Environmental Biotech Handout-PDF

ARBA MINCH UNIVERSITY
COLLEGE OF NATURAL SCIENCES

BIO-TECHNOLOGY PROGRAM
ENVIRONMENTAL BIOTECHNOLOGY (Biot4142)

For 4th Biotech. Students.
By:- Temam A. (Assistant Professor)
CHAPTER ONE
INTRODUCTION TO
ENVIRONMENTAL
BIOTECHNOLOGY

Environmental biotechnology
What was environmental biotechnology?
 Simple and traditional definition: use, in a controlled manner, of

microorganisms to degrade wastes.
 Solving environmental problems through biotechnology; e.g.

biosensor, Nanotechnologies and Biotreatments, etc.
 International Society for Environmental Biotechnology, since

1992. Two streams:
(1) microbial biotechnology for environmental improvement (sewage
treatments and bioremediation) and
(2) chemical engineering related to the environment. From waste
treatment to bioremediation.
3
Environmental biotechnology
What was environmental biotechnology?,…..Cont’d
 Use of molecular techniques to protect the environment,

including Risk assessments of GMOs
 Renewable energy and resources: engineering plants for the

production of clean energy, biofuel, biomass, etc.
 Environmental Biotechnology is the multidisciplinary

integration of sciences and engineering in order to utilize the
huge biochemical potential of microorganisms, plants and parts
thereof for the restoration and preservation of the
environment and for the sustainable use of resources
4
Waste Water Treatment (WWT)
General About WWT
What is wastewater treatment?

o Removing
contaminants/pollutants from
wastewater.
Therefore, Wastewater should be

collected and treated before its
ultimate disposal in order to :
 Reduce spread of
communicable diseases
 Prevent surface and ground
water pollution.
Fig. 1.1. Improperly disposed ww.
General About WWT
Some contaminants in WW.
 Suspended solids: lead to the development of sludge
deposits and anaerobic conditions
 Pathogens: cause diseases
 Nutrients: essential for growth (N, P,…).
 Refractory organics: resist conventional methods of
wastewater treatment.
 Heavy metals :may have to be removed if the
wastewater is to be reused
 Dissolved inorganic solids (calcium, sodium, and
sulfate): may have to be removed if the wastewater is
to be reused
6
General About WWT
 The wastewater can be broadly divided into two
categories:
1. Biodegradable wastewater:
 The wastes in general have a predominance of biodegradable
organic matter
 The stabilization of organic matter is accomplished biologically
using a variety of microorganisms.
 Based on bacterial relationship to oxygen the microorganisms
can be:
I. Obligate aerobes: utilise oxygen
II. Obligate anaerobes: without oxygen
III. Facultative anaerobes: utilise oxygène if présent and not
if not present
IV. denitrifiers
7
General About
General About WWTWWT
2. Non-biodegradable wastewater
 The wastewater are rich in non-biodegradable matter
consisting of solids and liquids in suspended or dissolved
form, including various inorganic and organic, many of
which may be highly toxic.
8
General About WWT
 Methods of treatment in which the application of
physical forces dominates are called unit operation
 Methods of treatment in which chemical or biological

activity are involved are known as unit process.
 WWT applies any of this operations, processes or

combination of both.
 is the combination of physical, chemical and biological
processes.
9
Objectives of WWT
 Removal of pollutants and the protection and

preservation of our natural resources.
 Protection of human health by the destruction of

pathogenic organisms prior to treated effluent being
discharged to receiving water bodies and land.
10
Wastewater Treatment Standards
 Environmental standards are developed to ensure that
the impacts of treated wastewater discharges into
ambient waters are acceptable.
Effluents from different establishments should be:
1. Free from materials and heat in quantities,
concentrations or combinations which are toxic or
harmful to human, animal, aquatic life.
2. Free from anything that will settle in receiving
waters forming putrescence or that will adversely
affect aquatic life.
3. Free from floating debris, oil, scum and other
materials;
11
Wastewater Treatment Standards
4. Free from materials and heat that alone, or in

combination with other materials will produce
color, turbidity, taste or odor in sufficient
concentration to create a nuisance or adversely
affect aquatic life in receiving waters;
5. Free from nutrients in concentrations that create
nuisance growths of aquatic weeds or algae in
the receiving waters.
12
Flow Sheets for WWT Systems
 The term "flow sheet" is used to describe a particular

combination of unit operations and processes used
to achieve a specific treatment objective.
 Is the combination preliminary treatment
primary
secondary
tertiary treatment system
13
Waste Water Treatment (WWT)
 WWT comprises of the following stages of treatment:
1. Conventional treatment
I. Preliminary treatment
II. Primary treatment
III. Secondary/biological
2. Advanced/tertiary treatment
 Remove chemicals using biological treatments on site (in situ) or
ex situ.
 Chemicals: heavy metals, trace organics or mixtures.
 Bacterial or fungal degradation of chemicals: Engineered
microbes for better and more efficient removal of chemicals on-site
14
Preliminary Treatment
15
Fig. 2: Layout of conventional wastewater treatment plant.
Flow Sheets for WWT Systems
17
Physical, Chemical and Bacteriological
Characteristic of Wastewater
Physical Characteristics
 Turbidity , color, odor, temperature, solids
Chemical Characteristics
 Alkalinity,pH, Chloride Contents, Dissolved gases,
Nitrogen compounds, Phosphorus, Presence of Fats, Oils
and Greases, Sulphides, Sulphates and Hydrogen
Sulphide Gas, Dissolved Oxygen (DO), Bio-Chemical
Oxygen Demand (BOD), Chemical Oxygen Demand (COD),
Total Organic Carbon
Biological Characteristics
18
What is the turbidity, color, odor, temperature of sewage?
 Turbid, grey/soapy/black after petrification, musty (not
offensive)/bad smell after H2S released, warmer
Solids
 sewage only contains about 0.05 to 0.1 percent solids
 Solids present in sewage may be in any of the 4 forms:
 suspended solids: solids which remain floating in sewage.
 dissolved solids: remain dissolved in sewage just as salt in
water
 colloidal solids: are finely divided solids remaining either in
solution or in suspension.
 settleable solids: solid matter which settles out.
19
 The proportion of these different types of solids is
generally found as:
 Inorganic matter consists of minerals and salts,
like: sand, gravel, debris, dissolved salts, chlorides,
sulphates, etc.
 Organic matter consists of:
I. Carbohydrates such as cellulose, cotton, fiber,
starch, sugar, etc.
II. Fats and oils received from kitchens, laundries,
garages, shops, etc.
III. Nitrogenous compounds like proteins and their
decomposed products, including wastes from
animals, urea, fatty acids, hydrocarbons, etc.
20
pH
 is a method of expressing the acid condition of the WW.
 For proper treatment, wastewater pH should normally be in
the range of 6.5 to 9.0.
 the fresh sewage is generally alkaline in nature ( pH > 7); but
as time passes, its pH tends to fall due to production of acids
by bacterial action in anaerobic or nitrification processes.
Chloride Contents
 derived from the kitchen wastes, human feces, and urinary
discharges, etc.
 when the chloride content of a given sewage is found to be
high, it indicates the presence of industrial wastes or
infiltration of sea water
21
Nitrogen compounds
 The presence of nitrogen in sewage indicates the
presence of organic matter, and may occur in:
a) Free ammonia, called ammonia nitrogen;
b) Albuminoid nitrogen, called Organic nitrogen;
c) Nitrites; and
d) Nitrates
Phosphorus
 is essential to biological activity and must be present
in at least minimum quantities or secondary treatment
processes will not perform.
 Excessive amounts can cause stream damage and
excessive algal growth.
22
Presence of Fats, Oils and Greases

 are derived in sewage from the discharges of animals
and vegetable matter, or from the industries like
garages, kitchens of hotels and restaurants, etc.
 Such matter form scum on the top of the sedimentation
tanks and clog the voids of the filtering media.
 Hence, need proper detection and removal.
23
Sulphides, Sulphates and Hydrogen Sulphide Gas
 reflects aerobic, and/or anaerobic decomposition.
 Sulphides and sulphates are formed due to the decomposition
of various sulphur containing substances
 this, decomposition also leads to evolution of H2S gas which
cause: bad smells and odours,
 corrosion of concrete sewer pipes.
 in aerobic digestion of, the aerobic and facultative bacteria
oxidize the sulfur and its compounds to sulfides, which
ultimately break down to form sulphate ions (𝑆𝑂4−2 ), which is a
stable and an unobjectionable end product
 In anaerobic digestion of, the anaerobic and facultative
bacteria reduce the sulphur and its compounds into sulphides,
with evolution of H2S gas along with methane and CO2, thus
causing very obnoxious smells and odours.
24
Bio-Chemical Oxygen Demand (BOD)
 used as a measure of the quantity of oxygen required
for oxidation of biodegradable organic matter by aerobic
biochemical action.
 The rate of oxygen consumption is affected by a number
of variables:
 temperature, pH, the presence microorganisms, and the
type of organic and inorganic material.
 The greater the BOD, the more rapidly oxygen is
depleted in the water body.
 The consequences of high BOD are the same as those
for low DO:
 aquatic organisms become stressed, suffocate, and die.
25
Chemical Oxygen Demand (COD)
 measures the total quantity of oxygen required for
oxidation of organics into carbon dioxide and water.
26
Biological Characteristics
 are due to the presence of bacteria and other living
microorganisms, such as algae, fungi, protozoa, etc.
 Most of the vast number of bacteria present in sewage
is harmless non-pathogenic bacteria.
 They are useful and helpful in bringing oxidation and
decomposition of sewage.
27
 The most important standard methods for analysis
of organic contaminants/oxygen demand are:
1. Theoretical Oxygen Demand (ThOD)
2. Chemical Oxygen Demand (COD)
3. Biochemical Oxygen Demand (BOD)
28
1. Theoretical Oxygen Demand (ThOD)
o This is the theoretical amount of oxygen required to
oxidize the organic fraction of the wastewater
completely to carbon dioxide and water.
o E.G. calculate the amount of oxygen required to oxidize
300mg/l of glucose is:
𝑪𝟔 𝑯𝟏𝟐 𝑶𝟔 + 𝟔𝑶𝟔 → 𝟔𝑪𝑶𝟔 + 𝟔𝑯𝟐 𝑶
(C = 12, H = 1 and O = 16), 𝐶6 𝐻12 𝑂6 is 180 and 6𝑂2 is
192
192; then, ThOD of, ∗ 300 = 321 mg/l .
180
o Because WW is so complex in nature its ThOD cannot
be calculated, but in practice it is approximated by the
chemical oxygen demand.
29
2. Chemical Oxygen Demand (COD)
 is determined by performing a lab. test with a strong oxidant like
dichromate solution.
 In order to perform this test, a known quantity of WW is mixed
with a known quantity of standard solution of potassium
dichromate, and the mixture is heated.
 The organic matter is oxidized by K2Cr2O7 (in the presence of
H2SO4 (helps to digest/break down the complex molecules).
 COD used more of to measure non-biodegradable matter.
 The advantage of COD measurements is that they are obtained
very quickly (within 3 hours)
 the disadvantages, they do not give any information on the
proportion of the WW that can be oxidized by bacteria.
30
3. Biochemical Oxygen Demand (BOD)
 Oxygen demand of WW is exerted by three classes of
materials:
1. Carbonaceous organic materials usable as a source of
food by aerobic organisms
2. oxidizable nitrogen derived from nitrite, ammonia, and
organic nitrogen compounds which serve as food for
specific bacteria (e.g., Nitrosomonas and Nitrobacter).
3. Chemical reducing compounds, e.g., ferrous ion (Fe2+),
sulfites (SO32-), and sulfide (S2-) which are oxidized by
dissolved oxygen.
For domestic sewage, nearly all oxygen demand is due to
carbonaceous organic materials.
31
 The carbonaceous BOD is the amount of oxygen
required by microorganisms to decompose carbonaceous
material that are subject to microbial decomposition.
 This is the first stage of oxidation and the corresponding
BOD is known as the first stage demand.
In the 2nd stage, nitrogenous

