Microbes in Bioremediation

Bioremediation

- procedure for the eradication of environmental pollutants from the

ecosystem through the application of microbes to restore the ecosystem to
its original form

- a unique method-cleaning polluted environments from the atmosphere

(industrial emissions and soil vent gases), solids (soils, sediments, sludge),
liquids (groundwater, industrial effluents), raw materials from industrial
processing.

- ability of certain microorganisms-convert, modify and utilize toxic

pollutants in order to obtaining energy & biomass production in the process
Bioremediation

-Bioremediation is a biological mechanism of recycling wastes into another form

that can be used and reused by other organisms. It is the process in which
microorganisms are used to degrade and remove pollutants from the
environment

-That means, it is a procedure for the eradication of environmental pollutants

from the ecosystem through the application of microbes to restore the
ecosystem to its original form

- It is a unique method-cleaning polluted environments from the atmosphere

(industrial emissions and soil vent gases), solids (soils, sediments, sludge),
liquids (groundwater, industrial effluents), raw materials from industrial
processing.

- Based on the ability of certain microorganisms-convert, modify and utilize

toxic pollutants to obtain energy & and biomass production in the process
Why microbes?
Why microbes?

- Microorganisms-widely distributed in the biosphere because their metabolic

ability is very impressive & they can be easily grown in a wide range of
environmental conditions
- “cheaper, simpler and more environmentally friendly ”

- microbiological well-organized procedural activity which is applied to break

down or transform contaminates to less toxic or nontoxic elemental and
compound forms.

- Interest in the microbial-based bioremediation of contaminants has

increased in recent years, as people endeavour to find sustainable ways of
remediating polluted environments

- biotransformation and bioremediation-based methods strive to harness the

naturally occurring microbial catabolic diversity-degrade, transform/
accumulate vast amounts of compounds, including radionuclides, metals,
pharmaceutical substances, polyaromatic hydrocarbons (PAHs), &
polychlorinated biphenyls (PCBs).
Bioremediators

Bioremediators are biological agents used for bioremediation;

Bacteria, archaea, fungi, algae, etc
How do they do it?
 How do they do it?

-Enzymes ~ oxidoreductases, hydrolases, lyases, transferases,

isomerases and ligases.
 Types of bioremediation
-Ex-situ bioremediation techniques:

- excavation of the samples & treatment is done away from the contaminated
sites->high cost & disruption of natural sites

- Factors-influence efficiency: cost, intensity, type, degree and depth of

pollution, geographical location and geology of the polluted site

- eg., composting which -degradation of organic waste products in p/o

microorganisms typically at elevated T-55C to 65C.
-In-situ bioremediation techniques:

- involves treatment of polluted substances at polluted site itself. This technique does
not disturb soil structure as does not require excavation.
-
these procedures seem to be less costly in comparison to ex situ, because no additional
cost is involved in excavation procedures but cost of designing and installation of
sophisticated instruments on-site for improvement in efficiency increases cost

-successfully used for treatment of heavy metals, dyes, hydrocarbons and chlorinated
solvents at polluted sites

-significant challenge-supply of nutrients/chemicals -microbes + appropriate mixing-

contaminants-to be degraded

This can be achieved through -Biostimulation and Bioaugmentation

Biostimulation refers to the stimulating Bioaugmentation is a technique which

activity of the microorganisms by providing involves a cluster of natural microbial
nutrients like phosphorus, nitrogen, oxygen strains or a genetically engineered strain
and other electron acceptors. for the treatment of contaminated soil or
water.
Types of microorganisms used in bioremediation

-Bacteria:

Bacillus cereus, B. subtilis, and Pseudomonas putida-Cr (VI)

- Sporosarcina ginsengisoli, Pseudomonas veronii, & Kocuria flava -potential -
eliminate As, Cu, Cd, and Zn
Types of microorganisms used in bioremediation

- Fungi (Mycoremediation):

- Strains of Rhizopus oryzae -biosorption ability of pentachlorophenol-

methylation and dechlorination.

- Zygomycetes and Aspergillus sp. have been validated to transmit the

property of decolorizing and detoxifying textile wastewaters.
-
- Penicillium chrysogenum, Scedosporium apiospermum, P. digitatum, &
Fusarium solani are also described for degradation capabilities of PCBs
Types of microorganisms used in bioremediation
- Algae (Cyanoremediation)

- vital role in the CO2 fixation & also very advantageous biomass for biofuel
production

- Selenastrum capricornutum & Monoraphidium braunii-potential-remove

bisphenol, benzene, toluene, and naphthalene from the polluted environment

- Chlorella sorokiniana, Nannochloris sp., and Chlamydomonas mexicana-

diclofenac, ibuprofen, paracetamol, metoprolol, carbamazepine, ciprofloxacin
carbamazepine triclosan and Carbamazepine.
Heavy Metal contamination
- different sources of heavy metals in the environment-natural, agricultural,
industrial solid waste, inland effluent, atmospheric sources, and so on.

