and Pedinophyceae represented about 2 and
0.3%, respectively but had 3.4 and 1.5% of the OTUs respectively.
In order to estimate the stage of novel diversity in each class, we
computed the fraction of the OTUs with less than 98% BLAST
similarity to any sequence from GenBank originating from
cultures. Without surprise, 100% of the OTUs from classes which
have not been brought in cultures met this criterion, such as
prasinophytes clade IX or VIII. In contrast, 100% of the
Chloropicophyceae, despite the fact that it is a newly created class
7, appear to match sequences from cultures suggesting that the
cultivation effort has been very exhaustive for this group in coastal
waters as it had been shown earlier to be in oceanic waters. In
contrast, a huge fraction of the diversity of abundant classes such
as Mamiellophyceae or Pyramimonadales remains to be brought
into culture. Dispersion of specifc Chlorophyta classes in coastal
waters. Mamiellophyceae were recovered at almost all stations
(120 out of 122) where more than 100 Chlorophyta reads where
recorded and were only absent at two oligotrophic stations OSD7
and OSD. Tey could reach up to 99% of Chlorophyta (OSD183 in
the North Sea of Belgium). The major Mamiellophyceae OTUs
(Supplementary Data S2) were dispersed to the three genera
Ostreococcus (80,988 reads), Micromonas (47,778 reads) and
Bathycoccus (22,305 reads). Pyramimonadales were also very
widespread (Fig. 5) and their maximal contribution reached 90%
(OSD108, Portugal coast). They were absent at some oceanic
infuenced stations in the Caribbean Sea (OSD28, Belize) or
Atlantic Ocean, in (OSD97, Azores). No clear dispersion pattern
appeared for Pyramimonadales. The major OTUs were allocated
to genera Pyramimonas and Pterosperma (Supplementary
Data S2). Chlorodendrophyceae were less prevalent than
Pyramimonadales, although being on average similar in relative
32
abundance. They represented up to 99% of Chlorophyta reads at
OSD93 (Atlantic Ocean, of Morocco) and were rich at
Mediterranean stations (OSD4 with 91%, 6 with 58%, 14 with
81%, 24 with 82%, 94 with 43% for example,
Chlorodendrophyceae contribution was lower along the North
American coasts (OSD28 with 16%, 41 with 3.9%, 58 with 4.6%,
60 with 12% for example, and they were absent in the sub-polar
North Atlantic (stations around Iceland, Greenland or Fram Strait.
Trebouxiophyceae correspond to more than 1% of the reads at 71
stations. Their maximum contribution (80% of Chlorophyta reads)
was found at OSD45 (Gulf of Mexico). They were recorded in
temperate coastal waters, especially of the USA and European
Atlantic coasts. Trebouxiophyceae were not present at high
latitudes nor at oligotrophic stations such as Hawaii, French
Polynesia or Azores. The 3 major OTUs (7,703, 1,423 and 1,317
reads, Supplementary Data S2) were dispersed to the highly
diversifed marine coccoid genus Picochlorum. Ulvophyceae
maximal contribution was evidenced at OSD169 (North Sea of
UK, 70%). Ulvophyceae were mostly present along the North
Atlantic European coast, at some stations of the Mediterranean
Sea (OSD78 in the Adriatic Sea and OSD 123 of Israel, for
example), in warm watered (OSD28, 124 and 147) and in
Antarctica (OSD187). The most abundant Ulvophyceae OTU
(Supplementary Data S2) was allocated to the macroalgal genus
Ulva, in particular matching with 100% similarity sequences from
U. fasciata and U. pertusa (synomyms of U. australis and U.
lactuca, respectively), both species being regarded as an invasive
having been carried by oysters, while the second one was assigned
to the marine green fagellate genus Oltmannsiellopsis, that is
extensively dispersed in coastal waters. Chlorophyceae were
always minor contributors to Chlorophyta and correspond to more
than 1% of Chlorophyta reads only at 28 stations located in the
Northern hemisphere. Their maximal contribution was reached in
the Arctic Ocean (Greenland Sea, OSD80 and OSD167, 95% and
40%, respectively) and the Mediterranean Sea (OSD90, Etoliko
lagoon, Greece, 57%). The major OTU (5,111 reads) was
allocated to a reference sequence corresponding to Carteria sp.
(RCC2487), a marine strain isolated from the Beaufort Sea. Te
second OTU (711 reads) was assigned to the very diversifed genus
33
Chlamydomonas, which sequences have been established in
almost all ecosystems from soil to marine waters. However this
OTU matched at 99.7% the sequence of strain NIES-1021 which
has been assigned to the marine species Chlamydomonas
kuwadae. The uncultivated prasinophytes clade IX represented
more than 1% of the Chlorophyta reads at 13 stations generally
situated in oligotrophic tropical and temperate stations (Figure 2).
Their highest contributions were found in the Pacifc Ocean
(OSD7, French Polynesia, 78%), Mediterranean Sea (OSD52 and
53, respectively 70 and 78%) and of Belize (OSD28, 34%). Major
OTUs (Supplementary Data S2) were allocated to the B clade.
Within Palmophyllophyceae, all OTUs were assigned to the order
Prasinococcales (genera Prasinococcus and Prasinoderma for the
major OTUs, Supplementary Data S2) and none to
Palmophyllales, which have only be evidenced from deep waters.
They contribute to more than 1% at 27 stations (Figure 2), mostly
in the Mediterranean Sea and along North Europe coasts. Maxima
evidenced were of Cyprus (OSD19, 77%) and in the Skagerrak
(OSD157, 37%). Interestingly they were existing at 62 other
stations signifying that they are possibly an ubiquitous but minor
component of the Chloropicophyceae correspond to more than 1%
at 20 stations (Figure 2) habitually situated in tropical oceanic
waters. They achieve their maximum contribution at the Azores
station OSD97 (45%) and of Bermuda (OSD8, 29%, Fig. S3). The
major OTUs (Supplementary Data S2) corresponded to the
species Chloroparvula pacifca and sp. (clades B2) and
Chloropicon roscofensis. Nephroselmidophyceae represented
more than 1% at 12 stations and their maximal contribution
between 5 and 6% of the Chlorophyta reads were evidenced in the
coastal North Atlantic Ocean (OSD106 of Iceland, 152 of Canada
and 157 of Norway.
The Nephroselmidophyceae also attained 2% at numerous
stations in the Eastern Basin of the Mediterranean Sea (such as
OSD123 of Israel). The two major OTUs belonged to the genus
Nephroselmis (Supplementary Data S2). Pedinophyceae
correspond to more than 1% of the Chlorophyta reads only at 9
stations and were frequently present at stations located of the USA
Atlantic coast (OSD35, 46, 143, 186) and in the Mediterranean
34
and Black Seas (OSD64 and 78). The highest contribution (7.1%)
was recorded in Chesapeake Bay (OSD35). The two major OTUs
belonged to the genus Marsupiomonas (Supplementary Data S2).
The order Pseudoscourfeldiales had more than 1% of the
Chlorophyta reads at only two stations (Figure 2)

Figure 2 Stations representing the algal class

from the Adriatic Sea (OSD 48 and 99, 1.8% and 1%,
respectively). Prasinophytes clade VIII was the least correspond
to group in this dataset with more than 1% at a single station of
the Iberic Atlantic coast (Figure 2). Finally, at 13 stations, more
than 1% of the Chlorophyta reads could not be classified in any
35
Chlorophyta class. The maximum fraction of unclassifed
sequences was found in the Mediterranean Sea of Cyprus
(OSD18, 16%), in the Atlantic Ocean of Belize (OSD 28,8.1%)
and of the East Coast of the US (OSD58, 7.5). Other unclassifed
reads were recovered from the Mediterranean Sea and of Iceland
(OSD128).
1.10 Fungi in Anoxic Marine Environments
A huge fraction of the marine biosphere is anoxic or partially
anoxic. In terrestrial ecosystem, fungi are frequently found in
saprotrophic and detritus habitats that are often low in oxygen.
Fungi have been shown to acquire a range of cellular and genomic
adaptations to life in anoxic environments. Moreover, many fungi
have been shown to play a function in anaerobic denitrification,
e.g., Fusarium oxyporum. SSU rDNA sequences closely related to
F. oxysporum have been recovered from marine anaerobic
environments and four marine isolates, including a Fusarium
species, have been established to grow in suboxic conditions,
utilizing nitrate for respiration and accumulating nitrite, and are
consequently able of participating in anaerobic denitrification in
marine ecosystem. Study by investigators observed the diversity
of fungi in oxygen-depleted regions of the Arabian Sea using
clone library methods. These researchers used multiple fungal-
specific SSU rDNA primer sets and one general eukaryotic-
specific primer set to amplify SSU sequences. Each primer set
revealed an overlapping subset of fungal diversity, with the
fungal-specific primers presenting a better diversity of fungi per
sampling effort than clone libraries constructed using universal
eukaryotic primers. This effects reveal the significance of using
different primers to control for PCR biases and imply that a
considerable portion of fungal diversity is missed when using
universal primer sets. The phylogeny of researchers identified 48
distinct fungal phylotypes (clustered at 99% sequence similarity):
27 branching within the ascomycete radiation, 20 branching
within the basidiomycetes, and only 1 unique phylotype branching
among the lower fungi. Several Dikarya sequences formed highly
novel branching positions in the phylogeny and clustered with
other environmental sequences improved from oxygen-depleted
36
habitats. Indeed, many sequences branched within a clade
previously termed the hydrothermal and/or anaerobic fungal
group, which branches with the Malassezia yeasts also recognized
from deep-sea eukaryotic environmental clone libraries. No
chytrid-like sequences were recognized from this study,
signifying the possibility that these taxonomic groups have a low
diversity in the marine environments examined or alternatively
that the PCR primers or DNA sampling process employed for this
study were collectively biased toward the Dikarya. Fungal-
specific clone library investigation of deeper water column
samples, sediments, and anoxic environments have considerably
increased the diversity of the fungi recovered from marine studies,
particularly when evaluated with surface water samples. Two of
these studies combined eDNA analysis with isolation and culture
research, indicating little overlap between the sequences
recovered from isolated cultures and eDNA and signifying the
possible presence of a more complex fungal community than
identified either by eDNA or culturebased analyses alone.
Nonetheless, marine fungal-specific clone library analyses reveal
a simple fungal community with relatively few phylotypes in total.
This is a stark contrast to terrestrial environments, where fungal
communities appear to be much more complex clearly verified by
the comparison of species accumulation curves from marine
environments with the results of clone library examination of
nonmarine habitats. Based on currently available data, it is
difficult to express the difference in the diversity and relative
abundance of fungal forms between terrestrial and marine
ecosystem. Yet the molecular data seem to be consistent with the
results of culturing efforts, although diverse diversity profiles are
revealed by these two technique, and imply that marine fungi are
relatively nondiverse and lower in abundance.
Molecular diversity of marine fungi
The kingdom Fungi was traditionally categorized as four
main groups: (a) Ascomycota, (b) Basidiomycota (which together
form the subkingdom Dikarya and have been the major focus of
experimental research and genome-sequencing initiatives), (c) the
zygomycetes, and (d ) the chytrids. This early model of fungal
taxonomy has been modified at a number of levels, including the
37
placement of the microsporidia with and potentially within the
Fungi and the division of the chytrids and zygomycetes into
multiple interbranching paraphyletic clades, followed by
subsequent taxonomic reclassifications There still remains much
uncertainty relating to the major divisions of the Fungi below the
Dikarya. Progress in drawing the fungal tree of life has been made
by studying fungi that have been isolated and cultured mainly
from terrestrial environments. These approach are limited because
they preferentially sample microbes that are easily cultured or that
acquire larger body sizes and/or distinctive morphologies.
Moreover, these approaches bring their own precise limitations
for studying fungi in marine ecosystem:
1. The culturing of fungal isolates from marine samples has
often led to the improvement of nonfungal microbes, which are
ecologically, morphologically, and trophically comparable to
fungi but are not true fungi.
2. The ecological preferences of most fungi recommend that
those in marine ecosystems are likely to inhabit in host organisms
or in benthic ecosystem, including deep-sea sediments. These
habitats are complicated to study by microscopy and in some cases
pose strict sampling complexity.
3. The majority of fungi harbor elevated levels of cryptic
diversity that is indistinguishable using microscopy of
environmental samples and/or culturing.
Additional obstacles arise because analogous fungal
morphotypes such as yeasts and flagellated zoospores branch in
distant and paraphyletic positions on the fungal tree of life making
classifications based on explanation of general morphological
characters difficult and often misleading. Molecular methods—
particularly the polymerase chain reaction (PCR) amplification of
taxonomically informative gene markers from eDNA samples
combined with clone library construction, sequencing, and
phylogenetic analyses—have established that microbial diversity
is much more complex than previously thought. The
environmental DNA, PCR, and clone library approach has been
employed for both prokaryotes and eukaryotes to place many
formerly unrecognized branches on the tree of life, in many cases
38
redefining our understanding of the evolutionary complexity of
the eukaryotes, although these consequences have been the
subject of much debate and revision. Molecular approaches have
also confirmed that poorly documented groups are important
ecosystem components. Yet most molecular surveys of microbial
eukaryotic diversity are recovered using only one primer set and
sample less than 500 clones, with the result that no eukaryote-wide
study has reached sampling saturation This has guided to the
implication that further sampling from the very similar
environments would expose more diversity and reveal that our
understanding of the complexity of the tree of life is still very
incomplete.
1.11 Viruses
The huge number of unknown viral populations in the marine
metagenome highlight the need for further isolation,
characterization and sequencing of definite marine viruses. This
special issue presents several new marine viruses of eukaryotes
Prymnesium parvum, and bacteria (Shewanella, Vibrio
anguillarum and Dinoroseobacter shibae, adding to the quickly
growing database of genome-sequenced and characterized marine
viruses. Numerous auxiliary metabolic genes and other functional
genes were recognized in the phage genomes, signifying a mutual
benefit for both phage and host that could possibly be
disseminated to other hosts by horizontal gene transfer (Figure 3).
Prophage-encode genes can thus supply to host functional
properties, including virulence, by so-called lysogenic
conversion, possibly expanding the niches engaged by the
lysogenized hosts (Figure 3). Further, prophage induction can
stimulate biofilm development by supporting the release of
extracellular DNA, which becomes a component in the biofilm
matrix. Investigations by Leigh revealed that lytic phage
infections also enhance biofilm formation in Shewanella, which
forms biofilms in the gut of the tunicate Ciona intestinalis.
Shewanella is part of a complex relationship between the C.
intestinalis and its related microbiome, and the study reveals that
phage interaction with its Shewanella host supply to the symbiotic
relationships between the gut microbiome and the tunicate host
39
 Figure 3

Figure 3. Schematic overview of important virus-host

interactions in the marine ecosystem covered in this special issue,
including viral infection of bacteria, phytoplankton, and fish.
Definite explanations of key interactions: (1) Bacteria can avoid
phage infection by mutational alteration of surface receptors or by
enzymatic degradation of the incoming phage DNA; (2)
Optionally, protection of cells in aggregates or biofilms can be a
protection strategy against phage infection; (3) Infection by
temperate phages can lead to the integration of the phage DNA in
the host genome as prophage; The integrated prophage can avoid
infection by similar phages (Superinfection exclusion mechanism)
and (4) contribute with important genetic information to the host
that may expand its metabolic or virulence properties. Prophage
induction leads to the discharge of new phages and may also
stimulate biofilm formation; (5) Phages can influence host gene
expression in cyanobacteria for improved infection efficiency,
either by exploiting the host genes or by encoding host
photosynthesis genes which are then expressed during infection;
(6) Phage interaction with their bacterial hosts add to shaping the
40
gut microbiome of invertebrates (e.g., tunicates), thus disturbing
the symbiotic relationship between gut microbes and their hosts;
(7) In the coccolithophorid phytoplankton Emiliania huxleyi the
diploid virally contaminated cells may undergo viral induced lysis
or re-emerge (dotted arrow) as haploid cells containing viral RNA
and lipids. These haploid cells are thought to resist virus infection
(as indicated with the X) and grow into the diploid cells by
karyogamy; (8) The huge and diverse group of nucleocytoplasmic
large DNA viruses (NCLDV) contaminate a series of
photosynthetic protists such as the prasinophytes Micromonas
pusilla and Ostreococcus tauri and, as exemplified in the figure,
the toxin-producing haptophyte Prymnesium parvum, thus
disturbing mortality, diversity and production of phytoplankton.
These connections are strongly controlled by environmental
feature such as temperature, nutrient availability and light. As for
bacteria, several methods of resistance (indicated with an X) to
viruses have been expressed in the photosynthetic protists
Viruses may also obtain accessory genes from their
eukaryotic or prokaryotic hosts (Figure 3). By expressing these
genes during contamination, the viruses may augment key steps in
cellular metabolism and ultimately amplify virus assembly. In
addition to using viral genes attained from the host, viruses may
also organize the expression of host genes during illness to
promote viral production or inhibit host defense systems. This was
confirmed by Fedida & Lindell, where expression patterns of
specific host genes in the cyanobacterium Synechococcus sp
strain WH8102 during cyanophage infection recommended that
the phage exploited the host genes for better infection efficiency.
The elevated local viral diversity acquired from oceanic
metagenomic data imply a high dispersal of viral genes across the
sampled ocean viral communities. Moebus had already
established that bacterial viruses with definite infectious
properties were dispersed across large spatial scales in the North
Atlantic. Later, a worldwide distribution of a virus contaminating
the picophytoplankton Micromonas pusilla was accounted by
Cottrell and Suttle, signifying that viruses are proficiently
multiplied in the marine environment. This is supported by the
study by Kalatzis, which reveal that H20-like vibriophages
41
infecting the fish pathogen Vibrio anguillarum are globally spread
either as free phages or as prophages inside bacterial genomes.
The researchers argue that selection for co-existence, rather than
arms race dynamics, might explain the global distribution of near-
identical H20-like bacteriophages and their incidence as
prophages in Vibrio genomes. Viral host cells have developed
multiple defense strategies against lytic viral infections (Figure 3).
These comprise both mutational changes in the cell surface
receptors providing resistance to phage adsorption, and various
mechanisms for devastating the viral DNA upon infection (e.g.,
restriction modification and CRISPR-Cas defense). Mordecai
recommend a diverse life cycle strategy of the dsDNA EhV
viruses infecting the coccolithophorid Emiliania huxleyi, where
the recognition of viral RNA in the virus-resistant haploid cell of
E. huxelyi recommended a new method of infection, and the co-
existence of viruses and host. Defense approaches are often
related with a fitness cost, as surface modification mutations may
have an influence on, e.g., substrate uptake or enzyme secretion,
and because virus inactivation method may be expensive to
maintain. Such trade-offs between resistance and fitness costs
were investigated in the two groups of eukaryotic phytoplankton,
Ostreococcus tauri, and E. huxleyi. Surprisingly, no direct cost of
resistance was identified in these systems, emphasizing the
complexity of interplay between virus-host co-evolution and the
environmental conditions. The large and diverse group of
nucleocytoplasmic large DNA viruses (NCLDV) consists of a
number of viral families infecting small photosynthetic protists,
thus affecting mortality, evolution and production of these
phytoplankton. In the present special issue, the research on
NCLDV infecting phytoplankton correspond to the Prasinoviruses
infecting the ubiquitous group of pico-sized Prasinophycea such
as Micromonas and Ostreococcus and viruses infecting bloom-
forming haptophytes such as Prymnesium parvum and Emiliania
huxleyi. These studies emphasize the progression in our
understanding of the function of viruses infecting eukaryotic
algae, offer a synthesis of the existing knowledge in the field, and
recognize gaps in our knowledge surrounding viral life history and
interactions with their hosts. The finding of the giant
Acanthamoeba polyphaga mimivirus encouraged a new line of
42
research, investigate the ecology and evolution of the group of
large DNA viruses infecting eukaryotic protists, including the
haptophyte Phaeocystis globosa. Researchers existing knowledge
and general characteristics of this collection of novel viruses and
their communications with their hosts, as well as their virophage
parasites. The compilation of papers included in the current
special issue emphasize the exploration of eukaryotic and
prokaryotic viruses, from discovery to complex interplays
between virus and host and the connections with ecologically
significant environmental variables. The findings of new viruses
and method underlying virus distribution and diversity exemplify
the fascinating world of marine viruses. The oceans significantly
shape Earth’s climate, hold 1.37 billion km3 of seawater, generate
half the half of the oxygen in the atmosphere, and are integral to
all known life. In a time where life in the oceans is under rising
threat (global warming, acidification, pollution, economic use), it
is pressing to understand how viruses influence host population
dynamics, biodiversity, biogeochemical cycling and ecosystem
efficiency.
1.12 Protozoa
Protozoan populations consisting of heterotrophic flagellates
and ciliates are usually considered to be the main grazers of
bacteria and picophytoplankton populations evenly in marine and
freshwater ecosystem. Additionally to these heterotrophic grazers,
there is rising support that many phytoflagellate species are able
of phagotrophy. Current laboratory studies of marine
phytoflagellates have predicted the possible implication of these
organisms in the marine ecosystem. Field investigation conducted
off the west coast of South Island, New Zealand revealed that
natural phytoflagellate populations could put forth a equivalent
grazing pressure to heterotrophic flagellates on both bacterial and
picophytoplankton populations. The diversity in size of ciliated
protozoa is reproduced in various range of feeding strategies and
prey items. Based on the structure of the feeding apparatus and
grazing rates on diverse particle sizes, Fenchel (1980) expected
that the majority of ciliates would depend on nanophytoplankton
and that bacterivorous ciliates would be lacking from open oceans
43
due to low bacterial numbers. Sherr & Sherr (1987), however,
confirmed that ciliates < 30 um are important bacterivores and
Borsheim (1984) revealed that particles of the same size as
picophytoplankton were preferred to larger phytoplankton cells by
two freshwater ciliates. Predaceous ciliates are also a common
component of ciliate communities, mainly in those which contain
haptorids. This group includes Didinium and Askenasia. Some of
these genera, such as Mesodinium, also display mixotrophic
capabilities. Such varied feeding strategies, even within the
similar genus, suggest that extrapolations of feeding rates from
literature values for similar genera, are of only limited value in
determining trophic interactions. Phytoplankton biomass in
waters off the west coast of the South Island is often dominated
by small picophytoplankton (< 2 jam) which can contribute 40-
80% of total biomass and 45-70% of the primary productivity.
Diatoms are the main plentiful inshore where nutrient levels are
prominent; the implication of phytoplankton cells > 2 fim
decrease with distance offshore. Significant correlations were
found between microzooplankton and potential food resources
(bacteria and picophytoplankton) based on spatial allotment off
the west coast of South Island. Direct measurements of grazing
rates in the New Zealand region are essential to determine whether
these correlations reflect trophic interactions.
In majority of the studies feeding behaviour of protozoa have
been restricted to a few species or one food resource. Our study
reveals that protozoans do opt for on the basis of food size and
that this selectivity varies with protozoan taxon. The clearance
rates of heterotrophic flagellates calculated in this study using
fluorescently labelled beads (0.03-7.9 nl ind"1 h"1) are
comparable with consequences from similar experiments. These
rates are, however, lesser than those accounted for heterotrophic
flagellates using disappearance type test, where rates ranged from
1.5 to 94 nl ind~' rr1. Some of the dissimilarity in these rates is
likely to cause from the active selectivity against ingesting
fluorescently labelled microspheres by some species as discussed
above. The clearance rates reported for 0.5|im microspheres
uptake in this study are, however, an order of magnitude below
those recorded in west coast waters in 1990 for both heterotrophic
44
and phytoflagellates. As the processes used were identical there is
no obvious explanation for this result. The clearance rates of 1.0
fim microspheres were elevated in our study with a mean of 1.0 nl
ind~' h~' for phytoflagellates and 3.0 nl ind~ ~' h~' for
heterotrophic flagellates, compared to 0.5 nl ind"1 rr1 for
phytoflagellates and 0.9 nl ind"1 h"1 for heterotrophic flagellates
in 1990. Despite disadvantages of the utilization of microspheres
(e.g., selection against artificial particles) clearance rates and food
preferences that have been determined by this process for a variety
of ciliates lie within the range of those determined by other
methods.
The benefit of fluorescent microspheres is that they are
clearly observable in protozoa, need minimum preparation, are
easily notable from symbiotic algae that are present in some
ciliates, and can be used to decide size-selectivity. Clearance rates
for choreotrichs (mostly Hakeria, Strombidium, and Strobilidium)
were considerably higher for 1.0 nm microspheres than for 0.5\xm
microspheres. The dissimilarity in clearance rates for the large
ciliates, Laboea strobila and Tintinnopsis, were even greater. This
preference is reliable with the examination of Fenchel (1980) and
Rassoulzadegan(1988), who found that ciliates < 30 (im fed
almost exclusively on picoflagellates and cyanobacteria whereas
larger ciliates ingested more nanoplankton size cells.

Figure: 4 Free swimming species

45
Several free swimming forms eat algae and you can see a green
color inside the cytoplasm (image left below); others eat smaller
protozoans like the species with a large cystostome and six
vacuoles, revealed in the righthand picture below. In existent life
the first vacuole near the 'mouth' contained a alive protozoan
which was moving.

Figure: 5 Tintinnids

Figure: 5 Tintinnids look like a French champagne glass.

They are chiefly marine organisms. Individuals can retract intoits
loric if troubled. When it comes out, the crown of cilia is used both
for locomotion and to grab food, The three same sample are most
likely from the genus Favella. Tintinnids are frequent in sea water
only throughout the summer months.
The effects from the current study recommend that diverse
taxa of protozoa also selectively ingest diverse particulate food
resources according to size. Unfortunately, clearance rates for
ciliates feeding on nanophytoplankton were not measured but
clearance rates on picophytoplankton-sized particles by
Tintinnopsis were in the middle of the range examined by Verity
(1985) for Tintinnopsis spp. feeding on prymnesiophycean
flagellates Table: 2.

46
 Table: 2 Clearance rates of microzooplankton. FLA,
fluorescent labelled algae; FM, fluorescent microspheres; FLB,
fluorescent labelled bacteria

1.13 Epiphytes – Coral Microbial association

Coral reefs are quickly degrading worldwide, with hard
corals normally being replaced by benthic algae. These changes
are triggered by a mixture of stressors, including overfishing of
herbivores, overloaded nutrient and organic matter input, global
climate variation, and evolving marine diseases. To recognize the
mechanisms implicated in these shifts, it is significant to examine
interactions between corals and algae. These interactions typically
engage a range of physical, microbial, and chemical mechanisms.
Algae can harm corals via direct physical interactions such as
shading and abrasion, the exudation of allelopathic substances,
and through indirect influences such as the liberation of dissolved
organic carbon (DOC). Microorganisms within coral mucus can
profit from discharged DOC and reduce oxygen concentrations in
proximity to coral tissue, which can consequently cause mortality
and disease. Furthermore, the effects of algae on corals depend on
the specific coral and algal species present. Crustose coralline
algae normally have positive effects on coral reefs, for instance,
through promoting coral recruitment and repressing macroalgal
47
recruitment, whereas turf algae can negatively influence coral
health through impeding coral recruitment success (and decrease
tissue thickness. Anthropogenic influence, mainly sedimentation
and terrestrial run-off, can enlarge the frequency of coral–turf
contacts and turf competitiveness. Brown macroalgae within the
genus Lobophora have been a focus of recent study because of
their rapid raise on many reefs worldwide. This genus can increase
following disturbances such as bleaching events and storms,
reaching benthic cover of up to 50%. Direct contact between
Lobophora and corals normally encourage coral bleaching and
mortality and results in decreased coral growth, reproduction, and
recruitment. Lobophora can also proceed as a substrate for various
epiphytic community. However, only some studies to date have
considered the function of epiphytes in coral–algal interactions or
have illustrated the effects of epiphytes from those of their algal
hosts. Epiphytes have a broad range of effects on their host, and
therefore their influence on host competitiveness is challenging to
predict. Epiphytes can stress their host through shading, can
decrease consumption of host algae by herbivores, and if
definitely attached can lead to tissue lesions and/or cause bacterial
infections. Some epiphytes are hemiparasitic and drain organic
carbon from their host using penetrating rhizoids. If epiphytes
perform as a stressor to their algal host and decrease algal growth
rates, they could potentially decline the competitiveness of their
algal hosts against corals. Alternatively, epiphytes could reinforce
competitiveness of their algal hosts by directly damaging corals.
For instance, a common epiphyte of Lobophora, Anotrichum
tenue, can overgrow living coral tissue. Furthermore, by varying
the composition and concentration of secondary metabolites in
their host, epiphytes can prevent herbivorous fishes from
consuming algal hosts and thus amplify host competitiveness. The
aim of this observational study was to evaluate the types and
outcomes of coral–algal interactions on coral reefs in Fiji and to
gain first insights into the relationship between the presence of
epiphytes and algal host competitiveness against corals. In
addition to characterizing the coral–algal interactions and
recording the genera concerned, herbivorous fish biomass and
sedimentation rates were calculated as explanatory variables. We
expected a negative relationship between herbivorous fish
48
biomass and the number of coral–algal interactions, and also
predicted that higher sedimentations rates would improve the
prevalence of turf algae implicated in interactions by deterring
herbivory
In this study, epiphytes were defined as all filamentous algae
growing on the surface of macroalgae (Figure. 6) and turf algae as
filamentous algae with a canopy height below two cm growing on
abiotic substrate. A coral–algal interaction was considered as
‘alga-winning’ if the coral was bleached below or next to the
interaction zone or if there was evident overgrowth of the alga
over the coral surface, as ‘coral-winning’ if the coral was visibly
overgrowing the algae, or as ‘neutral’ if neither was experimented
or each coral colony, the proportion of the colony perimeter in
contact with each type of contact category (i.e. algal genera, other
benthic organisms, or bare substrate) was calculated (hereon
referred to as ‘contact rate’). The two most regularly observed
algal groups (i.e. turf and Lobophora) were analysed in more
detail. For both types of algae, the relationship between contact
rate and site was analysed with a generalised linear model. For
associating with turf algae, a generalised linear mixed model
incorporating transect nested in site as a random effect was
applied to analyse the relationships between contact rate and
sedimentation rate, herbivorous fish biomass, coral genus, and
colony size (perimeter).