matter oxidized, and the
corresponding BOD is called
second stage BOD or nitrifying
demand.
32
 The BOD test results are used for the following purpose:
i. Determination of the quantity of oxygen required
ii. Determination of size of WW treatment facilities
iii. Measurement of efficiency of some treatment methods
iv. Determination of strength of sewage
v. Determination of quantity of clear water required for dilution
during disposal.
BOD test: dilution test method procedures
1. Sample is diluted with specially prepared dilution water
2. The water is aerated to saturate it with oxygen before
mixing it with sample
3. Initial DO of diluted sample is measured
4. Then, incubated for 5days at 20𝑜𝑐 , after 5days DO measured
33
again
PRELIMINARY AND PRIMARY
WASTEWATER TREATMENT METHODS
34
 consists solely in separating the floating materials
and heavy settleable inorganic solids.
 It also helps in removing the oils and greases, etc.
 reduces the BOD of the WW, by about 15 to 30%.
• The processes used

are:
1. Screening
2. communitors
3. Grit chambers or
Detritus tanks
4. Skimming tanks
35
1) Screening
 remove the floating matter, such as pieces of cloth, paper,
wood, cork, hair, fiber, kitchen refuse, fecal solids, etc.
 What if floating materials not removed?
 will choke the pipes, or adversely affect the working of the
sewage pumps.
Depending upon the size of

the openings, screens may be
classified as
 coarse screens,
 medium screens, and
 fine screens.
36
 Coarse screens (Racks): the spacing between the bars
(i.e. opening size) is about 50 mm or more.
 These screens help in removing large floating objects.
 collects about 6 liters of solids per million liter of WW
37
Fig. Fixed bar type fine screen
Medium screens: the spacing b/n bars is about 6 to
40 mm.
 These screens will ordinarily collect 30 to 90 liters of
material per million liter of sewage.
 The screenings usually contain some quantity of
organic material, which may putrefy and become
offensive,
Fine Screens: have perforations of 1.5 mm to 3 mm
in size.
 The installation of these screens proves very
effective, and remove 20% of the suspended solids.
 get clogged very often, and need frequent cleaning.
38
Disposal of Screenings
 screenings is material separated by screens.
 It contains 85 to 90% of moisture and other floating
matter.
 It may also contain some organic load which may
putrefy, causing bad smells and nuisance.
 To avoid such possibilities, the screenings are disposed
of either by:
 burning,
 burial, or
 dumping.
 The dumping is avoided when screenings are from
medium and fine screens,
39
2. Comminutors/shredder
• are the patented devices, which

break the larger sewage solids
to about 6mm in size,
Figure of Comminutor and shredder

• eliminate the problem of disposal of screenings, by reducing the
solids to a size which can be processed elsewhere in the plant.
40
3) Grit Removal Basins (grit chamber, detritus tank,..)
o are the sedimentation basins placed in front of the
wastewater treatment plant.
o The grit chamber remove the inorganic particles (specific
gravity about 2.65 and nominal diameter of 0.15 to 0.20mm
or larger) such as sand, gravel, egg shells, bones, and other
non-putresible materials
o Grit may clog channels or damage pumps due to abrasion,
and to prevent their accumulation in sludge digesters.
o These inorganic materials is removed by the process of
sedimentation due to gravitational forces.
o What is sedimentation process?
o The organic material is not allowed to settle in this process,
as they cause septicity of sewage.
41
Constant Velocity Horizontal Flow Grit Chambers
 is an enlarged channel or a long basin, in which the
cross-section is increased, so as to reduce the flow
velocity of sewage.
 why flow velocity reduction is required?
 to cause the settlement of the entire silt and grit
present in sewage.
 The important point in the design of the grit basins
is that the flow velocity should neither be too low
nor should it be so high. Why?
 if too slow lighter organic matter will settle down, septicity!!
 if too high, even silt and grit not settle down.
42
Primary Wastewater Treatment
 consists in removing large suspended organic solids.
 This is usually accomplished by sedimentation in
settling basins.
 What is sedimentation?
43
Sedimentation
 is the physical separation of suspended material
from water or wastewater by the action of gravity.
 SSs are separated by settling to the bottom of
sedimentation tanks by gravitational force.
 Sedimentation tanks are tank designed to remove
this organic matter from the sewage effluent coming
out from the grit chambers.
44
The principle behind sedimentation.
The very fundamental principle underlying the process of
sedimentation is that the organic matter present in sewage is
having specific gravity greater than that of water (i.e. 1.0).
 In still sewage, these particles will, tend to settle down by
gravity;
 in flowing sewage, particles are kept in suspension, because of
the turbulence in water.
 Hence, as soon as the turbulence is retarded by offering storage
to sewage, these impurities tend to settle down at the bottom
of the tank offering such storage.
45
Types of Settling
Depending on the particles concentration and the
interaction between particles, 4 types of settling can
occur: Discrete (type I), Flocculent (type II), Hindered
(type III), Compression (type IV)
1. Discrete particle settling: The particles settle
without interaction and occur under low solids
concentration.
2. Flocculent settling: particles initially settle
independently, but flocculate in the depth of the
clarification unit.
 The velocity of settling particles is usually increasing
as the particles aggregates.
46
hindered
3. Hindered/zone settling : Inter-particle forces are

sufficient to hinder the settling of neighboring particles.
47The particles tend to remain in fixed positions with respect
to each others.
4. Compression settling: occurs when the particle
concentration is so high that so that particles at
one level are mechanically influenced by particles
on lower levels. The settling velocity then
drastically reduces.
48
Biological Waste water treatment
 Biological treatment is an important and integral part of any
wastewater treatment plant that treats wastewater from either
municipality or industry having soluble organic impurities or a mix of
the two types of wastewater sources.
 Biological treatment have obvious economic advantage, both in terms

of capital investment and operating costs over other treatment
processes like chemical oxidation; thermal oxidation etc.
 Micro-organisms in sewage treatment plants remove the more

common pollutants from waste water before it is discharged into rivers
or the sea.
 The costs of waste water treatment can be reduced by the conversion

of wastes into useful products.
The Role of Microorganisms in Wastewater Treatment
 Micro-organisms (bacteria, fungi, protozoa etc.) are important in the

treatment of WW.
 They play an essential role in the conversion of organic waste to more
stable or less polluting substances.
How?
► Waste from humans is a useful food substrate for the micro-
organisms.
► And they require cellular building blocks, such as (carbon) C,
(hydrogen) H, (oxygen) O, (nitrogen) N, (phosphorus) P, and minerals
for growth.
► These can be obtained through consuming organic substances
containing these elements, or from inorganic materials, such as carbon
dioxide, water, nitrate and phosphate.
50
►Micro-organisms also require energy.
►They obtain this through respiration. In this process
organic carbon is oxidized to release its energy.
►Oxygen or other hydrogen acceptors is needed for the
respiration process.
►Algae and photosynthetic bacteria utilize energy from

sunlight, while certain types of bacteria can utilize energy
from chemical reactions not involving respiration.
►These building blocks and energy are used to synthesize
more cells for growth and also for reproduction.
51
 Three types of processes to represent the conversion of organic

wastes by micro-organisms.
1. Aerobic oxidation : utilize oxygen to oxidize organic substances to
obtain energy for maintenance, mobility and the synthesis of cellular
material.
2. Anaerobic oxidation: utilize nitrates, sulphates and other hydrogen

acceptors to obtain energy for the synthesis of cellular material from
organic substances.
52
 Methane (CH4) is a source of heat but, if released to the atmosphere

without being combusted, it contributes to the greenhouse gas effect.
 hydrogen sulphide (H2S) contributes to WW odour.
3. Photosynthetic:
53
Microbial Growth Kinetics
o Growth of a microbial population is defined as an increase

in numbers or an increase in microbial mass.
o Growth rate is the increase in microbial cell numbers or

mass per unit time.
o Microbial populations can grow as:
1. Batch cultures (closed systems) or
2. Continuous cultures (open systems).

54
1.Batch Cultures
 When a suitable medium is inoculated with cells, the growth of the
microbial population shows four distinct phases.
The lag phase is a period of cell

adjustment to the new environment
At log/exponential phase the number of
cells increases exponentially
At stationary phase microorganisms
cannot grow indefinitely, mainly because
of lack of nutrients and electron
acceptors, and the production and the
accumulation of toxic metabolites.
During death phase, the death (decay)
rate of the microbial population is higher
than
55
the growth rate.
Figure of Microbial growth curve
2. Continuous Culture of Microorganisms

 the exponential growth phase over a long period of time can be
achieved by growing continuously the cells in a completely mixed
reactor in which a constant volume is maintained.
 The most commonly used device is the chemostat
56
Physical and Chemical Factors Affecting Microbial

Growth
1. Substrate Concentration
2. Temperature
3. pH
4. Oxygen Level
57
B i o l o g i c a l Wa s t e wa t e r Tre a t m e n t
 To introduce WW contact with bacteria (cells) which feed organic

matter in WW.
 the purpose of biological treatment is BOD reduction.
Principle:
→ Simple bacteria (cells) eat the organic material.
→ Through their metabolism, the organic material is transformed into
cellular mass,
→ This cellular mass can be precipitated at the bottom of a settling tank
or retained as slime on solid surfaces or vegetation in the system.
→ Then, the WW exiting the system is much clearer than it entered.
→ Cells need oxygen to breath, so adequate supply of O2 should there
for the operation of biological WWT.
58
B i o l o g i c a l Wa s t e wa t e r Tre a t m e n t
The common methods of biological wastewater treatment are:

a) Aerobic processes such as trickling filters and activated
sludge process.
b) Anaerobic processes such as anaerobic digestion, and
c) Anoxic processes such as denitrification.
59
Aerobic & Anaerobic treatment
60
61
B i o l o g i c a l Wa s t e wa t e r Tre a t m e n t Te c h n o l o g i e s
1. TRICKLING FILTERS
 consist of tanks of coarser filtering media.
I. Principles of operation
 over tanks of coarser filtering media the WW is allowed to sprinkle
or trickle down, by means of spray nozzles or rotary distributors.
 The percolating sewage is collected at the bottom of the tank through
a well designed under-drainage system.
 sufficient quantity of oxygen is supplied by providing suitable
ventilation facilities in the body of the filter
 The purification is done mainly by the aerobic bacteria, which form a
bacterial film around the particles of the filtering media.
 The effluent must be taken to the secondary sedimentation tank for
settling out the organic matter oxidized while passing down the filter.
62
Aerobic B i o l o g i c a l Wa s t e wa t e r Tre a t m e n t
63 Figure of Photographic view of trickling filter with its rotary distributors

64
Figure of Typical section of a conventional circular trickling filter

Aerobic Biological
Aerobic B Treatment
i o l o g i c a l Wa s t e wa t e rTechnologies
Tre a t m e n t
2. Conventional Activated Sludge Process (ASP) System:
 This is the most common and oldest biotreatment process used to treat
municipal and industrial wastewater.
 Typically wastewater after primary treatment i.e. suspended impurities

removal is treated in an activated sludge process based biological
treatment system comprising aeration tank followed by secondary
clarifier.
 ACTIVATED SLUDGE PROCESS:
 Provides an excellent method of treating sewage.