- These deteriorators-either organic [e.g., PCBs, polycyclic aromatic

hydrocarbons (PAHs), fertilizers, pesticides] or inorganic pollutants including
various heavy metals (e.g., Cd, Cu, As, Zn, Hg, Pb) and other heavy metals.

- Microbes-play a necessary role in bioremediation of heavy metals

Why to stress on heavy metal contamination?

-Augmentation of agriculture & industries - harm to humans, livestock,

wildlife, flora, and fauna, causing ecological problems leading to disparity in
nature by delivering a wide range of xenobiotics

-Alleviating heavy metal conc. in water is vital-quality of aquatic living org.

- Zn, Cu, Ni, Fe, and Mn-essential trace elements. High-level accumulation of
these metals or ingested in greater amounts than the required conc.-
serious problems
Why to stress on heavy metal contamination?

- severe toxic effects-exposed to excessive conc.

- Ni, Cu, Fe and As-beneficial-human body-minimal amts but are

toxic/mutagenic/carcinogenic at a high conc.

- ability to accumulate in different parts of the human body.

- bind various biomolecules, such as proteins, nucleic acids-interfere with

their functions.
-Bacterial remediation:

- Microbial biomass

- Bacteria-important biosorbents due to their ubiquity, size, and ability to

grow under controlled conditions & resilience to environmental conditions.

- Flavobacterium, Pseudomonas, Enterobacter, Bacillus, and Micrococcus sp.

- great biosorption ability -high surface-to-volume ratios and potential active

chemisorption sites (teichoic acid) on the cell wall.
-Bacterial remediation:

- more stable and survive better-mixed cultures-consortia of cultures-

metabolically superior for the biosorption of metals and are more
appropriate for field applications.

- 78% reduction-(Cr) using-Acinetobacter sp. & Arthrobacter sp.

- Micrococcus luteus-remove Pb from a synthetic medium.

- biosorption of Pb, Cr, and Cd in tannery effluent using Bacillus subtilis, B.

megaterium, B. megaterium-highest Pb reduction followed by B. subtilis.

- B. subtilis exhibited the highest ability to reduce the concentration of Cd

followed by B. megaterium after 20 days.
Heavy metals Bioremediator
Cu Bacillus sp. Pseudomonas aeruginosa Micrococcus luteus, Citrobacter sp.
Enterobacter sp.

Ni P. aeruginosa P. valderianum, Desulfovibrio desulfuricans Enterobacter

Zn Bacillus sp. D. quercina Streptomyces rimosus Thiobacillus ferrooxidans

P. aeruginosa, Citrobacter sp

Cd P. valderianum Citrobacter, D. desulfuricans

Pb Citrobacter sp. D. quercina Burkholderia sp. Corynebacterium glutamicum

Bacillus firmus Pseudomonas putida

Hg Geobacter metallireducens
Au G. metallireducens Stenotrophomonas sp.
Ag G. metallireducens
Cr Desulfovibrio vulgaris D. desulfuricans Desulfuromonas acetoxidans
-Fungal remediation:

- Rhizopus, Penicillium & Aspergillus.

- Aspergillus sp. -removal of chromium in tannery wastewater.

- Coprinopsis atramentaria ability-bioaccumulate 76% of Cd2+ and 94.7% of

Pb2

- fungal biomass of A. niger, Rhizopus oryzae, Saccharomyces cerevisiae, and

Penicillium chrysogenum -convert toxic Cr (VI) to less toxic or non-toxic Cr
(III).

- Candida sphaerica produce biosurfactants with removal efficiencies of 95%,

90%, and 79% for Fe, Zn, and Pb, respectively.

- Several strains of yeasts, Hansenula polymorpha, S. cerevisiae, Yarrowia

lipolytica, Rhodotorula pilimanae, Pichia guilliermondii, and Rhodotorula
mucilage have been used to bioconvert Cr (VI) to Cr (III)
-Fungal remediation:
- Saccharomyces cerevisiae -ion exchange mechanism -acts as a biosorbent to
remove Zn (II) and Cd (II).