Figure: 6 Lobophora covered with filamentous epiphytes

49
 For contacts with Lobophora, the effect of the presence of
epiphytes were depicted. Furthermore, models were run
individually for each of the three most normally observed coral
genera. Following the same approach, the influence of the above
parameter on the competitiveness of turf algae and Lobophora was
tested for each coral genus incorporating a random factor for the
coral colony ID. Competitiveness was defined as the proportion
of the coral perimeter in contact with turf algae or Lobophora for
which the interaction was classified as ‘alga-winning’.
The enhanced competitiveness when epiphytes were present
on Lobophora could be driven by direct impacts of the epiphytes
themselves on neighbouring corals and/or via indirect
consequences on the algal host. In New Caledonia, bleaching of
coral perimeters in contact with L. herderacea has been projected
to be the reason by associated epiphytic filamentous algae.
Functionally, epiphytes are similar to turf algae, which can
damage corals. In this analysis, however, turf algae were related
with a relatively elevated proportion of neutral interactions,
signifying that epiphytic filamentous algae and filamentous turf
algae growing on abiotic substrate differ in their competitiveness,
possibly due to disparity in their species composition. Indirect
effects of epiphytic algae on coral–algal interactions could happen
via chemical alterations of the algal host. For instance, extracts of
Lobophora overgrown by epiphytes have a superior action against
human immunodeficiency virus than extracts from Lobophora
without epiphytes. As Lobophora can harm corals via different
allelochemicals, amplified production or concentration of any of
these chemicals in the incidence of epiphytes could considerably
influence host competitiveness in coral–algal interactions.
Another probable explanation for the elevated competitive
potential of Lobophora with epiphytes is that epiphytes prevent
grazing on algal host. Competition with corals can encourage
increased algal production of allelopathic chemicals at the cost of
the production of anti-herbivore substances, which can result in
superior algal palatability. If epiphytes release Lobophora from
grazing pressure, they could further assist in production of
allelochemicals that improve algal competitiveness without the
50
usual associated trade-off of increased palatability. Other
parameters such as the duration of the coral–Lobophora contact or
the species composition of the epiphytal community may also
have had an influence on the outcome of the competitions.
These results afford strong sign that the competitiveness
of Lobophora against corals, particularly those within the genus
Porites, is superior when epiphytes are growing on Lobophora.
These studies imply a need to consider related epiphytic algal
communities when investigating coral–algal interactions.
1.14 Sponge Microbial association
Sponges are evolutionary ancient metazoans belonging to the
phylum Porifera. They are sessile organisms and are mainly found
in marine and freshwater habitats. Sponges are extremely
proficient filter-feeding animals with porous internal canal system
for circulating water throughout their body, providing nutrients as
well as takes part in waste expulsion. About 15,000 species of
sponges have been depicted so far, but their true diversity may be
higher. There are mainly four classes of sponges namely the
Calcarea (five orders and 24 families), Hexactinellida (six orders
and 20 families), Homoscleromorpha (one order and two families)
and Demospongiae (15 orders and 92 families), being the major
populated class.
Sponge-algae symbiosis
The Haliclona-Ceratodictyon association was accounted as
an obligate symbiosis and in this sponge-macroalgal symbiosis,
neither sponge nor alga grow separately or in association with
other species. As the Haliclona caerulea/Jania adherens
association has been depicted as an obligatory and mutualistic
association.
Sponge-fungi symbiosis
The examination of fungal association with the sponge has
been initiated through the molecular detection of the sponges
namely Suberites zeteki and Mycale armata collected from
Kaneohe Bay situated on the northeastern coast of the island of
Oahu, HI and accounted for more than 20 fungal species from
51
each sponges. The fungal species reported includes Cladosporium
sp, Aureobasidium sp, Penicillium sp, Hypocreales sp, Candida
sp, Ascomycota sp, Phoma sp under the phylum of Ascomycota
and Schizophyllum sp, Phlebia sp, Malassezia sp, Basidiomycete
sp under the phylum of Basidiomycota. The study has been
continued with the usual isolation process of fungal communities
from sponge samples Amphimedon viridis, Dragmacidon
reticulate, Mycale laxissima, Mycale angulosa and around 61
species have been isolated and grouped under the genus of
Aspergillus, Atheliales, Arthtiniun, Botryosphaeria, Mucor,
Pestalotiopsis, Rhizopus, Cladosporium, Fusarium, Glomerella,
Phoma, Trichoderma, Verticillium. However among the fungal
communities, around 8–25% has been depicted as unidentified
cultures. It is important to note that the fungal symbionts of
sponges have the ability to accept the elevated salinity and pH
levels and reported for its potential of lignocelluloses degrading
enzymes. The fungus Scopulariopsis brevicaulis isolated from the
sponge has been depicted to be originally originated from the
terrestrial soil.
Sponge-yeast symbiosis
The sponge Chondrilla nucula proved as a habitant for
endosymbiotic fission yeast by the TEM examination of egg and
adult tissue. It supposed to participate in a significant role in
sponge metabolism. The novel yeast isolate Leucosporidium
escuderoifa., sp. nov., has been reported form the marine sponge
Hymeniacidon sp collected from Fildes Bay, King George Island,
Antarctica. This sponge yeast symbiosis and method of the yeast
association has not been assessed well.
Sponge-polychaetes symbiosis
The polychaetes are commonly found in close immediacy to
sponges due to the occurrence of holes, grooves, chambers and
channels that protect them as well as perform as a good source for
providing nutrients. Among the polychaetes, Haplosyllides has
been found to be an obligate endosymbiont of the sponge
Xetospongia muta. Sponges are unique marine sedentary filter
feeders not proficient in capturing prey size >5μm. Expulsion of
such huge food particles (prey) in filter feeding mechanism of
52
sponges is a power dependent process. The relation of polychaete
worms could be favorable to the host sponges as the worms could
prey such large organisms and the excreta of digested prey could
be used as a dietary source of host sponges.
Sponge-barnacles symbiosis
Barnacles are the sedentary crustacean animals, normally
found in the intertidal and shallow subtidal zones of oceans. They
occur as free-living as well as symbionts, and they primarily cause
marine fouling because of encrustation on ships and marine
engineering devices. Symbiotic barnacles are found to be related
with various marine fauna starting from motile organisms such as
whales, sirenians, sea turtles, crocodiles, sea snakes, crustaceans,
and molluscs to sessile organisms including sponges, cnidarians
and bryozoans. Ilan and co-workers reported that eight diverse
species of barnacles residing in nine species of sponges
investigated from the Red Sea. As inferred by them, barnacles
exist on sponges face the risk of being overgrown and engulfed by
the host tissue. Two sponge species Theonella conica and
Callyspongia sp., are slow grower hence, less possibility to be
engulfed. Although sponge-barnacle association has been found
to be mutualistic, barnacles gain extra benefits, since the sponge
(host) resistance protects the barnacles from predation and
competition.
Sponge-mangroves symbiosis
Mangrove forests are regarded as one of the most vegetated
estuarine ecosystem of the marine as they encompass of numerous
flora and fauna flourishing in the nutrient limited, drenched and
anoxic soils. A transplantation study of Ellison and co-workers
depicted the facultative mutualism between the red mangrove
Rhizophora mangle and its root-fouling sponges namely,
Tedaniaignis and Haliclona implexiformis in the mangrove cays
of Central America. These root fouling sponges profit the
mangrove by facilitating the transfer of inorganic nitrogen to
adventitious root. The ecological interaction between sponges and
mangroves remains unclear and require comprehensive
investigation. The study accomplished by Hunting and co-workers
concluded that the tannins play a essential role in recruitment of
53
associated sponge Tedania ignis. The tannin and polyphenol
production in roots of mangrove plant Rhizophora mangle and its
responsibility for the sponge colonization were also reported.
Since, it is being a appropriate example of plant-animal symbiosis.
Sponge and coral association
Evolutionary connection between sponge and coral reef has
been increasing significance in the functions of marine ecosystem.
Figure: 7 Sponges are playing a extremely vital, beneficial
function in coral reef and ecosystem construction process, rather
than destructive. Studies on the association between sponge and
corals are not well understood due to the complexity in long term
monitor, sampling and follow-up, however sustaining the reef
ecosystem dynamics is being obvious and beneficial function.
Sponges are known to help corals in many ways including
providing nutrient (sponge loop), making suitable atmosphere by
clearing water and providing substrate (stabilizing dead coral
rubbles) for new recruits.

Figure: 7 Sponge- Coral association

Marine sponges are sedentary aquatic animals harboring

microbial symbionts upto 50% of sponge biomass. Sponge-
microbial relationship is a subject of investigation since unique
useful effects of host-symbionts remain unresolved. Albeit reports
54
depicts that sponge-specific symbionts are accountable for
synthesis and sequestration of sponge-derived bioactive
molecules, it has been accounted that the sponge-microbiome
consortia are not reliable in diverse niches and geographical
locations. The idea of microbiome dynamics, particularly change
the diverse sponge microbiome to precise abundant microbiome
for the synthesis of bioactive molecules, perhaps take place as a
response to external pressure such as predation or competition and
it should be mainstream focus of sponge microbiome
investigation.

55
2 Unit-II: Cultivation of Marine microbes
and Nutrient cycling

2.1 Methods of studying marine microbes

i) Isolation and Identification
The sediment samples were processed by means of formerly
accounted technique in order to assist the growth of spore forming
Gram-positive bacteria which are related with the production of
bioactive chemical entities. They were processed using one of four
process: Method 1 (wet/stamp), under sterile conditions, 90% of
the sea water was poured off, and a sterile cotton swab employed
to lightly stamp the sediments onto selected agar media; Method
2 (dry/dilute), a small spatula of sediment (c 0.5 g) was transferred
to a sterile glass Petri dish, permitted to dry overnight, and then
diluted with sterile sea water (5 ml). This was stamped onto the
agar surface using a sterile cotton swab; Method 3 (dilute/heat),
dried sediment was diluted with sterile seawater (1:4 dilution) and
heated to 55°C for 6 min, and the resultant suspension stamped
onto the agar with a cotton swab; Method 4 (freeze/dilute), frozen
sediment was thawed, diluted with sterile seawater (1:4 dilution),
and the diluted sample plated onto agar using technique 2 and 3.
The progressed samples were inoculated onto chosen media and
the microbes permitted to grow at 20°C for 2–6 weeks.

ii) Isolation media of culturable isolates

The isolation media consisted of the following: nutrient agar
(23 g) with either seawater or distilled water (1 L); marine agar
(55.1 g) with either seawater or distilled water (1 L);
actinomycetes agar (22 g) with distilled water (1 L); M1, agar (18
g), starch (10 g), yeast extract (4 g), peptone (2 g), rifampin (5
μg/ml), and natural seawater (1 L); M2 (10% M1), agar (18 g),
starch (1 g), yeast extract (0.4 g), peptone (0.2 g), and natural
seawater (1 L); M3, agar (18 g), starch (2.5 g), yeast extract (1 g),
peptone (0.5 g), glycerophosphate (0.2 g), natural seawater (750
ml), and DI water (250 ml); M4 (100% seawater agar), agar (18
56
g), and natural seawater (1 L); ISP2 (M987), agar (20.0 g), yeast
extract (4.0 g), malt extract (10.0 g), dextrose (4.0 g), and distilled
water (1 L)

iii) Cultivation
Single pure colonies from each one of the preferred
microorganism were developed in liquid media (1 L) analogous to
the agar media on which it was initially isolated. The flasks were
shaken at 200 rpm at 27°C and the fermentation allowed to
proceed for 5 days. The fermentation broth was pooled and the
microbial cells were detached from the broth via vacuum
filtration. The broth was extracted with a 50:50 methanol:
methylene chloride (2 × 500 ml), and the cells were extracted in
50:50 methanol:methylene chloride (500 ml). The organic
solution from the broth was detached in vacuo and the aqueous
portion re-extracted with ethyl acetate (2 × 500 ml). The organic
solutions from the broth and cells were dried individually with
anhydrous sodium sulfate, filtered, and concentrated in vacuo to
afford crude extracts. Other liquid media fermentations (2 L) were
scaled up accordingly. Gram staining, nucleic acid extraction, 16S
rRNA amplification, sequencing, and phylogenetic analysis.
The Gram reaction of all pure cultures was depicted via the
nonstaining KOH technique. The nucleic acid extraction
implicated the standard PCR isolation conditions. The reaction
was boiled for 10 min (after the addition of nuclei lysis solution)
to lyse the cells and the DNA pelleted by centrifugation at 13,000
rpm for 3 min. DNA concentration and purity were determined
using a nanodrop spectrophotometer. Absorbances at 230, 260,
280, and 320 nm were recorded. DNA purity was assessed by
dividing A260 by A280, with pure DNA comprising an A260/280
ratio of 1.8 (with a ratio of 1.4–2.0 being an acceptable working
range). Samples were diluted by means of sterile water to achieve
a DNA concentration within the range of 1–5 ng/μl for PCR
processing. PCR was performed using the primers 27F (5′-
AGAGTT TGATCCTGGCTCAG-3′) and 1492R (5′-
TACGGCTACCTTGTTACG ACTT-3′), aiming the 16S rRNA
region of the domain Bacteria. The cycling conditions were:
primary denaturation at 94°C for 1 min; 30 cycles of 94°C for 45
57
s, 55°C for 1 min, 72°C for 1.5 min with a concluding extension
at 72°C for 10 min. Amplified 16S rRNA fragments were
examined using agarose gel electrophoresis. The correctly sized
PCR products were cut out of the gel with a sterilized scalpel and
the DNA was purified from the agarose using a Qiagen’s gel
extraction kit presuming a gel weight of 200 mg.
DNA yield were sequenced on ABI 3730XL capillary DNA
sequencers at Sequetech Corporation in Mountain View, CA and
at the Dana Farber/Harvard Cancer Center DNA Resource Core,
Boston, MA. While all 16S rRNA fragments were partly
sequenced using the 27F primer, the almost complete 16S rRNA
gene sequences of four isolates, 23MM, 38M1, 40M1, and 42M1,
were acquired using with five additional sequencing primers:
1492R, 936R (5ʹ-GTGCGGGCCCCCGTCAATT-3ʹ), 519F (5′-
CAGCAGCCG CGGTAATAC-3′), 519R (5ʹ-
GTATTACCGCGGCKGCTG-3ʹ), and 1114F (5ʹ-
GCAACGAGCGCAACCC-3ʹ).(Gontang et al., 2007; Mao,
Zhou, Chen, & Quan, 2012). All nucleotide sequences were
collected, evaluated, and manually edited using the Sequencher
software package (version 4.8; Gene Codes Co., Ann Arbor, MI)
and contrast to sequences within the NCBI database
(http://www.ncbi.nlm.nih.gov/) using the Basic Local Alignment
Search Tool (BLAST). The partial 16S rRNA gene sequences of
all isolates and their adjacent type strain were investigated using
the phylogeny.fr ‘one click’ phylogenetic analysis tool
(http://www.phylogeny. fr/version2_cgi/index.cgi)
Morphological manifestation of agarolytic bacteria isolated
from Central Lombok Coast are diverse. Colonies did not generate
diffusible pigment. Cell-free supernatant of these strains formed
various halos of clearing in diameter after 6 h of incubation at
29°C. Agarolytic bacteria produce observable alteration on agar
because of the cleavage of polysaccharide chains, ranging from
softening of gel to agar pitting and extensive liquefaction. The
ability of these agarolytic isolates to produce clearance zone on
agar medium are various forms.
Seventh isolates exhibit agarolytic activity that differ is based
on clearing zone in diameter size that is formed, least is attained
58
by Alg5.1 strain, and biggest is formed by Alg4.2 strain. All of
isolates show elevated levels of agarolytic activity with clearance
zone in diameter more than 30 mm, except Abn1.2 and Alg5.1
strains. This clear zone diameter size differentiation is caused by
ability isolates in forming agarase enzyme, completion enzyme
kind agarase that produced and also by amount of enzyme
molecule that vary. Vera et al. (1998) divide agarolytic bacteria
into 3 groups based on size of enzyme molecule in the connection
with degradation activity gel jelly. Group I molecule weighing 30-
35 kDa, consist of Pseudoalteromonas atlantica ATCC 19291, P.
antartica N-1, Streptomyces coelicolor and Pseudomonas sp.
strain PT-5; Group II molecule weighing 50-59 kDa, consist of
Alteromonas sp. strain C-1, Pseudomonas sp. strain W-7 and P.
atlantica t6c; and Group III molecule weighing more than 100
kDa that comprising of Vibrio species. Group I and II known has
elevated ability in softening and jelly pitting because has low
agarase molecule that can diffuse agar pore. Group III usually has
low agar degradation ability as it has big relative molecule size.
2.2 Physiological and Biochemical properties
The results of various biochemical and physiological test for
these strain are shown in Table 3. All of strains were sensitive to
ampicillin, tetracycline, vancomycin, and rifampicin, Gram-
negative, aerobic-anaerobic facultative, oxidase positive, agar,
agarose and starch hidrolysis. All but the Alg5.2 strain cannot
form indole.

59
 Table: 3 Phenotypic characteristics of marine agarolytic
isolates.

All strain not grow at 8°C, not at all at 42° C, develop well at
3% NaCl and defectively at lower concentration. All strain can
degrade and make use of a variety of complex polysaccharides,
such as agar, agarose, and starch. Although Alg3.1 can hydrolyze
carboxy methyl cellulose but can not use it as carbon source
solely. These effect suggest that agar-liquefying bacteria might be
a high-quality candidate as a producer bioethanol from
agarophyte, as if we mix this strain with new bacteria or yeast so
D-galactoses can be catabolytic into pyruvic acid via Tagatosa or
Leloir pathway, additionally the fermentation of pyruvic acid
generate huge amounts of alcohol, acetic and formic acids.
Growth rate of isolates
Growth curve that connect between cell total with incubation
period is depicted in figure 8. During 24h observation, seventh
isolates show growth pattern that preceed by phase lag, then
followed by exponential phase, static phase and death phase in
60
various isolates.
Figure: 8 Growth curve of marine agarolytic bacteria
depending on number of colony (log10 cfu/mL) for 24 h
incubation period at 29°C

Figure: 8

All strains exhibit various growth rate. Four strains that is

Abn1.2, Alg3.1, Alg4.2 and Alg5.1 show elevated growth rate,
when contrast with Abn1.1, Alg2.2 and Alg5.2. growth rate
dissimilarity is caused by enzyme ability dissimilarity existence
and enzyme in preparation substrate essential for growth. In Lag
or adaptation phase various agarolytic strain grow between 0 and
2 h (that are Abn1.2, Alg3.1, Alg4.2 and Alg5.1 strains), while a
new strains (Abn1.1, Alg2.2 and Alg5.2) happen around 0 and 6
h. Long or the fast this adaptation phase is acquired in exponential
phase and bacterial growth rate. Exponential phase begun at with
time turn that differ. In Abn1.1 logarithmic phase happen around
6 up to clock 20, Abn1.2, Alg4.2 and Alg5.1 happen clock 2 up to
clock 12, Alg2.2 at 6 up to clock 16, and clock 2 up to clock 16 in
Alg3.1 strain. Extensive phase logarithmic period array from 10 -
14 clocks. Time deepness logarithmic phase to Abn1.1, Alg3.1
and Alg5.2 that is around 14 h, while Alg2.2, Abn1.2, Alg4.2 and
Alg5.1 that is 10 h. Normally unicellular microorganisms grow at
exponential phase rapidly because it is inclined by environment
situation likes temperature, chemical composition and genetics is
the characteristic of microorganism. Time differentiation that
wanted to initiate logarithmic phase and long period logarithmic
61
phase explain diversity of isolates speedily with the metabolism is
utilized for nutrition availability. Ability liquefaction agar towards
seventh isolates done to investigate the ability of liquefying agar
in marine agar medium in reaction tube. Liquefaction strength is
calculated based on liquefaction depth in millimeter. Three strains
has high ability in liquefies jelly, that is Alg3.1, Alg4.2 and Alg
5.1 of the size 10, 7.5 and 3.3 mm during 7 incubation days. On
the divergent all strain that come by gastrointestinal tract only can
pit jelly or form puncture around of colony. The pitting or
liquefying capacity vary in every agarolytic bacteria strain, based
on agarase enzyme that formed by isolate and degree of gene
expression of agarase enzyme that is quantifiable based on total
protein enzyme that generated. More complete agarase enzyme
ware that has more ideal jelly hydrolysis and concentration
excelsior monomer that formed. Such strain also can generate
agarase enzyme with high concentration will lead to high jelly
hydrolysis degree. In case Alteromonas sp. strain C-1 demonstrate
that strain this can liquefy jelly even though large the enzyme
molecule size (59 kda) because strain C-1 can to produce high
concentration of agarase enzyme.
2.3 Preservation of microbes
Five different process were compared, including the MO
(storage of macerates covered by mineral oil at 4°C) and GC
(storage of macerates at 20°C in 50% glycerol) methods, the RS
method (storage of macerates at 20°C using the commercial
ROTI-STORE system, and the SGT (storage of macerates in
sintered glass beads and 5% trehalose at 20°C) and SD (storage of
sections in 5% dimethyl sulfoxide [DMSO] at 70°C) methods
developed by us. Prior to preparation of material for storage, the
macroorganisms were soaked three times for 30 min each in 1 liter
of sterile SME, resultant in at least a 104 -fold reduction of
cultivatable bacteria from the medium contrast with original
seawater. To evade problems of survival of microorganisms
arising from frequent freezing and thawing, a total of four samples
were equipped for each storage process in parallel and employed
only once for each time point evaluation. For the SD method, an
aliquot of the sponge was cut off and flooded for an additional 30
62
min in 500 ml of sterile SME, comprising 5% DMSO. From this
DMSO-impregnated material, a core of 6-mm diameter was
detached with the use of a flame-sterilized cork borer. Two pieces
of ca. 5-mm total length were placed into a 2-ml screw-cap vial
containing ca. 3 mm of compressed dry ice. The vial, placed into
a box comprising crushed dry ice, was packed to the top with
crushed dry ice to result in quick freezing. The frozen vials were
accumulated in a deep-freezer at 70°C. For the SGT, RS, GC, and
MO methods, one single macerate was equipped from the SME-
washed sponge by cutting the material with the aid of a sterile
scalpel into 1-mm pieces and improving most of the liquid (10 ml)
by gentle pressure. Coarse debris was detached by filtering
through sterilized cotton, and this macerate was employed for
subsequent preparation of the SGT, RS, GC, and MO samples. In
the case of SGT samples, 500 l of macerate was transported into a
vial containing 500 l of filter-sterilized 10% trehalose in SME and
ca. 20 sinter glass spheres of 2- to 3-mm diameter. The amount of
glass spheres was adjusted to result in complete coverage after
mixing; the samples were stored up at 20°C. In the case of GC
samples, 500 l of macerate was added to a vial holding 500µl of
sterilized glycerol; after mixing, storage was again done at 20°C.
For preparation of RS samples, 500 l of macerate was added to
one ROTISTORE vial and assorted. The protocol suggested by
Roth Company asks for elimination of the liquid contained in the
vials and storage of dry glass beads in the vials. We could not
detect a difference in survival if the glass beads were used in the
recommended dry form or if the cryopreservation liquid added by
the supplier was not removed. For both cases storage was at 20°C.
In the case of MO samples, 500 l of macerate was overlaid by 1
ml of heat-sterilized (120 min at 160°C) paraffin oil, and trial
samples were stored at 4°C.
The MO method (as expected) should not be employed for
storage of macroorganismic material, because it very clearly
selects for a few phylotypes of recoverable bacteria. Selection will
take place by development of bacteria during storage, which was
experimented clearly. The MO method, indeed, was included in
our study only to be able to analyze which influence a assortment
force might exert on the diversity of recoverable bacteria. The GC
63
method did result in a survival of bacteria which is roughly 10%
in contrast to that for the other methods. In addition, the amount
of different colonies we could isolated from samples stored that
way also was little, which obviously speaks against the use of the
GC method for the reason of recovering a immense diversity of
bacteria. The RS method resulted in the recovery of 32 and 20%
diverse bacteria recovered after 1 and 6 months of storage,
respectively. These information are lower than those for the SGT
(37 and 32%) and SD methods (41 and 32%), but still the RS
process permitted us to recover many more different isolates than
the GC and MO methods. Consequently, the RS method well
might be considered to be used for storage. With respect to
handling, less hands-on time is required for the RS method and
the SGT method (2 h) than for the SD method (2 h versus 3 h).
The SD and SGT methods had been employed by us with the
rationale that addition of a cryoprotectant to biological samples
should assist in survival of microorganisms contained therein. The
SD method uses a quick-freezing protocol to further reduce cell
damages; a certain inhibitory effect of DMSO, though, was
observed. Trehalose— which we used in the SGT protocol—is
another extensively used cryoprotectant, not only for conservation
of microorganisms (5, 16, 18) but also for constant protein
preparations. In the case of the SGT method, the addition of the
SIRAN-Carrier glass spheres was essential; in parallel
experiments, survival of microorganisms was condensed by at
least fivefold if no glass spheres were added. Both the SD and
SGT methods were more or less equally proficient with respect to
recovery of different bacteria and also did not show great
dissimilarity with respect to survival of bacteria after storage (but
note that only the SD method allowed us to recover lower
eukaryotes). By macroscopic observation (difference in colony
types), the SD process seemed to result in the highest diversity of
bacteria to be recovered; this was also reflected to a certain degree
by the 1-month storage value. It has to be taken into account,
however, that the SD method asks for a more complicated
handling of samples and their storage at 70°C. The SGT method
in this respect is much less demanding by asking for normal
freezer temperatures of 20°C. We conclude that the SD and SGT
methods for storage of macroorganismic material result in equal
64
or elevated survival levels of total bacteria and allow recovery of
a higher diversity of bacteria than the other three process tested.
The choice of which of these two methods should be utilized
possibly will depend on the equipment accessible during
collection of macroorganisms
2.4 Role of microbes in carbon cycle
Marine ecosystems supply half of all biological carbon
fixation on Earth. Though, in order for long-term carbon
sequestration, photosynthetically fixed carbon required to be
transported to and accumulated in deep ocean waters and
sediments. The biological carbon pump (BCP) facilitate to fulfill
this function, transferring particulate organic carbon (POC) from
the ocean surface to its interior and thus causative agent for
climate modulation on geological time scales. The microbial
carbon pump (MCP) is another biological carbon sequestration
method. The essence of MCP is the microbial conversion of labile
dissolved organic carbon (LDOC) into recalcitrant suspended
organic carbon (RDOC) that is resistant to more biological
degradation and thus sustained in the ocean for decades to
millennia. Both structural recalcitrance and low concentration of
DOC molecules contribute to their persistence. However, there are
discussions on the relative assistance of these two distinct method
to the development of the huge RDOC pool in the ocean. These
debates advance study investigating the marine DOC molecular
composition. The MCP generates both structural recalcitrance and
a vast molecular diversity of DOC compounds each present at
picomolar or subpicomolar concentrations (i.e., below microbial
uptake thresholds) to avoid being further consumed.
Microorganisms shape the marine ecosystems and drive the
biogeochemical cycles. The carbon sequestration competence of
both BCP and MCP is mainly synchronized by microbial
communities. POC and dissolved organic carbon (DOC) are the
two diverse forms of organic carbon in the ocean, supporting
distinct carbon sequestration processes, correspondingly. The
formulation of the BCP concept started more than 35 years ago,
and investigated on this front has been being extremely active ever
since. Although the concept of MCP is quite new, its research is
65
gaining identification and momentum. In spite of the great study
efforts, neither MCP nor BCP has attained full understanding,
concerning their respective quantitative involvement to climate
modulation and the environmental and biological features that
may control their contributions and dynamics.
Organic matter offer the basis for the BCP and MCP to
function. Most organic matter in the surface ocean is formed by
primary producers. Thus, the marine primary production, which is
subject to both top-down and bottom-up controls, is a key feature
influencing BCP and MCP. Zooplankton grazing and viral lysis
influence the biomass, productivity, and partitioning of produced
organic matter (particulate vs. dissolved) of the primary producer
communities. The accessibility and chemical speciation of
nutrients participate a critical function in depicting the
composition, abundance, and productivity of the marine
photosynthetic microbial communities also, and different
phytoplankton may have diverse carbon export potentials.
Warming and nutrient scarcity may shift the phytoplankton
communities, favoring taxa with minute cell sizes, for instance
picocyanobacteria. Prochlorococcus are the most rich and
productive picocyanobacteria in oligotrophic oceans. They can
scarcely sink quickly enough on their own to straightly contribute
to BCP. However, this characteristic view is recently challenged.
Prochlorococcus are a possible source of transparent exopolymer
particles (TEPs) that develop marine aggregate formation and thus
assist the BCP. Heterotrophic bacteria may also play a function in
prompting TEP generation and aggregate formation of
Prochlorococcus. DOC released from Prochlorococcus via viral
lysis and other methods may fuel the MCP for RDOC production.
The whole ecosystem structure has newly been planned to mainly
set the carbon sequestration effectiveness. These instance
highlight the need of methodical and mechanistic examinations on
the marine ecosystem key players and interactions, chiefly in
terms of their carbon sequestration function and quantitative
contributions. Chemolithoautotrophic bacteria and archaea supply
organic carbon to the ocean as well. They not only play a key task
in sustaining the chemosynthetic ecosystems connected to deep-
sea hydrothermal vents and cold seeps but also may contribute
66
significantly to food web and energy transfer in non-extreme
environments. Dark carbon fixation may offer significant primary
production in definite marine water. Microbial degradation and
remineralization of marine particulate organic matter (POM)
considerably lower the BCP effectiveness. However, the identical
process regenerate nutrients and energy resource, likely playing a
task in fueling chemolithoautotrophy and partly offsetting fixed
carbon loss during sinking POM remineralization.
Chemolithoautotrophy may help increase the MCP as well.
Ammonia-oxidizing archaea, regularly leading in mesopelagic
and bathypelagic marine waters, liberate DOC to partially support
in situ prokaryotic heterotrophy. The role of
chemolithoautotrophy to the global ocean’s primary production,
BCP, and MCP warrants further examination. Even though the
ocean’s total primary production is at elevated levels (up to 50 Gt
C/year), only a minute fraction (90%). Heterotrophic microbes
uptake and respire DOC, and numerous particle-associated
microbes secrete extracellular enzymes to hydrolyze POC into
DOC. Respiration not only extensively lowers the BCP efficiency
but also might lead to deoxygenation and acidification in the
affected marine waters. Respiration may constrain the MCP as
well. Although respiration is a essential metabolic method and the
balance between respiration and primary production controls the
ecosystem carbon storage capacity, respiration is much less
investigated than primary production in the ocean. This condition
hinders our understanding of the ocean’s carbon cycle. For
instance, the subtropical gyres cover ∼40% of the Earth surface.
Though, whether these oligotrophic open oceans are overall
autotrophic (i.e., net CO2 sinks) or heterotrophic (i.e., net CO2
sources) is still being discussed, let alone convinced prediction of
their climate modulation potentials.