 BOD removal is up to 80 - 95%, and bacteria removal is up
to 90 - 95%.
66
Principle of operation:
 The WW effluent from primary sedimentation tank mixed with
20 to 30% of volume of activated sludge (from ASP unit).
 This activate sludge contains a large concentration of highly active
aerobic microorganisms.
 the sewage are intimately mixed together with a large quantity
of air for about 4 to 8 hours.
 the moving organisms will oxidize the organic matter and the
suspended and colloidal matter tends to coagulate and form a
precipitate which settles down readily in the secondary settling
tank.
 The settled sludge (containing microorganisms) called activated sludge is then
recycled to the head of the aeration tank to be mixed again with the sewage being
treated.
 The mixture of WW and activated sludge is called mixed liquor.
67
Units of an Activated Sludge Plant

1. Aeration Tanks 2. secondary clarifier
68 Figure 9: of a conventional AS plant giving high degree of treatment

Activated Sludge
The separated biomass is returned to the aeration tank by means of return activated
sludge (RAS) pump.
Excess biomass (produced during the biodegradation process) is wasted to the sludge
handling and dewatering facility.
Use of genetically engineered organisms
 Nowadays organisms can also be supplemented with additional genetic
properties for the biodegradation of specific pollutants if naturally
occurring organisms are not able to do that job properly or not quickly
enough.
 Combining different metabolic abilities in the same micro-organism for

restoration and preservation of environment.
 Developing new microbial properties in response to the degradation of

xenobiotics (that is, man- made chemicals that are normally not found in
nature).
 Currently, some genetically modified bacteria have been approved for

71
bioremediation purposes.
Use of genetically engineered organisms...
 In the late 1970s and early 1980s, bacterial genes encoding catabolic
enzymes for recalcitrant compounds started to be cloned and
characterized.
 Soon, many microbiologists and molecular biologists realized the

potential of genetic engineering for addressing biodegradation .
 Genetically engineered microorganisms (GEMs) have shown potential

for bioremediation of soil, groundwater and activated sludge, exhibiting
the enhanced degrading capabilities of a wide range of chemical
contaminants
72
Biosensors /monitor pollution/
 Biosensors are detecting devices that rely on the specificity of cells
and molecules to identify and measure substances at extremely low
concentrations.
 Measurement of mutagenic activity (microtox and mutatox tests with
lux gene from Vibrio)
 Detection of pathogens by multiplex-PCR
 Detection of toxins (Ciguatoxin)
 A biosensor is composed of a biological component, such as a cell,
enzyme or antibody, linked to a tiny transducer.
 Biosensors can, for example,
 measure the nutritional value, freshness and safety of food.
 locate and measure environmental pollutants.
 detect and quantify explosives, toxins and biowarfare agents.
73
1) Bio Detection Systems
 CALUXR Bioassay
 A sensitive bioassay for exposure to
dioxins and related compounds
 Synthetic gene promoter was created

and linked to a reporter gene which
gives color when the gene promoter is
turned on.
 The synthetic gene promoter contains

multiple cis-acting elements responsible
for dioxin (DRE) and dioxin receptor
(Ah receptor) binding.
 The reporter gene is transfected into a

cell-line for the bioassay.
74
2) Pathogen detection
 Bacteria: coli form bacteria, salmonella, Legionella, Vibrio, etc.
 Virus: Influenza, hepatitus, polio, etc.
 Algae: dinoflagellates, diatoms, toxic algae, ciguatoxin, etc.
 Multiplex technology is being developed: one run for many
pathogens.
 Collection with minimal amount of samples: water, soil, or air.
 Use PCR or real-time PCR techniques
75
3) Stress Proteins
 Metallothionein for exposure to heavy metals
 Cytochrome P450 (CYP) IA1 for exposures to trace organics
 Vitellogenin (an egg yolk protein) for exposure to environmental
estrogens
 Heat shock protein for general stress conditions
 These biomarkers are NOT biomarkers of toxic effects.
 They are biomarkers of exposures. = Still controversial
 Biomarkers have biological relevance and usually less expensive
than chemical analyses. Data could be diagnostic and indicative.
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4) Bacterial Biosensors
 Bacterial sensors can be used to test for environmental
pollutants.
 Bacteria with bioluminescent are good candidates for sensors.
 In the presence of pollutants the bioluminescent decreases.
 The structural genes (luxCDABD) encodes the enzyme for

bioluminescent was cloned into the soil bacteria
Pseudomonas fluorescens.
77
.
Cont’d…
 To screen water samples for pollutants (metal or organic)
a suspension of P. fluorescens was mixed with the
solution to be tested.
 After a 15 min incubation the luminescence of the
suspension was measured.
 When the solution contained low to moderate levels of
pollutants the bioluminescence was inhibited.
 The procedure is rapid, simple, cheap and a good screen
for pollutants.
78
79
Membrane Bioreactors
 The combination of membrane separation technology and
activated sludge process
 Membrane bioreactor (MBR’s) - A wastewater treatment
system that combines the use of filters (membranes) and
bacterial processes (bioreactor) to treat wastewater.
 Bioreactor - The section of the wastewater treatment system
which contains microorganisms or cells to remove
biodegradable pollutants.
 Membrane - Filter to remove solid waste.
 this technology employs a mixed-culture of aerobic
microorganisms.
 it is a wastewater treatment technology which has expanded
rapidly in the last 5 years.
Figure 6.8 Schematic membrane bioreactor
Membrane Bioreactors
 Urban wastewater is usually treated by conventional activated sludge processes
(CASP’s), which involve the natural biodegradation of pollutants by heterotrophic
bacteria (i.e. activated sludge) in aerated bioreactors. Activated sludge could be
separated by gravitational setting. The treatment efficiency is usually limited by the
difficulties in separating suspended solids (SS’s).
 Membrane bioreactor (MBR) is an improvement of the CASP, where the traditional
secondary clarifier is replaced by a membrane unit for the separation of treated water
from the mixed solution in the bioreactor.
 Membrane Bioreactor Technology is based on Biological Treatment followed by
membrane separation, system comprising of an intense activated sludge process with
the biomass separation stage carried out by membrane cassettes located outside the
aeration tank in a separate membrane tank.
 The Membranes replace the settlement stage in conventional activated-sludge systems
and effectively revolutionize the process.
 The separation of biomass from treated water using membranes provides filtered
quality final effluent, offering possibilities of re-use
 It allows very high biomass mixed liquor suspended solids (MLSS) concentrations to
be developed in the bioreactor without the detrimental effects usually associated with
traditional settlement techniques.
3 - Membrane bioreactor (combined main
and post- treatment step for wastewater)
 The membrane bioreactor (MBR)

couples the activated sludge process
with membrane filtration for sludge
separation
 Construction and O&M costs are
relatively high (high energy demand
for aeration)
 Regarded as a “high-tech” solution
 Produces excellent effluent quality,
with extremely low pathogen
concentrations
 Useful also for industrial wastewater
Source of photos: http://www.water-

technology.net/projects/carnation/carnation6.html
Advantages:
1) The membrane bioreactor can offer a greater degradation
capacity for persistent chemicals, making possible the
biological removal of benzene, nitrobenzene and
polyaromatic hydrocarbons (PAHs), for example, which
represent a significant risk, both to the environment and
human health, due to their high toxicity.
 Removal efficiency for these substances can approach 99%.
2) Not all of the contaminants present in the effluent are typically

completely converted into carbon dioxide and water, a certain
percentage being turned into metabolic byproducts instead,
though this can amount to less than 5% in a well-managed
bioreactor system (produce a much smaller quantity of excess
sludge).
 These systems are, of course, more expensive than the

conventional activated sludge or trickling filters.
B i o l o g i c a l Wa ste wate r Tre at m e nt
3. Waste Stabilization Pond
 Is large shallow basins enclosed by earthen embankments in which
wastewater is biologically treated by natural processes involving pond
algae and bacteria.
Types of Pond
o Anaerobic Ponds
o Facultative and
o Maturation/aerobic pond
 WSP comprise a single series of these anaerobic, facultative and
maturation ponds or several of such series in parallel.
 A long hydraulic retention time is necessary because of the slow rate at
which the organic waste is oxidized.
 Typical hydraulic retention times range from 10 -100 days depending on
the temperature of a particular region.
86
I. Anaerobic Ponds
 Anaerobic ponds are unmixed basins designed to enhance the
settling and biodegradation of particulate organic solids by
anaerobic digestion.
 Pond depth is usually between 3 to 5 meters and
 HRT for municipal sewage is b/n 1 - 3 days and for industrial WW
may increase to 20 days.
 In cold climates, anaerobic ponds mainly act as settling ponds,
whereas higher sewage temperatures enhance the anaerobic
degradation process.
 At higher temperatures BOD is therefore more effectively removed.
 Typical TSS removal percentages range between 50 and 70%.
 BOD removal rate is increase with temperature and range b/n 30
and 75%.
87
Treatment Mechanisms
 BOD removal is the combined effect of sedimentation and
biological degradation.
 Biological degradation is due to the anaerobic degradation of
complex organic material.
 Anaerobic ponds require some preliminary treatment of municipal
WW.
 Usually coarse screening and grit chamber is applied
88
II. Facultative Ponds
 are the second treatment step in a pond system.
 In facultative ponds the anaerobic pond effluent is further treated,
aimed at further BOD, nutrient and pathogen removal.
 Facultative ponds are usually 1.5 - 2.5m deep.
 The HRT of this ponds is varies b/n 5 and 30 days.
89
Processes In facultative ponds
 the top layer of facultative ponds is aerobic due to oxygen
production by algae and the bottom layer is anaerobic due to the
absence of algae activity.
 The three main mechanisms for BOD removal are aerobic digestion,
sedimentation and anaerobic digestion.
 Sedimentation results only in temporary storage of BOD in the
sludge layer.
 This BOD (in sludge) is removed while the pond is de-sludged. Part
of the sludge BOD is however anaerobically transformed into
methane gas.
90
Figure of BOD removal mechanisms in a facultative pond