- Cunninghamella elegans, Stachybotrys sp., Allescheriella sp., Botryosphaeria

rhodina, Phlebia sp., Pleurotus pulmonarius and Botryosphaeria rhodina.

- Aspergillus parasitica and Cephalosporium aphidicola can biodegrade soils

contaminated with Pb (II) via biosorption process.

- Neocosmospora vasinfecta, Verticillum terrestre, Hymenoscyphus ericae,

etc. having ability to convert mercury (II) to a nontoxic oxidation state of
mercury.
 Heavy metals
Bioremediator
Cr Aspergillus sp. Rhizopus sp. A. lentulus A. foetidus Rhizopus
oligosporus Termitomyces clypeatus

Zn Trametes versicolor¸ Rhizopus arrhizus Penicillium spinulosum

Cu R. arrhizus P. spinulosum Penicillium chrysogenum Ganoderma
lucidum, Aureobasidium pullulans Aspergillus niger

Ni Rhizopus arrhizus R. nigricans

U Pleurotus mutilus Aspergillus terreus

Cd R. arrhizus R. nigricans Phanerochaete chrysosporium Pleurotus

sapidus P. chrysogenum Aspergillus sp.

Pb R. arrhizus R. nigricans P. ostreatus P. chrysogenum A.

niger A. lentulus
Hg P. sapidus
Th A. terreus
 Yeasts
Heavy Metals Bioremediator

Pb Saccharomyces cerevisiae

Cd S. cerevisiae Candida tropicalis

Cr S. cerevisiae

Ni S. cerevisiae

Cu Pichia guilliermondi C. tropicalis

Zn S. cerevisiae C. tropicalis
-Algal remediation:

- Phycoremediation - use of various types of algae and cyanobacteria for the

remediation of heavy metals by either removal or degradation of toxicants.

- Algae -chemical moieties –surface, hydroxyl, carboxyl, phosphate, and

amide, which act as metal binding sites.

- dead cells of Chlorella vulgaris to remove Cd2+, Cu2+, and Pb2+ ions from
aqueous solutions under various conditions of pH, biosorbent dosage, and
contact time.
Heavy Metals Bioremediator

Cr Spirogyra sp. Spirulina sp.

Cu Spirogyra sp. Spirulina sp. Chlorella vulgaris

Pb C. vulgaris Nostoc sp.

Cd C. vulgaris Nostoc sp.

Zn Nostoc sp.

Ni C. vulgaris Nostoc sp.

Fe Nostoc sp.
-Oil spills:
- defined as the uncontrolled release of crude oil hydrocarbons into the environment.

- 3 million metric tons of oil contaminants spill into the ocean annually.

- Common sources-spills - tankers, pipelines, storage tanks, refineries, drilling rigs,

wells, and platforms, bunkering activities, accidental spills-collision, ramming,
fire/explosion, tank failures, well blowout & so on………….

- significant harm to humans and the environment-endanger public health - direct

exposure and through contamination of drinking water, interfere with the livelihood of
fishermen and disrupt the local economy, and severely harm organisms and ecosystems
-Oil spills:

- detrimental effects-can persist over time.

- environmental recovery 2 to 10 years, a shoreline survey in Alaska found

over 55,000 kg of relatively unweathered oil from the Exxon Valdez spill
remained in subsurface sediments over a decade after the spill, continuing to
increase mortality in local wildlife
-
The Exxon Valdez spill (Mar, 24-1989)-hundreds of millions of dollars in damages &
killed hundreds of thousands of organisms including seabirds, otters, and whales
Thank you!
-Land Oil spills:
Biodegradation of hydrocarbons by naturally occurring populations of microorganisms is
one of the primary mechanisms by which petroleum is removed from the environment

- Pseudomonas and Mycobacterium-degrade PAH’s.

- Geobacter, Achromobacter, Bacillus, Arthrobacter, and Phanerocheate genus

-degrade petroleum products.

- Pseudomonas, Aeromonas, Moraxella, Beijerinckia, Flavobacteria,

Chrobacteria, Nocardia, Corynebacteria, Modococci, Streptomyces, Bacilli,
Arthrobacter, Aeromonas, & Cyanobacteria and some yeasts.