67
2.5 Role of microbes in Nitrogen Cycle

Figure: 9
Figure: 9 Nitrogen species implicated in N cycling and its
transformations. Each gray circle signify a N species, and the
number next to each N species designate its oxidation state.
Colored arrows characterize each N transformation and the main
marker genes involved: N2 fixation (nifH) in red, nitrification
(amoA, hao, nxrA, nxrB) in light blue, nitrate reduction (narG,
napA) in violet, DNRA (nrfA) in magenta, assimilatory nitrate and
nitrite reduction to ammonia (nasA, nasB, narB, nirA) in brown,
ammonium assimilation (glnA, gdhA) in gray, remineralization
(gltB) in dark blue, denitrification (nirK, nirS, norB, nosZ) in
aquamarine, anammox (nirS, hzsA, hzoA, hzoB) in orange, and
N-damo (nirS, nod) in green
Nitrogen is present in diverse oxidation states in the ocean,
ranging from -III in reduced forms like ammonium (NH4+) and
organic N to +V in fully oxidized nitrate (NO3−), which highlights
its significance as both an electron acceptor and donor for power
68
metabolism in marine ecosystems (Figure 9). Microorganisms
chiefly mediate the redox transformations of N, altering the
concentrations of N compounds in the ecosystem. The main
sources of fixed N for the ocean are biological N2 fixation (BNF)
and atmospheric deposition, while the main sinks are
denitrification and anaerobic ammonium oxidation (anammox).
Because modification of this balance caused by anthropogenic
action may pose major impact on marine ecosystem health,
biodiversity and climate change, the study of microbial
communities implicated in marine N cycling has gained great
awareness in current years. Microbial communities related to
marine N cycling have been studied extensively using both
culture-dependent and independent method. These technologies
have permitted the accomplishment of a wealth of genomic data,
enlightening enormous metabolic versatility within N-
transforming microorganisms. Additionally, the study of genes
encoding key metabolic proteins along with rate measurements of
N processes has offered significant discoveries about the genomic
possibility of microorganisms contributing in N processes and
their activity in marine system.
Marine N2 fixers (diazotrophs) alter dissolved N2 gas into
bioavailable ammonia (NH3). This is an extremely energy
necessitating process that only a small but various group of
bacteria and archaea are able to carry out. Marine diazotrophs
mainly comprise non-heterocystous filamentous cyanobacteria
(e.g., Trichodesmium, Lyngbya), heterocystous filamentous
cyanobacteria (e.g., Aphanizomenon, Nodularia), diatom
symbiotic cyanobacteria (e.g., Richelia), and unicellular
cyanobacteria (Ca. Atelocyanobacterium thalassa [UCYN-A],
Crocosphaera watsonii [UCYN-B] and Cyanothece [UCYN-C].
UCYN-A is additionally divided into at least four sublineages;
two of them (UCYN-A1 and UCYN-A2) live symbiotically with
discrete prymnesiophyte microalgae. Other marine diazotrophs
include heterotrophic bacteria (e.g., Klebsiella, Vibrio),
phototrophic bacteria (e.g., Chlorobium, Chromatium,
Rhodospirillum), strict anaerobes (e.g., Clostridium,
Desulfovibrio), iron (Fe) oxidizers (e.g., Thiobacillus),
methanogenic Euryarchaeota, and even members of
69
Planctomycetes. These microorganisms share a widespread
characteristic: the nitrogenase complex, which catalyzes N2
fixation. Nitrogenase is composed of two metalloproteins: the
molybdenum (Mo)–Fe protein (dinitrogenase) encoded in the
nifDK genes; and the Fe protein (dinitrogenase reductase)
encoded in the nifH gene. Alternative nitrogenases replace Mo
with vanadium or solely comprise Fe, and are encoded in the vnf
and anf genes, correspondingly. The nifH gene is the preferred
biomarker in the study of diazotroph diversity because it remains
highly conserved. Factors influencing BNF in marine systems
Oxygen (O2), light, temperature, inorganic N forms, phosphorus
(P), Fe, and organic matter are the chief feature that affect marine
diazotroph distribution. Nitrogenase is sensitive to O2, and
diazotrophs have developed numerous protective strategies
against this. For instance, several cyanobacteria generate
specialized N2-fixing cells called heterocysts that offer an almost
anoxic environment. Temporal separation is another protection
mechanism; most photosynthetic diazotrophs and certain
proteobacterial diazotrophs fix N2 at night, whereas
Trichodesmium and heterocyst-forming cyanobacteria fix N2
during the day. The ability of Trichodesmium to fix N2 during
daytime remains enigmatic. Several method have been
considered, such as its nitrogenase is restricted to specialized cells
called diazocytes, the photoreduction of O2 to H2O in the
photosystem I (the Mehler reaction), the uncoupling of CO2 and
N2 fixation in cyanophycin granules, or the down-regulation of
photosynthesis throughout the period of maximum nitrogenase
activity at midday, which recommend that light may also be a
determinant aspect for BNF. Interestingly, the symbiosis between
UCYNA (which lacks genes for oxygenic photosynthesis and C
fixation, Zehr et al., 2008) and its algal host has led to facilitate
daytime BNF, although nifH expression of UCYN-A has also
been experimented at night. Temperature is an vital feature
determining the distribution of diverse diazotrophs in the ocean.
Trichodesmium appears to be dispersed mostly in (sub)tropical
surface waters; while small diazotrophs have been found in a
broad latitudinal span, from colder surface waters to (sub)tropical
marine waters. It is significant to note that temperature may be
correlated with other feature that control the distribution patterns
70
of marine diazotrophs such as light, NO3− or O2. There is rising
evidence that BNF may not be as receptive to inorganic N as
previously thought, especially when P is not limited. P availability
influences BNF and the distribution of diazotrophs. For example,
cyanobacterial diazotrophs have been associated with high P
concentration in the Arctic Ocean and the Baltic Sea. High BNF
might be also related with denitrified waters in oxygen minimum
zones (OMZs), which are limited in N relative to P. Thus, N:P
ratio may play a critical function, as BNF rates at high inorganic
N concentrations can be offset when P is existing.
Diazotrophs need much more Fe for growth than other
microbes, and its bioavailability directly affects BNF in many
regions of the ocean. Fe is normally depleted in surface waters of
the open ocean, and Fe additions can arouse diazotrophic activity;
thus the delivery of dust rich in Fe to the ocean may finally
manage the rate and distribution of marine BNF. For example,
diazotrophs, particularly Trichodesmium, are rich in the North
Atlantic Ocean, in which dissolved Fe concentrations are
comparatively high as dust inputs are greater than in the South
Atlantic Ocean, where dissolved Fe concentrations are extremely
low. In addition, direct experimental measurements have verified
that marine BNF can be co-limited by both Fe and P availability.
At last, dissolved organic matter seems to arouse heterotrophic
diazotrophs in aphotic environments and coastal waters due to the
high-energy necessities of the reaction. Additionally, the interior
of C-rich particles may be appropriate for heterotrophic BNF.
Additionally, organic matter could encourage the mixotrophic
nutrition of Trichodesmium when inorganic nutrients are limited.
Distribution of diazotrophs in marine environment
BNF is particularly significant in extremely oligotrophic
environments such as open-ocean gyres, in which bioavailable N
is limited. Much of the studies on diazotroph distribution has
payed attention on Trichodesmium, which is chiefly found in the
North and Tropical Atlantic Ocean and the Arabian Sea, where it
often forms massive surface blooms. The domination of
Trichodesmium in warm oligotrophic waters of the Tropical
Atlantic Ocean seems to be due to a reduction of the N:P ratio by
71
an amplified uptake of inorganic N forms by non-diazotrophic
cyanobacteria, while its domination in the Northern Atlantic
Ocean seems to be due to elevated levels of dissolved Fe
concentrations. Diatom symbiotic cyanobacteria have a much
patchier distribution, possibly as the diatom hosts require silicon
to build their cell walls. They predominate in the warm ocean,
with the largest densities in the plumes of Amazon, Congo and
Mekong rivers.
Heterotrophic diazotrophs are also significant N2 fixers in the
global ocean, where they are omnipresent in the marine sunlit
layer. Heterotrophic Proteobacteria are prevalent in oceanic
environments, but govern in the Pacific coastal upwelling
systems, the South Pacific Gyre, the Indian Ocean, and the
Arabian Sea. Diverse and active heterotrophic diazotrophs are
also found in sinking particles, aphotic waters, OMZs, NH4+ rich
sulfidic-anoxic waters of the Baltic Sea, and colder waters such as
those in the Arctic Ocean, where nifH sequences associated to
anaerobic bacteria.
2.6 Role of microbes in Phosphorus cycle
Planktonic microbes in low- P environments (e.g., the
Sargasso Sea) are particularly frugal with P that they obtain from
their environments. Phospholipids and nucleic acids appear to be
the main reservoirs of P within the planktonic cells in the open
ocean, and recent investigation shows that plankton have evolved
method to economize on these biochemical P requirements. For
instance, using genomic evidence and direct interpretation, Van
Mooy(2006) show that Prochlorococcus and Synechococcus, the
picocyanobacteria that often govern low-P environments, mainly
synthesize a type of membrane lipid that contains sulfur and sugar
rather than more general lipid forms that contain phosphate (e.g.,
phospholipids). This switch from P-lipids to S-lipids reduce
cellular demand for P and may be an significant adaptation of
picocyanobacteria to the low-P environments in which they
regularly thrive.
An additional genome-enabled examination about P
limitation responses is the frequent presence of genes associated
72
to the high-affinity uptake of phosphate in the Sargasso Sea
environmental genome (SSEG) sequencing project and the
genomes of marine cyanobacteria, viruses, and eukaryotic
phytoplankton. For instance, there are numerous copies of the
gene for the high-affinity, phosphate-binding protein PstS in the
SSEG. There are also multiple copies of PstS in the marine
cyanobacterial genomes sequenced to date, including diverse
Prochlorococcus ecotypes and the nitrogen-fixing genera
Trichodesmium and Crocosphaera. Two cyanophage myoviruses
(P-SSM2 and P-SSM4) that contaminate Prochlorococcus also
both have copies of PstS. This specify the potential significance
of P to cyanophages and their hosts, with PstS expression probably
serving to enhance P acquisition during the infection of P-limited
hosts. P-related genes are not exclusive to the cyanophages. In
fact, a putative phosphate-repressible permease (ehv117) is
existing in the genome of the coccolithovirus EhV-86. The
coccolithophore host for this virus, Emiliania huxleyi, also has a
putative phosphate-repressible permease, which is up-regulated
under low-P conditions
Importance of Dissolved Organic Phosphorus
While Pi is normally regarded as the most bioavailable form
of P, its concentration in the surface waters of the open ocean is
much lesser than that of DOP (Figure 10). Yet, we know
surprisingly slight about the chemical composition or
bioavailability of DOP. Not unlike DOC and DON, only a small
fraction of the chemicals that compose DOP are amenable to
direct molecular characterization. For instance, dissolved
nucleotide triphosphates, which are implicated in cellular energy
storage and several biosynthetic pathways, can be directly isolated
and calculated

73
 Figure 10. A conceptual representation of suspended P pools,
their bioavailability, and P transformations in transversely of the
prokaryotic cell membrane

The phosphate pool and pathway is specified in black,

phosphoesters in orange, and phosphonates in green. Note the
comparative range of the different P pools; their likely
bioavailability is designated on the right of the figure. In this
presentation, we point out the possibility for microbial
metabolism of phosphate (through a high affinity system), general
phosphoesters (via hydrolysis by nucleases or phospholipases, for
instance), phosphomonoesters (via hydrolysis by alkaline
phosphatase – AP), and phosphonates (via a C-P lyase). We
emphasize that these are illustration routes, and the existence and
the localization of the transporters and enzymes revealed here may
vary significantly among microbes. For instance, the method of
phosphonate transport, hydrolysis, and accumulation as either Pi
or LMW DOP is not well characterized. We also emphasize here
several significant areas of P biogeochemistry that are poorly
understood and deserve extra consideration: (1) the occurrence
and efficient task of viral P-related genes; (2) the reactivity of
HMW phosphoesters, their form of hydrolysis, and their
transportation into the cell; (3) the cause and cycling of dissolved
phosphonates; (4) the composition and bioavailability of
74
LMWDOP; and (5) the frequency and specificity of microbial
phosphonate metabolism.
Even though there remains a woefully insufficient perceptive
of the linkages among organic P composition and bioavailability,
current molecular biological studies show that the comparative
bioavailability of P, whether exist as Pi, DOP, or PP, is a
potentially significant driver of microbial niche partitioning in the
sea. For instance, there appear to be substantial differentiation in
the presence, topology, and regulation of genes thought to be
concerned in P acquisition among strains of Prochlorococcus,
which may be a purpose of their adaptation to diverse P regimes.
Another example of niche partitioning is our recent perceptive of
the diversity of strategies for the use of P from DOP, such as from
phosphomonoesters or phosphonates (Figure 10). In the
picocyanobacteria, such as Prochlorococcus, some strains appear
to be able to hydrolyze phosphomonoesters, whereas others do
not. Furthermore, even when microbes may have the capacity to
hydrolyze phosphomonoesters, this action may be regulated
differently between diverse genera, and even different strains of
the same species over small spatial scales. With the resolution to
recognize alkaline phosphatase activity in single cells, it appears
that there may be significant heterogeneity in the niche
partitioning of planktonic microbes with regard to
phosphomonoester hydrolysis. This may also be the case for
phosphonate hydrolysis. A combination of genomic, culture, and
field interpretation imply that Trichodesmium has the genetic
capacity to metabolize phosphonate compound. Phosphonates
were normally considered to be an unavailable form of P for
phytoplankton growth until the discharge of the marine
cyanobacterial genomes revealed genes putatively concerned in
phosphonate metabolism. The ability to metabolize phosphonate
compounds may explain why Trichodesmium is so successful in
low-phosphate environments. Although Synechococcus may be
able to utilize definite phosphonate compounds, the strains and
phosphonate compounds tested to date recommend that other
picocyanobacteria (Crocosphaera and Prochlorococcus) are
unable to metabolize exogenous phosphonate as a sole P source.
As such, Trichodesmium appears to inhabit a unique niche with
75
regard to P metabolism along with marine cyanobacteria.
2.7 Role of microbes in sulphur cycle
Sulfate reducing microorganisms
The sulfate reducers encompass a very varied group of
anaerobic microorganisms, regularly of the Bacteria domain, with
catabolic capacities for a broad spectrum of fermentation
products. These contain primarily H2 and VFAs but also many
other substrates such as hydrocarbons or aromatic compounds.
Many Sulfate reducing microorganisms (SRM) belong to the
Deltaproteobacteria, including members of the Desulfovibrionales
and Desulfobacterales orders. The Desulfotomaculum are Gram-
positive bacteria, distinguished by the ability to form endospores.
The SRM found in marine sediments mostly belong to uncultured
groups that are only distantly associated to cultivated sulfate
reducers. Among the abundant SRM, a number of of which do
have cultured relatives, are the deltaproteobacteria
Desulfobacteraceae (in particular from the Desulfococcus and
Desulfosarcina cluster) and Desulfobulbaceae. Deeper in the
sediments, other taxa of SRM become predominant, such as the
phyla Firmicutes, Chloroflexi, and Atribacteria.
Recent genomic data from marine and terrestrial subsurface
environments have discovered the potential capacity for sulfate or
sulfite reduction in many other bacterial and archaeal phyla that
were not previously connected with this process.

Figure: 11
Figure: 11 Metabolic pathway of dissimilatory sulfate
reduction presenting the lively uptake of SO42− by a membrane-
bound sulfate transporter, the four steps in the reduction pathway,
76
and the passive release of H2S. Abbreviations: Sat, ATP
sulfurylase; APS, adenosine-50 -phosphosulfate; Apr, adenylyl-
sulfate reductase; Dsr, dissimilatory (bi)sulfite reductase; ETC,
membrane-bound electron transfer complex. Sulfur is green
whereas electron transfers to sulfur are specified in red
The functional significance of this broad diversity of SRM
for the marine sulfur cycle is currently not known. The known
SRM share a common pathway for DSR, which is illustrated in
Figure 11.
Sulfate is taken up from the environment by low- or high
affinity sulfate transporters and becomes stimulated with ATP in
the cytoplasm by the enzyme ATP sulfurylase (Sat) to form
adenosine-50 -phosphosulfate (APS). The APS is condensed to
sulfite by adenylyl-sulfate reductase (Apr), which accept electrons
from a membrane-bound electron transfer complex (ETC). The
(bi)sulfite is further reduced to H2S by the dissimilatory (bi)sulfite
reductase (Dsr) complex via a DsrC-bound trisulfide. The formed
H2S diffuses passively out all the way through the cell membrane.
Whereas this is the main forward direction of microbial sulfate
reduction, each step has a definite reversibility determined by the
intermediate substrate and product concentrations, which
mutually generate the forward thermodynamic drive. This enables
a partial back-reaction, which offers a mechanism for sulfur
isotope fractionation. Functional marker genes for the key
enzymes of DSR are employed to investigate the diversity of SRM
and to verify their distribution and abundance in the environment.
The SRM communities in the upper, bioturbated zone of the
seabed change distinctly from the deeper subsurface communities.
For instance, in studies from Aarhus Bay, between the Baltic Sea
and the North Sea, the microbial communities, including the
SRM, were found to have high diversity within the upper 5–10 cm
of bioturbated sediment.
The diversity decline with depth and age in the sediment,
concomitantly with a shift of the total community from strong
predominance of Bacteria to practically equal abundance of
Bacteria and Archaea at depth. Prominently, the assembly of the
subsurface communities, i.e., the establishment of their
77
assortment and community structure, was found to take place at
the base of the bioturbated zone, underneath which unusual
community members from the surface sediment persevere and
became leading as the community was gradually buried and
became isolated. The genetic and physiological diversity of the
subsurface communities was thus a effect of purifying selection
rather than of mutation. Such a purifying selection imply a gradual
loss, throughout many generations, of the less competitive species
and rising domination of the new competitive species beneath the
environmental conditions in the subsurface sediments. The
resultant reduction in species wealth of microbial communities
may persist for hundreds of thousands of years as the sediment is
steadily buried deeper.
SRM are dispersed throughout all biogeochemical zones in
the seabed, from the heterogeneous and chemically fluctuating
surface sediment all over the sulfate zone and deep into the
sulfate-depleted methane zone. The large quantity of
microorganisms decline with depth and age in the sediment and
so does the number of SRM cells. This was revealed in extracted
DNA by targeting diagnostic single-copy genes such as those
encoding for the alpha or beta subunit of dissimilatory sulfite
reductase (dsrAB). The decrease in abundance of SRM is even
steeper than that of the entire microbial community. In the top 5–
10 cm of sediment, which comprise a heterogeneous and variable
environment due to mixing (bioturbation) and irrigation by
burrowing macrofauna, the SRM may in fact contain up to 25%
of all microbial cells, while down through the sulfate zone this
number steadily drops below 5% and approaches 2– 3% at depth.
The relatively large quantity of SRM is elevated in the SMT where
the community feeds on methane in addition to the buried organic
matter. In the methane zone, SRM are also present, but in small
numbers. The relative SRM abundances cited here for subsurface
sediment are lesser than data acquired for the same sediments a
decade earlier by researchers. The dissimilarity may be ascribed
to new DNA extraction methods and to a larger diagnostic gene
sequence database for SRM, which has lead to more precise qPCR
primers for dsrB gene quantification.
Analyses of 16S rRNA and functional marker genes in
78
amplicon and metagenomic data reveal a huge diversity of
potentially sulfide oxidizing microorganisms in sediments. Of the
cultivated genera, for instance Thiobacillus and Thiomicrospira,
many are autotrophic or mixotrophic and couple the oxidation of
sulfide with chemoautotrophic CO2 assimilation. Yet, these
genera do not visible to be the chief, active sulfide oxidizers in
marine sediments. Examination with non-phototrophic CO2
assimilation in sediments have used 14C-microautoradiography or
13C incorporation into bacterial phospholipid fatty acids (PLFA)
to recognize which cells are implicated in dark, sulfide-dependent
CO2 fixation. By grouping of 14CO2 assimilation and gene
analyses, uncultured Gammaproteobacteria were recommended to
play the most vital function and to comprise 40–70% of the CO2-
fixing sulfide oxidizers. Researchers found that dark CO2 fixation
corresponded to 15–30% of the sediment oxygen uptake in a
coastal sediment, which would imply an extremely high growth
yield of the sulfide oxidizing bacteria, even elevated than that
found in pure culture.
The most prominent microorganisms responsible for sulfide
oxidation are the huge specialist sulfide oxidizers of the
gammaproteobacterial family Beggiatoaceae, such as the
filamentous Beggiatoa and Thioploca or the spherical
Thiomargarita. It should be distinguished that, through single-cell
sequencing of morphologically recognized sulfur bacteria, this
taxonomy was revised by Salman et al. (2011) who proposed to
divide the family Beggiatoaceae into seven novel Candidatus
genera. These large bacteria are normally limited to the surface
layer of organic-rich sediments where they can make use of steep
chemical gradients of sulfide and oxidants (nitrate and oxygen).
The bacteria have developed exciting adaptations to bridge the
spatial or temporal gap between sulfide and oxidants, for instance
motility and storage of nitrate and elemental sulfur. The nitrate is
accumulated in vacuoles up to several hundred mM concentration
and might maintain cellular respiration for days to months.
Elemental sulfur, produced as an intermediate during sulfide
oxidation, is accumulated in membrane invaginations in the
cytoplasm and supply an energy-rich electron donor for, similarly,
long periods. The filaments glide up and down in the several-cm
79
thick, seemingly oxidized surface sediment by casual (Beggiatoa)
or oriented (Thioploca) patterns of movement. Thiomargarita, in
contrast, is practically immotile, but the particularly large cells of
several hundred µm diameter have adequate storage capacity to
endure starvation from sulfide or nitrate for months.

80
3 Unit-III: Marine Extremophiles and
Bioremediation

3.1 Thermophilic microorganism

Importance of thermophiles
Most of the biorefineries utilize enzymes to generate the
preferred products. These processes need higher working
temperatures as they assist easy mixing, high mass transfer rate
and diffusion coefficient as well as enlarge the solubility of
compounds. It also declines the threat of contamination by
common mesophiles. The enzymes from thermophiles unlike the
normal enzymes are capable to withstand these elevated
temperatures and pressures of these bioreactors. Hence,
thermophiles have become a significant topic of concern due to
their capacity to endure at extremely high temperature and to
generate enzymes that can survive the harsh conditions of these
industrial processes. Another cause for the extensive awareness to
this group is because it may lead us to biomolecule stability and
the universal ancestor. From the view of evolutionary
mechanisms, knowledge about these organisms can assist us
understand the evolution of life on Earth. The possibility of life
elsewhere in the solar system and the universe can also be better
investigated as the circumstances are just as harsh. Many formerly
indefinite metabolic processes and novel compounds can also be
revealed that could have profitable applications one day.
Different types of thermophiles
The vast genetic and metabolic diversity present in elevated
temperature atmosphere reflect the array of pH, oxidation-
reduction states, solute concentrations, gas compositions, and
mineral composition that illustrate these surroundings.
Thermocrinis ruber, an aerobic, facultatively chemolithotrophic
bacterium, cultivate in alkaline hot spring in the Lower Geyser
Basin of Yellowstone National Park, USA between 44 and 89ºC
by oxidizing hydrogen, elemental sulfur, thiosulfate, formamide.
Deep-sea hydrothermal systems at a depth of 2600 m on the East
81
Pacific Rise maintain anaerobic autotrophic methanogens such as
Methanococcus jannaschii which grows optimally at 85ºC.
Meanwhile, the interaction of volcanic gases and seawater at
volcano in the Aeolian Islands and the solfatara fields of Naples,
Italy that generates acid solutions host acidophilic Archaea,
including Acidianus infernus, Thermoplasma volcanium, and
Metallosphaera sedula, which grow optimally at a pH near 2.8
Microorganisms which reside in these high temperature
surroundings are categorized into numerous groups depending on
their optimum temperature:

 Organisms which can stay alive underneath 45ºC are

facultative thermophiles.
 Organisms which have an most favorable growth
temperature less than or equal to 45°C but can develop at
temperatures better than 45°C as well are identified as
thermotolerant.
 Organisms with a best growth temperature between 45
and 60ºC are moderate thermophiles. Strict thermophiles
are those which have possible growth at temperatures
between 60 and 90ºC.
 Organisms which cultivate at temperatures superior than
90ºC are extreme thermophiles or hyperthermophiles

Thermophiles in marine environment

It is increasingly evident that surface thermal features and the
organisms they sustain are giving investigators a glance of what
life may be resembling in the deep subsurface. The metabolic
diversity observed in the deep biosphere depicts that
chemosynthetic organisms can take benefit of many forms of
energy that are enough to sustain life. These energy sources can
be associated to photosynthesis at the surface, as in the case of
heterotrophs that utilize organic compounds in sediments that are
the residue of photosynthetic organisms, or they can be entirely
independent of photosynthesis, as in the case of autotrophs that
gain energy and organic carbon by reacting CO2 and H2 provided
by geologic processes.
82
 The areas around hydrothermal vents are biologically more
useful in contrast to the bulk of the deep sea. They host complex
communities sustain by the chemicals dissolved in the vent fluids.
Chemosynthetic bacteria and archaea appears as the base of the
food chain, sustaining diverse organisms, as well as giant tube
worms, clams, limpets and shrimp.
The chemosynthetic bacteria develop into a thick mat and
attract other organisms, such as amphipods and copepods, which
nourish upon the bacteria. Superior organisms, such as snails,
shrimp, crabs, tube worms, fish, and octopi, form a food chain of
predator and prey relationships above the main consumers. The
major families of organisms existing around seafloor vents are
annelids, pogonophorans, gastropods and crustaceans.
Tube worms are a vital part of the community around a
hydrothermal vent. They may develop to over two meters tall and
have no mouth or digestive tract but as an alternative have a
special organ which houses chemosynthetic bacteria. They attract
nutrients formed by the bacteria in their tissues similar to parasitic
worms. The worm utilizes its plume of gills to accumulate the
chemicals the bacteria need. In return, the bacteria supply the
worm with chemosynthetically formed carbon compounds. The
two species that reside in hydrothermal vents are Tevnia
jerichonana, and Riftia pachyptila. Although invertebrates lead
vent communities, various communities are known to house
vertebrates like eels. For instance, Eel City located near Nafanua
volcanic cone, American Samoa.
Other model of the unique fauna which dwell in this
ecosystem are the scaly-foot gastropod Crysomallon
squamiferum, a species of snail with a foot reinforced by scales
made of iron and organic materials, and the Pompeii worm
Alvinella pompejana, which is capable of withstanding
temperatures up to 80°C.
Over 300 novel species have been revealed at hydrothermal
vents, many of them "sister species" to others accessible in
geographically separated vent areas. The instance of convergent
evolution seen involving distinct hydrothermal vents is seen as
main support for the assumption of natural selection and of
83
evolution as a whole.

Metabolism
Most of the life on our planet relies on plants by means of
sunlight energy to exchange water and CO2 into sugar
(photosynthesis). The sugar is then consumed by animals, moving
along food chains, eventually being decomposed back to water
and CO2. Hydrothermal vent communities are able to maintain
such vast sum of life because vent organisms depend on
chemosynthetic bacteria for food. The water from the
hydrothermal vent is wealthier in dissolved minerals and supports
a huge population of chemoautotrophic bacteria. These bacteria
utilize sulfur compounds, particularly hydrogen sulfide, and a
chemical highly toxic to most known organisms, to produce
organic material through the process of chemosynthesis.
Adaptations
The proteins of thermophilic organisms are capable to sustain
stable folds at elevated temperatures. The alteration required to
sustain stability at high temperature have been a focus of much
curiosity. The mechanisms experimented include an raise in
secondary structure propensity, changes that decline unfolded
state entropy such as the introduction of Pro residues, reduction of
Gly residues, smaller loops and the calculation of disulfide bonds,
an amplification of hydrogen bonds and salt bridges and better
optimized hydrophobicity. The most prominent distinction we
examine between the membrane proteins in mesophiles and
thermophiles is a general increase in the hydrophobicity of the
thermophile transmembrane helices. One observation made by
ressearchers is the amplification in small amino acids in
thermophiles. This could have two steady effects: (1) packing
small residues rather than huge residues lowers the entropy of
packing which could be predominantly significant at elevated
temperature; and (2) small residues can permit for more intimate
interactions among helices.

84
3.2 Haloalkaliphiles
The most remarkable examples of naturally occuring alkaline
environments are soda deserts and soda lakes. Particularly
alkaline lakes, for instance, Lake Magadi in Kenya and the Wadi
Natrun in Egypt, are possibly the most stable extremely alkaline
environments on Earth, with a reliable pH of 10.5 to 12.0
depending on the site. Several organisms isolated from alkaline
and highly saline environments such as soda lakes also need
elevated salinity, which is accomplished by adding NaCl to the
isolation medium. In particular, hypersaline soda lakes are
colonized by alkaliphilic representatives of halophilic archaea.
These red-pigmented microbes reach numbers of 107 to 108 /ml in
soda lake brines. A new haloalkaliphilic archaeon was isolated
from Lake Magadi. Cells of this organism have large gas vacuoles
in the stationary phase of growth, and colonies formed by these
archaea are bright pink. The GC content of the DNA is 62.7 mol%.
The name Natronobacterium vacuolata sp. nov. is proposed. The
type strain is chosen as NCIMB 13189. Lodwick determined the
nucleotide sequence of the 23S and 5S rRNA genes from the
haloalkaliphilic arachaeon Natronobacterium magadii. A
phylogenetic tree for the halobacteria was formed by alignment of
the 23S rRNA sequence with those of other archaea.
Consequently, Kamekura studied the diversity of alkaliphilic
halobacteria on the source of phylogenetic tree reconstructions,
signature bases precise for individual genera, and sequences of
spacer regions between 16S and 23S rRNA genes. They projected
the following changes: Natronobacterium pharaonis to be
transferred to Natronomonas gen. nov. as Natronomonas
pharaonis gen. nov. comb. nov.; Natronobacterium vacaolatum to
be transferred to the genus Halorubrum as Halorubrum
vacuolatum comb. nov.; and Natronobacterium magadii to be
transferred to the genus Natrialba as Natrialba magadii. Recently,
Xu et al. (200) isolated two haloalkaliphilic archaea from a soda
lake in Tibet. The two strains were gram negative, pleomorphic,
flat, nonmotile, and strictly aerobic. Growth needs at least 12%
NaCl and takes place among pH 8.0 and 11 with an optimum at
pH 9.0 to 9.5. On 16S rRNA phylogenetic trees, the two strains
produced a monophyletic cluster. They vary from their closest
85
neighbors, Halobacterium trapanicum and Natrialba asiatica, in
polar lipid composition, in addition to physiological and
phenotypic characters. DNA-DNA hybridization signifies that the
two strains belonged to diverse species of the same genus. The
results indicated that the strains should be categorized in a novel
genus, Natronorubrum gen. nov. Recently, Kevbrin isolated an
alkaliphilic, obligately anaerobic, fermentative, asporogenous
bacterium with a gram-positive cell wall structure from soda
deposits in Lake Magadi, Kenya. 16S rDNA sequence study of
this bacterium depicted that it corresponds phylogenetically to
cluster XI of the low-GC gram-positive bacteria. On the oringin
of its diverse phylogenetic position and exclusive physiological
properties, they projected a novel genus and species, Tindallia
magadii, for this strain. A novel osmolyte, 2-sulfotrehalose, was
revealed in numerous Natronobacterium species of
haloalkaliphilic archaea. The concentration of this novel
disaccharide amplified with increasing concentrations of external
NaCl, behavior reliable with its character as an osmolyte. Other
common osmolytes (glycine, betaine, glutamate, and proline)
were not accumulated or utilized for osmotic balance in position
of the sulfotrehalose by these halophilic archaea.
3.3 Osmophiles
Osmophilic organisms are microorganisms adapted to
environments with high osmotic pressures, such as high sugar
concentrations. Osmophiles are similar to halophiles (salt-loving
organisms) in that a critical aspect of both types of environment is
their low water activity, aW. High sugar concentrations represent
a growth-limiting factor for many microorganisms, yet
osmophiles protect themselves against this high osmotic pressure
by the synthesis of osmoprotectants such as alcohols and amino
acids. Many osmophilic microorganisms are yeasts; a variety of
bacteria are also osmophilic.
Osmophilic yeasts can cause spoilage of honey, chocolate
candy with soft centers, jams, molasses, corn syrup, flavored
syrups and toppings, concentrated fruit juices, and similar
products. Many of the common spoilage yeasts in this group
belong to the genus Zygosaccharomyces. Techniques for the
86
enumeration of osmophilic microorganisms have been reported by
numerous investigators. However, wide acceptance of standard
methods has not been attained. Generally, the enumeration of
osmophilic yeasts requires a consideration of the aw of both the
diluents and the plating media. Inaccurate results may be obtained
if a high aw diluent or agar medium is used or if the agar medium
has a reduced aw but the diluent does not. The type of solute used
to reduce the aw may also play an important role in enumeration
techniques. Glycerol, glucose, and sucrose are the most
appropriate solutes. Because of the particulate material in many
food products, the pour plate technique is the method of choice for
enumeration. Membrane filtration techniques have been
suggested for products having suspected low counts, low
viscosity, or that may require the isolation of contaminants. A
simple presence-absence test for the detection of small numbers
of osmotolerant yeasts in high-sugar foods may be useful for
qualitative purposes
Media
Dichloran with 18% glycerol (DG 18) developed by Hocking
and Pitt for xerophilic molds can also be appropriate for the
enumeration of moderate osmophilic yeasts. For the most
osmotolerant yeasts, malt extract-yeast agar with various amounts
of glucose depending on the types of foods and the species of
osmophilic yeasts suspected to be present has been recommended.
For example, Lenovich and Konkel recommended malt extract-
yeast extract 40% wt/wt glucose agar (MY40G), whereas Pitt and
Hocking suggested MY50G for the purpose of isolating and
enumerating osmotolerant yeasts.
Analysis of products by the membrane filter technique
requires that the sample be diluted with distilled water. Although
dilution may facilitate sample analysis, it may cause osmotic
stress to the mycoflora present by altering the aw of the sample.
The sterilization of high-sugar media may result in compounds
that are toxic to osmophiles. Steaming media containing high
sugar at 100uC for 30 min will destroy all microorganisms except
bacterial spores, which are unlikely to grow in media with more
than 60% hexose. Because of the high viscosity of many of these
87
plating media, it is important to swirl the plates more than one
would with standard plating media to ensure that the sample is
uniformly distributed throughout the agar. Incubation of plates
with low aw media in a forced-draft incubator will cause plates to
dry and prevent colonies from fully developing. Placing a shallow
pan of distilled water in the bottom of the incubator will prevent
drying.
3.4 Barophilic microbes
Barophile is a bacterium which desire to grow or entirely
develops at reasonably elevated hydrostatic pressures such as the
Challenger Deep in the Marianas Trench which has a intensity of
10,994 m. It is deeper than the height of the uppermost mountain
in the world, Mt. Everest. The Marianas Trench was created when
two oceanic plates converge with one plate downward into the
mantle to appear as a trough known as an oceanic trench. The
usual atmospheric pressure at the surface of earth is 0.1 MPa
(1.013 X10 ^5Pa). The base of the trench has enormously elevated
hydrostatic pressure, 1000 times greater than 0.1 MPa due to the
large column of water sitting above it. Barophilic bacteria are
finest modified with growth pressure superior than 40MPa
whereas reasonably barophilic bacteria cultivate ideally above 1
atm but less than 40MPa.
Physical Environment
The Challenger Deep is a extremely cold, exceedingly
pressurised environment with hydrothermal vents on the ocean
floor which liberate hydrogen sulfide and other minerals as energy
basis for barophilic bacteria. The vents supply energy for growth
to microorganisms for chemosynthesis as microbes are not
capable to perform photosynthesis due to lack of sunlight in deep
ocean. Temperatures can attain 300 degrees Celsius near vents.
The venting fluid is extremely acidic but surrounding water is
basic. The ocean floors consist of pelagic sediment which is
viscous as it contains shells, animal skeletons, decaying plants and
microorganisms. Despite the harsh environment, microbial flora
composed of extensive range of taxa for instance actinomycetes,
fungi, non-extremophilic bacteria and various extremophilic
88
bacteria has been revealed in the mud of Mariana Trench.