91
III. Maturation Ponds
 Maturation ponds are shallow ponds (1 - 1.5m deep).
 An active algal biomass is maintained throughout the entire depth
of the system
 so that during daytime large amounts of oxygen are produced.
 BOD removal is much slower than in facultative ponds, since the
most easily degradable substances consumed already.
 The major application for maturation ponds is to polish or upgrade
facultative pond effluents and achieve substantial microbial
reductions to allow safe use of the effluents in agriculture or
aquaculture
92
93
Photo of Kaliti (A.A)WSP- effluent diversion weir for irrigation
Removal of Pathogenic Microorganisms in maturation pond
 Pathogen removal occurs in anaerobic, facultative and maturation
ponds, but only maturation ponds are designed on the basis of
required removal rates for pathogens.
 Four groups of pathogenic micro-organism can be distinguished in
WW: bacteria, viruses, protozoa and helminthes.
 Both helminth eggs and protozoan cysts are removed by
sedimentation.
 Removal bacteria (fecal coliform) and virus is due to a combination
of several processes:
 Adsorption to particles and subsequent sedimentation
 Grazing by other micro-organisms (protozoa)
94
 Natural decay
95
Figure of Typical scheme of a waste stabilization system
CHAPTER TWO
REMOVAL OF SPECIFIC POLLUTANTS
By: Temam A. (Assistant Professor)

Sources of Heavy Metals
Heavy metals are naturally occurring elements that have a high atomic
weight and a density at least 5 times greater than that of water.
Their multiple industrial, domestic, agricultural, medical and
technological applications have led to their wide distribution in the
environment; raising concerns over their potential effects on human health
and the environment.
Their toxicity depends on several factors including the dose, route of
exposure, and chemical species, as well as the age, gender, genetics, and
nutritional status of exposed individuals.
Because of their high degree of toxicity, arsenic, cadmium, chromium,
lead, and mercury rank among the priority metals that are of public health
significance.
These metallic elements are considered systemic toxicants that are
known to induce multiple organ damage, even at lower levels of exposure.
 In addition to the natural occurrence of heavy metals, they are widely
used in industry, agriculture, and military operations.
 These processes have led to the continuous accumulation of heavy metals

in the environment, which raises threats to public health and ecosystems.
 The high concentrations of heavy metals in the environment were also
attributed to several life-threatening diseases, including cancer and
cardiovascular ailments
 Because of their high degree of toxicity, arsenic, cadmium, chromium,
lead, and mercury rank among the priority metals that are of public
health significance.
 These metallic elements are considered systemic toxicants that are
known to induce multiple organ damage, even at lower levels of
exposure.
 Heavy metals  present abundance in nature.
 Metals added by human activities:
 Manufacturing
 Construction
 Agriculture
 Transportation
 Effects of Heavy Metals:
 Potential adverse effects of soluble metal compounds on human health
and on the health of organisms, treatment inhibition, aesthetics of the
environment.
 Certain metals in low concentrations are not only harmless , but traces
are essential for good nutrition.
Co, Cu, Fe, Se, Zn.
 Some metal salts on the other hand may be toxic.
Evaluation of toxicity:
Acute toxic effects: show up quickly upon ingestion of, or contact
with a metal compound.
Soluble copper  gastroenteritis symptoms with nausea
Chromium lung tumors, skin sensitizations, inflammation of
the kidneys
Selenium Poison in high concentrations, carcinogen, cause
tooth decay.
 Chronic poisoning e.g. cadmium and lead accumulate in body
tissue.
 Mutagenic / teratogenic toxicity  when certain metals combine
with organic compounds, these substances may produce changes in
genetic makeup or cause abnormal tissue development in embryos
(teratogenicity)
Some heavy metals and their environmental
and physiological effects
*
*
*
*
Potential Strategies for Bioremediation
(Microbial system for heavy metal accumulation)
 Microorganisms are mainly used in bioremediation to eliminate heavy

metals from the polluted environment.
 The elimination of heavy metals requires their concentration and
containment as they cannot be degraded by any biological,
physical, or chemical processes .
 Therefore, employing microorganisms in heavy metal elimination and
environmental cleaning is an effective approach due to their varied
ability of interacting with heavy metals.
 For instance, microorganisms can transform heavy metals from one
oxidative state or organic complex to another.
 Mainly, microorganism-based remediation depends on the resistance of
the utilized microbe to the heavy metal that is either activated
independently or through metal stress. Microorganisms perform the
remediation of heavy metals through different processes (Figure 4):
(Biosorption and Bioaccumulation )
 Biosorption and bioaccumulation are processes by which the microorganisms,
or biomass, bind to and concentrate heavy metals and contaminants from the
environment. However, both biosorption and bioaccumulation work in distinct
ways.
 During biosorption, contaminants are adsorbed onto the sorbent’s cellular
surface in amounts that depend on the composition and kinetic equilibrium of
the cellular surface. Thus, it is a passive metabolic process (Figure 4(1)) that
does not require energy/respiration.
 Bioaccumulation, on the other hand, is an active metabolic process that
needs energy and requires respiration .
 Since contaminants (such as heavy metals) bind to the cellular surface of
microorganisms during biosorption, it is a revisable process. In contrast,
bioaccumulation is only partially reversible.
 Biosorption was also shown to be faster and to produce a greater number of
concentrations.
 Figure4: Mechanisms of heavy metal biosorption by bacterial
cells. Bacterial biosorption of heavy metals through (1) cell surface
adsorption, (2) extracellular precipitation, (3) intracellular
accumulation through special components, such as metallothioneins
(MT) or, (4) intracellular accumulation into vacuoles.
104
(Biosorption)
 Biosorption is an emerging method that came into practice about two
decades ago.
 It holds outstanding potential as a cost-efficient method for
environmental cleaning and reducing heavy metal pollution resulting
from industrial and agricultural sources.
 This method depends on the sequestration of toxic heavy metals by
the moieties of biosorbent cell surfaces (Figure 4(1)) such as those
found in fungi/yeast, bacteria, and algae.
 Applications of biosorption in bioremediation include heavy metal
elimination from soil, landfill leachates and water as well as several
other roles.
 Several living organisms have been tested as potential biosorbents.
This includes bacteria such as Bacillus subtilis and Magnetospirillum
gryphiswaldense, fungi such as Rhizopus arrhizus, yeast such
as
105
Saccharomyces cerevisiae and algae such as Chaetomorpha linum and
marine microalgae (seaweed).
(Genetic Engineering in Biosorption)
 The introduction of genetic engineering of bacteria and other
microorganisms opened up new horizons for designing tailored
organisms with engineered metal-binding peptides that possess
improved affinity and selectivity for target metals to be used as heavy
metal biosorbents.
 In bacteria, the surface display systems in various species such
as Staphylococcus xylosus and S. carnosus were shown to express two
different polyhistidyl peptides, i.e., His3-Glu-His3 and His6. The
encoded gene products of the chimeric surface proteins were targeted
to improve functionality in terms of metal binding as well as surface
accessibility.
 Similarly, Escherichia coli and Pseudomonas putida showed improved
phosphate biosorption through the immobilization of an intracellular
phosphate-binding protein (PBP) onto its cell surface .
106
(Bioaccumulation)
 Bioaccumulation takes place when the absorption rate of the
contaminant is higher than the rate of losing it.
 Thus, the contaminant remains contained and accumulated inside

the organism .
 Bioaccumulation is a toxicokinetic process that affects the
sensitivity of living organisms to chemicals.
 Organisms can normally resist concentrations of chemicals up to

certain levels, beyond which these chemicals become toxic and
endanger the organism.
 The sensitivity of organisms to chemicals is highly variable

depending on the types of organisms and chemicals involved.
107
(Bioaccumulation)
 Bioaccumulation candidate organisms should have a tolerance
ranging between one or more contaminants to higher levels.
 Furthermore, they may demonstrate superior biotransformational

capabilities, changing the toxic chemical to a non-toxic form
which enables the organism to reduce the toxicity of the
contaminant while keeping it contained.
 Several different organisms are used for the study of

bioaccumulation and as indicators for increased levels of
pollutants, including plants, fungi , fish, algae and bacteria.
108
(Genetic Engineering in Bioaccumulation)
 In order to identify suitable bioaccumulation organisms, investigating
the mechanisms and genes involved in the bioaccumulation process as
well as the genes responsible for sensitivity/tolerance and the
tolerated concentrations of different chemicals is crucial.
 Molecular biology techniques have been broadly used in such
investigations.
 The DNA microarray technique was also used to identify genes

regulated in response to exposure to high concentrations of heavy
metals.
 Whole genome sequencing of microorganisms with potential
bioaccumulation capacities helped in investigating candidate genes to be
109targeted for improving the bioaccumulation efficiency of the organism.
(Bioleaching )
 The use of electrical and electronic equipment (EEE) has significantly
increased in recent decades throughout the world, generating large amounts of
waste electrical and electronic equipment (WEEE).
 WEEE contains toxic components such as Pb, Cd, Hg, Cr VI, and
polybrominated biphenyls but also valuable materials such as plastic, Fe, Cu,
Al, Au, Ag, Pd, and other metals.
 Among WEEE, the gold-plated finger integrated circuits found in computer

motherboards (GFICMs) and the printed circuit boards of cellular phones
(PCBs) are a rich secondary source of metals such as Cu, Au, Pd and Ag.
 Their recovery is typically achieved by pyrometallurgical and

hydrometallurgical processes, which have certain energy and
110
environmental disadvantages.
(Bioleaching )
 Therefore, some microbiological processes have been proposed as
alternatives to chemical processes for treating such electronic waste.
 For instance, bacteria such as Thiobacillus ferrooxidans and T. thiooxidans and

fungi such as Aspergillus niger and Penicillium simplicissimum are able to
mobilize high percentages of Cu, Sn, Al, Ni, Pb and Zn powder from
WEEE.
 Currently, 55 bacterial and 36 archaeal complete genomes from

microorganisms that are present in bioleaching processes are publicly
available according to the NCBI database
111
(Bioleaching )
 The solubilization of metals can be accomplished by various species of
bacteria and fungi and is based on three main mechanisms that can
occur simultaneously.
I. Acidolysis : by far the most important one and is quite similar to the
mechanism of conventional acid leaching. Organic and inorganic acids
are produced by the microorganisms, which in turn leach the metals
from the solids by protonation.
II. Complexolysis: biogenic agents are excreted from the microbes, and
these solubilize metal ions through ligand formation. Citric acid, for
instance, is a powerful natural chelating agent .
III. Redoxolysis: oxidation and reduction reactions occur that set metals
free from the mineral.
112
(Bioleaching )
 Bioleaching can be an interesting route for metal recovery from
industrial waste materials, such as incinerator ashes, metallurgical
slag, and mineral tailings.
 It can also be applied in the detoxification of soils and groundwater

contaminated with heavy metals at various deposit sites
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Bioremediation
Bioremediation: is the use of natural and recombinant
microorganisms for the cleanup of environmental toxic pollutants
or Use of living organisms to transform, destroy or immobilize
contaminants.
Goal: Detoxification of the parent compound(s) and conversion to

products that are no longer hazardous to human health and the
environment.
Bioremediation
Bioremediation
 Bioremediation is considered a cost-effective and
environmentally friendly approach.
 It relies on improved detoxification and degradation of toxic

pollutants either through intracellular accumulation or via
enzymatic transformation to lesser or completely non-toxic
compounds.
 Many naturally or genetically modified microorganisms possess the

ability to degrade, transform, or chelate various toxic chemicals
and hence provide better strategies to combat environmental
pollution.
Forms of Bioremediation
In situ Bioremediation:
Bioventing
In situ biodegradation
Biostimulation
Biosparging
Bioaugmentation
Natural Attenuation
Ex situ Bioremediation:

Land farming
Composting
Biopiles
Bioreactors
In Situ Bioremediation
 Bioventing
One of the most common approaches in soil
Supply air and nutrients via wells
Takes advantage of indigenous microorganisms
 In situ biodegradation
Supply air and nutrients by circulating aqueous solutions through
contaminated soils or groundwater
 Biosparging
Injection of air below the water table  increases groundwater
oxygen concentrations and mixing in saturated zone
 Bioaugmentation
Addition of indigenous or exogenous microorganisms
Limits to use: competition and necessity
 Biostimulation
 Natural Attenuation or Intrinsic Bioremediation
Bioventing
Biosparging
Natural Attenuation
Using the biodegradative capacity of indigenous microbes
without additional enhancement.
Relies on combined biological (i.e., biodegradation), physical
(i.e., volatilization, dispersion, sorption), and chemical
processes (e.g., hydrolysis, oxidation, reduction)
Feasible only when the biodegradation rate is faster than

the rate of contaminant migration.
In Situ Bioremediation
Five Steps of In Situ Bioremediation
1. Site investigation
2. Treatability studies
3. Recovery of free product and removal of the

contamination source
4. Design and implementation of the in situ

bioremediation system
5. Monitoring and performance evaluation of the in situ bioremediation

system
Ex Situ Bioremediation
 Land farming
 Contaminated soil is excavated and spread over land
 Soil is periodically tilled to improve aeration
 Remediation due to indigenous microorganisms, as well as chemical and
physical processes
 Generally limited to the superficial 10–35 cm of soil
 Can reduce monitoring and maintenance costs
 Composting
 Combines contaminated soil with nonhazardous organic amendants (e.g.
manure or agricultural wastes)
 Biopiles
 Combination of land farming and composting
 Control physical losses of contaminants
 Bioreactors
 Soil and water pumped up from a contaminated plume and processed
through an engineered containment system
 Degradation in a bioreactor is generally greater than in situ because the
contained environment is more controllable and predictable
Ex situ bioremediation
 Ex situ bioremediation method involves the digging of the contaminated media like
soil, sediment, or sludge pushing of groundwater to facilitate microbial degradation
of pollutants. Ex situ bioremediation can take place in two ways:
(1)Slurry-phase bioremediation and (2) solid-phase bioremediation.
Slurry-Phase Bioremediation
 Slurry-phase bioremediation is a biological procedure where the contaminated soil,
sludge, or sediment is blended with H2O and other chemicals within a bioreactor, a
container in which three phases are created like liquid, solid, and gas (three-phase)
and blending conditions are maintained to elevate the biodegradation rate of water-
soluble and soil-connected pollutants present in the water slurry of the contaminated
soil, sludge, or sediment and biomass of indigenous microbes.
 It is blended so as to keep the microbes in association with the hazardous
contaminants present in the substrates. Then oxygen and nutrients are supplemented
to the reactor to establish the ideal environmental conditions for microbes to degrade
the specific pollutants.
 Once the process gets completed, the H2O is withdrawn from the soil, and the soil is
assessed and replenished in the environment.
 This method is relatively rapid as compared to other bioremediation methods. In
slurry-phase bioremediation, the rates of pollutants disintegration are effective in a
bioreactor treatment system than in solid-phase systems in situ since the enclosed
environment is more manageable, controllable, and predictable.
Solid-Phase Bioremediation
 Solid-phase bioremediation is a method which treats the contaminated
soil within an aboveground treatment area.
 Conditions within the treatment areas are monitored in order to ensure
optimum treatment is taking place.
 This kind of treatment is easy to uphold, but it needs a lot of space, and
the course of decontamination takes longer as compared to slurry-
phase bioremediation.
 The theory of solid-phase bioremediation is based on the mechanical
breakdown of polluted soil by abrasion and by an intensive blend of
the components in an enclosed vessel.
 This confirms that the nutrients, microbes, pollutants, O2, and H2O are
in permanent contact.
 Solid-phase soil treatments include soil biopiles, land farming, and
composting practices for detoxification and disintegrating the
hazardous toxic contaminants.
 Land farming, also known as land application/land treatment, is an ex situ treatment method
in which contaminated soil, sediment, or sludge is digged and dispersed on a prepared bed
and cyclically turned over (tilled) for aerating the mixture till contaminants gets degraded
via stimulated aerobic microbial activities in the soils due to aeration and/or the
supplementation of moisture, minerals, and nutrients.
 This practice is restricted for the treatment of superficial 10–35cm of soil. The land farming
method has been verified to be efficient in lowering the concentrations of all the components
of petroleum products usually found on under-ground storage tank sites.
Composting
 Composting is a biological disintegration process in which organic wastes are transformed into
humus-like matters by microbes, which is stable organic end product (compost).
 In composting, the contaminated soil is dug out and blended with a bulking agent and organic
materials (such as animal wastes, wood chips, vegetative wastes, etc.).
 The existence of these organic constituents aids the proliferation of a rich microbial
community which changed the organic matter into compost via their enzymatic activity.
 Usually, composting is the anaerobic, thermophilic procedure of microbiological
disintegration of polluting agents (organic wastes) into stable end product (usually compost)
which can be disposed safely into the environment
 Under normal environmental circumstances, earthworm; soil
insects, i.e., mites, sow bug, ants, springtails, and beetles; and
nematodes start the degradation of organic material into minute
particles, thus intensifying their bioavailability for the microbial
community, whereas, under regulated environmental conditions,
composting machinists disintegrate the large waste entities via
chopping or grinding.
 A huge number of microbes are involved in the disintegration of
organic contaminants that are readily available in the wastes.
 Soil microbes like bacteria, fungi, actinomycetes, and protozoa
are acquainted when the wastes are blended with soil or
inoculated with finished compost.
 The composting process is executed by three classes of microbes:

(1) psychrophiles, (2) mesophiles, and (3) thermophiles. For
profitable composting, microbes require nutrients, moisture,
temperature, and oxygen.
Biopiling
Biopiling, also called biocells, biomounds, or bioheaps, is an ex situ
bioremediation method in which burrowed soil, sludge, or sediments are
blended with soil amendments, placed on a treatment area, and
remediated using forced aeration.
 This method encompasses the stacking of contaminated soil, sludge, or dried
sediments into piles and accelerating the biodegradation activity of aerobic
microbial community by forming ideal proliferating conditions within the
pile.
 Biopiles are generally 2–3 meters in height, and contaminated soil, sludge, or
sediment is conventionally laid on top of the treated soil.
 This methodology is extensively employed for the remediation of wide range
of diesel, crude, and lubrication oil contaminated soils, sludge, or sediments
 The primary procedure of toxic waste eradication in biopiles is by
stimulating the metabolic activities of pollutants degrading microbes by the
supplementation of nutrients (carbon and nitrogen) and diffusion of oxygen
within the soil.
 Soil microbial activity can also be augmented in biopile soil through direct
addition of pollutants degrading microbes.
Ex Situ Bioremediation
Microorganisms involved in Bioremediation
 Aerobic bacteria:
 Examples include: Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus, and
Mycobacterium
 Shown to degrade pesticides and hydrocarbons; alkanes and polyaromatics
 May be able to use the contaminant as sole source of carbon and energy.
 Methanotrophs:
 Aerobic bacteria that utilize methane for carbon and energy
 Methane monooxygenase has a broad substrate range
 active against a wide range of compounds (e.g. chlorinated aliphatics such as
 trichloroethylene and
 1,2-dichloroethane)
Bioremediation
Microorganisms involved in Bioremediation..
 Anaerobic bacteria:
 Not used as frequently as aerobic bacteria

 Can often be applied to bioremediation of polychlorinated biphenyls
(PCBs) in river sediments, trichloroethylene (TCE), and chloroform
 Fungi:
 Able to degrade a diverse range of persistent or toxic environmental

pollutants
 Acclimatization and lag phase

Bioremediation
Phytoremediation
 Phytoremediation refers to the use of plants in polluted sites to
promote biological, biochemical, physical, microbiological and
chemical interactions to attenuate the toxicity of contaminants.
 Depending on the type of pollutant, it occurs through different
mechanisms, namely biodegradation, vaporization, filtration,
among others.
 Elemental contaminants like heavy metals or radioactive
elements are mainly extracted, transformed and sequestered,
 while organic contaminants are eliminated mainly through
rhizodegradation, biodegradation, vaporization or stabilization.
 Within phytoremediation, there are many different ways that
plants interact with contaminants
132
Cont’
 Phytoextraction: This mechanism involves removal of the contaminant from the soil
and its accumulation in some part of the plant (e.g., root, stem or leaf).
 Phytotransformation or Phytodegradation: After the absorption of the contaminant by
the plant, it is transformed into a less toxic form. Such a transformation can also
occur on the plant’s own surface.
 Phytovolatilization: In this process, the plant removes the pollutant from the soil and
then manages to convert it into a volatile product, releasing it into the atmosphere.
 Phytostimulation: Plant roots release substances (exudates) that can accelerate
degradation of certain pollutants. When microorganisms contribute to degradation,
the whole process is called phytostimulation
 Phytostabilization: This process consists in the immobilization of pollutants in the
soil, thus avoiding erosive processes and allowing the association with humus and
lignin.
 Rhizofiltration: It is the process in which plant roots promote absorption,
precipitation and removal of organic and inorganic contaminants from wastewater,
groundwater and surface water.
 Rhizodegradation: The metabolic activity of microorganisms in the rhizosphere
(fungi, bacteria, and yeasts) is 10 to 100 times greater than in soil, which allows them
to effectively degrade contaminants together with natural organic nutrients to
extraction: This mechanism involves removal of the contaminant from the soil and its
accumulation in some part of the plant (e.g. root, stem or leaf).
133
Phytoremediation
 Heavy metals such as cadmium and lead are not readily absorbed
or captured by organisms.
 The assimilation of metals such as mercury into the food chain