- A recombinant strain of Acinetobacter baumannii was found to magnify

degradation rates at crude oil-contaminated sites
-Land Oil spills:

- Bacteria- Yokenella sp., Alcaligenes sp., Roseomonas sp., Stenotrophomonas

sp., Acinetobacter sp., Flavobacter sp., Corynebacterium sp., Streptococcus
sp., Providencia sp., Sphingobacterium sp., Capnocytophaga sp., Moraxella
sp., Bacillus sp., and Enterobacter

- Fungi from Penicillum, Aspergillus, and Fusarium have also been described
with PAH degrading properties

- Fungi-Neosartorya, Graphium, Talaromyces, Aspergillus, and Amorphoteca

-Marine Oil spills:
- damage to the brain, liver, and kidneys of fish, marine mammals, and
terrestrial species.

- Sea turtles are at risk

- can be a threat to aquatic, marshland, and coastal ecosystems that are

often impacted by the oil.
-
- Eg, The Exxon Valdez oil spill-> contamination of fish along with their
embryos and juvenile larvae

- chronic effects among sediment foraging marine birds resulted in a decline in

their abundance

- About 10% of the total input is from the devastating oil spills, which cause
both ecological and economical damage.
-Bacteria-dominant hydrocarbon degraders in aquatic systems

- Pseudomonas, Achromobacter, Acinobacter, Alcaligenes, Arthrobacter,

Bacillus, Brevibacterium, Cornybacterium, Flavobacterium, Nocardia,
Planomicrobium, Sphingobium, Stenotrophomons, Vibrio.

- Moreover, Alcanivorax, Alteromonas, Cycloclasticus, Erythrobacter,

Halomonas, Marinobacter, Microbacterium, Micrococcus, Muricauda,
Oleibacter, Oleispira, Oleiphilus, Rhodococcus, Rheinheimera, Thalassospira,
Thalassolituus-unique skills to live solely on Hydrocarbons in marine
ecosystems

- It has been reported that hydrocarbon degrading microorganisms would

comprise less than 0.1% of the indigenous microbial population in an
unpolluted environment but it would reach to 100% in an oil-polluted one
Mycoremediation
Fungi-reported to degrade-structurally diverse range of oil derived compounds
by employing a variety of mechanisms-extracellular & some intracellular
enzymes-cytochrome P450 monooxygenases, nitroreductases, transferases
such as laccases and fungal peroxidases.

- Yeast and fungi: Aspergillus, Candida, Cladosporium, Penicillium, Rhodotorula,

Sporobolomyces, Trichodorerma

- Hassanshahian et al. (2012)-isolation and characterization of two crude oil-

degrading yeast strains, Yarrowia lipolytica PG-20 and PG-32, from the
Persian Gulf-characterized by high emulsifying activity and cell
hydrophobicity
Mycoremediation

- Aspergillus and Penicillium-transform the aliphatic and aromatic crude oil

components & 11 species of Trichoderma were reported to grow on different
crude oil fractions (Harms et al., 2011)

- wood-inhabiting marine fungi-good producers of ligninolytic enzymes-

degrade several aromatic and recalcitrant environmental pollutants
Microbes in Space Research
 Microbes in Space Research

The Aerospace Microbiology Research Group studies how microbes survive & and
adapt to natural & simulated space conditions, s.as-microgravity, radiation,
vacuum, extreme temperature fluctuations, desiccation, etc.

- Space microbiology study involves analysis of the samples collected outside the
Earth’s biosphere

or pure cultures of terrestrial microorganisms that were evaluated either

during or after exposure to components of the space flight env

or studies of auto flora of crew members or microflora of recoverable

spacecraft which were performed to evaluate changes in populations of
microorganisms.
 Microbes in Space Research
- The vast, cold, & radiation-filled conditions of outer space presents an
environmental challenge for any form of life

- Earth’s biosphere-evolved for more than 3 billion years shielded by the

protective blanket –atmosphere.

- Within the last 50 years, space technology has provided tools for
transporting terrestrial life beyond this protective shield in order to study in-
situ responses to selected conditions of space.
 Space Environment

The majority of experiments on microorganisms in space were performed

using Earth-orbiting robotic spacecraft e.g., the Russian Foton satellites

- European Retrievable Carrier (EURECA)

- Human-tended spacecraft, such as space shuttles and space stations, e.g.,

MIR and the International Space Station (ISS).

- Space: extremely hostile environment: intense radiation field of galactic and

solar origin, high vacuum, extreme temperatures, and microgravity.
 Space Environment

-survival of some microorganisms exposed to outer space has been studied using
both simulated facilities and low Earth orbit exposures.

- Bacteria-first organisms-investigated, when in 1960 a Russian satellite carried

Escherichia coli, Staphylococcus, and Enterobacter aerogenes into orbit.