Proteobacteria gamma
Phylogenetic study of psychrophilic and barophilic deep-sea
bacteria from a variety of geographical locations are
constantly place them under one of the 5 Proteobacteria
gamma genera: Shewanella, Photobacterium, Moritella and
CNPT3 group. An unmanned submersible named Kaiko entered
the world’s deepest bottom deepth and improved sediment
sample. DNA was extracted from the sample, sequenced and
analysed. Three kinds of bacteria groups were revealed which
belong to the Proteobacteria gamma subgroup. The first two type
were directly related to Pseudomonas. The detection
of Pseudomonas verifies a diverse, prior examination
whereby Pseudomonas bathycetes was the primary bacterium
isolated from the sediment at Marianas Trench. The additional
bacterium belongs to the genus Shewanella.
Actinobacteria
Actinobacteria were selectively isolated from sediment
by inoculating in selective media: the isolates can be due to
the species Dermacoccus, Micromonospora, Streptomyces,
and Williamsia. The actinomycetes effectively cultivated in lab
consist of strains that were dissimilar from the terrestrial species
which had a reduced amount of development at elevated
pressures.
Geobacillus
Geobacillus include mostly of thermophilic
bacteria. Geobacillus sp. Strain HTA-462 which is not only
thermophilic but also barophilic was established at the bottom of
the Challenger Deep.
Microbial process
The influence of pressure can normalize the gene expression
of bacteria in deep sea ocean. Early investigations propose that
barophilic bacteria amplify the number of unsaturated fatty acids
89
in cell membrane to keep their cell membrane fluid at high
pressure. Though, later studies are recognizing the existence of
cell components in cell membrane. High-pressure- modified
bacteria Shewanella and Photobacterium sp. SS9 were isolated
from the deep-sea sediment.
Identification of pressure regulated genes in Shewanella
In the study of pressure-regulated genes from Shewanella,
downstream of promoter of barophilic strain, opening reading
frames (ORFs 1 and 2) forms the pressure-regulated operon.
Maximum transcript levels of these genes were experimented at a
elevated pressure of 70 MPa. Analogous sequences of ORFs 1 and
2 were also established in other deep-sea bacteria. Downstream of
this operon is ORF3 which is also stimulated by high pressure.
ORF3 encodes the CydD protein. CydD is accountable for
assembly of cytochrome bd complex which is one of the vital
components of Electron Transport Chain (ETC) in inner
membrane and hence may be significant for development at high
pressure.
Gene expression regulated by pressure in Photobacterium
sp.SS9
OmpH and OmpL encode porins or outer membrane
proteins. The ompH and ompL gene expression is inversely
regulated by pressure. There is high ompH expression
when Photobacterium is in elevated optimum pressure while high
OmpL protein expression at usual atmospheric pressure. OmpH
enables better uptake of nutrients which is vital in a low nutrient
environment like deep sea. Omp gene expression is regulated by
ToxR/S which senses pressure changes and can perform as an
activator or repressor depending on the pressure.
Ecological significance of barophilic bacteria
Barophilic bacteria increasing in sediment support life by
supplying source of carbon to benthic animals which ingest
bacteria. Barophilic bacteria also maintain each other
development by recycling nutrients. Some barophilic bacteria
decay organic matter, therefore releasing a variety of minerals
such as sulfates, phosphates and nitrates which are utilized by
90
other bacteria with diverse respiratory processes.

Potential applications of barophilic bacteria

Enzymes formed by barophilic bacteria can function at high
pressure; hence these enzymes may be utilized in elevated
pressure bioreactors, toxic clean-up in deep sea and high-pressure
food processors.
3.5 Psychrophiles
Earth is chiefly a cold marine planet: 90% of the water in
the oceans has temperatures of 5°C and 20% of the terrestrial
region of the Earth is permafrost (frozen soils), glaciers and ice
sheets, polar sea ice and snow covered regions. These regions
have cold-adapted microorganisms which have a limited
temperature range for growth. Stenopsychrophiles (formerly true
psychrophiles) have a higher temperature limit of 20°C for
growth. Though, the majorities of isolates are eurypsychrophile
(formerly psychrotolerant) and have a wider temperature series,
tolerating warmer environments. Psychrophile microorganisms
regularly exist in other extremely cold environments such as deep
oceans, caves, and land surfaces and even in the upper
atmosphere. They have been depicted in performing DNA
synthesis at −20 °C and lively metabolism at −25 °C. A variety of
stenopsychrophiles isolated from Antarctic have been analysed;
for instance the genera Arthrobacter, Gelidibacter, Halobacillus,
Halomonas, Marinobacter, Planococcus, Pseudoalteromonas,
Pseudomonas, Psychroflexus, Psychroserpens and Shewanella.
Methanogens, members of Archaea, are the only group recognized
to have individual species capable to cultivate in a very broad
temperature ranging from subzero to 122°C. The adaptations and
mechanisms associated to life in icy environments comprise
responses to cold shock and RNA chaperones. Cold shock
proteins (CSPs) act as cold-adaptive proteins in psychrophiles.
They are small proteins that bind to RNA to conserve its single-
stranded conformation and contain a nucleic-acid-binding
domain, known as the cold shock domain (CSD), and also they
have additional function besides serving as RNA chaperones.
91
Small RNA-binding proteins (RBPs) can assist cold adaptation
but together with the CSPs they have other functions in bacteria.
RNA helicases are regulated during cold growth and are able of
unwinding secondary structures in an ATP-dependent manner in
some psychrophiles. The presence of dihydrouridine can boost up
tRNA flexibility and is elevated in some psychrophilic bacteria
and archaea. Other features concerned in cold adaptation are the
assembly of secondary cold active metabolites, enzymes that are
activated and induced by cold, antifreeze proteins, and the
formation of pigments and membrane fluidity. From the structural
point of analysis, the proteins of these organisms have a superior
content of α-helix relative to the β-sheets, which is considered to
be a significant aspect to maintain flexibility even at low
temperatures. Also the cytoplasmic membranes of these
microorganisms contain an elevated proportion of unsaturated
fatty acids (52%) compared to mesophilic (37%) and thermophilic
(10%) organisms, support the maintenance of the semi-fluid state
of membranes. Marine psychrophiles take part in biogeochemical
cycling, polar food web and generate a wide variety of enzymes
including amylases, cellulases, peptidases, lipases, xylanases and
other classes of enzymes. High rates of catalysis at low
temperatures are usually achieved by the flexible structure and
low stability of cold-active enzymes. The most widespread
adaptive feature of cold-active enzymes is a reaction rate (kcat)
that is mostly independent of temperature. Furthermore, enzymes
adapted to lower temperatures permit efficient production at low
cost, save energy, and are significant in thermal protection,
consequential in progress in the quality of a range of products.
Enzymes suitable to low temperatures are utilized in the food,
cosmetics, pharmaceutical, and biofuels industries; also they are
functional to substances for molecular biology, nanotechnology,
in the manufacturing of household detergents, in the cleaning of
animal wastes, with peptidases for cleaning contact lenses and
pectinases to extract and clear fruit juices. An obvious illustration
of the profit produced by the industrial application of cold adapted
microorganisms is the utilization in the hydrolysis of lactose at
low temperatures in the process of milk storage. This bioprocess
has enabled the production of milk for patients with milk
intolerance and is now being patented for employing on a large
92
scale.
3.6 Hyperthermophiles
Marine biotopes of hyperthermophiles consist of a variety of
hydrothermal systems located at shallow to abyssal depths.
Analogous to ambient sea water, submarine hydrothermal systems
usually include high concentrations of NaCl and sulfate and reveal
a slightly acidic to alkaline pH (5-8.5). Otherwise, the main gasses
and life-supporting mineralic nutrients may be similar to that in
terrestrial thermal areas (Table 4). Shallow submarine
hydrothermal systems are established in various parts of the
world, mostly on beaches with active volcanism, like at Vulcano
island, Ischia island and Stufe di Nerone, Naples (all: Italy);
Ribeira Quente, Sao Miguel, Azores; Sangeang island, Indonesia;
Obock, Gulf of Tadjura, Dijbouti; and Kunashiri, Japan. The
hydrothermal system at Volcano is located close to the Porto di
Levante harbour among the Fossa and Vulcanello volcanoes. It
consists of H2S-containing submarine fumaroles, hot springs and
hot sandy sediments located in a depth of one to ten meters with
temperatures between 80 and 105°C. A variety of novel groups of
marine hyperthermophiles had been revealed at this place, some
members of which were found later to survive in other shallow
and abyssal marine hydrothermal systems, too. A further newly
revealed shallow submarine hydrothermal system is situated at a
depth of 105 meters at the Kolbeinsey Ridge (67°05'29"N;
18°42'53"W) which correspond to the northern continuation of the
Mid Atlantic Ridge. It had been visited earliest by the German
diving vessel Geo. Hot fluids with temperatures between 40 and
131°C and pH 6.5 are venting out from cracks and holes at the sea
floor.
Most impressive are the deep sea "smoker" vents, where mineral-
laden hydrothermal fluids with temperatures up to about 400° C
escape into the cold (2.8°C) surrounding deep sea water and build
up huge rock chimneys. Even though these hot fluids are sterile,
the surrounding porous smoker rock material appears to contain
very steep temperature gradients which supply zones of suitable
growth temperatures for hyperthermophiles. Some smoker rocks
are teeming with hyperthermophiles (for example 108 cells of
93
Methanopyrus per gram of rock inside a Mid Atlantic “Snake Pit“
hot vent chimney). Deep sea vents are situated along submarine
tectonic fracture zones and are known from several places which
can be visited for sampling by deep dive submersibles like Alvin
(U.S.A.), Nautile (France), and Mir (Russia). Powerful big black
smoker systems do exist at the Mid Atlantic Ridge deep sea floor
in a depth between 3000 and 4000 meters at the "TAG", "Snake
Pit", "Broken Spurr" and the "Moose" sites.
Type of thermal area
Characteristics Terrestrial Marine
Locations Solfataric Submarine
fields: steam- hot springs, hot
heated soils, mud sediments and
holes, surface vents ("black
waters; intensely smokers"); lively
originating hot seamounts
springs;
subterranean oil
stratifications
Temperature Surface upto Up to ~400
100°C; depth, °C ("black
above 100 °C smokers")
Salinity Frequently Generally
low (0.1–0.5% salt) ~3% salt
pH 0–10 5–8.5
(rarely: 3)
Major H2O, CO2, CO, CH4, H2, H2S, S0 ,
lifesupporting S2O32-, SO32–, SO42-b), NH4+ , N2, NO3– ,
gasses and nutrients Fe2+, Fe3+, O2 (surface)

Table: 4 Biotopes of hyperthermophiles

The temperature of the smoker fluids is generally between
200 and 360° C and the pH about 9. Additionally to regular black
smoker vent chimneys, beehive-shaped smoker vents are found in
94
the middle of the Mid Atlantic Ridge from which fluids with
temperatures of up to 244° C are seeping out. At the base of the
Mid-Atlantic smokers are frequently hydrothermally heated lava
rocks and minor sediments with much lesser temperatures
between 20 and 100° C. Based on the gasses and mineral nutrients
of the cooled-down venting fluids, an enormous chemosynthetic
production of microbial biomass by thermophilic and mesophilic
vent bacteria and archaea is going on, which correspond to the
starting point at an (aphotic) food web which includes a tight
assembly of a variety of higher life forms like deep sea shrimps
(e.g. Rimicaris excoculata), crabs, mussels and fishes. In contrast,
the surrounding deep sea floor is less in nutrients and,
consequently very poor in animals. Further deep sea vents are
located in the Pacific, for example at 21° N at the East Pacific Rise
in a depth of about 2700 meters. Many burning smokers with
temperatures upto 400° C are found there, at a lava-covered deep
sea floor without major sediments. Again, based on microbial
biomass there is an "island" of very rich deep sea life as well as
giant clams (Calyptogena magnifica), giant tube worms, up to 4
meters long (Riftia pachyptila) and many crabs and fishes.
Another widespread abyssal vent system is within the
Guaymas Basin. It is situated at the southern continuation of the
San Andreas fault in a depth of 1500 meters between the Baja
California peninsula and the main land of Mexico. The underneath
of this ocean arm is covered with organic sediments up to 400
meters in thickness, which are heated hydrothermally. The
temperature gradients within the sediments are vertical. For
instance, we had calculated at some sites temperature of 85, 105,
132, and 150° C at a sediment depth of 5, 10, 15 and 35 cm,
respectively. Additionally to the heated sediments, assemblies of
active black and white smoker chimneys and smoker flanches are
present; it shows the evidence of fluid temperatures of between
308-326° C (pH 7). The heated flanch rocks, analogous to walls
of smoker vents, contain a variety of species of
hyperthermophiles.
As we recognized recently, a further type of submarine high
temperature environment is provided by active sea mounts. Close
to Tahiti, there is an assembly of active sea mounts which
95
represent hot spot volcanoes located upon the Pacific tectonic
plate. At a depth of 1500 meters, there is the summit crater of
Teahicya Seamount which abunds in ferric iron hydroxide
minerals and harbours extensive hydrothermal venting systems. In
the same area, there is a new gigantic abyssal volcano, Macdonald
Seamount (28°58.7' S; 140°15.5'W) the summit of which is to be
found approximately 40 meters below the sea surface. In 1989,
during a French-German expedition, just when we approached the
Macdonald area, the seamount began to erupt. We, therefore, were
capable to discover for the initial time an erupting submarine
seamount and its surroundings for the existence of
hyperthermophilic archaea. On the first day of eruption
(24.01.1989) we took hydrocast samples about 2 to 4 kilometers
away from the active site. pH of the sea water was between 8.30
and 8.02 and methane concentration varied between 41.5 and 62.2
nanoliters per liter, which are usual values for surface open sea
water. After one quiescent day eruptions began again, and huge
patches of greyish slick of metallic appearance floated at the
surface. Some of this material was sampled. Next day there were
further strong eruptions, broken up by periods of silence.
Hydrocast samples were taken from water showing green
discoloration at the surface. They had unusually low pH values of
around 6 and enormously high methane concentrations of up to
7800 nanoliters per liter, representing that they came from the
submarine plume of the eruption. On the same day during a quieter
period, sampling within the active crater was challenged by the
submersible Cyana. Some lava rocks with orange-red
hydrothermal precipitates from the crater wall were composed,
but sampling ended when the volcano erupted again. No
hyperthermophiles could be supplemented from seawater samples
taken on the initial day at a distance of two kilometers from the
active zone. In distinction, samples taken from the floating
metallic grey-looking slick, the sea water from the submarine
eruption plume, and from rocks from the active crater contained
elevated concentrations of viable hyperthermophiles. As
expected, none of the hyperthermophiles supplemented was able
to grow at 60° C or below, and therefore within ambient sea water.
This reveals the existence of these organisms in active seamounts
and their liberation during submarine volcanic eruptions.
96
3.7 Halophiles
Halophiles are microorganisms that need salt (NaCl) for
growth, and they can be found in lakes, oceans, salt pans or salt
marshes. Additionally about 25% of the available land on Earth is
in the form of saline deposits. According to the best possible salt
concentration for growth, they are categorized in three categories:
(i) extreme halophile— develops in an environment with 3.4–5.1
M (20% to 30%) NaCl; (ii) moderate halophile—Grows in an
atmosphere with 0.85–3.4 M (3% to 25%) NaCl; and (iii) slightly
halophile—multiply in an surrounding with 0.2–0.85 M (1% to
5%) NaCl. Halotolerant microorganisms do not show total
requirement for salt to grow but develop well in elevated salt
concentrations.
Members of the family Halobacteriaceae have been isolated
from diverse habitats as well as alkaline and salt lakes, marine
salterns, the Dead Sea and saline soils. Deep hypersaline anoxic
basins (DHABs) or deep-sea hypersaline anoxic lakes (DHALs)
are intense habitats that have been revealed on the sea floor in
diverse oceanic regions, such as the Gulf of Mexico, the Red Sea
and the Eastern Mediterranean Sea. DHABs are composed by
dissolution of evaporitic deposits, captured in the sea floor,
forming extremely constant brine and harshly stratified in water
columns, a chemocline. The brines enclosed in these basins are
distinguished by hypersalinity, 5–10 times higher than seawater,
be deficient in oxygen and extremely reducing conditions,
elevated pressure (around 350 atm–35 MPa) and absence of light.
These physicochemical factors make the DHABs one of the most
severe environments of the planet and they have also make sure
that those habitats have been maintain isolated for thousands of
years.
Since the detection of the first Mediterranean DHAB named
Tyro in 1983, six others have been unveiled: l’Atalante, Bannock,
Discovery, Medee, Thetis, and Urania, and these habitats are a
cause of anaerobic halophilic microorganisms. Prokaryotes from
the Bacteria and Archaea domains belonging to novel taxonomic
lineages were revealed in high abundance in DHABs by 16S
rRNA libraries and fluorescent in situ hybridization (FISH).
97
Microbiologically DHABs of the Eastern Mediterranean are the
majority experimented. Halorhabdus utahensis constitutes 33%
of the total archaeal community and tolerates up to 0.8 M MgCl2.
In the Mediterranean Ridge, a DHAL named Kryos has been
identified. This lake is filled with MgCl2-rich, athalassohaline
brine (salinity > 470 practical salinity units). Two groups of
halophilic euryarchaeal divisions (MSBL1 and HC1) account for
~85% of the rRNA-containing archaeal clones analyzed in the
2.27–3.03 M MgCl2 layer. An earlier study implicit that 2.3 M of
MgCl2 was the upper limit of concentration for life to survive,
despite the fact that halophilic archaea have been identified in
deep hypersaline anoxic basins composed of saturating
concentrations of MgCl2.
In addition, some halophiles are thermostable and tolerant to
a wide range of pH. The metabolic diversity of halophiles is
widely spread, consist of the anoxic phototrophic, aerobic
heterotrophic, fermenter, denitrifying, sulfate reducers and
methanogenic organisms.
Halophiles have developed diverse adaptive strategies to
sustain the osmotic pressure stimulated by the high NaCl
concentrations in the surroundings they inhabit. Some extremely
halophilic bacteria gather inorganic ions (K+, Na+, Cl−) in the
cytoplasm, which is a kind of “salt-in” strategy to balance the
osmotic pressure of the environment, and they have also
developed specific proteins that are constant and energetic in the
presence of salts. Also, the halophile organisms contain enzymes
that sustain their activity at high salt concentrations, alkaline pH
and high temperatures. Majority of the proteins and enzymes
denature when suspended in elevated levels of salt concentrations.
This feature is reliant on the number of acidic amino acids on the
surface of the protein.
The role of electrostatic interactions in the stability and
folding of halophilic proteins has been examined and is an
significant determinant of haloadaptation. The intracellular K+
ions of haloarchaea have been found to be extremely high, near to
5 M. Based on the relative analyses of halophile and non-halophile
proteomes, the amino acid composition of halophilic enzymes is
98
in common distinguished by an rich content of acidic amino acid,
a maximum proportion of aspartic and glutamic acids, a little
frequency of lysine, and elevated incidence of amino acids with a
low hydrophobic character. Structural investigation between
halophilic and mesophilic proteins expose that the main
dissimilarity are concentrated on the surface of the protein. This
distinctiveness allows cooperation with electrostatic interactions
and the occurrence of a higher number of salt bridges. The
stability of the enzymes depends on the negative charge on the
surface of the protein owing to acidic amino acids, the
hydrophobic groups in the presence of high salt concentrations
and the hydration of the protein surface due to carboxylic groups
occuring in aspartic and glutamic acids. In addition, negative
surface charges are thought to be significant for the solvation of
halophilic proteins, to avoid denaturation, aggregation and
precipitation.
One of the alteration mechanism developed is the lipid
composition. Structural adaptations have been experimented in
the S-layers of halophiles. The extreme halophile includes
sulfated glucuronic acid residues and a higher degree of
glycosylation, leading to an increased density in surface charges.
This characteristic reveal an adaptation in reaction to the higher
salt concentrations experienced by Halobacterium salinarum.
Moreover, in Haloarchaea, some S-layer glycoproteins are
enhanced in acidic residues.
The halophiles have been utilized in biodegradation of
organic pollutants, in desalinization of wastewater, in
nanotechnology, in fabrication of biopolymers and as
osmoprotectors. The halotolerance of hydrolases resultant from
halophilic bacteria can be exploited wherever enzymatic
transformations are essential to function under physical and
chemical conditions, such as in the existence of organic solvents
and extremes in temperature and salt content. Many halophiles can
emit extracellular hydrolytic enzymes, such as amylases, lipases,
peptidases, xylanases and cellulases that are thermostable and
adjustable to an extensive range of pH.

99
3.8 Bioremediation of heavy metals
Microorganisms including bacteria and fungi play a
significant function in bioremediation of heavy metal
contamination. Microbial remediation of heavy metal
contamination is environment-friendly, proficient, cost-effective,
self reproducible and recycles bioproducts. Involvement of
microbial processes epitomizes reasonable and long-term solution
for remediation. Microbial processes are foremost to the
solubilization and immobilization of heavy metals play a essential
role in bioremediation. While removal of metal contaminants
from solid matrices like sediment and soils is assisted by
solubilization, immobilization is implicated in in situ
transformation of heavy metals and is mostly applicable to heavy
metal removal from aqueous solutions. Figure 12 depicts a variety
of microbial mechanisms implicated in detoxification and
transformation of metals.
As depicted in Figure 12, volatilization is one of the
significant mechanisms involved in heavy metal transformation.
Vidal and Vidal investigated arsenic metabolism (As(V)) in
marine yeast Rhodotorula rubra and based on qualitative
examination of metabolism products, reported generation of
As(III), methylarsonic acid [CH3AsO(OH)2], dimethylarsinic acid
(CH3)2AsO(OH) and volatile alkylarsines. It was experimented
that some of the produced As (III) was transported to culture
medium and the residual got methylated. Further methylation of
the dimethylarsenic acid eventually led to the formation of volatile
alkylarsine product. Researchers also accounted volatalization of
15.75% supplied trivalent arsenic from the culture medium by
Aspergillus sydowii.

100
 Figure: 12 A variety of microbial mechanisms concerned in
detoxification and transformation of heavy metals

In a case study, investigations elucidated the importance of

biofilm-forming bacteria in bioremediation of heavy metals,
particularly mercury. The authors recommended the bacteria like
Bacillus cereus BW-03 are proficient of synthesizing EPS like
polysaccharides, amyloids, biosurfactants and extracellular
enzymes that make them promising candidates for remediation of
inorganic mercury.
Heavy metal removal by Bacteria
Several microorganisms from the marine ecosystem have
been accounted to acquire heavy metal tolerance and removal
efficiency. Many of the reports also established multimetal
removal efficiency of either microbial cultures or their products.
Shirdam isolated three marine bacteria, Pseudomonas putida
PTCC 1664, Bacillus cereus PTTC 1665 and Pseudomonas
101
pseudoalkaligenes PTCC 1666 from the East Anzali wetland
sediments of the Caspian Sea. The isolates were resistant to
cadmium (Cd), nickel (Ni) and vanadium (V) and accumulated
about 40-50% Cd, 5-6% Ni and 10-12%.
Immobilized cells were experimented to be more proficient
in biosorption of heavy metals than free cells. The uptake of metal
ions was accounted to be in two stages viz. rapid and slow stage.
Metal ions adsorb onto the microbial surface through the rapid
stage while in the slow stage transportation of metal ions across
the cell membrane occurs.
Dash and Das isolated two extremely mercury resistant
isolates, Bacillus thuringiensis PW-05 (from the marine
environment) and Bacillus sp. SD-43 (from steel industry waste)
and experimented that the marine isolate reveal more mercury
volatilization ability. They recommended that marine bacteria
could be a enhanced choice for improved bioremediation of
mercury-contaminated sites.
Researchers experimented multimetal resistance in marine
bacteria extremely resistant to mercury (BHRM). The bacteria
supplied with 100 ppm initial metal concentration could eliminate
70% Cd (72h) and 98% Pb (96h). Volatilization (for Hg),
entrapment in extracellular polymeric substances (for Hg, Cd, and
Pb) and/or precipitation as sulfide (for Pb) were recommended to
be accountable for heavy metal detoxification.
A marine biofilm-forming mercury-resistant bacterium B.
cereus BW-03 was accounted as a suitable candidate for
remediation of mercury-contaminated waste. Improved
bioremediation capability of biofilm-forming bacteria was
recognized to the incidence of EPS, extracellular enzymes and
biosurfactants. The authors recommended that for mercury
bioremediation priority should be given to biosorption feature
than to volatilization aspect
Heavy Metal Removal by Fungi
Among inshore microbiota, fungi are vital members that
come across metal ions and complexes. Evaluation of mycobiota
for remediation of metal pollution is an stimulating area of
102
research. In India, Gujarat state encompasses the country’s 22%
coastline. Mycobiota from marine coastal habitats of Gujarat state
have been discovered to possess diverse ecologically important
potentials including removal of arsenic.
Taboski evaluated the toxicity of Cd and Pb to two fungal
species Corollospora lacera and Monodictys pelagica from the
marine environment by investigating their radial growth rate and
biomass accumulation. Biosorption of metals was also assessed.
Radial growth of fungi was not affected by lead, though,
increasing cadmium concentration condensed the radial growth of
fungi, particularly, M. pelagica. About 93% of lead sequestration
by C. lacera was found to be extracellular. M. Pelagica
bioaccumulated over 60mgg-1 Cd and over 6 mgg1 Pb. Over
7mgg-1 Cd and up to 250 mgg-1Pb was bioaccumulated by C.
lacera.
Khambhaty observed dead fungal biomass of four marine
Aspergillus species for Hg(II) biosorption and observed
Aspergillus niger as the most efficient Hg(II) biosorbent. Dead
biomass of A. niger exhibited 40.53 mgg-1 Hg(II) elimination
under optimized situation. Assessment of possible cell-metal ion
interaction discovered involvement of –OH and NH2 groups
present on the cell surface in Hg(II) biosorption.
Hexavalent chromium tolerant strain of Trichoderma viride
was isolated from water samples from the Mediterranean Sea. The
fungus possibly will eliminate 4.66 mgg-1 Cr(VI). It was
experimented by transmission electron microscopic assessment
that chromium accumulation by the fungus did not influence its
mycelial and conidial structures. Upon optimization of Cr(VI)
removal, factors like biosorbent dosage, contact time, initial metal
concentration and pH of the solution were known as influential
parameters.
Two marine fungal strains of Dendryphiella salina were
experimented to absorb 80–92% Hg2+ from the liquid media.
Strain Den32 had advanced absorption efficiency than strain. The
study discovered the possible application of both the strains for
bioremediation of mercury, particularly through biosorption.