may worsen matters.
Phytoremediation is useful in these situations, because

natural plants or transgenic plants are able to bioaccumulate these
toxins in their above-ground parts, which are then harvested for
removal.
Phytoremediation
 Effective and low cost
 Pollutants are absorbed in roots, thus plants removed could be

disposed or burned.
 Sunflower plants were used to remove cesium and strontium

from ponds at the Chernobyl nuclear power plant.
 Transgenic plants with exogenous metallothionein (a metal

binding protein) used to remove metals.
135
Types of Phytoremediation
 Phytoextraction or phytoaccumulation
 Plants used to accumulate contaminants in the roots and aboveground
biomass
 Can be a relatively low cost option for a large area
 Results in biomass that must be properly disposed of or reused
 Phytotransformation or phytodegradation
 Uptake of contaminants and transformation to more stable, less toxic, or
less mobile forms
 Eg. metal chromium can be reduced from hexavalent to less mobile (and
non-carcinogenic) trivalent chromium
 Phytostabilization
 Mobility and migration of contaminants are reduced through sorption onto
or into the plant
 Rhizodegradation
 Breakdown of contaminants through activity of the rhizosphere
 Rhizofiltration
 Water remediation technique
 Used to reduce contamination in natural wetlands and estuary areas.
Bioremediation
Advantages of Phytoremediation
 The cost of the phytoremediation is lower than that of traditional processes
both in situ and ex situ
 Can be employed in areas that are inaccessible without excavation
 The plants can be easily monitored
 The possibility of the recovery and re-use of valuable metals (by companies
specializing in “phyto mining”)
 It is potentially “the least harmful” method because it uses naturally

occurring organisms and preserves the environment in a more natural state
CHAPTER THREE
XENOBIOTIC COMPOUNDS
Recalcitrants, Hazardous wastes, Genetic engineering

approach for biodegradation, detoxification methods.
Solid waste - sewage sludge - disposal and utilization)

Types of pollutants
Types of pollutants
 Organic pollutants → catabolized …Naturally occurring
 Xenobiotics - substances foreign to an entire biological

system, i.e. artificial substances, which did not exist in
nature before their synthesis by humans
 Metals from ore extraction and manufacturing
141
Contaminants Potentially Amenable to
Bioremediation
Readily Somewhat Difficult to Generally

degradable degradable degrade recalcitrant
____________ _____________ _____________ _____________
_
fuel oils, gasoline creosote, coal chlorinated dioxins
tars solvents (TCE)
ketones and pentachloro- some pesticides polychlorinated

alcohols phenol (PCP) and herbicides biphenyls (PCB)
monocyclic
aromatics
bicyclic aromatics
(naphthalene)
Biological solution
Biodegradation of xenobiotics
 BIODEGRADATION OF XENOBIOTICS HYDROCARBONS,
PLASTICS & PESTICIDES
 XENOBIOTICS:
 It is derived from a greek word “XENOS” meaning ‘foreign or strange’.
Xenobiotics are those chemicals which are man-made and do not occur
naturally in nature.
 They are usually synthesized for industrial or agricultural purposes e.g.
aromatics, pesticides, hydrocarbons, plastics , lignin etc.
 They are also called RECALCITRANTS as they can resist degradation to
maximum level.
 BIODEGRADATION: According to the definition by the International
Union of Pure and Applied Chemistry, the term biodegradation is
“Breakdown of a substance catalyzed by enzymes in vitro or in vivo.
 In other words, defined as the ability of microorganisms to convert toxic
chemicals (xenobiotics) to simpler non-toxic compounds by synthesis of
certain enzymes
 Biodegradation of xenobiotics can be affected by substrate specificity,
145
nutrition source, temperature, pH etc.
Sources of Xenobiotics
1. Petrochemical industry : -oil/gas industry, refineries. -
produces basic chemicals e.g. vinyl chloride and benzene .
2. Plastic industry : - closely related to the petrochemical
industry - uses a number of complex organic compounds -such
as anti-oxidants, plasticizers, cross-linking agents
3. Pesticide industry : - most commonly found. -structures are
benzene and benzene derivatives.
4. Paint industry : - major ingredient are solvents, - xylene,
toluene and so on.
5. Others : - Electronic industry, Textile industry, Pulp and Paper
industry, Cosmetics and Pharmaceutical industry, Wood
146
preservation
Biodegradation of Pesticides
 Pesticides are substances meant for destroying or mitigating any pest.
They are a class of biocide. The most common use of pesticides is as plant
protection products (also known as crop protection products).
 It includes: herbicide, insecticide, nematicide, termiticide, molluscicide,
piscicide, avicide, rodenticide, insect repellent, animal repellent,
antimicrobial, fungicide, disinfectant, and sanitizer.
 DIFFERENT METHODS:
A. Detoxification: Conversion of the pesticide molecule to a non-toxic
compound. A single moiety in the side chain of a complex molecule is
disturbed(removed), rendering the chemical non-toxic.
B. Degradation: Breakdown or transformation of a complex substrate
into simpler products leading to mineralization. E.g. Thirum
(fungicide) is degraded by a strain of Pseudomonas and the
degradation products are dimethylamine, proteins, sulpholipids, etc
147
Biodegradation of Pesticides
C. Conjugation (complex formation or addition reaction): An organism
makes the substrate more complex or combines the pesticide with cell
metabolites. Conjugation or the formation of addition product is
accomplished by those organisms catalyzing the reaction of addition of
an amino acid, organic acid or methyl crown to the substrate thereby
inactivating the pesticides.
D. Changing the spectrum of toxicity: Some pesticides are designed

to control one particular group of pests, but are metabolized to yield
products inhibitory to entirely dissimilar groups of organisms, for e.g.
the fungicide PCNB is converted in soil to chlorinated benzoic acids
that kill plants.
148
Biodegradation of xenobiotics
 There are many mechanisms involved on the biodegradation of pesticides
and other contaminants. These may be summarised as follows:
Dehalogenation- nitrofen, DDT, cyanazine, propachlor.

 Deamination- fluchloralin
Decarboxylation- DDTc, biofenox, dichlorop-methyl
Methyl oxidation- bromacil
Hydroxylation- benthiocarb, bux insecticide
149
Biodegradation of Plastics
 Plastic is a broad name given to different polymers with high molecular
weight, which can be degraded by various processes.
 The biodegradation of plastics by microorganisms and enzymes seems to
be the most effective process.
 It consist of two steps- fragmentation and mineralization. But at
the core, reaction occurring at molecular level are oxidation and
hydrolysis.
 The decomposition of major condensation polymers (e.g. polyesters and
polyamides) takes place through hydrolysis, while decomposition of
polymers in which the main chain contains only carbon atoms (e.g.
polyvinyl alcohol, lignin) includes oxidation which can be followed by
hydrolysis of the products of oxidation.
150
METHOD HYDROLYSIS:
 The process of breaking these chains and dissolving the polymers into
smaller fragments is called hydrolysis. E.g. Pseudomonas sps
 Polymeric Chains is broken down into constituent parts for the energy
potential by microorganisms. Monomers are readily available to other
bacteria and is used.
 Acetate and hydrogen produced is used directly by methanogens.
 Other molecules, such as volatile fatty acids (VFAs) with a chain length
greater than that of acetate is first catabolized into compounds that can
be directly used by methanogens.
1. ACIDOGENESIS:
 This results in further breakdown of the remaining components by
acidogenic (fermentative) bacteria into ammonia, ethanol, carbon
dioxide, and hydrogen sulfide. E.g Streptococcus acidophilus.
METHOD HYDROLYSIS:
2. ACETOGENESIS:
 Simple molecules created through the acidogenesis phase are further
digested by Acetogens to produce largely acetic acid, as well as carbon
dioxide and hydrogen.
3. METHANOGENESIS:
 Methanogens use the intermediate products of the preceding stages and
convert them into methane, carbon dioxide, and water.
 These components make up the majority of the biogas emitted.
 Methanogenesis is sensitive to both high and low pHs and occurs
between pH 6.5 and pH 8.
 The remaining, indigestible material the microbes cannot use and any
dead bacterial remains constitute the digestate.
Biodegradation of Plastics
 Some of the microorganism that can degrade plastics are:-
 Aliphatic Polyesters PolyEthylene Adipate (PEA)- lipases

from R. arrizus, R. delemar, Achromobacter sp. and Candida cylindracea
 Poly (β-Propiolactone) PPL - estereases from Acidovorax sp.,
Variovorax paradoxus, Sphingomonas paucimobilis.
 Poly-3-Hydroxybutyrate (PHB) – estereases from
Pseudomonas lemoigne, Comamonas sp. Acidovorax faecalis, Aspergillus
fumigatus
 Poly Lactic Acid (PLA) - proteinase K from Tritirachium album,
Amycolatopsis sp Strains of Actinimycetes has been reported to
153 degrade polyamide (nylon), polystyrene, polyethylene.
Biodegradation of Hydrocarbons
 A hydrocarbon is an organic compound consisting entirely of hydrogen
and carbon.
 The majority of hydrocarbons found on earth naturally occur in crude
oil.
 Aromatic hydrocarbons (arenes), alkanes, alkenes, cycloalkanes and
alkyne-based compounds are different types of hydrocarbons.
 BIODEGRADATION OF PETROLEUM:
 Petroleum compounds are categorized into 2 groups
1. Aliphatic hydrocarbon e.g. alkane, alcohol, aldehyde
2. Aromatic hydrocarbon e.g. benzene, phenol, toluene,
catechol Aromatic hydrocarbons are degraded aerobically and
anaerobically.
154
Biodegradation of Hydrocarbons
 AEROBIC DEGRADATION:
 Are metabolized by a variety of bacteria, with ring fission. Accomplished by mono-
and dioxygenases.
 OTHER MECHANISMS
1. Photometabolism : in bacteria, this light-induced “bound oxygen” (OH ) is used to
oxidize substrates
2. under nitrate-reducing condition : Nitrate-reducing bacteria couple the
oxidation of organic compound with water to the exergonic reduction of nitrate via
nitrite to N2.
3. dissimilation through sulfate respiration: Sulfate- reducing bacteria couple the
oxidation of organic compound with water to the exergonic reduction of sulfate via
sulfite to sulfide.
 Some microorganisms involved in the biodegradation of hydrocarbons includes;
Acinetobacter, Arthrobacter, Azotobacter, Flavobacterium, Pseudomonas putida Candida
tropicalis Trichosporon cutaneoum Aspergillus & related Arthrobacter, Bacillus spp.,
Micrococcus, P. putida.
 Genetic Regulation of Xenobiotic Degradation plasmid-borne mostly in the genus
155 Pseudomonas
Polycyclic Aromatic Hydrocarbons (PAH):
 Bacteria, fungi, yeasts, and algae have the ability to metabolize both
lower and higher molecular weight PAHs found in the natural
environment.
 Most bacteria have been found to oxygenate the PAH initially to form
dihydrodiol with a cis-configuration, which can be further oxidized to
catechols.
 Most fungi oxidize PAHs via a cytochrome P450 catalyzed mono-
oxygenase reaction to form reactive arene oxides that can isomerize to
phenols.
 White-rot fungi oxidize PAHs via ligninases (lignin peroxidases and
laccase) to form highly reactive quinones.
Polycyclic Aromatic Hydrocarbons (PAH):
Compound Organisms Metabolite:

Naphthalene: Acinetobacter calcoaceticus , Alcaligenes
denitrificans, Mycobacterium sp. , Pseudomonas sp., Pseudomonas
putida ,
 Naphthalene cis -1,2 – dihydrodiol, 1,2 –
dihydroxynaphthalene, 2 - hydroxychromene - 2 – carboxylic
acid, trans – o – hydroxybenzylidene pyruvic acid,
salicylaldehyde, salicylic acid, catechol, gentisic acid,
naphthalene trans – 1,2 – dihydrodiol : Pseudomonas putida,
Pseudomonas fluorescens, Pseudomonas cepacia.
Fluoranthene: Alcaligenes denitrificans - Mycobacterium sp. ,
Pseudomonas putida , Pseudomonas paucimobilis, Pseudomonas
cepacia , Rhodococcus sp.
157
Polychlorinated Biphenyls (PCBs):
 Synthesized chemicals from petro-chemical industry used as
lubricants and insulators in heavy industry.
 First manufactured in 1929 by Monsanto.
 Used because- Low reactivity, Non-flammable, High electrical
resistance, Stable when exposed to heat and pressure, Used as
Hydraulic fluid, Casting wax, Carbonless carbon paper, Compressors,
Heat transfer systems, Plasticizers, Pigments, Adhesives, Liquid cooled
electric motors, Fluorescent light.
 RISKS: Causes reproductive disabilities in animals, human, birds;

Carcinogenic; Bioaccumulation; Soluble in almost all the solvents, fats,
oils; Nervous system damage and Endocrine gland malfunction
158
METHODS FOR PCB REMOVAL:
 Natural Attenuation:
 Microbes already in the soil are allowed to degrade as they can
naturally and the site is closely monitored.
 Biostimulation:
Microbes present in the soil are stimulated with nutrients such as

oxygen, carbon sources like fertilizer to increase degradation.

 Bioaugmentation:
Microbes that can naturally degrade PCB’s are transplanted to the

site and fed nutrients if necessary.
METHODS FOR PCB REMOVAL:
 PATHWAYS FOR PCB REMOVAL:
1. FUNGAL DEGRADATION:
Aspergillus niger: fillamentous with cytochrome p450 that
attacks lower chlorinated PCB’s
 Phanerochaete chrysosporium: White rot fungi can
attack lignin (PCB) at low concentration with the help og
ligninases.
2. BACTERIAL DEGRADATION: Soil bacteria breaks down PCBs

via dioxygenase pathways.
 Most identified seem to be Pseudomonas species, Achromobacter,
Acinetobacter, Alcaligenes, Arthrobacter, Corynebacterium, Rhodococcus,
Burkholderia .
Introduction to sludge
161
162
Source of sludge
163
Objective of sludge management
164
Main type of sludge treatment
165
o Sludge Thickening
 is a procedure used to remove water and increase the solids content.
o Sludge Digestion Process
 The sludge withdrawn from the sedimentation basins contains a lot of

putrescible organic matter, and if disposed of without any treatment,
the organic matter may decompose, producing foul gases and a lot of
nuisance, pollution and health hazards.
 In order to avoid such pollutions, the sludge should stabilized by
decomposing the organic matter under controlled anaerobic
conditions, and then disposed of suitably after drying on drying beds,
etc.
166
 Factors Affecting Sludge Digestion and Their Control
 Temperature, pH value, Seeding with digested sludge, Mixing and
stirring of the raw sludge with digested sludge.
 Dewatering
Disposal of
Dewatered Sludge
 Dumping into the Sea

 Burial into the Trenches
 Incineration
167
168
Conditioning
169
CHAPTER FOUR
APPLICATION OF BIOTECHNOLOGY IN
INDUSTRY:

INDUSTRY
 CLEAN TECHNOLOGY AND SUSTAINABILITY :
 Clean technology is a conceptual and procedural approach to

industrial activities that demands that all phases of the life cycle of a
product or of a process should be addressed with the objective of
prevention or minimization of short- and long-term risks to human
health and to the environment.
 Sustainable development: is strategies and actions that have the

objective of meeting the needs and aspirations of the present
without compromising the ability to meet those of the future.
171
APPLICATION OF BIOTECHNOLOGY IN INDUSTRY
 Figure 4.1: Process flow chart for enzyme production and purification
APPLICATION OF BIOTECHNOLOGY IN INDUSTRY
 Figure 4.2: Flow chart summarizing application of enzymes and clean production strategy.
PESTICIDES INDUSTRY
 Chemical pesticides: the problem
 The production of crops is significantly reduced by invertebrate pests, plant
diseases, and weeds.
 At present, crop protection relies heavily on chemical pesticides.

 However, consumers are deeply suspicious of the possible health effects of
pesticide residues on food, a concern that is picked up by retailers who are in
many cases pushing for levels of reduction in pesticide use that go beyond what
is required by regulators.
 It is important to recognize that the elimination of pesticides would have a

substantial impact on the quantity and quality of food available and its price.
174
PESTICIDES INDUSTRY
 How can one reconcile the need to sustain levels of food production and farm
income with the declining availability and acceptability of chemical pesticides?
 The broad solution is to use the ecologically based pest control management
strategy known as Integrated Pest Management (IPM) where ‘the basic
goal is to use control tactics against pests only when necessary.’
 IPM suggests using methods for control only if the pest population is causing
damage above the economic injury level. IPM thus always requires a good
understanding of the pest system.’
 There is a role for chemical pesticides in IPM when infestations cannot be
controlled by any other means, but they should be used as the last rather than
the first resort.
 There is also scope for increased use of alternatives to chemical pesticides such
as biological controls and in particular biopesticides.
175
PESTICIDES INDUSTRY
 Categories of Biopesticides
1. Microbial pesticides consist of microorganism (e.g.,
bacterium, virus, fungus and protozoan) as the active ingredient.
 Microbial pesticides can control many different kinds of pests although
each separate active ingredient is relatively specific for its target pests.
 For example, there are fungi that control certain weeds, and other fungi
that kill specific insects. The most widely used pesticides are subspecies and
strains of Bacillus Thuringiensis or Bt.
 Each strain of bacterium produces different mix of proteins and specifically
kills one or a few related species of insect larvae.
 While some Bt’s control moth larvae found on plants, other Bt’s are
specific for larvae of flies and mosquitoes. The target insect species are
determined whether the particular Bt produces protein larvae that can
176
bind to larval receptor thereby causing insect larvae to starve.
PESTICIDES INDUSTRY
 Categories of Biopesticides….
2. Plant Incorporated protectants (PIP) are pesticidal

substances that plants produce from genetic material that has been added
to the plant.
 For example, scientists can take the gene for the Bt pesticidal protein,
and introduce the gene into the plant's own genetic material.
 Then the plant, instead of the Bt bacterium, manufactures the substance

that destroys the pest.
177
PESTICIDES INDUSTRY
 Categories of Biopesticides…
3. Biochemical pesticides are naturally occurring substances that

control pests by non-toxic mechanisms.
 Biochemical pesticides include substances, such as insect sex pheromones,
which interfere with mating, as well as various scented plant extracts that
attract insect pests to traps.
 Plants, including major crops such as maize and cotton, release volatile
signalling chemicals when attacked by insect pests. These chemicals act as
attractants of other, parasitic insects.
4. Conventional pesticides, by contrast, are generally synthetic

178 materials that directly kill or inactivate the pest.
Biopesticide Production: Use of New
Genetic-Engineering Technology
 Biological control is the most important alternative to chemical pesticides in
protecting crops from pests, pathogens, and weeds.
 Valuable genes from completely unrelated organisms can now be utilized for
biological control purposes.
 Biological control using recombinant DNA (genetic engineering) technology
can be achieved in several different ways: control agents may be improved; or
crop plants can be engineered to carry better resistance genes to provide
protection.
 All these approaches have successfully been used in several different ways
experimentally.
 Product development has been very active in the area of incorporating
resistance genes mainly from BT-directly into plants. Successes include potato,
tomato, tobacco, and cotton.
179
 General root colorizing bacteria of plants have also been engineered to
produce insecticidal toxins, which protect against pests such as the corn
rootworm.
 Another bacterium living in the vascular tissues of corn has also been modified
to give protection against the corn borer.
 None of these modified plants or associated organisms is available
commercially yet. Similar approaches are used for the biological control of
plant pathogens and weeds, but research has been most active in the area of
insect control.
 How safe are these organisms for actual use? How do they affect the
environment or humans? Therefore, a very critical approach is necessary
toward the use of genetic-engineering technologies in agriculture.
180
 In principle, genetic engineering can be used for biological pest control in two
ways:
1. Engineering Biological Control Agents : The genetic improvement of
biological control agents, is a relatively new concept. For this, a great deal
must be known about the biology, ecology, and behavior of the organism. This
is a very crucial step.
2. Engineering Crop Plants : engineering crop plants to be resistant to pests
 The first published reports of successful engineering of crop plants to produce
insecticidal or antifeedant proteins appeared in 1987.
 The crop plants were tobacco and tomato, producing the delta endotoxin of
Bacillus thuringiensis to make them resistant against caterpillars.
 To date, transgenic crop plants have been produced of at least 27 different
species, including potato, cabbage, sugar beet, rice, soybeans, corn, rapeseed,
sunflower, walnut, and poplar.
TANNARY INDUSTRY
 LEATHER PROCESSING Leather Tanning with Enzymes:
 Dehairing, Bating Hides and skins have hair attached to them that must be removed
for their use as leather.
 The conventional way to remove hair from hides is to use harsh chemicals such as lime
and sodium sulfide. These chemicals completely dissolve the hair and open up the
fiber structure.
 With enzyme-assisted dehairing, it is possible to reduce the chemical requirements
and obtain a cleaner product and a higher area yield with fewer chemicals in the
wastewater.
 Since the enzyme does not dissolve the hair as the chemicals do, it is possible to filter
out the hair, thus reducing the chemical and biological oxygen demand of the
wastewater.
 Additionally the hides and skins contain proteins and fat between the collagen fibers
that must be all or partially removed before the hides can be tanned.
 To make the leather pliable, it is necessary to subject the hide to an enzymatic
treatment before tanning to selectively dissolve certain protein components. This is
called bating.
TANNARY INDUSTRY
 Traditionally, dog or pigeon dung was used as the bating agent. This was a difficult, unreliable
and smelly process.
 Obviously, this was a very unpleasant environment to work in. Since “dung bates” owed their
softening effect to the action of a protease enzyme, during the 20th century, the Leather
Industry has switched over to using bacterial proteases and pancreatic trypsin.
 Environmental Benefits: Lower chemical load to waste system. Lower odor during
processing,
 Consumer Benefits: Better leather, lower odor process for workers and the factory
neighbors.
 Degreasing of Leather Traditionally, the degreasing of sheepskins is done by solvent-
extraction using paraffin solvent systems.
 A new process based on the enzymatic breakdown of fats by a lipase enzyme has been
introduced to the leather industry. The enzymatic degreasing process replaces the solvent-
based process.
 Since the enzyme interferes less with the skin structure, the enzymatic process also results in a
product with improved quality, for example improved tear strength and more uniform color.
183
Waste treatment of Food industry
 Wastewater generated from agricultural and food operations has
distinctive characteristics that set it apart from common municipal
wastewater managed by public or private sewage treatment plants
throughout the world:
 It is biodegradable and non-toxic, but has high concentrations