- Many kinds of microorganisms have been selected for exposure experiments

since then.
 The International Space
Station as it was
configured on August 20,
2001, Credit: NASA
Credit: NASA
Credit: NASA
 MICROBES IN THE SPACE

-On April 20, 1967, the unmanned lunar lander Surveyor 3 landed near Oceanus
Procellarum on the surface of the moon.

- One of the things aboard was a television camera.

- On November 20, 1969, Apollo 12 astronauts-recovered the camera

- When NASA scientists examined it back on Earth, they were surprised to

find specimens of Streptococcus mitis that were still alive.

- These bacteria had survived for 31 months in the vacuum of the moon’s
atmosphere- radiation exposure, deep freeze, no water or nutrient source
 MICROBES IN THE SPACE
-Spores of Bacillus subtilis - exposed to selected factors of space (vacuum, solar
UV radiation, heavy ions of cosmic radiation), and their response was studied by
Horneck (1981) after recovery.

- Horneck (1993) studied spores of B. subtilis onboard several spacecraft (Apollo

16, Spacelab 1, and LDEF). Microbes - exposed to selected parameters of
space like space vacuum, different spectral ranges of solar UV radiation, &
cosmic rays & their survival and genetic changes were studied after retrieval.

- Horneck et al. (2010) reported responses of microorganisms (viruses, bacterial cells,

bacterial and fungal spores, and lichens) to selected factors of space (microgravity,
galactic cosmic radiation, solar UV radiation, and space vacuum) in space and laboratory
simulation experiments.
Deinococcus radiodurans R1 is about 5 times more resistant to ionizing radiation-
than of B. subtilis

- It has also been reported that species of Methylobacterium taken from samples across the
space station -helpful to plants, promoting their growth and fighting pathogens that
affect them.
 Microbes with ability to grow in space conditions (Experimental)

Bacteria Fungi
Anabaena cylindrica, Aspergillus oryzae A.niger, A.terreus,
A.versicolor
Bacillus subtilis, B.thuringiensis, B.pumilus
Chaetomium globosum
B.mycoides, B. megaterium, B.cereus
Cryomyces antarcticus
Actinomyces erythreus
C.minteri
Azotobacter chroococcum
Penicillium roqueforti
A.vinelandii
A Trichoderma koningii
Chroococcidiopsis
Trichophyton terrestre
Deinococcus aerius
Cladosporium herbarum
D. aetherius , D. radiodurans
Sordaria fimicola
Escherichia coli
Micrococcus luteus Yeasts
Pseudomonas aeruginosa Rhodotorula rubra, R. mucilaginosa
Streptococcus mitis Saccharomyces cerevisiae, S.ellipsoids
Hydrogenomonas eutropha Zygosaccharomyces bailii
 Archaea
Halorubrum chaoviatoris

Viruses
T7 phage
Canine hepatitis
Influenza PR8
Tobacco mosaic virus
Microbial research in space is being conducted for almost 50 years now
-The closed system of the International Space Station (ISS) - acted as a microbial
observatory- microbial survivability upon exposure to space conditions began .50 years ago,
conducting research on adaptation and survivability of microorganisms exposed to space
conditions.

- Initial experiments: 1935-balloon flights and rocket payloads.

- in 1966-National Aeronautics and Space Administration (NASA), during the Gemini IX

and XII missions, began experiments-bacteriophage T1 & spores of the fungi Penicillium
roqueforti were directly exposed to space conditions (microbial survivability -almost
negligible)

- This finding was later learned to be due to non-penetrating radiation in space, including
solar UV radiation or soft X-rays, because covering the samples with a thin layer (0.4 mm)
of aluminium resulted in 3,000-fold higher survival of bacteriophage T1 and nearly 100%
survival of fungal spores. This study was one of the first to assess the survival limits of
microorganisms upon exposure to space conditions.
Microbial research in space is being conducted for almost 50 years now
- Several studies have been conducted to understand how microbes adapt to these space
conditions, but these studies are limited to a few species and also require researchers to
further understand changes at the genetic level.

- The microbial species that have been isolated from the ISS include both potentially
pathogenic and industrially important microorganisms. Therefore, understanding how
these microorganisms adapt to space conditions will aid in developing strategies to mitigate
the risk posed by pathogenic microorganisms to the health of crew members, who might be
in an immunocompromised state in the ISS

- In addition, understanding how space conditions alter microbial processes in industrially

important microorganisms will provide us with information on how to engineer these
microbes for efficient production of important compounds

- . The knowledge gained from these studies will be of utmost importance in implementing
safety measures for long-duration spaceflights and interplanetary explorations involving
humans.