103
3.9 Bioremediation of Oils
Hydrocarbon degrading bacteria and fungi are extensively
distributed in marine, freshwater, and soil habitats. Similarly,
hydrocarbon degrading cyanobacteria have been reported, even
though different reports specify that growth of mats built by
cyanobacteria in the Saudi coast led to conservation of oil
residues. Distinctive bacterial groups already known for their
capability to degrade hydrocarbons contain Pseudomonas,
Marinobacter, Alcanivorax, Microbulbifer, Sphingomonas,
Micrococcus, Cellulomonas, Dietzia, and Gordonia groups.
Molds corresponding to the genera Aspergillus, Penicillium,
Fusarium, Amorphoteca, Neosartorya, Paecilomyces, Talaro‐
myces, Graphium and the yeasts Candida, Yarrowia and Pichia
have been concerned in hydro‐ carbon degradation. Though,
reports in literature on the definite numbers of hydrocarbon
utilization are at discrepancy with one another because of the
methodological dissimilarity employed to specify petroleum-
degrading microorganisms.
Diverse petroleum-degrading bacteria reside in marine
environments. They have often been isolated as degraders of
alkanes or of such aromatic compounds as toluene, naphthalene
and phenanthrene. A number of marine bacteria proficient of
degrading petroleum hydrocarbons have been newly isolated.
These are bacteria of the genera Alcanivorax, Cycloclasticus,
Marinobacter, Neptunomonas, Oleiphilus and Oleispira within the
γ-Proteobacteria, and of the genus Planococcus within Gram-
positive bacteria. These bacteria, with the probable exemption of
Marinobacter and Neptunomonas, utilize limited carbon sources
with a preference for petroleum hydrocarbons and are thus
‘professional hydrocarbonoclastic’ bacteria. For instance,
Alcanivorax strains grow on n-alkanes and branched alkanes,
however cannot utilize any sugars or amino acids as carbon
sources. Likewise, Cycloclasticus strains grow on the aromatic
hydrocarbons, naphthalene, phenanthrene and anthracene,
whereas Oleiphilus and Oleispira strains develop on the aliphatic
hydrocarbons, alkanoles and alkanoates. Many ‘nonprofessional’
hydrocarbonoclastic bacteria have been isolated: for instance,
104
Vibrio, Pseudoalteromonas, Marinomonas and Halomonas have
been isolated as marine bacteria capable of degrading
phenanthrene or chrysene.
Fundamental reactions of aerobic degradation
The enter step of hydrocarbon degradation is the addition of
one oxygen atom, in several cases, two oxygen atoms, to the
hydrocarbon molecule, which is then transformed to an alkanol (in
the case of aliphatic hydrocarbons) or to a phenol (in the case of
aromatic molecules). In some species, an epoxide is the primary
intermediate. This activation formulate the hydrocarbon more
soluble in water, marks a reactive site, and introduces a reactive
site for the next reactions. The reaction need energy, which is
naturally generated via the oxidation of a reduced biological
intermediate such as NADH, which itself is reoxidized by an
electron acceptor. For the degradation of alkanes, diverse enzyme
systems are known which carry out the principal attack. An
omega-hydroxylase system consisting of three proteins (the
rubredoxin reductase, a rubredoxin and an omega-hydroxylase)
was isolated and characterized from Pseudomonas. In several
bacterial or fungal species as well as in mammalian cells, there are
enzyme systems which are reliable on cytochrome P450 acting as
a terminal oxidase. The major intermediates of the alkane
degradation are fatty acids, which are formed from the alkanols
via aldehydes. These acids can be further decayed by the pathway
typical of physiological carboxylic acid degradation, in which the
molecule is shortened. However, fatty acids can also be excreted
by the cells and collected in the environment
Once released, they can create ambiguous effects. On the one
hand, fatty acids can provide carbon source for bacteria of a
community, thus enhancing the hydrocarbon degradation. On the
other hand, fatty acids (chain length 14 C) can hinder growth and
hydrocarbon metabolism as they interfere with the cell membrane.
This aggravates a toxic effect and decrease growth. Diverse
degradative pathways have been confirmed for aromatic
substrates. The choice of the pathway depends on the category of
the organism and/or on the type of the aromatic molecule,
particularly on its substituents and (in the case of polyaromatic
105
molecules, PAH) on the number of rings. For an indication of the
fundamental possibilities of PAH biodegradation, three dissimilar
metabolic routes are well thoughout to be the main pathways.
Biodegradation strategy for crude oil removal from marine
environment. Seeding with genetically engineered
microorganisms (bioaugmentation with GEMs)
Although it was not obviously greater to native organisms
and has not at all been tested in the field, the frost organism still
patented was a microorganism genetically engineered to degrade
oil. The rationale for producing such organisms is that they might
probably be designed either to be more proficient than obviously
occurring species or to have the capacity to degrade fractions of
petroleum not degradable by obviously occurring species. To be
efficient, such microorganisms would have to overcome all of the
troubles related to seeding a spill with nonindigenous microbes.
EPA has not yet carried out any GEM product reviews for
commercial applications, although at least two companies are
making an allowance for using genetically engineered products
for remediating hazardous waste. Since the development and
utilization of GEMs are still restricted by scientific, economic,
regulatory, and public perception obstacles, the imminent
utilization of bioengineered microorganisms for environmental
cleanup is unlikely. Lack of a strong research infrastructure, the
predominance of small companies in the biore‐ mediation field,
lack of data sharing, and regulatory obstacles are all barriers to the
profitable utilization of genetically engineered organisms. The
progress of GEMs for application to marine oil spills does not
have high priority. Many individuals, together with EPA officials,
believe that we are so far away from recognizing the potential of
obviously occurring microorganisms to degrade marine oil spills
that the amplified problems related with GEMs make them
unnecessary at this time.
3.10 Biofouling and their control
Common fouling organisms in aquaculture
Although spatial and temporal dissimilarity survive in the
overall composition and biomass of fouling communities, in
106
general, a universal suite of sessile, suspension-feeding organisms
govern. These contain varied organisms including barnacles,
bivalves, bryozoans, polychaetes, ascidians, hydroids, sponges
and algae (Figure 13, Table 5).

Figure: 13 General fouling organisms related with

aquaculture operations: (A) Ciona intestinalis (vase tunicate);
(B) Ectopleura crocea (pink mouthed hydroid); (C) Mytilus
edulis (blue mussel); (D) Ectopleura larynx (ringed tubularia).

A normally occurring trait across the majority of these taxa

is their ‘invasiveness’. Many are international in their distribution,
being regularly transported around the globe by shipping, and
acquire a capacity to survive and replicate in a new location. An
instance of ‘invasiveness’ is the vase tunicate Ciona intestinalis,
107
now one of the mainly challenging fouling organisms in global
mussel culture, mainly in North America. Even though its native
range is indefinite owing to its unresolved taxonomic status, C.
intestinalis has extended to aquaculture industries in temperate
and tropical regions worldwide. It was primarily recognized on
the west coast of North America in the 1930s, and invaded the east
coast of North America in 2004. New populations have emerged
over the past 50 years along the coastline of Australia, New
Zealand, Asia, South Africa and South America. Given the
ongoing change in global climate and features that have assist the
spread of C. intestinalis and other invasive biofouling organisms,
there are compelling forecast for an amplification in their spread.
The impact of biofouling on aquaculture
While fouling community structure is spatially and
temporally variable, the impact of fouling is, in practically all
cases, extremely harmful to aquaculture. Surprisingly, though,
there are situation where biofouling is useful, or at least, does not
affect production. For instance, fouling can improve shellfish
growth, amplify primary production of phytoplankton and
consequently food availability to shellfish, provide shellfish with
security against predation facilitate the settlement of
commercially farmed shellfish or alleviate disease risk. These
examples, though, are the exemption, and biofouling is mainly
harmful to the cost effective production of shellfish and fish.
Shellfish

108
 Table: 5 An overview of common fouling organisms in
marine finfish culture and their documented adverse impacts

The effects of biofouling of shell surfaces and equipment fall

into five main categories: (1) physical injure by invasive
organisms that bore into the shell (endoliths) or epibiotic
calcareous organisms rising on the shell surface, affecting
aesthetics; (2) mechanical obstruction of shell function due to
colonisation of shells, mainly around the hinge and lip, affecting
feeding capacity and susceptibility to predators; (3) biological
competition for resources for instance food and space, affecting
development and condition; (4) environmental alteration due to
colonisation of culture infrastructure, leading to reduced water
flow, waste build-up, decline oxygen levels and condensed food
109
availability. In addition, biodeposition and the extend of non-
indigenous organisms can have toxic effects on surrounding
natural ecosystems; (5) amplified weight from biofouling biomass
on stock and equipment (eg panels, nets, ropes and floats), leading
to larger production costs related with extra maintenance
requirements and loss of stock and equipment.
Biological competition
Fouling species often fight with shellfish for food and space,
resultant in decreased recruitment success, inhibited development
and reduced product value. Food availability strongly manipulate
shellfish growth and competition for food resources between
shellfish and biofouling organisms can be considerable. Fouling
organisms are primarily filter feeders and even though the strength
of their competitive interaction depends upon resource limitation
and the species present, many foulers struggle with farmed
shellfish for food. The tunicate C. intestinalis and the mussel
Mytilus edulis are suspension feeders with overlapping inclination
in the mass range of particles they consume. These organisms
have very analogous clearance rates and as such food competition
may be considerable. Similarly, in oyster aquaculture, tunicates
struggle for phytoplankton, causing reduced growth. In addition
to food competition, shellfish may experience competition for
space and are mainly susceptible to interference competition from
overgrowth. For example, Ectopleura larynx can smother scallop
shells and attach their shells together, and overgrowth by colonial
ascidians can diminish the feeding ability of mussels.
The control of biofouling in aquaculture
The negative and important impacts that biofouling has on
viability and profitability of aquaculture has demand a long and
determined effort in biofouling control. Historically, the
aquaculture industry has borrowed antifouling (AF) technologies
from new marine industries which focus on chemical AF
technologies. AF paints to control biofouling are normally used
on surfaces in marine transport, oil and gas industries. These
paints leach biocidal compounds such as heavy metals and organic
biocides onto the surface, producing a thin, toxic layer which
avoids the onset of biofouling. However, many of the chemicals
110
and heavy metals concerned are documented as dangerous in the
environment, with harmful effects on the survival and
development of shellfish and fish and this has encouraged an
effort to avoid or mitigate biofouling in aquaculture through
alternative methods. As a result, biofouling control remains one
of the mainly complicated challenges and valuable production
issues facing the aquaculture industry.
Biological control
Biological control, where predation of pest species by other
marine organisms is employed to manage fouling levels, is a
functional management strategy in small-scale shellfish culture.
Natural control mechanisms negate the require for costly physical
and chemical treatments, and are safer for the health and wellbeing
of the shellfish and the growers. Examples from oyster culture
comprise the use of periwinkles, crabs and sea urchins. Though,
ensuring mobile organisms such as these remain on culture
infrastructure for extended time periods is challenging, mainly in
mussel culture. This may require modification of culture
techniques such as the addition of protective cages approximately
mussel socks for retention. The utilization of biocontrol in huge
scale shellfish culture is therefore tenuous.

111
4 Unit –IV:Sea food Microbiology

4.1 Distribution of pathogenic microorganisms

To effectively decide the human health risk related with
coastal and estuarine sediments, it is significant to measure the
size of the pathogen pool. The profusion of FIOs such as
Escherichia coli and Enterococcus spp. has been well studied,
however, further concentration is necessary for pathogens such as
Campylobacter spp., Salmonella spp., E. coli O157:H7 and
norovirus, which may be the reason for illness through shellfish
consumption or exposure to recreational water. Earlier
investigation has chiefly focused on them presence/absence of
these microorganisms in sediments, except for an apportionment
of risk, a quantitative approach is necessary. The reported quantity
of fecally related bacteria in coastal and estuarine environment is
characteristically between 0 and 104 colony forming units (CFU)
or most probable number (MPN)/100 ml for water and 101 to 106
CFU or MPN/100 g wet weight for sediment (Table 7 & 8).
Similar trends have been experimented in viral abundance in
marine and estuarine sediment (Table 7 & 8), though; the relative
difference in water/sediment abundance cannot be evaluated due
to the small sample size.

Table: 7 Fecal bacteria and viruses related with coastal and estuarine
sediments

112
 Table: 8 Abundance of fecal bacteria and viruses associated
with coastal and estuarine sediments

Nonetheless, Staggemeier directly evaluated the

concentrations of adenoviruses in equivalent water and sediment
samples resultant from freshwater streams, dams, and springs and
established that the viral abundance in sediment was considerably
higher than in the overlying water. Significantly, they established
that adenoviruses may be existing in sediment in the absence of
the virus in the water column. Researchers found that sediment
had better spatial changeability in bacterial abundance than water,
and that populations of enteric organisms can persevere in the
environment. The elevated natural variability in the sediment
fraction for both bacteria and viruses, has been related to
methodological dissimilarity in dissociation from sediment
particles which may effect in inconsistent enumeration. Pathogens
and FIOs also relate with suspended solids (flocs) in the overlying
water column. The floc fraction is prone to resuspend easily and
is a significant but poorly calculated contributor to bacterial
loading for water quality monitoring. However, flocs are
ephemeral and prone to break up on disturbance, which afford a
technical challenge to enumeration. Several studies, have reported
113
a decrease in the quantity of bacteria and viruses with sediment
depth. Current investigation showed a two-log reduction in
culturable E. coli from the sediment surface (top 1 cm) to 4 cm in
depth. Generally, the top 2 cm of sediment is considered to have
high FIO profusion whereas below 2 cm has considerably lower
abundance. Discrete seasonality of bacteria in sediments has been
experimented, with better abundance in autumn-winter months in
contrast to spring-summer months. In distinction, researchers
established that summer to autumn had better abundance in soils
and winter to spring had the lowest profusion. Investigations noted
a different diurnal pattern in E. coli abundance in the water
column, perhaps due to UV light inactivation, while the better
stability and shield from stressful conditions could decrease short
term changes in abundance. Physio-chemical conditions such as
temperature, turbidity, salinity, nutrient and oxygen
concentrations and water profundity are all significant feature
controlling the distribution of bacteria. The weather, season,
disease occurrence in the community; tides and freshwater inputs;
time of day; sediment type (sand/mud) and deposition rates;
distance from the shore; and predation by, and competition with,
the intrinsic microbial community also affects the profusion and
distribution of bacteria and viruses. The complexity of interacting
feature that influence pathogen and FIO survival in sediments
frequently restricts direct assessment between studies. Effective
surveillance alongside sufficient site/sediment categorization may
enable more insights into the influence of the sediment fraction on
bathing water quality. Reports recommend that the number of
infectious or culturable pathogens may associate poorly with the
number distinguished by molecular approaches. Consequently,
integrated surveillance schemes using both molecular recognition
of bacterial/viral genomes by PCR and culture-based methods
(e.g., bacterial culture or viral infectivity cell culture tests) may be
needed. However, high degree of inhibition at either the extraction
or genome quantification stages recommend that optimization and
standardization of molecular methodology in sediments is also
necessary. Enteric phages (e.g., F+ RNA coliphages) have been
employed as general markers of fecal pollution. Advantages of
this approach consist of, target specificity (each phage is typically
specific to one host) and their greater environmental persistence
114
in comparison to FIOs; typically 3-fold longer under controlled
conditions. In addition, source apportionment can be undertaken
using various genogroups of F+ RNA coliphages (e.g., I, IV for
animal and II and III for human). Concentrations of F+ RNA
coliphages were among 9 and 20 fold higher in sediments than the
overlying water column. Under controlled conditions, F+ RNA
coliphages show reduced correlation with E. coli, therefore cannot
be readily in contrast to larger historic datasets (usually E. coli or
intestinal enterococcus). However, coliphages correlate better
with disease occurrence and concentrations of pathogens (e.g.,
norovirus). Characteristically, next generation approaches are
being utilized for microbial source tracking (See Section
Outlook), however, F + RNA coliphages still offer a functional
indicator of viral culturability.
4.2 Indicator Organisms in Marine Environment
Indicators are needed for application in the marine ecosystem
to help guard the quality of shellfish beds and primary contact
recreational waters, and to avoid the spread of enteric disease.
Sewage treatment and subsequent dilution obtained upon entry
into rivers or by direct disposal into coastal waters via marine
outfalls and sludge dumping is monitored by bacteriological
methods. Unfortunately, marine and estuarine waters present
special problems, since the behavior of pathogens and indicators
differs from that in fresh water. Indicators used in freshwater
environments are not always appropriate for salt water.
The coliform group, i.e., total coliforms, are aerobic and
facultative anaerobic, Gram-negative, non-spore-forming, rod-
shaped bacteria that ferment lactose with gas formation within 48
h at 35°C. Total coliforms are the most universally used indicator
group, but include bacteria, in addition to Escherichia coil, that
are not specifically associated with fecal pollution, i.e., Klebsiella
spp., Citrobacter spp., and Enterobacter spp. Fecal coliforms are
differentiated from other coliforms 65 by their ability to produce
gas from lactose at 44.5°C within 24 h. Several techniques are
available for enumeration of total and fecal coliforms, including
tube enrichment and membrane filter methods, which are
continually being compared and modified.
115
 Enterococci have been proposed as indicators of fecal
pollution for coastal bathing waters. Enterococci are Gram-
positive cocci which grow at temperatures between 10-45°C, and
survive exposure to 60°C for at least 30 min, grow at pH 9.6 and
also grow in 6.5% sodium chloride. The enterococci are part oft
he genus Streptococcus, and include certain group Q streptococci
(e.g., Streptococcus avium), Streptococcus faecalis, Streptococcus
faecium and Streptococcus durans. The enterococci group
excludes Streptococcus bovis and Streptococcus equinus, which
are not usually found in human feces. Recently, Schleifer and
Kilpper-Balz proposed that S. faecalis and S. faecium be
transferred to the genus Enterococcus nom. rev. as Enterococcus
faecalis comb. nov. and Enterococcus faecium comb. nov. Fecal
streptococci comprise all these organisms, as well as
Streptococcus mitis, and Streptococcus safivarius, as a result, less
indicative of human fecal pollution. In fact, Moore and Holdeman
found that S. salivarius ranked 38th on a list of human fecal
bacteria, making it the most frequently encountered fecal
streptococcus in human feces, and placing just behind E. coli
which ranked 37th (the most abundant species was Bacteroides
vulgatus ).
Fecal streptococci are characterized by their ability to grow
in the presence of azide and ethyl violet at 35°C, in Azide
Dextrose Broth and Ethyl Violet Azide Broth, or to produce red
or pink colonies on KF streptococcus agar, lack the cataiase
enzyme, and develop in bile broth at 35°C. The enterococci have
a fecal source, stay alive in sewage treatment better than
coliforms, and are present in concentrations of 10-100 times less
than E. coli in treated sewage. The endurance of enterococci in sea
water is longer than that of E. coli and total coliforms, but
development does not usually take place in sea water, regardless
of the total salt concentration. Enterococci have not been
considered to be ubiquitous in the natural environment or to
multiply in water, even though biotypes of S. faecalis have been
isolated from insects and plant surfaces. The above cited qualities,
in the view of some investigators, make enterococci as valuable
indicators of fecal pollution.
The use of fecal coliform-fecal streptococcus ratios was
116
proposed by Geldreich and Kenner to differentiate between
human and other animal fecal pollution. Since fecal streptococci
endure longer than fecal coliforms in fresh water, the changing
ratio of fecal coliform-fecal streptococcus makes the use of this
ratio of limited and questionable significance
4.3 Prevention and control of marine pollution
Installing Overflow Pipe in Fuel Tank
When bunkering, if fuel is contributed to the fuel tank of a
ship beyond capacity, there is an opportunity of fuel flowing out
of the ship through the air-vent of the fuel tank.
In order to avoid this situation, an overflow pipe in the overflow
tank is installed in order that fuel oil should spill out of the fuel
tank flows into the overflow tank, and moreover, a flow detection
sensor in the pipe or a high-level alarm sensor in the tank in order
to detect overflow immediately.
Using Air Seal for the Stern Tube
Air seal for the stern tube on ships. An air seal is a machine
that continuously sends compressed air into the space in the
section where the propeller shaft pierce from the inside to the
outside of the ship. This creates a sealed area inside the stern tube,
which separates lubricating oil and seawater.
Use of electric powered deck equipment
We have commenced to our new ships electrically driven
deck equipment, such as a mooring winch (*1) and a ramp way
(*2) that used to be hydraulic powered equipment. This has
reduced the risk of leakage of hydraulic oil used for hydraulic
driving.
*1 mooring winch: a device for winding up a rope or a wire
to moor a ship.
*2 ramp way: a sloping path to be stretched to a quay when
cars are loaded onto a car carrier ship and when they are landed.
It is stored during a voyage.

117
Indirect Cooling System (Central Cooling System)
It is a machine for cooling the engine and lubricating oil by
indirectly exchanging heat with seawater via dedicated
freshwater. Use of this system avoid leakages or overboard spills
of lubricating oil owing to trouble in the cooling system, as
thermal exchange is not directly made between lubricating oil and
seawater in this system.
When sea creatures attach to the hull, the resistance of the
hull increases, and fuel consumption increases. This
outcome results in an increase in CO2 emission.
When those attached sea creatures are carried into other sea
areas during voyage, it will affect the ecosystem (of those
areas). Our companies support acceptance of low friction paint
for new ships to decrease fuel consumption and avoid
attaching of sea creatures, and are also trying to reduce
CO2 emissions and sustain the biological diversity.
We also support use of low friction paint, as well as existing paint,
for ships that are previously in service.
International and National standards of water quality
The most frequent and traditional approach for deriving
WQOs has been measurements of physical and chemical
parameters, and assuming that if these physical and chemical
factor can be sustained at certain level, the aquatic atmosphere will
be protected. However in more recent years it has been predictable
that these are largely indirect measures of the state or health of the
surroundings, and the substitute way is to monitor the biology of
the environments directly (e.g., ANZECC and ARMCANZ).
Nevertheless, the WQOs still play an important role in preserving
the health of aquatic ecosystems, as the factors concerned are
easier to measure and monitor than most bioindicators.
Water quality objectives can be an significant factor of any
framework for water resources management. In very extensive
terms, there are three diverse approaches to water resources
management adopted by overseas jurisdictions (CCME):
1) The technology-based approach: where limits on the
release of chemicals are based on some definition of what can
118
reasonably be attained technically/economically. As such the
standard for discharge into the receiving waters primarily depends
on the effectiveness of the treatment technology and the dilution
capacity available, whilst little or no consideration is given to
establish WQOs. This approach is normally adopted by
jurisdictions such as Germany, Japan, Malaysia, etc.
2) The use-protection approach: that essentially engage the
designation of beneficial uses/ environmental values to a water
body, and a proper mix of management options are useful to
ensure these uses/values are not compromised. In this approach
WQOs are the source for assess whether the designated
uses/values are being adversely affected. They can also be used to
back calculate to a corresponding effluent concentration. This
approach is generally adopted by jurisdictions such as Australia,
Canada, Europe, and US.
3) The non-degradation approach: where discharge limits are
recognized based on the natural background levels of substances
of concern at the site. This approach is in fact the strictest form of
the “use-protection approach”, and has normally been limited to
waters of high environmental value.
5) Most jurisdictions distinct beneficial uses (or intended
uses or functional uses or environmental values) to some extent
and recognized associated WQOs for a number of parameters.
This highlights the significance and distinction of the use-
protection approach and justifies in particular the setting of
WQOs.
6) In application, a mix of management approaches is usually
utilized. WQOs can be useful in benchmarking individual
technology-based approaches, are likely to be incorporated in the
mix of management approaches used to attain WQOs. WQOs can
also appear as part of the non–degradation approach if the
framework for establishing WQOs is broad and flexible, as in the
case of Australia (ANZECC and ARMCANZ) and the EU. In the
Australian state of NSW, and a lot of other jurisdictions, all three
approaches can be utilized at the similar time depending on the
location and condition.

119
 7) Throughout all of the jurisdictions, diverse types of
methodologies are utilized for deriving WQOs, for each of the
three type of factor of interest: toxic chemicals, physicochemical
characteristics (including nutrients), and microbiological
indicators. While there appears to be a consensus in the technique
used for derivation of WQOs for the latter factors (physical,
nutrients and microbiological), there are further disparate view
and ways of estimating WQOs for toxic substances. This fact
imitates the many gaps in knowledge about ecotoxicology, and
consequently translates in many uncertainties that formulate the
task of regulation and water policy quite difficult.
Microbiology of processed Finfish
Fish is a highly perishable food which requests proper
handling and preservation if it is to have a long shelf life and
maintain an attractive quality and dietary value. The central
concern of fish processing is to avoid fish from deteriorating. The
most evident method for conserving the eminence of fish is
to keep them alive until they are prepared for cooking and eating.
For thousands of years, China accomplished this through the
aquaculture of carp. Other process used to maintain fish and fish
products include

Iikejime process of fish slaughter

 management of temperature by means of ice, refrigeration

or freezing
 management of water activity by drying,
salting, smoking or freeze-drying
 the physical power of microbial loads through microwave
heating or ionizing irradiation
 the chemical management of microbial loads by adding
acids
 oxygen deprivation, for instance vacuum packing.
Usually more than one of these methods is utilized. When
chilled or frozen fish or fish foodstuffs are transported by road,
rail, sea or air, the cold chain must be preserved. This need
insulated bottle or transportation vehicles and sufficient
120
refrigeration. Modern shipping containers can unite refrigeration
with a controlled atmosphere.
Fish processing is also concerned with proper waste
management and with adding worth to fish products. There is a
rising demand for ready to eat fish foodstuffs, or goods that do not
need much preparation.
When fish are captured or produced for commercial
purposes, they require some preprocessing so they can be
transported to the next part of the marketing chain in a fresh and
undamaged circumstance. This means, for instance, that fish
caught by a fishing vessel require handling so they can be
accumulated safely until the boat lands the fish on shore. Typical
handling processes are:

 transporting the catch from the fishing gear (such as

a trawl, net or fishing line) to the fishing vessel
 holding the catch before further handling
 sorting and grading
 bleeding, gutting and washing
 chilling
 storing the chilled fish
 unloading, or landing the fish when the fishing vessel
returns to port
The number and order in which these operations are
undertaken differs with the fish species and the sort of fishing gear
utilized to catch it, in addition to how large the fishing vessel is
and how long it is at sea, and the nature of the market it is
supplying. Catch processing operations can be physical or
automatic. The apparatus and measures in modern industrial
fisheries are designed to decrease the rough handling of fish,
heavy manual lifting and inappropriate working positions which
might result in injuries.
Preservation techniques
Preservation methods are required to avoid fish spoilage and
extend shelf life. They are intended to inhibit the action of
spoilage bacteria and the metabolic alteration that result in the
loss of fish quality. Spoilage bacteria are the definite bacteria that
121
generate the unpleasant odours and flavours related with spoiled
fish. Fish normally host lots of bacteria that are not spoilage
bacteria, and the majority of the bacteria present on spoiled fish
participate no role in the spoilage. To flourish, bacteria require the
right temperature, adequate water and oxygen, and atmosphere
that are not too acidic. Preservation methods work by interrupting
one or more of these needs. Preservation techniques can be
classified as follows:
Control of temperature
If the temperature is declined, the metabolic activity in the
fish from microbial or autolytic processes can be condensed or
stopped. This is accomplished by refrigeration where the
temperature is dropped to about 0 °C, or freezing where the
temperature is dropped below -18 °C. On fishing vessels, the fish
are refrigerated automatically by flow of cold air or by packing
the fish in boxes with ice. Forage fish, which are frequently fixed
in large numbers, are regularly chilled with refrigerated or chilled
seawater. Once chilled or frozen, the fish require further cooling
to preserve the low temperature. There are in key concern with
fish cold store design and administration, such as how great and
energy proficient they are, and the way they are insulated
and palletized.
An efficient method to conserve the freshness of fish is to
chill with ice by distributing ice consistently around the fish. It is
a safe cooling process that keeps the fish moist and in a simply
stored form suitable for transport. It has become extensively used
since the progress of mechanical refrigeration, which makes ice
easy and cheap to generate. Ice is produced in a variety of shapes;
crushed ice and flake Ice, plates, tubes and blocks are normally
used to cool fish. Particularly effective is slurry ice, made from
micro crystals of ice produced and suspended within a solution of
water and a freezing point depressant, for instance common salt.
A recent development is pumpable ice technology. Pumpable
ice flows like water, and because it is homogeneous, it cools fish
quicker than fresh water solid ice process and reduces freeze
burns. It complies with HACCP and ISO food security and public

122
health standards, and utilizes less energy than conventional fresh
water solid ice technologies.
Control of water activity
The water activity, aw, in a fish is defined as the ratio of
the water vapour pressure in the flesh of the fish to the vapour
pressure of pure water at the similar temperature and pressure. It
ranges between 0 and 1, and is a factor that measures how
accessible the water is in the flesh of the fish. Available water is
essential for the microbial and enzymatic reactions implicated
in spoilage. There are a number of techniques that have
been or are utilized to tie up the accessible water or eliminate
it by reducing the aw. Traditionally, methods such
as drying, salting and smoking have been used for thousands of
years. These processes can be very easy, for instance, by using
solar drying. In more recent times, freeze-drying, water
binding humectants, and fully mechanical equipment with
temperature and humidity control have been added. Frequently a
combination of these techniques is utilized.
Physical control of microbial loads
Heat or ionizing irradiation can be used to destroy
the bacteria that is the reason for decomposition. Heat is applied
by cooking, blanching or microwave heating in a manner that
pasteurizes or sterilizes fish products. Cooking or pasteurizing
does not completely inactivate microorganisms and may required
to be followed with refrigeration to conserve fish products and
raise their shelf life. Sterilised products are stable at ambient
temperatures up to 40 °C, but to make sure they remain sterilized
they require packaging in metal cans or retortable pouches before
the heat treatment.
Chemical control of microbial loads
Microbial growth and proliferation can be inhibited
by a method called biopreservation. Biopreservation is
attained by adding antimicrobials or by increasing
the acidity of the fish muscle. Most bacteria stop
multiplying when the pH is less than 4.5. Acidity is
increased by fermentation, marination or by directly
123
adding acids (acetic, citric, lactic) to fish products. Lactic
acid bacteria generate the antimicrobial nisin which
further increase preservation. Other preservatives
comprise nitrites, sulphites, sorbates, benzoates and essential
oils.

Control of the oxygen reduction potential

Spoilage bacteria and lipid oxidation usually require oxygen,
so reducing the oxygen around fish can increase shelf life. This is
done by controlling or altering the atmosphere around the fish, or
by vacuum packaging. Controlled or altered atmospheres have
definite combinations of oxygen, carbon dioxide and nitrogen, and
the method is often combined with refrigeration for more effective
fish preservation.
4.4 Rapid diagnosis of contamination in sea
foods
Introduction
Rapid recognition of pathogens is vital for efficient disease
control in aquaculture. Discovery of pathogens is significant not
only in contaminated fish (clinically and sub-clinically), but also
in the environment e.g. among harvesting and re-stocking, and as
an 'early warning system. The function of antibody probes and
DNA primers/probes in pathogen detection and detection has
made a major impact on the development of such fast diagnostic
methods. Standardisation and corroboration of these methods,
though, has in most cases not been addressed.
Antibody-based tests
A range of antibody-based tests and molecular tests have
been developed to distinguish mainly bacterial and viral fish
pathogens, though tests have also newly been accounted for
parasites and fungal agents. The antibody-based tests comprise of
slide agglutination, co-agglutination/latex agglutination,
immunodiffusion, direct and indirect fluorescent antibody tests
(FAT and IFAT), immunohistochemistry (IHC) and enzyme
linked immunosorbent assay (ELISA), dot blot/dip stick and
western blot (WB). The antibody-based test chosen for the
124
recognition of pathogens depends on a diversity of features since
each method has its merits and disadvantages. Although such
process are useful for the findings of pathogens in pure culture
or/and in infected fish tissue, their sensitivity thresholds limit use
in environmental samples, particularly where pathogen levels are
enormously low. DNA detection methods, however, such as
polymerase chain reaction (PCR) and in situ hybridisation are
preferably suited.
DNA detection methods
Many bacterial pathogens can now be distinguished in
samples of a variety of kinds without the need to first culture the
organism. PCR techniques are not only highly precise and quick
but they also can lead to the discovery of 'non-culturable'
bacteria. PCR and in-situ hybridisation methods are
currently being developed for the discovery of numerous
fish pathogens. These consist of tests to recognize
numerous bacterial pathogens such as Renibacterium
salmoninarum; Aeromonas salmonicida; Vibrio
anguillarum, Photobacterium damsela subspecies pisicida,
formerly Pasteurella piscicida, and many others are in progress.
Examples of antibody and DNA based methods currently in use.
A variety of quick technique for pathogen recognition and
discovery has been developed at the University of Stirling. These
will be employed to demonstrate the application of such tests, and
provide to pin-point areas necessitate standardisation. All the tests
need validation in the field. The tests developed comprise
IFAT, IHC, ELISA (detection of pathogen/antibodies), PCR
(one cycle/nested/reverse cross blot hybridisation/RT-
PCR/quantitative PCR), and in-situ hybridisation. These
assays are carry out on a diversity of pathogens, as
well as bacteria (Aeromonas salmonicida, Aeromonas
hydrophila, Photobacterium damsel subspecies piscicida,
Renibacterium salmoninarum, Mycobacterium spp., Vibrio
anguillarum and Flavobacterium psychrophilum; parasites,
viruses (infectious salmon anaemia virus), and fish rickettsia. In
various instances the antibody-based methods are adequate and
attain the essential sensitivity and specificity. On other juncture,
125
for example in water samples and sub-clinical disease, the
amplified sensitivity of DNA-based methods is necessary. Again,
the process chosen depends on a variety of features.
PCR and in-situ hybridisation
PCR is used for the discovery of pathogens in blood,
water, sediment and tissue samples owing its high sensitivity
and specificity. In our laboratory, we employed this
technique to identify Mycobacterium spp., F. psychrophilum, R.
salmoninarum and ISAV. This involves detection to species
level in some cases e.g. M. marinum, M. fortuitum and M.
chelonae can be recognized using reverse cross blot PCR. This is
usually achieved on fresh or frozen samples. Fixed samples on the
other hand are analysed using in situ hybridisation. Both PCR
and in situ hybridisation are employed in preference to antibody-
based technique for the detection of the parasite PKX as
recognition with antibodies is life cycle dependent.
Detection of PCR products
PCR can be achieved as a single or nested assay, and the
products can be recognized using a variety of methods. Our
present tests include single/nested PCR (PKX, R.
salmoninarum), reverse cross blot hybridisation to recognize
multiple species in a single test (Mycobacterium spp.), RT-PCR
for RNA viruses (ISAV), and quantitative PCR (F.
psychrophilum). Each assay has been optimised and the technique
selected according to the type of pathogen and the kinds of
samples requiring examination. For instance, the detection of F.
psychrophilum is necessary in environmental samples and fish
tissue. Quantitation is also desired so that upward trends in
pathogen titre can be identified. This is accomplished with this
particular assay by linking an ELISA to the PCR to detect the
products.
Standardisation, validation and interpretation of results
Clearly, the standardisation of technique is critical. The
extraction method and primers used will influence the results of
the PCR and these should be clearly defined. The use of PCR
beads minimises the threat of contamination and extreme concern
126
should be taken with nested PCR. The inclusion of the entire
appropriate positive (sometimes a series of DNA positive
standards is required) and negative controls is necessary if the
results are to be interpreted properly. The investigation of results
may be difficult in some cases, particularly with tissue samples
where tissue may reduce the reaction. Table 8 demonstrates the
interpretation of results from a typical PCR test in laboratory. As
well as having the apparent positive and negative controls it is
required to include spiked samples i.e. a known positive control is
added to the sample.

Table: 8 Interpretation of results from a typical PCR test

This controls for inhibition by the tissue and false negative

results. Generally the non-spiked samples are run in duplicate,
while the spiked samples are run singly. Thus, three results are
produced from each sample. A decision is then taken on whether
to recognize the results as positive or negative, or to repeat the
test. A positive result is predicted from the spiked sample on each
occasion, regardless of the result from the actual test sample (the
non-spiked sample). A negative result can, though, sometimes be
produced by the spiked sample if the DNA level is high, resulting
in inhibition. If this is the case then assessment of the spiked
results with those of the test sample (run in duplicate) results
should guide to a definitive elucidation; whether this is to report
positive, negative or to replicate the test. As shown in Table 8 the
127
test would need to be repeated if all three samples were negative
in the test, or if the non-spiked duplicate samples resulted in one
positive and one negative.
The PCR tests developed in our laboratory are proficient of
detecting the existence of specific pathogen DNA. As long as the
test is optimised, standardised and all the suitable controls are
included, the results can be interpreted and appear to be reliable.
The actual meaning of the effect with regard to disease, however,
has not been established as the tests have not been fully
authenticated in the field. It is significant to note that both the
antibody and DNA-based methods do not essentially detect live
pathogen. A positive result will also be attained with non-viable
pathogen, or even merely divisions of a dead pathogen. Therefore,
justification of the technique in the field to see how a positive
result associates with disease and illness is vital. Blind trials need
to be carried out to confirm that a negative is always negative in
the test, and assessment conducted with other methods. Some
preface work has been conducted on the validation of the F.
psychrophilum, Mycobacterium spp. and PKX tests in the
laboratory. There are also many reports of the application of PCR-
based techniques to the discovery of fish pathogens and these also
need validation.

128
5 Unit – V: Marine Microbial Products

5.1 Properties of carrageenan

The chemical reactivity of carrageenans is primarily due to
their half-ester sulfate groups which are strongly anionic, being
comparable to inorganic sulfate in this respect. The free acid is
unstable, and commercial carrageenans are available as stable
sodium potassium and calcium salts or, most commonly, as a
mixture of these. The associated cations together with the
conformation of the sugar units in the polymer chain establish the
physical properties of the carrageenans. For instance, kappa- and
iota-carrageenans form gels in the existence of potassium or
calcium ions whereas lambda-carrageenan does not. The
functionality of carrageenans in various applications depends
mainly on their rheological properties. Carrageenans, as linear,
water-soluble, polymers, characteristically form highly viscous
aqueous solutions. Viscosity depends on concentration,
temperature, the existence of other solutes, and the type of
carrageenan and its molecular weight. Viscosity amplifies nearly
exponentially with concentration and decline with temperature.
Carrageenans are at risk to depolymerisation through acid-
catalysed hydrolysis. At elevated temperatures and low pH this
might quickly lead to complete loss of functionality.
Anticoagulant and antithrombotic activity
Carrageenan is classified into different types, all containing
22–35% sulphate groups. Several information exists on the
anticoagulant activity of carrageenan. Along with the carrageenan
types, λ-carrageenan has about twice the activity of unfractionated
carrageenan and four times the activity of κ-carrageenan. Though,
the most active carrageenan has roughly onefifteenth the activity
of heparin. The major basis of the anticoagulant activity of
carrageenan appears to be an anti-thrombic property. λ-
Carrageenan showed greater anti-thrombic activity than κ-
carrageenan possibly due to its higher sulphate content, while the
activity of the unfractionated material was somewhere between
the two. λ-Carrageenan consistently extended the clotting time
129
and was more poisonous than κ-carrageenan. The dissimilarity in
sulphate content between the two carrageenans did not correspond
directly to dissimilarity in anticoagulation action and toxicity. As
declared above, the mechanism underlying the anticoagulant
activity of carrageenan invloves thrombin inhibition. λ-
Carrageenan has been revealed to potentiate the inactivation of
thrombin by ‘anti-thrombin BM’. These interpretation would
therefore imply that there is either anti-thrombin potentiation via
heparin co-factor II (HC-II) and/ or a direct anti-thrombin effect.
Antiviral activity
Carrageenan is a discerning inhibitor of a number of
enveloped viruses, as well as human pathogens as human
immunodeficiency virus, herpes simplex virus (HSV), human
cytomegalovirus, human rhinoviruses and others. Carrageenan
acts mainly by avoiding the binding or the entry of virions into
cells. This discovery is consistent with the fact that carrageenan is
similar heparan sulfate, an HPV cell-attachment factor.
Carrageenan extracted from seaweed, is an extraordinarily potent
inhibitor of papillomavirus infectivity in vitro. It was found to be
active against a range of common sexually transmitted HPV types
that can cause cervical cancer and genital warts. Carrageenan is
too active in vitro and in murine model systems in opposition to
other viruses, as well as herpes simplex viruses and some strains
of HIV-1. However, in vitro IC50 values for carrageenan
inhibition of herpes simplex virus and HIV-1 infectivity are about
a thousand-fold superior than the IC50s experimented for
carrageenan inhibition of genital HPVs in vitro. Researchers
reported that the λ-carrageenan and partially cyclised μ-/ι-
carrageenan from Gigartina skottsbergii demonstrate strong
antiviral result against diverse strains of herpes simplex virus
(HSV) type 1 and type 2. Analogous results were accounted by
researchers who described the ability of carrageenan solutions
(lambda, kappa, or iota) to prevent HSV-2 infection.
Anti-tumour and immunomodulatory activities
Numerous studies have illustrated that carrageenans have
antiproliferative activity in cancer cell lines invitro, in addition
with the inhibitory actvity of tumor growth in mice. As well as,
130
they have antimetastatic activity by blocking the connections
among cancer cells and the basement membrane, hinder tumor cell
proliferation and tumor cell adhesion to a variety of substrates, but
their accurate mechanisms of action are not yet completely
understood. In 1986 Yamamoto reported that the oral
administration of several seaweeds is the reason for significant
decline in the occurrence of carcinogenesis in vivo. Researchers
investigated the altered effects of carrageenan on colonic
carcinogenesis in male rats. No treated related changes in clinical
signs and body weights were found. Histopathological
examination did not demonstrate any enhancement by
carrageenan carcinogenesis, carrageenan does not own any
promoting activity at the maximum dietary level of 5.0% for
colorectal carcinogenesis under the present experimental
circumstances.
Tetracycline and chlorotetracycline production
Tetracyclines signify one of the most considerable groups of
antibiotics and the process regularly used for their industrial
production is conventional fermentation. Researchers employed
Streptomyces aureofaciens immobilised in κ-carrageenan in an
effort to progress the production of tetracycline and
chlorotetracycline.
Industrial applications
A proficient integrated nitrogen removal system was
developed by the co-immobilisation of Nitrosomonas europaea
and Pseudomonas sp. in κ-carrageenan, taking advantage of the
oxygen gradient inside the entrapment beads. The immobilisation
of M. aurum in κ-carrageenan and its utilization in an air-bubble
fermenter resulted in a development of its morpholine-degrading
capacity. The carrageenan gel and immobilised microbial cells
technique was used for 4-chlorophenol degradation. Aerobic and
anaerobic microbial communities have also been co-immobilised
into κ-carrageenan/gelatin gel beads. Under air-limited conditions
these immobilisates catalyse the degradation of 2,4,6-
trichlorophenol. Pentachlorophenol pesticide degradation in
contaminated soil was tried with the use of Pseudomonas sp.
UG30 cells immobilised in κ-carrageenan.
131
 Carrageenans are used to gel, thicken, or suspend;
consequently they are used in emulsion stabilisation, for syneresis
control, and for bodying, binding and dispersion. The main uses
are in foods, particularly dairy applications. Furcellaran generally
finds applications analogous to those for kappa-carrageenan.
Traditionally, furcellaran has dominated two major European
application fields: tart or cake glaze powders and flan powders.
Today the special properties of excellent gel texture and flavour
discharge make furcellaran a favoured product for utilizimg in
milk pudding powders.
In toothpastes carrageenans function as a “binder” to impart
the desired rheological properties to the paste and to provide the
cosmetic quality of “sheen”. Toothpastes consist of ingredients
which interact in complex and in poorly understood ways and the
carrageenan often must be carefully tailored to achieve
satisfactory performance in a particular formulation. Carrageenan
suffers severe competition in the U.S. domestic market from
sodium carboxy methyl cellulose, a much cheaper gum. Despite
this, business has been retained – and regained – due to the
superior quality and appearance carrageenan imparts to
toothpastes. Outside the United States carrageenan has maintained
a strong position in this application, due, among other factors, to
its immunity to degradation by enzymes which attack cellulose
gums. Gelatinous extracts of the Chondrus crispus (Irish Moss)
seaweed have been used as food additives for hundreds of years.
Carrageenan is a vegetarian and vegan alternative to gelatin.
When used in food products, carrageenan has the EU additive
E-number E407 or E407a when present as “processed eucheuma
seaweed”, and is commonly used as an emulsifier and also known
as Carrageen Moss. It is boiled in milk and strained, before sugar
and other flavourings such as vanilla, cinnamon, brandy, or
whisky are added. The end-product is a kind of jelly similar to
pannacotta, tapioca, or blancmange.
5.2 Agar Agar
Agar or agar-agar, is a jelly-like substance, acquired
from red algae. Agar is a mixture of two components: the linear
132
polysaccharide agarose, and a heterogeneous mixture of smaller
molecules called agaropectin. It forms the supporting structure in
the cell walls of certain species of algae, and is released on boiling.
These algae are known as agarophytes, and belong to
the Rhodophyta (red algae) phylum.
Agar has been utilized as an ingredient in desserts throughout
Asia, and also as a solid substrate to contain culture
media for microbiological work. Agar can be used as a laxative,
an appetite suppressant, a vegetarian substitute for gelatin, a
thickener for soups, in fruit preserves, ice cream, as a clarifying
agent in brewing, and for sizing paper and fabrics.
The gelling agent in agar is an unbranched polysaccharide
acquire from the cell walls of some species of red algae, primarily
from tengusa (Gelidiaceae) and ogonori (Gracilaria). For
commercial purposes, it is resultant primarily from ogonori. In
chemical terms, agar is a polymer made up of subunits of the
sugar galactose.
Composition
Agar comprises a mixture of two polysaccharides: agarose
and agaropectin, with agarose making up about 70% of the
mixture. Agarose is a linear polymer, made up of repeating units
of agarobiose, a disaccharide made up of D-galactose and 3,6-
anhydro-L-galactopyranose. Agaropectin is a heterogeneous
mixture of smaller molecules that occur in lesser amounts, and is
prepared up of alternating units of D-galactose and L-galactose
heavily modified with acidic side-groups, such as sulfate and
pyruvate.
Agar exhibits hysteresis, melting at 85 °C (358 K, 185 °F)
and solidifying from 32–40 °C (305–313 K, 90–104 °F). This
property lends an appropriate balance among easy melting and
good gel stability at relatively high temperatures. Since many
scientific applications need incubation at temperatures close to
human body temperature (37 °C), agar is more suitable than other
solidifying agents that melt at this temperature, such as gelatin.

133
Uses
Culinary
Agar-agar is a normal vegetable gelatin counterpart. It is
white and semi-translucent when sold in packages as washed and
dried strips or in powdered form. It can be used to make
jellies, puddings, and custards. When making jelly, it is boiled in
water until the solids dissolve. Sweetener, flavoring, coloring,
fruits and or vegetables are then added, and the liquid is poured
into molds to be served as desserts and vegetable aspics or
incorporated with other desserts such as a layer of jelly in a cake.
Agar-agar is approximately 80% dietary fiber, so it can serve
as an intestinal regulator. Its bulking quality has been behind fad
diets in Asia, for example the kanten diet. Once
ingested, kanten triples in size and absorbs water. This results in
the consumers feeling fuller. This diet has newly received some
press coverage in the United States as well. The diet has shown
promise in obesity studies
Microbiology
An agar plate or Petri dish is used to offer a growth
medium utilizing a mix of agar and other nutrients in which
microorganisms, including bacteria and fungi, can be cultured and
experimented under the microscope. Agar is indigestible for many
organisms so that microbial growth does not affect the gel used
and it remains constant. Agar is typically sold commercially as a
powder that can be mixed with water and equipped similarly to
gelatin before use as a growth medium. Other constituents are
added to the agar to meet the nutritional needs of the microbes.
Many microbe-specific formulations are accessible, because some
microbes favour certain environmental circumstances over others.
Agar is frequently dispensed using a sterile media dispenser.
Motility assays
As a gel, an agar or agarose medium is porous and
consequently can be used to determine microorganism motility
and mobility. The gel's porosity is directly related to the
concentration of agarose in the medium, so various levels of
134
effective viscosity (from the cell's "point of view") can be
selected, depending on the experimental objectives.
A common detection assay involves culturing a sample of the
organism deep within a block of nutrient agar. Cells will attempt
to cultivate within the gel structure. Motile species will be able to
migrate, albeit slowly, throughout the gel and infiltration rates can
then be depicted, whereas non-motile species will show
development only along the now-empty path introduced by the
invasive initial sample deposition.
Another setup normally used for measuring chemotaxis and
chemokinesis employs the under-agarose cell migration assay,
whereby a layer of agarose gel is placed between a cell population
and a chemoattractant. As a concentration gradient develops from
the diffusion of the chemoattractant into the gel, various cell
populations need diverse stimulation level to migrate can then be
visualized over time using microphotography as they tunnel
upward through the gel against gravity along the gradient.
Plant Biology
Research grade agar is utilized extensively in plant biology
as it is possibly enhanced with a nutrient and/or vitamin mixture
that permits for seedling germination in Petri dishes under sterile
conditions (given that the seeds are sterilized as well). Nutrient
and/or vitamin supplementation for Arabidopsis thaliana is
standard across most experimental circumstances. Murashige &
Skoog (MS) nutrient mix and Gamborg's B5 vitamin mix in
general are utilised. A 1.0% agar/0.44% MS+vitamin dH2O
solution is appropriate for growth media among normal growth
temperature.
While utilising agar, within any growth medium, it is
significant to know that the solidification of the agar is pH-
dependent. The most favorable choice for solidification is
between 5.4–5.7. Frequently, the purpose of KOH is needed to
increase the pH to this range. A general guideline is about 600 µl
0.1M KOH per 250 ml GM. This entire mixture can be sterilized
using the liquid cycle of an autoclave.

135
 This medium lends itself to the function of specific
concentrations of phytohormones etc. to encourage specific
growth patterns in that one can effortlessly arrange a solution
containing the preferred amount of hormone, add it to the known
volume of GM, and autoclave to both sterilize and evaporate off
any solvent that may have been utilised to dissolve the often-polar
hormones. This hormone/GM solution can be spread across the
surface of Petridishes sown with sprouted and/or etiolated
seedlings. Investigations with the moss Physcomitrella patens,
however, have revealed that selection of the gelling agent – agar
or Gelrite – does influence phytohormone sensitivity of
the plant cell culture.
5.3 Seaweed fertiliser
Seaweed and its derived products are employed as fertilizer
in the coastal areas throughout the globe. In India it is utilised for
coconut plantations especially in Tamil Nadu and Kerala. The
elevated amount of water soluble potash, other mineral and trace
elements present in seaweeds are voluntarily absorbed by plants
and they manage nutrient scarcity in plants. Carbohydrates and
other organic matter present in seaweeds vary the nature of the
soil and recover its moisture retaining capacity. Hence, large
quantities of seaweeds including sea grasses such as Cymodocea,
Diplanthera, Enhalus and Halophila are employed as manure in all
parts of the country either directly or in form of compost.
Effect of Seaweed Liquid Fertilizer on Plant Growth
Seaweeds have been utilised as a food and manure for
plantation crops by coastal people in many countries. Recent
studies recommend that application of seaweed extract as seed
treatment and/or foliar spray helps in considerable growth of
plants. The extract contains micro-nutrients, auxins and
cytokinins and other growth promoting substances. These
hormones take part in a significant role in enhancement of cell size
and cell division, and together they complement each other as
cytokinins are efficient in shoot generation and auxins in root
progress, while micro-nutrients recover soil health. The quantity
of plant growth regulators like auxin and cytokinin contents was
recorded up to 150ì g/L and 235ì g/L respectively in Sargassum
136
wightii. Seaweed extract is viewed superior because the organic
matters aids in maintaining moisture and minerals needed for the
plants. The liquid extract of Ulva lactuca, Sargassum wightii and
Gelidella acerosa induced maximum sprouting, root and shoot
growth in Ragi. Extract of Enteromorpha intestinalis result in
elevated levels of seed germination, root, shoot length and
chlorophyll content of Sesamum indicum. Gracilaria edulis
extract maintain higher growth, fruiting and flowering in
Abelmoschus esculentus. When 1% of Gracilaria edulis applied to
soil bed maximum germination, growth and progress in Zea mays
and Phaseolus mungo was obtained. SLF prepared from
Hydroclathrtus and applied to soil drench exhibited maximum per
cent raise the growth parameters of Sorghum. Researchers
observed a positive effect on the vegetative growth of black gram,
brinjal and tomato when liquid extract from Gracilaria verucosa
and Chaetomorpha linum was applied as foliar spray.
Effect of Slf on the Biochemical Composition of Crop Plants
Researchers observed a linear amplification in the pigment
concentrations, protein, total soluble sugar, reducing sugar, starch,
phenols, lycopen and vitamin C content of Lycopersicon
esculentum upon treatment with liquid extract of Sargassum sp.
The liquid extract of Ulva fasciata, Sargassum ilicifolium and
Gracilaria corticata inclined the photosynthetic pigments,
carbohydrate, proteins and free aminoacids content of Trigonella
foenum - graecum. Different concentrations of liquid extract of
Ulva lacuta, Caulerpa scalpelliformis, Padina tetrastromatica and
Sargassum linearifolium increased the amount of protein,
carbohydrate, and aminoacid of Brassica nigra. Application of
liquid extract from Ulva lacutca and Sargassum sp. to the soil bed
enhance the photosynthetic pigment composition, soluble protein
and starch, aminoacid content of Phaseolus mungo, Zea mays and
Cyamopsis tetragonoloba. The biochemical composition of Vigna
radiata was amplified by applying 10% liquid extract of
Sargassum wightii. Ramasubramanian reported the effect of
seaweed extract of Gracilaria edulis illustrated an amplifcation in
the pigment concentration, protein and enzyme activity of
Abelmoschus esculentus. Researchers in a comparative study on
the impact of the liquid extract of seaweed and sea grasses of
137
Mandapam coast found promoting the chlorophyll content of Zea
mays. Liquid extract of Padina pavonia helped in increasing the
pigment, total soluble sugar, protein and lipid content Cyamposis
tetragonoloba
Seaweed Liquid Fertilizer on Quality and Fertility of Soils
Application of seaweed extract has enhanced the moisture
holding capacity of the soil. Brown seaweeds are rich in
polysaccharides coupled with their hydrophilic property which
makes the compound significant in the agricultural and
pharmaceutical industries. Alginate occurs in the cell walls of
seaweeds as a mixed salt. Alginic acid combine with ions in soil
to form high molecular weight complex that absorb moisture,
swell, maintain soil moisture and progress crump formation
resulting in better aeration and capillary activity of soil pores,
which in return encourage the growth of plant root system and
microbial activity. Application of seaweeds and seaweed extract
triggers the development of beneficial microbes and secretion of
soil conducting substances by these microbes. Seaweed liquid
extract improved soil fertility by improving the moisture holding
capacity and also assist in the growth of soil micro-biota. Some of
the beneficial microbes like Rhizobium when applied in
consortium with seaweed extract, it improved the early growth
and yield quality properties in legume plants like Arachis hypogea
and Vigna mungo and the response was 12-25% higher than that
of control.
Immune Activity of Seaweed Extract
Researchers reported that the methanol and ethanol extract of
some commonly occurring green algae like Codium adherens,
Ulva reticulata and Halimeda tuna in Tamil Nadu were more
effective than that of the commercial medicine against Escherichia
coli and Staphylococcus sp. There are also information that the
seaweed species Ulva lactuca, Padina gymnospora, Sargassum
wightii and Gracilaria edulis revealed antibacterial activity and
defence mechanism against human bacterial pathogens
Staphylococcus aureus, Shigella dysentriae, Salmonella
paratyphi, Pseudomonas aeruginosa and Klebsiella pneumonia.
The organic extracts of three marine macroalgae viz.,
138
Chaetomorpha linum, Enteromorpha compressa and Polysiphonia
subtilissima of Chilika Lake, Odisha showed definite activity in
inhibiting the growth of three Gram-negative bacteria Shigella
flexneri, Vibrio cholerae and Escherichia coli and two Gram
positive bacteria Bacillus subtilis and Bacillus brevis.
Experiments reported that ethanol extract of marine red
macroalga, Gracilaria verrucosa showed antioxidant activity. The
liquid extract of Palmaria palmate, Macrocystis integrifolia,
Nereocystis leutkeana gave a positive result in anti-proliferative
and antioxidant activity to mammals
Under this situation SLF may work as a good inducer for
sustainability in agricultural production coupled with preservation
of soil health. In India seaweeds are not used widely except for
invention of phycocolloids. But being a wealthy source of
vitamins, minerals and growth promoters, they can be of vast help
to the coastal farmers for their utilization as a source of organic
fertilizer.
5.4 Astaxanthin
It is a keto-carotenoid. It fits in to a superior class of chemical
compounds known as terpenes (as a tetraterpenoid) assembled
from five carbon precursors, isopentenyl diphosphate,
and dimethylallyl diphosphate. Astaxanthin is classified as
a xanthophyll (originally derived from a word meaning "yellow
leaves" since yellow plant leaf pigments were the first recognized
of the xanthophyll family of carotenoids), but currently employed
to describe carotenoid compounds that have oxygen-containing
components, hydroxyl (-OH) or ketone (C=O), such
as zeaxanthin and canthaxanthin. Indeed, astaxanthin is a
metabolite of zeaxanthin and/or canthaxanthin, containing both
hydroxyl and ketone functional groups
Astaxanthin is present in most red-coloured aquatic
organisms. The content varies from species to species, but also
from individual to individual as it is highly dependent on diet and
living conditions. Astaxanthin, and other chemically related asta-
carotenoids, has also been found in a number of lichen species of
the arctic zone.
139
The primary natural sources for industrial production of
astaxanthin comprise the following:

 Euphausia pacifica (Pacific krill)

 Euphausia superba (Antarctic krill)
 Haematococcus pluvialis (algae)
 Pandalus borealis (Arctic shrimp)
 Xanthophyllomyces dendrorhous, formerly Phaffia
rhodozyma (yeast)
Algae are the primary natural source of astaxanthin in the
aquatic food chain. The microalgae Haematococcus
pluvialis seems to accumulate the highest levels of astaxanthin in
nature and is currently, the primary industrial source for natural
astaxanthin production where more than 40 g of astaxanthin can
be obtained from one kg of dry biomass. Haematococcus
pluvialis has the productional advantage of the population
doubling every week, which means scaling up is not an issue.
Specifically, the microalgae are grown in two phases. First, in the
green phase, the cells are given an abundance of nutrients to
promote proliferation of the cells. In the subsequent red phase, the
cells are deprived of nutrients and subjected to intense sunlight to
induce encystment (carotogenesis), during which the cells
produce high levels of astaxanthin as a protective mechanism
against the environmental stress. The cells, with their high
concentrations of astaxanthin, are then harvested.
Phaffia yeast Xanthophyllomyces dendrorhous exhibits
100% free, non-esterified astaxanthin, which is considered
advantageous because it is readily absorbable and need not be
hydrolysed in the digestive tract of the fish. In contrast to synthetic
and bacteria sources of astaxanthin, yeast sources of astaxanthin
consist mainly of the (3R, 3’R)-form, an important astaxanthin
source in nature. Finally, the geometrical isomer, all-E, is higher
in yeast sources of astaxanthin, as compared to synthetic sources.
In shellfish, astaxanthin is almost exclusively concentrated in
the shells, with only low amounts in the flesh itself, and most of it
only becomes visible during cooking as the pigment separates
from the denatured proteins that otherwise bind it. Astaxanthin is
140
extracted from Euphausia superba (Antarctic krill) and from
shrimp processing waste. 12,000 pounds of wet shrimp shells can
yield a 6–8 gallon astaxanthin/triglyceride oil mixture.
Astaxanthin is used as a dietary supplement and feed
supplement as food colorant for salmon, crabs, shrimp, chickens
and egg production.
Biosynthesis
Astaxanthin biosynthesis starts with three molecules of
isopentenyl pyrophosphate (IPP) and one molecule of
dimethylallyl pyrophosphate (DMAPP) that are combined by IPP
isomerase and converted to geranylgeranyl pyrophosphate
(GGPP) by GGPP synthase. Two molecules of GGPP are then
coupled by phytoene synthase to form phytoene. Next, phytoene
desaturase creates four double bonds in the phytoene molecule to
form lycopene. After desaturation, lycopene cyclase first forms γ-
carotene by converting one of the ψ acyclic ends of the lycopene
as a β-ring, then subsequently converts the other to form β-
carotene. From β-carotene, hydrolases (blue) are responsible for
the inclusion of two 3-hydroxy groups, and ketolases (green) for
the addition of two 4-keto groups, forming multiple intermediate
molecules until the final molecule, astaxanthin, is obtained
For seafood and animals
The primary use of synthetic astaxanthin today is as an
animal feed additive to impart coloration, including farm-raised
salmon and chicken egg yolks. Synthetic carotenoid pigments
colored yellow, red or orange represent about 15–25% of the cost
of production of commercial salmon feed. Today, almost all
commercial astaxanthin for aquaculture is produced synthetically.
Class action lawsuits were filed against some major grocery store
chains for not clearly labeling the astaxanthin-treated salmon as
"color added". The chains followed up quickly by labeling all such
salmon as "color added". Litigation persisted with the suit for
damages, but a seattle judge dismissed the case, ruling that
enforcement of the applicable food laws was up to government
and not individuals.

141
Dietary supplement
The primary human application for astaxanthin is as a dietary
supplement, although as of 2018, there was insufficient evidence
from medical research to show that it affects disease risk or health
of people, and it remains under preliminary research. In 2018,
the European Food Safety Authority sought scientific information
from manufacturers of dietary supplements about the safety of
astaxanthin.
5.5 Beta carotene
Plant carotenoids are the primary dietary source of
provitamin A worldwide, with β-carotene as the best-
known provitamin A carotenoid. Others include α-
carotene and β-cryptoxanthin. Carotenoid absorption is restricted
to the duodenum of the small intestine and dependent on class B
scavenger receptor (SR-B1) membrane protein, which is also
responsible for the absorption of vitamin E (α-tocopherol). One
molecule of β-carotene can be cleaved by the intestinal
enzyme β,β-carotene 15,15'-monooxygenase into two molecules
of vitamin A.
Absorption efficiency is estimated to be between 9 and 22%.
The absorption and conversion of carotenoids may depend on the
form of β-carotene (e.g., cooked vs. raw vegetables, or in a
supplement), the intake of fats and oils at the same time, and the
current stores of vitamin A and β-carotene in the body.
Researchers list these factors that determine the provitamin A
activity of carotenoids:

 Species of carotene
 Molecular linkage
 Amount in the meal
 Matrix properties
 Effectors
 Nutrient status
 Genetics
 Host specificity
 Interactions between factors

142
Dietary sources
Beta-carotene is found in many foods and is sold as a dietary
supplement. β-Carotene contributes to the orange color of many
different fruits and vegetables. Vietnamese gac (Momordica
cochinchinensis Spreng.) and crude palm oil are particularly rich
sources, as are yellow and orange fruits, such
as cantaloupe, mangoes, pumpkin, and papayas, and orange root
vegetables such as carrots and sweet potatoes. The color of
β-carotene is masked by chlorophyll in green leaf
vegetables such as spinach, kale, sweet potato leaves, and
sweet gourd leaves. Vietnamese gac and crude palm oil have the
highest content of β-carotene of any known plant sources, 10
times higher than carrots, for example. However, gac is quite rare
and unknown outside its native region of Southeast Asia, and
crude palm oil is typically processed to remove the carotenoids
before sale to improve the color and clarity. The average daily
intake of β-carotene is in the range 2–7 mg, as estimated from a
pooled analysis of 500,000 women living in the US, Canada, and
some European countries.
The U.S. Department of Agriculture lists these 10 foods to
have the highest β-carotene content per serving Additionally,
supplemental β-carotene may increase the risk of prostate
cancer, intracerebral hemorrhage, and cardiovascular and total
mortality in people who smoke cigarettes or have a history of
high-level exposure to asbestos.
Marine Carotenoids: Application
Carotenoids have been usually used in food and animal feed
owing to their color properties. The natural carotenoids are
utilised to reinforce fish color, which amplify consumers’
perception of value. An instance is the adding up of carotenoids
to fish feed to impart colour to farmed salmon. The nutraceutical
assets of carotenoids also involved attention of the food industry.
Huge numbers of scientific studies have established the benefits
of carotenoids to health and application for this function is
growing rapidly. Besides, carotenoids have been projected as
added-value compounds that could supply to make microalgal
biofuel production economically feasible.
143
 Among all accessible natural carotenoids, five can be
considered to be the most applicable ones in economical terms.
The key function of carotenoids is currently as dietary
supplements, fortified foods, food color, animal feed and
pharmaceuticals and cosmetics. β-carotene, the most extensively
known of the carotenoids, is known to be a vitamin A precursor,
likely several other carotenoids. Carotenoids have antioxidant
properties and a large number of studies have confirmed their
benefits to health. In particular, carotenoids are considered to
decrease the threat of degenerative diseases and cancer
particularly in elderly people.
The health industry utilized carotenoids in over-the-counter
(OTC) dietary supplements and fortified foods. This is one of the
best ever growing segments of the industry but is still relatively
small in contrast to the color segment. The pharmaceutical and
cosmetics industries also utilize carotenoids mostly for their
coloring properties, while their use by the pharmaceutical and
cosmetics companies is increasing quickly due to their
nutraceutical properties. An illustration of a new product from this
segment is a ‘beauty pill’ containing the carotenoid lycopene. This
product belongs to a novel market segment known as
‘cosmeceuticals’, which intend to combine cosmetics and
nutraceutical food ingredients to generate products to recover skin
and hair.
Chemically synthesized nature identical carotenoids govern
the market but naturally extracted carotenoids are increasing in
popularity due to increasing demand for natural products from
consumers. Natural carotenoids can be extracted from plant
material such as tomatoes, algae and fungi. Individual carotenoids
are existing in a variety of forms. The most common forms are
cold water soluble powder, oil emulsion and beadlets.
Concentrations range from 0.2 to 100%. The most common
concentration is 10%. Blends or mixed carotenoids are also
accessible containing two or more diverse carotenoids. Like the
individual carotenoids, blends are available in a variety of forms
including, water dispersible powder, oil suspension and beadlet
forms. The concentration of blends ranges from 1 to 30%, with the
most common concentration being 10%.
144
 In addition, if carotenoids are disposed within the microalgal
matrix (carotenoid enriched dry biomass) also a quantity of
minerals whose existence is intrinsic to the algal biomass is
provided in the formula. These mineral have positive effects to
human health, particularly in enhancing anabolic activities.
Carotenoids have also been employed as preservatives in
cosmetics and solar protection products. Because of the content of
carotenoids, the commercial value of microalgae increased and
their use extended generally into many function of the food
market. That includes the utilization of Arthrospira, Chlorella,
Dunaliella, Spirulina and Aphanizomenon as functional foods
which can be found in the market in the form of pills, tablets and
capsules. These microalgae have also been incorporated in
nutritional prescription of pasta, snacks, sweets, drinks and bubble
gum. Microalgae are also utilised in fish color quality
improvement in aquaculture. Salmonids are supplied with
astaxanthin-enriched microalgae species, in particular
Haematococcus pluvialis.
5.6 Enzymes
Marine microorganisms have been attracting more and more
consideration as a resource for new enzymes, as the microbial
enzymes are comparatively more firm and active than the
corresponding enzymes resultant from plants or animals. The
marine environment range from nutrient-rich regions to
nutritionally sparse locations where only a small number of
organisms can survive. The complexity of the marine environment
concerning high salinity, elevated pressure, low temperature and
particular lighting conditions, may contribute to the significant
dissimilarity between the enzymes produced by marine
microorganisms and homologous enzymes from terrestrial
microorganisms, leading to the enhance marine microbial enzyme
technology in recent years and the resultant valuable products.
Protease
In 1972, Nobou Kato isolated a novel type of alkaline
protease from marine psychrobacter, and since then quite a few
proteases have been frequently acquired from marine
microorganisms. An alkaline protease, formerly isolated from a
145
symbiotic bacterium found in the gland of Deshayes of a marine
shipworm, was evaluated as a cleansing additive. Researchers
preferred 30 kinds of marine bacteria from the sea water, mud, fish
and other samples; after UV mutagenesis they isolated the N1-35
strain, this strain formed protease that had considerable
advantages in contrast with the terrestrial ones. Both proteases
showed elevated stability towards non-ionic surfactants. In
addition, both of them showed tremendous firmness and
compatibility with a broad variety of profitable liquid and solid
detergent.
Chitinase
As marine zooplankton are regularly supposed to shed, there
is a large quantity of abandoned chitin, which could be a
prosperous resource of carbon and power for development and
reproduction of chitin-degrading microorganisms. The entire
fabrication of chitin in the whole marine biocycle is at least 2.3
million metric tons per year. At the present, researchers have
found a wide range of microorganisms that can produce chitinase
or chitosanase, including Aspergillus, Penicillium, Rhizopus,
Myxobacter, Sporocytophaga, Bacillus, Enterobacter, Klebsiella.
In addition, a mixture of chitinase genes were already cloned from
marine bacteria and fungi. Suolow and Jone inserted two chitinase
genes (ChiA, ChiB) into E. coli, and subsequently these genes
ware transferred into Pseudomonas; finally they acquired four
high-yielding chitinase strains.
Alginate lyases
The brown alga is one of the largest marine biomass
resources. Alginate has a extensive range of applications;
additionally, the degraded low-molecular fragment shows more
potential. Alginate lyases, characterize as either mannuronate or
guluronate lyases, are a complex copolymer of α-L-guluronate
and its C5 epimer β-D-mannuronate. They have been isolated
from a broad range of organisms, as well as algae, marine
invertebrates, and marine and terrestrial microorganisms. In
current years, the marine microbial alginate lyases have been
significantly developed. Discovering and characterization of
alginate lyases will improve and increase the use of these enzymes
146
to engineer fresh alginate polymers for applications in a variety of
industrial, agricultural, and medical fields.
Carrageenases
Carrageenan and carrageenin are a family of linear sulfated
polysaccharides, which are extracted from red seaweeds. 80% of
the carrageenan is utilised in food and food-related industries, and
it can be used as a coagulant, adhesive, stabilizer and emulsifier.
In addition, it has also been extensively applied in the
pharmaceutical and cosmetics industries. The oligosaccharides
acquired from carrageenan degradation show a variety of specific
physiological activities, such as anti-viral, anti-tumor, anti-
coagulation, etc. Researchers using carrageenan containing
medium, cultured cytophaga lk-C783, and obtained extracellular
κ-carrageenase with a molecular weight of 10 kD. In 2004,
isolation of an extracellular κ-carrageenase with a molecular
weight of 30 kD was obtained from marine cytophaga MCA-2.
Xylanase amd Hydrolase
Xylanases are hydrolases depolymerizing the plant cell
component xylan, the second most abundant polysaccharide.
Xylanases could be formed by fungi, bacteria, yeast, marine algae,
etc., however the principal commercial source is filamentous
fungi. Xylanase could be utilised on semi-cellulose to produce
products with elevated economic value, such as xylitol. In the
paper and pulp industry, utilization of xylanase can develop the
lignin dissolution rate and decrease the usage of Cl 2 and ClO2,
thereby reducing pollution and recover pulp properties. Xylanase
can also degrade some polysaccharides in juice or beer, thus it
could contribute to beverage clarification. Indian researchers
acquired several fungal isolates from marine habitat revealed
alkaline xylanase activity.
Amylases
Amylases were found in bread making, and they can
breakdown complex sugars such as starch into simple sugars such
as glucose, maltose and dextrin. They can be classified into α-
amylase, β-amylase and γ-amylase. Unlike the other forms of
amylase, γ-amylase is most proficient in acidic environments. To
147
date, researchers have established some terrestrial
microorganisms which can form extracellular amylase, such as
Arxula adeninivorans, Lipomyces, Saccharomycopsis,
Schwanniomyces, Candida japonica and Filobasidium
capsuligenum. With the progress of marine science and
technology, researchers reported more and more microorganisms
from marine habitats proficient of producing amylase. The marine
yeast strain Aureobasidium pullulans N13d, producing an
extracellular amylase, was isolated from the deep sea sediments
of Pacific Ocean. Investigations reported a novel α-amylase from
marine Streptomyces sp. D1 by using media containing 2%
sucrose, 0.35% peptone and 0.15% of malt extract. Researchers
isolated a novel amylase from the Mucor sp. related with the
marine sponge Spirastrella sp., this enzyme has an optimum pH of
5.0 and an optimum temperature of 60 ºC.
Antibiotics derived from marine sources
Marine microorganisms signify a major source for the
discovery and growth of new antibiotics due to their rich
biodiversity and genetic capacity to produce unique metabolites.
In particular, marine bacteria derived from deep-sea sediments
have revealed to be a prosperous source of secondary metabolites
with novel structures and excellent biological activities, including
antimicrobials. Majority of natural antibiotics are biosynthesized
by bacteria belonging to the high GC Gram-positive bacteria. In
particular, actinomycetes correspond to the most vital source of
bioactive natural products with clinical or pharmaceutical
applications. Majority of the antimicrobial compounds were
isolated from deep-sea derived actinomycetes.
Researchers first revealed and categorized the type-strain of
the new marine actinomycete, Marinactinospora thermotolerans
SCSIO 00652, isolated from a sediment collected from site E410
(1◦58.7420 N 11◦00.2280 E; black soft mud at 3865 m depth) in
the northern South China Sea. Thereafter, researchers purified a
novel polythiazole cyclic peptide, referred to as marthiapeptide A
(1), from this organism. Marthiapeptide A (1) revealed strong
antibacterial action against Micrococcus luteus, Staphylococcus
aureus ATCC 29213, Bacillus subtilis ATCC 6633, and B.
148
thuringiensis, with MIC values of 2, 8, 4, and 2 µg/mL,
respectively. Three novel cyclic hexapeptides, named
desotamides B, C, and D were recognized from Streptomyces
scopuliridis strain SCSIO ZJ46, which was isolated from a South
China Sea sample sediment compilation at a depth of 3536 m
(120°0.2500 E, 20°22.9710 N). Among the novel compounds
identified, desotamide B (2) revealed an microbicidal assessment
against S. aureus ATCC 29213, Streptoccocus pneumoniae NCTC
7466, and methicillin-resistant Staphylococcus epidermidis
(MRSE) shhs-E1, with MIC values of 16, 12.5, and 32µg/mL.
New cyclic congeners, marfomycins A (3), B (4), and E (5), were
isolated from the South China Sea-derived Streptomyces
drozdowiczii SCSIO 10141. The producer strain of marfomycins
A, B, and E was isolated from a sediment collected at a depth of
1396 m (118◦58.24750 E, 22◦2.36890 N). Unique N-terminally
formylated side chain and five nonproteinogenic amino acid
residues characterize marfomycins A (3), B (4), and E (5).
Marfomycins A (3), B (4), and E (5), display a selective anti-
infective activity against M. luteus, among a panel of Gram-
positive and Gram-negative bacteria, with MIC values of 0.25, 4,
and 4 µg/mL, respectively. The microbicidal marthiapeptide A
(1), desotamides B (2), and marfomycins A (3), B (4), and E (5)
(Figure 14) represent new deep-sea derived cyclic peptides.
Cyclic peptides and depsipeptides are secondary metabolites of
microorganisms and plants, with a renowned broad spectrum of
biological properties.
This class of NP represents a valuable source for the
discovery of new therapeutics, due to their favorable properties,
such as resistance to enzymatic degradation; a large surface area,
which provides high affinity and selectivity for the target; and
limited conformational flexibility, which enhances binding
properties. Moreover, they often possess better membrane
penetration properties. As a result, they have much longer half-
lives in vivo than their acyclic counterparts, and are thus of great
interest in the field of drug discovery. Cyclic antimicrobial
peptides (AMPs) have emerged as good antimicrobial candidates
due to their aforementioned characteristics and high activity.

149
An additional family of microbial metabolites with effective
antitumor and antibiotic properties is communally designated as
spirotetronate polyketides. Three new spirotetronate polyketides
with microbicidal activity isolated from deep-sea Gram-positive
bacteria are shown in Figure 15

Figure: 14 Molecular structure of new cyclic peptides

Compound (1), marthiapeptide A, Compound (2), desotamide B,
Compounds (3–5), marfomycins A, B, and E

Two diverse research groups recognized the already-known

lobophorins B and two new spirotetronate antibiotics, designated
lobophorins F (6) and H (7), from actinomycetes isolated from
deep-sea sediments of the South China Sea.

150
 Both lobophorins F (6) and H (7) were extracted from
Streptomyces sp. strains (SCSIO 01127 and 12A35, respectively).
The two strains were isolated from a sediment sample collected at
the depth of 1350 m (111◦54.6930 E, 08◦56.0030 N) and 2134 m
(17°59.9280 N, 111°36.1600 E), respectively. Lobophorin F (6)
revealed antibacterial assesment against S. aureus ATCC 29213
and Enterococcus faecalis ATCC 29212 with MIC values of
8µg/mL for both of the strains.

Figure: 15 Structures of new spirotetronate polyketides

Lobophorin F (6) Lobophorin H (7)
Lobophorin H (7) displayed antibacterial activity against B.
subtilis CMCC63501 with a MIC value of 3.13 µg/mL. Based on
the inhibitory action displayed against Gram-positive bacteria,
lobophorins F (6) and H (7) may potentially discover relevance in
antibacterial drug development. Moreover, the detection of more
spirotetronate antibiotics analogs helps to reveal the structure–
activity relationships and possible applications of these
compounds. Lobophorin F and H are analogs of lobophorin B,
which was formerly isolated from an alga-associated
actinobacterium; lobophorin B is structurally related to another
spirotetronate antibiotic, named kijanimicin. New studies specify
that kijanimicin binds to the TetR family of transcriptional
regulators that control the expression of a variety of cytoplasmic
proteins in prokaryotes.
151
5.7 Antitumour agents derived from marine
sources
Bacteria
Marine microorganisms are a resource of novel genes, and
development lead to the detection of new drugs and targets.
Secondary metabolites formed by marine bacteria have yielded
pharmaceutical products such as new anti-inflammatory agents
(e.g., pseudopterosins, topsentins, scytonemin, and manoalide),
anticancer agents (e.g., bryostatins, discodermolide, eleutherobin,
and sarcodictyin), and antibiotics (e.g., marinone). The
involvement of probiotic bacteria, such as lactobacilli and
bifidobacteria, is mostly in the management of pathogenic
microbes, through manufacture of antibacterial protein namely,
bacteriocin and anticancer substances. The dietary supplements of
lactobacilli are apparently decreasing the induction of
experimental colon cancer. They arouse and modulate the
mucosal immune system by reducing the production of
proinflammatory cytokines through measures on NFκB pathways,
increasing manufacture of anti-inflammatory cytokines such as
IL-10 and host resistance peptides such as β-defensin 2, enhancing
IgA defenses and influencing dendritic cell maturation as well as
modulation of cell proliferation and apoptosis through cell
responses to short chain fatty acids.
Majority of the marine animal phyla generate toxins and
some studies show that these marine toxins may be formed by
marine bacteria associated the animals. The microbial toxins are
functional in neurophysiological and neuropharmacological
studies. For instance, bacteria present in Noctiluca scintillans are
accountable for causing red tides. The major metabolite,
macrolactin-A, inhibits B16-F10 murine melanoma cancer cells,
mammalian herpes simplex virus (HSV) (types I and II), and
guard T lymphocytes against human immunodeficiency virus
(HIV) replication.
Kahalalide F (KF) is a depsipeptide isolated from the mollusk
Elysia rubefescens from Hawaii and the compound is supposed to
be synthesized by microbes associated with the animal. KF
152
induces cytotoxicity and blocks the cell cycle in G1 phase in a
p53-independent manner. In vitro, KF displays action against
solid tumors with an interesting pattern of selectivity in prostate
cancer cell lines. In addition, wide in vivo work expresses that the
agent has action in breast and colon cancers.
Only a few marine bacteria can be isolated under laboratory
conditions and there is an vital requirement to extend novel culture
techniques to isolate slow-growing bacteria and also to isolate the
bacteria that are exclusive in production of novel natural products.
Actinomycetes
For more than 50 years, the soil-derived actinomycetes of
terrestrial source have provided a main pharmaceutical resource
for the detection of antibiotics and related bioactive compounds.
Though, marine actinomycetes established new attention.
Gutingimycin is a highly polar trioxacarcin derivative from
Streptomyces species, isolated from sediment of the Laguna de
Terminos, Gulf of Mexico. The same Streptomyces species also
yields trioxacarcins D–F, in addition to the known trioxacarcins
A–C. Among the antibiotic-producing microbes, marine
actinomycetes within the family Micromonosporaceae are very
capable. These microbes are found to be effective sources of
anticancer agents that target proteasome function and their
industrial potential is authenticated by several pharmaceuticals.
Thiocoraline is a new bioactive depsipeptide isolated from
Micromonospora marina, a marine microorganism situated in the
Mozambique Strait that inhibits RNA synthesis. The bioactive
compound is also selectively cytotoxic against lung and colon
cancer cell lines as well as melanoma. Interestingly, the compound
has preferential antiproliferative effects in colon cancer cell lines
with defective p53 systems. Thiocoraline signify a model of an
anticancer agent obtained from marine microorganisms and
exemplify how the problems of drug supply can be conquered by
artificial culture. More than 50% of the marine cyanobacteria are
potentially exploitable for extracting bioactive substances which
are proficient in either killing the cancer cells by inducing
apoptotic death, or affecting the cell signaling through activation
of the members of protein kinase-c family of signaling enzymes.
153
The cell extracts of Calothrix isolates deter the progress in vitro
of a chloroquine-resistant strain of the malarial parasite,
Plasmodium falciparum, and of human HeLa cancer cells in a
dose-dependent manner. Bioassay directed fractions of the
extracts have lead to their isolation and structural characterization
of Calothrixin A (I) and B (II), pentacyclic metabolites with an
indole [3, 2 – j] phenanthridine alkaloids which put forth their
development inhibitory effects at nanomolar concentrations.
Another compound, Curacin-A, isolated from the organic extracts
of Curacao collections of Lyngbya majuscula is an extraordinarily
potent antiproliferative agent as it hinder the polymerization of the
tubulin and it also demonstrate the inhibitory assesment
selectively on colon, renal, and breast cancer-derived cell lines.
Largazole is an exclusive chemical scaffold with inspiring
antiproliferative activity derived from Symploca sp. The
apratoxins are another class of cyanobacterial compounds that
hinder a diversity of cancer cell lines at nanomolar concentrations.
The parental compound, apratoxin A, isolated from a strain of
Lyngbya boulloni shows cytotoxicity to an adenocarcinoma. The
coibamide A is a compound resultant from a strain of
Leptolyngbya, and it exhibits significant cytotoxicity against
NCIH460 lung and mouse neuro-2a cells. The cytotoxicity is a
widespread means of action for numerous cyanobacterial
compounds.
In current times, the most noteworthy detection is of
borophycin, cryptophycin 1 & 8, and cyanovirin. Borophycin is a
boron-containing metabolite, isolated from marine cyanobacterial
strains of Nostoc linckia and N. spongiaeforme var. tenue. The
compound displays potent cytotoxicity against human epidermoid
carcinoma (LoVo) and human colorectal adenocarcinoma (KB)
cell lines. Borophycin is related both to the boron containing
boromycins isolated from a terrestrial strain of Streptomyces
antibioticus and to the aplasmomycins isolated from a marine
strain of Strepetomyces griseus (actinomycetes). Seaweeds are
significant sources of protein, iodine, vitamins, and minerals and
hence, their metabolites have revealed activities against cancer
incidences. Edible seaweed like Palmaria palmate is shown to be
efficient antioxidant, proficient of inhibiting cancer cell
154
proliferation. The alcoholic extract of the red alga Acanthophora
spicifera exhibits tumoricidal activity on Ehrlich’s ascites
carcinoma cells developed in mice at a dose of 20 mg/kg,
analogous to the standard drug, 5-flurouracil. This is confirmed
by increase in the mean survival time, decline in tumor volume,
and viable cell count. The smear study displays membrane
blebbing, vacuole formation, and reduction in staining intensity,
which further ascertains the tumoricidal activity. The seaweeds
Acanthaphora spicifera, Gracilaria foliifera, and Padina
boergesenii of the Gulf of Mannar region demonstrate cytotoxic
activity in their alcoholic extracts.
5.8 Polysaccharides derived from marine
microbes
Marine bacteria, such as Bacillus, Planococcus, Enterobacter,
Pseudoalteromonas, Vibrio, Rhodococcus, etc, are the main EPS
producers and have been widely studied till date. Most of the EPS
producing marine bacteria are Gram-negative in nature, while
very little are Gram positive. It was experimented that marine
bacterium Saccharophagus degradans produced EPS in elevated
quantity from numerous carbohydrates sources including starch
and xylose. Thus, the production of EPS from S. degradans was
improved by nutritional limitation. Vibrio furnissii strain VB0S3
was isolated and characterized from coastal regions of Goa and
showed to produce maximum EPS in batch cultures during the late
exponential growth phase. Planococcus maitriensis Anita I was
isolated from the coastal sea water area of Bhavnagar, India. This
bacterium was able to generate an EPS which can be further
utilized for bioremediation, improved oil recovery and cosmetic
applications. Enterobacter cloacae, isolated from marine
sediments in India produced an acidic EPS that revealed excellent
emulsifying properties as comparable to other commercial gums.
EPS production by Pseudoalteromonas CAM025 and CAM036,
isolated from Antarctica sea water and sea ice were depicted.
Marine bacteria such as Halomonas maura, Halomonas ventosae,
and Halomonas alkaliantarctica were isolated and evaluated for
the EPS production. Some exopolysaccharide producing marine
bacteria have been tabulated in Table: 9
155
 Probable applications of marine bacterial EPS have been
concerned in industries such as pharmaceutical, biomedical, food,
bioremediation and so on due to their strict physical and chemical
parameters. The applications of EPS in industries are mainly
depicted by their physical and chemical properties.
Rheological, emulsifying, solidifying properties of bacterial
exopolysaccharides have been the key property for the diverse
applications. Additionally to EPS extracted from terrestrial
bacteria, marine bacteria have established their function in the
pharmaceutical and biomedical industries (Table 9).

Table: 9 Some potential biotechnological applications of

marine bacterial EPS
Medical and pharmaceutical applications
EPS secreted by B. licheniformis and Geobacillus
thermodenitrificans are evaluated and it is found that they are
dominantly inspiring Th1 cell mediated immunity. Thus, these can
be probably utilised as immunomodulatory agent for beneficial
activities. Subsequently sulfation and depolymerisation of EPS
formed by A. infernus, the EPS derivatives can be used in the
treatment of lipemia and arteriosclerosis. HE800 EPS produced by
Vibrio diabolicus was found to be strong bone healing material
lacking any inflammatory and hypersensitivity reactions. It was
confirmed that HE800 EPS improved collagen structuring in
engineering connective tissue model and encourage fibroblast
settled in extracellular matrix. Additionally, it was experimented
156
that through collageneous matrix building, the addition of HE800
EPS, amplified and accelerated collagen fibrils formation with 67
nm periodic striations. Polysaccharide B1 from the marine
bacterium, Pseudomonas sp., was found to be further
cytotoxically dynamic to the central nervous system and lung
cancer cell lines since the EPS induced apoptosis in the cells. It
has been established that the EPS produced by marine bacteria
have strong affinity for heavy metals and thus EPS can be utilised
for bioremediation of heavy metals from the environment. It was
found that the powerful interaction of EPS produced by
Altermonas sp. strain 1644 between divalent and monovalent
cations. The EPS produced by A. infernus also showed a very
strong affinity for lead, cadmium and zinc.
Recent advances
In vitro studies performed in Italy found that the EPS1-T14
formed by marine bacterium, B. licheniformis T-14 inhibited the
biofilm formation of clinical isolates Escherichia coli 463,
Klebsiella pneumoniae 2659 , Pseudomonas aeruginosa 445 and
Staphylococcus aureus 210 to a substantial extent reliant on the
dosage and concentrations of the EPS1-T14. According to them,
this antibiofilm activity of EPS1-T14 was owing to the surfactant
properties of EPS1-T14 which could influence bacterial cell
surface hydrophobicity and thereby hinder with the initial
adhesion step, which was important for the biofilm formation.
Authors also recommended that the existence of fructose and
fucose in the EPS1-T14 could hinder with the surface lectins of
various bacteria such as Pseudomonas aeruginosa, thereby
interfering with the congregation of adhesions in the cell wall.
Therefore, EPS1- T14 could be interesting anti-adhesive drug in
medical and nonmedical prospects, which desires further
researches and studies.
5.9 Biosurfactants derived from marine
microbes
Several biosurfactants have been accounted to exhibit
microbicidal activity against diverse human pathogens; besides,
these compounds regularly display anti-adhesive and anti-biofilm
157
activities, making them functional to decrease the adhesion and
colonization by pathogenic microorganisms, in addition to to
eliminate pre-formed biofilms. The most significant
biosurfactants produced by marine microorganisms are
represented in Table 10.
A number of of these biosurfactants are efficient against a
broad spectrum of human pathogens, including Gram-positive and
Gram-negative bacteria, as well as the yeast Candida albicans.
Additionally, in some cases they are also efficient against MDR
clinical isolates. Therefore, they can be an substitute to the
existing drugs to treat infections caused by those pathogens.

Table: 10 Biosurfactants produced by marine microorganisms

158
 It should be renowned that, with the exemption of the isolate
Serratia marcescens described by Dusane and co-workers, all the
other microorganisms reported in Table 10 were isolated from
marine samples collected in the coastal waters of India, which
recommends a elevated levels of investment of this country in the
exploration of the marine resources. Concerning the phylogeny of
the different isolates, more than half of them are actinomycetes.
Furthermore, most of those microorganisms were isolated from
marine macro-organisms.
Concerning the lipopeptide biosurfactant formed by the
Bacillus circulans strain reported by Das, six diverse fractions
were acquired from the crude extract using reverse phase HPLC,
and only one of them was accountable for the antimicrobial
activity revealed by the crude biosurfactant. It should be pointed
out that, contrary to other lipopeptide biosurfactants such as
surfactin, this biosurfactant did not confirm haemolytic activity,
which could assist its use as a therapeutic agent. Likewise, the
lipopeptide biosurfactant produced by B. circulans DMS-2 was
recognized as a mixture of diverse fengycin isoforms (including
C15-, C16- and C17-fengycin). Four diverse surface-active
fractions were resolved and purified through HPLC, and only one
of them (containing C16- and C17-fengycin) was accountable for
the antimicrobial assessment observed in the crude biosurfactant.
The HPLC-purified fractions of the lipopeptide biosurfactant
produced by B. circulans displayed lower MICs and MBCs
against S. marcescens, Proteus vulgaris and Enterobacter cloacae
(between 10 and 60 µg/ml-1) when contrast with the conventional
antibiotics penicillin and streptomycin (between 40 and 900
µg/ml-1). Concerning the MDR Escherichia coli, Klebsiella
pneumoniae and Staphylococcus aureus (which were resistant to
penicillin and streptomycin at concentrations up to 1000 µg/ml-1),
MICs and MBCs between 60 and 800µg/ml-1 and 200–1000
µg/ml-1, respectively, were acquired for this biosurfactant. The
glycolipid biosurfactant formed by Streptomyces sp. MAB36
possessed a analogous inhibitory activity against Aspergillus
niger and C. albicans to the conventional antifungal nystatin. The
biosurfactant produced by Nocardiopsis dassonvillei MAD08 was
more effective against E. coli and Staphylococcus epidermidis
159
than chloramphenicol. At last, the biosurfactant produced by S.
marcescens displayed an elevated inhibitory effect against C.
albicans and Pseudomonas aeruginosa in contrast to the
conventional antimicrobials fluconazole and streptomycin,
respectively.
In the case of the isolate B. circulans, the microbicidal
assesment of the biosurfactant was reliant on the carbon source
used, owing to the production of diverse isoforms in various
media; the biosurfactant formed using culture media containing
glycerol, starch or sucrose revealed a superior antimicrobial
activity when compared with the one produced in a medium
containing glucose. Though, in the case of marine
microorganisms, which are modified to the marine environment
circumstances, their cultivation in the laboratory or in industrial
fermenters can be hard. One substitute is to produce those
biosurfactants in heterologous hosts. In the case of the lipopeptide
biosurfactant formed by Bacillus licheniformis NIOT-AMKV06.
The strain Streptomyces sp. ISP2-49E, isolated from
marine sediment samples acquired from Galveston Bay
(Texas) must be mentioned. This isolate formed the rhamnolipid
biosurfactant L-rhamnosyl-L-rhamnosyl-β-hydroxydecanoyl-β-
hydroxydecanoate (Rha-Rha-C10-C10), being the first report on a
rhamnolipid-producing Streptomyces strain. Rhamnolipids have
been accounted to possess a wide spectrum of antimicrobial and
anti-adhesive activities. However, the main rhamnolipid
producers are P. aeruginosa strains, an opportunistic human
pathogen. Consequently, the utilization of alternative non-
pathogenic rhamnolipid producers can contribute to the protected
use of rhamnolipids as therapeutic agents. That can be
accomplished using either non-pathogenic natural rhamnolipid-
producing strains, or engineered non-pathogenic hosts expressing
the genes necessary for the synthesis of rhamnolipids. The
antagonistic activities displayed by these biosurfactants against
human pathogens (including MDR pathogens) make them
candidates to be used as a substitute to traditional antibiotics.

160
5.10 Pigments produced by marine microbes
Pigmented compounds formed by marine microbes are the
scope of this study, precise examples will still be reveal for
comparative purposes, it is essential to outline common biological
activities or because the same pigments were isolated from a
variety of microorganisms.
Prodiginines
There are many research reports and reviews regarding
prodiginines and their biological activity investigations. In
addition to the Serratia, several species of marine bacteria
of the genera Streptomyces, Actinomadura, Pseudomonas,
Pseudoalteromonas, and others have also been reported to produce
prodigiosin and related compounds. In particular, Alteromonas
denitrificans, which was isolated from the fjord systems off the
west coast of Norway and later reclassified as Pseudoalteromonas
denitrificans, has been reported to produce cycloprodigiosin. This
compound has immunosuppressive, antimalarial, and apoptosis-
inducing activities. Pseudoalteromonas rubra, found in the
Mediterranean coastal waters, also produces cycloprodigiosin, in
addition to prodigiosins. α-Proteobacteria isolated from a marine
tunicate collected in Zamboanga, Philippines, was reported to
produce heptyl prodigiosin. In vitro antimalarial activity against
Plasmodium falciparum 3D7 (IC50 = 0.068 mM and SI = 20) was
about 20 times the in vitro cytotoxic activity against L5178Y
mouse lymphocytes. In vivo experiments using Plasmodium
berghei-infected mice, at concentrations of 5 mg/kg and 20 mg/kg,
significantly increased their survival, while also causing sclerotic
lesions at the site of injection.
Carotenes.
Carotenes are polyunsaturated hydrocarbons that contain 40
carbon atoms per molecule and are exclusively synthesized by
plants. They are orange photosynthetic pigments significant for
plant photosynthesis. Recently, an unusual halophilic bacterium,
which need 15–25% salt for its regular development, was found
in Santa Pola near Alicante and on the Balearic island of Mallorca,
Spain. Oren and Rodr´ıguez-Valera investigated red-pigmented
161
saltern crystallizer ponds in these region of Spain and
demonstrated that the pigments were carotenoid or carotenoid-like
compounds formed by halophilic bacteria related to the
Cytophaga-Flavobacterium Bacteroides group. Therefore, it has
been shown that Salinibacter is an vital constituent of the
microbial community that contributes to the red coloration of
Spanish saltern ponds. Astaxanthin is one of the carotenoids that
have profitable value as a food supplement for humans and as food
additives for animals and fish. A carotenoid biosynthesis gene
cluster for the production of astaxanthin has been isolated from
the marine bacterium Agrobacterium aurantiacum. Recently,
another astaxanthin-producing marine bacterium was isolated and
identified as Paracoccus haeundaensis.
Violacein
This bacterial strain, Chromobacterium marinum, was
isolated from open ocean waters and formed a blue pigment that
was recognized as violacein on the basis of physicochemical
characteristics. Later, Gauthier described 16 violet-pigmented
heterotrophic bacilli isolated from Mediterranean coastal waters
and proposed the name Alteromonas luteo-violaceus for these
strains. Another six bacterial species were also isolated by
researchers from neritic waters on the French Mediterranean coast
and were very similar to Alteromonas species. These species
produced characteristic pigmentations ranging from pinkish beige
with reddish-brown diffusible pigment, lemon yellow, bright red
turning carmine in old cultures, and orange to greenish-brown.
Light violet, dark violet, or almost black pigments were also
formed and later recognized as violacein. The strains revealed
antibiotic activity against Staphylococcus aureus.
Quinones
Quinones are additional colored compounds with an aromatic
ring structure that have been isolated from marine surroundings.
Quinone derivatives range in color from yellow to red, exhibit
antiviral, antiinfective, antimicrobial, insecticidal, and anticancer
activities, and have many commercial applications as natural and
artificial dyes and pigments. Streptomyces sp. B6921 strain
produced glycosylated pigmented anthracycline antibiotics,
162
including fridamycin D and two new compounds, named
himalomycin A and B, each of which displayed similar levels of
strong antibacterial assesment against Bacillus subtilis,
Streptomyces viridochromogenes, S. aureus, and Escherichia
coli. This strain also produced rabelomycin, N-benzylacetamide,
and N-(2 - phenylethyl) acetamide. Two novel pigmented
antitumor antibiotics, chinikomycin A and B, together with
manumycin A, were isolated from a marine Streptomyces sp.
strain M045.
Melanins
Vibrio cholerae, Shewanella colwelliana, and Alteromonas
nigrifaciens were some of the primary marine bacterial strains
depicted to produce melanin or melanin like pigments. The
pigment synthesized by Vibrio cholerae was reported to be a type
of allomelanin derived from homogentisic acid. Melanin
formation in V. cholera is a effect of alterations in tyrosine
catabolism and not from the tyrosinase-catalyzed melanin
synthetic pathway. Cellulophaga tyrosinoxydans was reported to
have tyrosinase activity and form a yellow pigment recommended
to be a pheomelanin. The most descriptive example of melanin-
producing marine bacteria is the actinomycetes. This is
particularly the case for the genus Streptomyces, from which most
compounds with known biological activity have been isolated. All
Streptomyces strains are reported to use tyrosinases in the
synthesis of melanin pigments. Another important melanin-
synthesizing bacterium is Marinomonas mediterranea, which
forms black eumelanin from L tyrosine
5.11 Preservation methods of Sea foods
Ozone Treatment
Ozone treatment, either by gaseous or dissolved forms, is
among one of the mainly powerful oxidizing and food contact
sanitizing treatments approved by the U.S. Food and Drug
Administration (FDA). Ozone treatments oxidize a variety of
cellular components leading to membrane leakage and eventually
cell death; it has elevated levels of biocidal activity, requires short
contact times, and can take place at the aquaculture level or on the
163
final product. As an example of the former, applying 0.07 mg/L
of ozone directly to seawater at shrimp hatcheries has been shown
to allow the survival of shrimp, but eradicate pathogenic vibrios.
Trout filets treated with ozone for two hours, were evaluated by
(TVB-N) measures, and found to have a shelf-life of 6 days, as
compared to 4 days for untreated filets.
Natural organic treatments
Adding essential oils, tea polyphenols, and organic acids to
seafood products has been recommended to widen shelf-life, limit
pathogen proliferation, and preserve a synthetic preservative free
marketing status. Essential oils such as thyme, oregano, rosemary,
turmeric, and shallots have been shown to decline the levels of
non-pathogenic spoilage bacteria in seafood, when used in
concentrations as low as 0.05 mg/mL. A diversity of polyphenols
including catechins, epigallocatechin gallate (EGCG),
epigallocatechin, epicatechin gallate, and epicatechin, can be
extracted from tea and have been revealed to have antioxidant and
antimicrobial properties. Dipping treatments of dried-seasoned
jumbo squid in a mixed tea phenol solution also showed a
protective effect against bacterial spoilage, moisture loss,
oxidation of lipids, and degradation of lipids. Tea phenol
treatment has also been shown to have a synergistic effect when
combined with ozone treatment to widen shelf-life, and decrease
nucleotide breakdown and lipid oxidation. Organic acids such as
citric acid (300 mg/mL) and lactic acid (150 mg/mL) have been
shown to decrease growth of spoilage organisms in freshly
shucked oyster sample; in addition, dipping treatments of oysters
in each of these organic acids prove a reduction of potentially
pathogenic V. vulnificus below the detection level of 1.0 log/g
from an initial artificially inoculated concentration of 6.0 log/g
Phage Treatment
Two different phage groups have shown assurity
in controlling populations of V. parahaemolyticus in raw
oysters: a Siphoviridae phage pVp-1, and VPp1a phage isolated
from V. parahaemolyticus. Depuration is a control process in
which molluscs are held in potable water that has been treated with
chlorine, ozone or UV light, for a few hours before consumption
164
to reduce their bacterial loads through the process of filter feeding.
While this method is extremely effective in reducing coliform
counts, if not it is carried out at low temperature and for a period
of days, depuration is normally not efficient against vibrios.
However, depuration in the presence of the phage VPp1a was able
to reduce V. parahaemolyticus concentrations by 2.35–2.76 log
CFU/g over a period of 36 h at 16°C. The phage pVp-1 was
investigated for its capacity to eradicate V. parahaemolyticus
contamination as applied as both a bath immersion and directly to
contaminated oyster meat.
High pressure processing
It is commercially used at a range between 200 and 600 MPa,
as an substitute to thermal processing. HPP treatment as short as
1–2 min on oysters increases shelf-life by as many as 11 days, by
lessening the overall bacterial load and in the process kills the
oyster causing the adductor muscle to liberate and making
shucking easier. Particular awareness has been paid to the ability
of HPP to reduce V. parahaemolyticus and V. vulnificus in
oysters. At yield, the density of V. parahaemolyticus is normally
less than 103 MPN/g; however, pathogen levels quickly amplify
to >106 MPN/g if the storage temperature is not properly
controlled, and these levels are dangerous to human health.
Storage temperatures of seafood prior to HPP do not appear to
influence resistance of V. parahaemolyticus to HPP; however,
cold storage may increase the resistance of V. vulnificus to HPP
treatment by increasing the percentage of polyunsaturated fatty
acid in the cell membrane. HPP also leads to meat becoming more
opaque, and consequences in the cooked appearance of numerous
types of fish, two features that may bound its acceptance by
consumers.
Irradiation
Irradiation of food products has become an rising technology
with hopeful features to develop the safety and shelf-life of many
different food types. Irradiation suggests several unique
characteristics including direct inactivation of organisms in frozen
foods. The utilization of gamma irradiation and X-rays to
eradicate pathogenic strains of bacteria such as vibrios in live
165
oysters is becoming a popular substitute to thermal treatment.
Gamma irradiation dose levels from 0.5–3.0 kGy have been tested
on live oysters with studies showing that the maximum dose of
3.0 kGy did not destroy the oysters or affect any of their sensory
attributes. Although, reductions of 6-log V. parahaemolyticus
were observed when dosage levels as low as 1.0 kGy were used.
X-ray treatments on laboratory inoculated V. parahaemolyticus
ready to consume shrimp products treated with 0.1–4 kGy X-ray
levels revealed a 6-log reduction in CFUs at 3 kGy. To accomplish
a 6- log reduction of V. vulnificus in oysters 1.0 kGy was
necessary for half shell oysters and 3.0 kGy for whole shell
oysters.
5.12 Quality control for microbial quality of
fishes
A. Sensory evaluation
Sensory evaluation measures the freshness of fish and fish
products with respect to the five distinct senses including taste,
smell, feel and appearance. Sensory evaluations of freshness are
generally used feature for ensuring quality of fish. Sensory
evaluation can either be objective or subjective but for successful
marketing, both are measured. In case of objective sensory
evaluation, trained personnel are used to categorize freshness,
whereas, in subjective sensing, one is based upon consumer
fulfillment and market investigation of fish markets
B. Microbial assessment
Fish decomposition starts after catch starts and alteration
occur in pH, atmosphere and nutrient composition have effects on
micro flora. Raw fish consists of its own unique flora, determined
by the microbial content of the surrounded water, which still
remain in spite of food processing and subsequent cooling.
Classification of fish with alike microflora is considered when
declaring the quality of Fish and its products. Microbes play a
essential role in the shelf life of fish as gram-negative,
fermentative bacteria (such as Vibrionaceae) spoil unpreserved
fish, whereas psychrotolerant gram negative bacteria
166
(Pseudomonas spp. and Shewanella spp) can continue
development in chillers.
Total viable counts or TVC‟s are utilized generally as a part
of microbiological allowing one to recognize the active number of
growing/dividing microbes. TVC ranges from 102 – 106 cfu/g in
case of fish and the range verify its acceptability. TVC limit varies
greatly upon its conditions (temp, atmosphere, pH etc.). Among
exports are the jew fish (bhola, bhetki), queen fish (talang), tongue
sole fish, ribbon fish (baala), and cuttle fish (katla) marine fishes
from Bangladesh. The highest microbiological limit for the TVAC
is 5 × 105 cfu/g and TVAC mostly ranged from 2.8 × 105 to 4.9 ×
105 cfu/g for exported frozen fish which was below the utmost
acceptable.
C. Biochemical assessment
Sensory evaluations in assessing fish of high maxima or
minimal evaluation values. Biochemical tests come into use when
production is with marginal quality of fish. Usually the chemical
evaluation either increases or decreases with respect to microbial
spoilage or autolysis. Total volatile basic amines (TVB) is one of
has been used as measurements of fish quality. The quantity of
trimethylamine (produced by spoilage bacteria), dimethylamine
(produced by autolytic enzymes during frozen storage), ammonia
(produced by the deamination of amino-acids and nucleotide
catabolites) or other volatile basic nitrogenous compounds in fish,
amplify in concentration. Examination of quality can also be done
by detection of Primary (PV) and secondary (TBARS) lipid
oxidation products formed by oxidation. PV counts amplify in rate
at the preliminary stage of growth curve, and this becomes
reversed at later stages. PV counts include the total quantity of
hydroperoxides by chromatographic techniques in details. PV
measurements could be completed by iodometric titration, ferric
ion complex measurement spectrophotometry, and infrared
spectroscopy.
D. Biosensor Detection
Biosensors are developing as appealing instrumentation
answers for fast detection of food borne pathogens, toxins,
pesticide and medication deposits, toxic metal particles. A
167
biosensor is an quantitative, investigative device consisting a
biological sensing element (such as an enzyme, antibody,
receptor, or microbe) coupled to a chemical or physical
transducer. The transducer surface is employed to fix the
biological sensing element. Transducers are in charge for
converting the particular biological response electrochemical e.g
electrodes), micro mass (quartz microbalances or surface acoustic
wave devices), light (optical fibres), or thermal (thermistors) into
an electrical impulse, which can be detected by a software. After
fatality, oxygen contribution in fish meat ceases, metabolically
lively muscles de hydrolyse ATP and begin to degradation of
forming compounds.
E. Toxin detection
Studies reveal that fishes are most vulnerable to heavy metal
contamination in contrast to other marine life. Toxic heavy metals
come into the human body through food sources, particularly fish.
Lead contaminates fish through water pipes, spilled paint and
leaking gasoline in the sea. Stumpy levels of Pb slowly affect the
brain and high exposures causes poisoning. Methyl mercury (Hg)
is normally found in fish such as shark, swordfish and tuna.
Mercury poisoning is the reason for difficulties in movement in
infants. Bangladesh has elevated levels of Arsenic (As), which is
a carcinogenic, whereas cadmium has side effects in the male
reproductive systems. Histamin level discovery during the
HACCP execution could be done by enzyme kits or elisa kits.
Tilapia fish undergo flourishing heavy metal screening which was
revealed by using commercial Elisa kits and veterinary drug
residual screening in Bap. Benzo(a)pyrene (BaP) is one of
Polycyclic Aromatic Hydrocarbons (PAHs) which leads to harm
in physical condition such as red blood cell damage (leading to
anemia), DNA damage, genotoxicity, lung cancer and
reproductive effects and the best recognized of the carcinogenic
PAH.
F. Spectroscopy method
Quality Inspection spectroscopes are planned to identify dark
colorings in white fish. Dark colorings in the fish body relay to
spoilage, insufficient bleeding and browning, bruising having
168
harmful impact in consumer preference. Grade E quality fish must
not contain deficiency such as scars, bruises or clots and
discoloration is allowed. For grade A, quality fish, no evident
bruising is allowed. Evaluation of bruises in pacific pink salmon
(Oncorhynchus gorbuscha) by means of visible spectroscopy by
partial least-squares (PLS) modeling is a dependable technique
utilised these days. QIM scores could be calculated by using
visible spectroscopy in quantifying the freshness of cod. Storage
changes and frozen-thawed differences of salmon fillets using VIS
spectrometry are also depicted. Near Infra Red Spectrophotometry
was employed for real-time quantification of bacterial loads on
fish.
G. Machine vision
Machine vision has initiated the automatic operations in fish
quality inspection and guarantee in terms of size, weight,
numbers, grading, species recognition and monitoring. Deficiency
in fish cause quality degrading along with a more composite
procedure for programmed grading. Deficiency like injury,
bruises and dissections required numerous imaging modes in a
single system could also improve the visibility of superficial
defects in Atlantic salmon fillets. X-ray machine vision identified
fish bones in salmon and trout fillets using X-ray images with
precision of 99%.
5.13 Marine resources used as food and drugs
Microalgae, the most chief and simply-organized members
of marine plant being, are prosperous sources of food ingredients,
such as β-carotene, Vitamins C, A, E, H, B1, B2, B6 and B12,
astaxanthin, polysaccharides and polyunsaturated fatty acids. As
such, bioactive molecules from microalgae are profitably
produced, employed as food additives and also incorporated into
infant milk formulations and dietary complement.
Macroalgae are a significant source of vital nutrients and
novel bioactive molecules for human nutrition. For instance, red
and brown seaweeds are substitute sources of vitamins, minerals
and proteins and are good sources of essential fatty acids. They
have been utilised to prepare bioactive peptides and to develop
169
protein digestibility. Macroalgae are a significant source of
essential nutrients and novel bioactive molecules for human
nourishment. For instance, red and brown seaweeds are choice
sources of vitamins, proteins and minerals and are fine sources of
essential fatty acids. They have been used to prepare bioactive
peptides and to develop protein digestibility.
Marine Fish
Fish inhabit the utmost position in marine animal
consumption and are significant to the world economy. In 2012
fish provided approximately 16% of the world’s protein
requirements with herring, cod, salmon, tuna, flounder, mullet and
anchovy being the most ordinary species of fish utilised for food.
One of the major commercially canned fishery products in the
world is tuna. The dietary benefits of fish consumption are due to
the existence of proteins, unsaturated essential fatty acids,
minerals (for instance, calcium, selenium, iron and zinc), and
vitamins, namely Vitamin A, B3, B6, B12, E and D. Research has
also revealed that peptides derived from fermented fish following
enzymatic treatment may be of use in therapeutics for the
management of many common acute and chronic diseases such as
viral infections, hypertension, cancer and Alzheimer’s disease.
Fish collagen may also be utilised in bone treatment as an
substitute to mammalian collagen which is known to be
immunogenic.
Marine Invertebrates:
Sponges
Sponges are sessile metazoans that consist of a gelatinous
material, mesophyl, and are the simplest form of multicellular
animals. Their hollow pitcher-like body is reinforced by spicules
(a needle-like silica or calcium structure) and spongia (collagen
fibers). There are various chemically-diverse compounds like
alkaloids, terpenoids, polyketides, macrolides, polyphenolic
compounds, peptides and sterols that are isolated from sponges
with the prospective to cure a variety of ailments.

170
Molluscs and Echinoderms
Bioactive peptides acquired from the fermented blue mussel
and oyster sauces considerably decrease hypertension whilst
ground abalone and its shells are utilised for curing eye diseases.
Many Asian populations eat cuttlefish, squid, octopus and
nautiloids owing to their therapeutic effects, for instance rickets
are cured with the bones of cuttlefish, in addition to
gastrointestinal disorders and ear inflammation.
Nutrient composition of marine crustaceans like shrimp and
krill was analysed and found to reduce the total blood lipids in
humans, and develop Vitamin A levels, specific proteins and
eicosapentaenoic acid, an omega-3 fatty acids. These are
recommended to be used in the development of value-added
health food products and for human consumption owing to
elevated nutritional value.
Marine derived bioactive component:
Proteins
Proteins from marine sources including fish (cod, herring,
tuna, Pollock, hake and haddock), crustaceans, molluscs,
extremophiles like Dunaliella and seaweeds have distinctive
properties such as gel-formation, film and foaming capacity,
anticoagulant, antioxidant and microbicidal activity. For instance,
collagen, gelatin and albumin are widespread marine proteins
used in foods and are enzymatically hydrolyzed for the production
of bioactive peptides which may have the prospective to be used
as nutraceuticals. The marine protein protamine is also utilised as
a natural bactericidal preservative in the food industry.
Peptides
Many researchers have focused on the progress of
pharmaceutical compounds from marine-derived peptides
particularly for ACE inhibition and antihypertensive function.
Marine proteins from fish, molluscs and crustaceans are amongst
the prosperous sources of bioactive molecules. Researchers
reported that fish protein hydrolysates have some novel peptides
that can bind to cell surface receptors and improve calcium
absorption. The therapeutic application of these peptides is the
171
management of osteoporosis and Paget’s disease. Collagen is a
precious part of bovine and porcine meat and is utilised in various
industries like cosmetics, pharmaceutics, food and biomedicine.
Fatty Acid
Marine fish species and algae have been recognized as
sources of polyunsaturated fatty acids which are rich in ω-3 or ω-
6 fatty acids. The occurence of these unsaturated fatty acids in
marine-derived foods raise their applicability as nutraceuticals in
the food industry. The most widespread sources of marine oils are
fungi (Phycomycetes), fish (salmon, tuna, sardines, and herring),
microalgae, extremophiles, macroalgae (Bryophyta, Rhodophyta)
and krill. Consumption of marine oils supply numerous health
benefits like visual and neurodevelopment, amelioration of
diseases such as hypertension and arthritis and a reduced threat of
cardiovascular trouble.
Prebiotics
Prebiotics are non-digestible, selectively-fermented
compounds that encourage the growth and activity of beneficial
gut microbiota which, in turn, offer a health profit to the host.
Frequently, prebiotics are oligosaccharides such as chitosan
oligosaccharides, while certain other algal polysaccharides are
also identified to have a prebiotic activity. Bifidogenic benefits
have been also accounted from the exopolysaccharides formed by
marine lactic acid bacteria. Additionally, the cyanobacterial
biomass of Spirulina platensis is able to stimulate both
Lactobacillus and Bifidobacterium species, encourage their
prebiotic effect. These pigments offer nutraceutical agents, natural
food coloring, anticarcinogenic, anti-inflammatory and
antioxidant compounds.
Enzymes
Enzymes derived from marine sources are lipase, chitinolytic
enzymes, polyphenol oxidase (Catecholase, tyrosinase, cresolase,
polyphenolase, catechol oxidase, phendase), and transglutamase,
and red algal enzymes implicated in a starch degradation pathway
(for instance, α-1, 4-glucanase). Enzymes are also utilised as food
ingredients. They are employed in food processing due to their salt
172
tolerance, specificity, diverse properties and high activity at mild
pH. Biomolecules such as enzymes isolated from extremophiles
can be extremely utilised in the food industry owing to their
unique activities under abnormal conditions, and it has been
generally accepted that extremophiles have strong prospective to
be precious resources for utilisation in biotechnology.
Vitamins and Minerals
Vitmains and minerals carry out many essential functions in
the body, for instance, they provide transport inside cells and also
supply as cofactors during metabolic processes. Seaweeds are
prosperous sources of vitamins and minerals including iron,
iodine, manganese and zinc. Some types of seaweed could be
employed as natural sources of iodine.

173
6 References
 Cristiana Cravo-Laureau and Robert Duran. Marine coastal
sediments microbial hydrocarbon degradation processes:
contribution of experimental ecology in the omics’era,
Frontiers in Microbiology, 2014, 39(5): 1-8.
 El-Tarabily, K.A. Total microbial activity and microbial
composition of a mangrove sediment are reduced by oil
pollution at a site in the Arabian Gulf. Canadian Journal of
Microbiology, 2002, 48: 176–182.
 Gregory, J. Dick, Karthik Anantharaman, Brett J.
Baker, Meng Li, Daniel C. Reed and Cody S. Sheik. The
microbiology of deep-sea hydrothermal vent plumes:
ecological and biogeographic linkages to seafloor and water
column habitats, 2013, 4(124): 1-16

 Winn, C.D., Karl, D.M., and Massoth, G.J. Microorganisms

in deep-seahydrothermalplumes. Nature,1986, 320: 744–746.

 Kadko, D.C., Rosenberg, N.D., Lupton, J.E., Collier, R.W.,

and Lilley, M.D. Chemical-reaction rates and entrainment
within the Endeavor Ridge hydrothermal plume. Earth Planet
Science Letters, 99, 315–335.

 Baharum, S.N., Beng, E.K., and Mokhtar, M.A.A. Marine

Microorganisms: Potential Application and Challenges, 2010,
10(6): 555-564.

 Malin Bomberg, Leone Montonen, Uwe Münster and German

Jurgens. Diversity and function of archaea in freshwater
habitats, Current trends in Microbiology, 2008, 1-30.

 Charpy, L., Casareto, B.E., Langlade, M.J., and Suzuki, Y.

Cyanobacteria in Coral Reef Ecosystems: A Review, Journal
of Marine Biology, 2012, 259571: 1-9.

174
 Margot Traugin, Daniel vaulot. Green Microalgae in marine
coastal waters: The Ocean Sampling Day(OSD) Dataset,
Scientific reports, 2018, 14020(8): 1-12.

 Thomas, A., Richards, Meredith, D.M., Jones, Guy Leonard,

and David Bass. Marine Fungi: Their Ecology and Molecular
Diversity. Annual Reviews of marine science, 2012, 4, 495-
522.

 Mathias Middelboe, I.D., Corina, P.D., Brussaard. Marine

Viruses: Key Players in Marine Ecosystems. Viruses, 2017,
302(9): 1-6.

 Mark, R., James, Julie, A., Hall, Paul Barrett, D. Grazing by

protozoa in marine coastal and oceanic ecosystems off New
Zealand. New Zealand Journal of Marine and Freshwater
Research, 1996, 30: 313-324.

 Andreas Eich, Amanda, K., Ford, Maggy, M., Nugues, Ryan,

S., McAndrew, Christian Wild and Sebastian, C.A., Ferse.
Positive association between epiphytes and competitiveness
of the brown algal genus Lobophora against corals. Peer J, 1-
14.

 Seghal Kiran G, Sivasankari Sekar, Pasiyappazham

Ramasamy, Thangadurai Thinesh, Saqib Hassan, Anuj
Nishanth Lipton, A.S., Ninawe, Joseph Selvin. Marine sponge
microbial association: Towards disclosing unique symbiotic
interactions. Marine environmental research, 2018, 1-11.

 Anna Christensen, Glenroy D.A., Martin. Identification and

bioactive potential of marine microorganisms from selected
Florida coastal areas, 2017, 6, 1-10.

 Faturrahman, Anja Meryandini, Iman Rusman.

Morphological and physiological of marine agarolytic
bacteria isolated from central lombok coastal, 2013, 79-84.

175
 Hongyue Dang. Grand Challenges in Microbe-Driven Marine
Carbon Cycling Research. Frontiers in Microbiology, 2020,
1039(11): 1-6.

 Silvia Pajares and Ramiro Ramos. Processes and

Microorganisms Involved in the Marine Nitrogen Cycle:
Knowledge and Gaps, 2019, Frontiers in marine science, 6,
739, 1-33.

 Sonya T. Dyhrman, James W, Ammerman and Benjamin,

A.S, Van Mooy. Microbes and the Marine Phosphorus Cycle.
Oceanography, 2007, 20(2):110-116.

 Bo Barker Jørgensen, Alyssa J., Findlay and André Pellerin.

The Biogeochemical Sulfur Cycle of Marine Sediments.
Frontiers in Microbiology, 2019, 849(10): 1-27.

 Sherene Mathai, Keerthana Rachel Roy, Rajendran N.

Importance of Marine Thermophiles in Biotechnological
Applications. International Journal of Pharmaceutical
Sciences Review and Research, 27(2): 153-160.

 Koki Horikoshi. Alkaliphiles: Some Applications of Their

Products for Biotechnology, Microbiology and Molecular
Biology Reviews, 1999, 63(4): 735-740.

 https://microbewiki.kenyon.edu/index.php/Barophilic
bacteria in Marinas Trench, Challenger Deep

 Gabriel Zamith Leal Dalmaso, Davis Ferreira and Alane

Beatriz Vermelho. Marine Extremophiles: A Source of
Hydrolases for Biotechnological Applications. Marine Drugs,
2015, 13, 1925-1965.

 Karl O. Stetter. Hyperthermophilic microorganisms.

Encyclopedia of Life Support Systems, 1, 1-10.

176
 Haresh Z Panseriya, Haren B Gosai, Bhumi K Sachaniya,
Anjana K Vala and Bharti P Dave. Marine Microbial Mettle
for Heavy Metal Bioremediation: A Perception. Marine
Ecology: Current and Future Developments, 2019, 1, 410-
435.

 Mehdi Hassanshahian and Simone Cappello. Crude Oil

Biodegradation in the Marine Environments. Biodegradation-
Engineering and Technology, 101-135.

 Isla Fitridge, Tim Dempster, Jana Guenther & Rocky de Nys.

The impact and control of biofouling in marine aquaculture: a
review. Biofouling: The Journal of Bioadhesion and Biofilm
Research, 28, 7: 649-669.

 Francis Hassard, Ceri L. Gwyther, Kata Farkas, Anthony

Andrews, Vera Jones, Brian Cox, Howard Brett, Davey L.,
Jones, James E. Mc Donald and Shelagh K., Malham.
Abundance and distribution of enteric bacteria and viruses in
the coasta and Estuarine sediments- a review. Frontiers in
Microbiology, 2016, 1692 (7): 1-31.

 Elisa L. Eliott, Ritta R. Colwell. Indicator Organism for

estuarine and Marine waters. FEMS Microbiology Reviews,
2985(32): 61-79.

 https://www.epd.gov.hk/epd/wqo review/en/pdf/Technical
Note S4.pdf

 Necas, J., Bartosikova, L. Carrageenan: a review, Veterinarni

Medicina, 58, 2013 (4): 187–205.

 Mohanty, D. Seaweed liquid fertilizer and its role in

agriculture productivity. An international Journal of
Environmental Sciences, 3, 23 – 26.

 Chen Zhang, Se-Kwon Kim. Research and Application of

Marine Microbial Enzymes: Marine Drugs 2010, 8, 1920-
1934.
177
 Emiliana Tortorella, Pietro Tedesco, Fortunato Palma
Esposito, Grant Garren January, Renato Fani, Marcel Jaspars
and Donatella de Pascale, Marine Drugs, 2018, 355, 1-16.

 Sithranga Boopathy, N., and Kathiresan, K. Anticancer

Drugs from Marine Flora: An Overview, Journal of
Oncology, 2010, 214186, 1-18.

 Devesh Parkar, Rahul Jadhav, Mukesh Pimpliskar, Marine

bacterial extracellular polysaccharides: A review. Journal of
Coastal Life Medicine, 2017; 5(1): 29-35.

 Eduardo, J. Gudiña, José, A., Teixeira and Lígia R.,

Rodrigues. Biosurfactants Produced by Marine
Microorganisms with Therapeutic Applications. Marine
Drugs, 2016, 38(14): 1-15.

 Azamjon B., Soliev, Kakushi Hosokawa, and Keiichi

Enomoto. Bioactive Pigments from Marine Bacteria:
Applications and Physiological Roles. Evidence-Based
Complementary and Alternative Medicine, 2011, 670349, 1-
17.

 Jennifer Ronholm, Fiona Lau and Swapan K. Banerjee.

Emerging Seafood Preservation Techniques to Extend
Freshness and Minimize Vibrio Contamination. Frontiers in
Microbiology, 2016, 350, (7): 1-6.

 Hafiz Ansar Rasul Suleria, Simone Osborne, Paul Masci and

Glenda Gobe. Marine-Based Nutraceuticals: An Innovative
Trend in the Food and Supplement Industries, Marine
Drugs, 2015, 13, 6336-6351.

178

View publication stats