of biochemical oxygen demand (BOD) and suspended solids (SS).
 The constituents of food and agriculture wastewater are often

complex to predict, due to the differences in BOD and pH in
effluents from vegetable, fruit, and meat products and due to the
seasonal nature of food processing and post-harvesting.
184
Waste treatment of Food industry
 Processing of food from raw materials requires large volumes of high grade
water.
 Vegetable washing generates waters with high loads of particulate matter and
some dissolved organic matter. It may also contain surfactants.
 Animal slaughter and processing produces very strong organic waste from
body fluids, such as blood, and gut contents. This wastewater is frequently
contaminated by significant levels of antibiotics and growth hormones from the
animals and by a variety of pesticides used to control external parasites.
 Processing food for sale produces wastes generated from cooking which are
often rich in plant organic material and may also
contain salt, flavourings, colouring material and acids or alkali. Very significant
quantities of oil or fats may also be present.
Pulp and paper industry
Biotechnological techniques like molecular biology,
enzymes engineering, genetic engineering genomics,
proteomics, metabolomics and bioinformatics etc. have
helped in developing economically feasible and well
designed methods in production of pulp and paper sector.
Biotechnology increases the cost efficiency, develop

environmentally feasible processes and improves the
product quality.
186
Conventional way of making paper pulp
Bark of wood is removed. The logs obtained are cut into smaller
pieces called chips.
The chips are cooked by heating under pressure using caustic
soda and sulfur.
By this lignin that binds the cellulose fibers are removed.
This is the chemical pulping process.
It gives 30% yield lesser than by mechanical pulping.
 In mechanical pulping, debarked logs are forced through
rotating toothed steel discs. The discs tear the logs and remove the
lignin. The lignin degraded gives paper a brownish tinge which are
used for printing newspapers.
 Making pulp using enzymes (Biopulping)
 Biopulping is the treatment of wood chips with lignin- degrading fungi. It is

used prior to mechanical pulping of wood. It eases subsequent mechanical and
chemical pulping.
 It improves penetration and effectiveness of chemicals during the “cooking” of

wood chips for separating the cellulose fibers from the lignin.
 Biopulping reduces the demand for energy and chemicals, improves paper
quality, and decreases the environmental impact of pulp production.
 Biotechnology can be used to enhance the properties required in cellulose
fibers .
 Genetically engineered microorganisms and enzymes can displace many of the

environmentally adverse practices used in pulp processing.
 Fungi such as O. piliferum can degrade lignin and is used in a fermentation
process. This is applied before carrying out the normal mechanical or chemical
pulping.
 It reduces the overall energy requirements for mechanical process and reduces
the quantum of chemicals for chemical pulping.
Pulp bleaching using enzymes (Biobleaching)
 Chlorine is used for bleaching process which has a huge polluting potential.
Pulp is usually tinged with brown color due to lignin content. Enzyme
enhances this bleaching process by two ways.
 Xylase breaks down the carbohydrate xylan (this entraps pulp lignin) to
reduce the need for chlorine in bleaching but gives numbers of byproducts
dioxins and PCBs.
 Lipases are used to control deposits of pitch. Cellulases are used to improve
rates of dewatering of pulp; and pectinases for digesting pectins. Cellulases are
used for drainage , deinking and fiber modification.
 Hydrophobicity of fiber surfaces can be altered by the enzyme laccase .
 The enzymatic process saves water and energy and the effluent is ecologically
harmless.
 The laccase is used for direct delignification of pulp, enabeling replacement of
current bleaching chemical stages such as oxygen or ozone stage.
 Biobleaching of pulp with enzymes have several advantages like: reduction of
chlorine consumption; pulp dewatering; deinking; removal of pitch;
degradation of dissolved and suspended organics in concentrated effluents of
mills.
 Biobleaching eliminates few of processing steps, thereby simplify and reduce
the severity of treatment of wastewater.
Pulp and paper industry Effluent
 Effluent from the pulp and paper industry is generally high in suspended
solids and BOD.
 Plants that bleach wood pulp for paper making may generate chloroform,
, dioxins , furans, phenols and chemical oxygen demand (COD).
 Stand-alone paper mills using imported pulp may only require simple primary
treatment, such as sedimentation or dissolved air flotation.
 Increased BOD or COD loadings, as well as organic pollutants, may require

biological treatment such as activated sludge or upflow anaerobic sludge
blanket reactors. For mills with high inorganic loadings like salt, tertiary
treatments may be required, either general membrane treatments
like ultrafiltration or reverse osmosis or treatments to remove specific
contaminants, such as nutrients.
191
DISTILLERY EFFLUENT
(IMPACT OF DISTILLERY EFFLUENT ON ENVIRONMENT)
 Following are the impacts of distillery effluent on environment.

 Discharge of wastewater with high TDS would have adverse impact on aquatic
life and to make unsuitable water for drinking purpose, if used for irrigation
reduce the crop yield ,corrosion in water system and pipe line.
 Suspended solids in wastewater reduce the light penetration and plant
production as a result in receiving water by increasing turbidity it can also clog
the fish gills.
 High amount of BOD in the wastewater leads to the decomposition of organic
matter under the anaerobic condition that produces highly objectionable
products including Methane (CH4), Ammonia (NH3), and Hydrogen Sulphide
(H2S) gas.
 Low Dissolved Oxygen (DO) in water bodies affect the aquatic life as DO
drops fish and other species are threatened and may get killed.
IMPACT OF DISTILLERY EFFLUENT ON ENVIRONMENT
 Fall in DO levels causes undesirable odours, tastes and reduce the

acceptability of water for domestic purpose.
 In steam generation, DO is one of the most important factors causing
corrosion of the boiler material.
 Generally, industrial wastewater changes pH level of the receiving water body.
Such changes can affect ecological aquatic system; excessive acidity particularly
can result in release of hydrogen sulphide (H2S) to air.
 Alkaline nature of wastewater causes declination in plant growth and crop
growth.
 Colour and odour of the effluent of distillery was red brown in colour with
unpleasant odour of Indol, Sketol and other sulphur compounds.
 Spent wash is a complex, multi component stream that is known to cause
considerable fouling.
Treatment of Distillery industry effluent
 Distilleries are one of the most polluting industries generating
enormous amount of wastewater from which an average of 10–15 L
of effluent is released with the production of 1 L of alcohol.
 The distillery wastewater known as spent wash is characterized
by its dark brown color, high temperature, low pH, and high
percentage of dissolved organic and inorganic matter.
 It also contains nearly 2% of the dark brown recalcitrant pigment
called melanoidin which imparts dark brown color to the
effluent.
 Various physical, chemical, and biological treatment
methods have been adopted for the removal of color from this
wastewater. But these methods only change the form of
contaminants rather than degrading them completely.
 Biological methods produce relatively little amount of product after
treatment by resolving a large amount of organism elements into carbon
dioxide to be stabilized, or by removing organic matters contained in
wastewater with the generation of methane gas.
 In the biological treatment methods, pollutants in wastewater can be

resolved, detoxified, and separated by using mainly microorganisms.
 Due to the relatively low cost and the variations of work progress, the
biological methods have been most widely used all over the world.
 A number of fungi, bacteria, yeast, and algae have been reported to

have effluent treatment capabilities by the process of absorption,
adsorption, and enzymatic degradation techniques.
 Bioremediation: Generally, methods of treating wastewater
include physical–chemical methods and biological methods.
 Methods such as sedimentation, flotation, screening, adsorption,

coagulation, oxidation, ozonation, electrolysis, reverse osmosis,
ultrafiltration, and nanofiltration technologies have been used for
treatment of suspended solids, colloidal particles, floating matters,
colors, and toxic compounds .
 The drawbacks of the physical–chemical methods include high costs

and the need to re-treat the products, which further increases the
cost of treatment.
Treatment of Distillery Spent Wash
 Biological treatment can be divided into aerobic and anaerobic

depending on the availability of oxygen.

 Aerobic treatment involves activated sludge treatment, aerated lagoons, and
aerobic biological reactors.
 Anaerobic filter, upflow sludge blanket (UASB), fluidized bed,

anaerobic lagoon, and anaerobic contact reactors are anaerobic
processes, that are commonly used to treat distillery mill effluents.
 Among these treatments one thing is common, use of microbes . These

microbes have effluent-treatment capabilities.
TYPES OF BIOREACTORS IN EFFLUENT TREATMENT
1. Anaerobic Reactors:
 Wastewater treatment using anaerobic process is a very promising reemerging

technology, produces very little sludge, requires less energy, and can become
profitable by cogeneration of useful biogas.
 However, these processes have been sensitive to organic shock loadings, low
pH, and show slow growth rate of anaerobic microbes. This often results in
poor performance of conventional mixed reactors.
1. Anaerobic Reactors….
 In recent years, the UASB process has been successfully used for the
treatment of various types of wastewaters performed a comparative
study of UASB and anaerobic fixed film reactors for treatment of
molasses wastewater.
 The UASB technology is well suited for high strength distillery

wastewaters only when the process has been successfully started up and
is in stable operation.
 However, the conventional UASB reactors showed severe limitations
mainly related to mass transfer resistance or the appearance of
concentration gradients inside the systems and slow primary startup
requiring several weeks.
A. Fixe film reactors: In stationary fixed film reactors, the reactors has a biofilm
support structure(media) such as activated carbon, PVC(polyvinyl Chloride)
support, hard rock particle or ceramic rings for biomass immobilization. The
wastewater is distributed from above/below the media.
B. Upflow Anaerobic Sludge Blanket Reactor(UASB): UASB technology is being
used extensively for effluents from different sources, such as distelleries, food
processing units, tannaries and municipal wastewater. The active biomass in the
form of sludge granules is retained in the reactor by direct settling.
C. Anaerobic fluidized bed reactor: In the anaerobic fluidized bed , the mdi for
bacterial attachment and growth is kept in the fluidized state by drag force exerted
by the upflowing wastewater. The media used are small particle sized sand,
activated carbon, etc. under fluidized state, each media provided a large surface
area for biofilm formation and growth.
D. Anaerobic filter reactor: In anaerobic filter,reactors, the entrapment of sludge
aggaragates between packing material supplied to the reactor, e.g down flow and
upflow anaerobic filter
A. Upflow Anaerobic Sludge Blanket Reactor(UASB): UASB technology is being
used extensively for effluents from different sources, such as distelleries, food
processing units, tannaries and municipal wastewater. The active biomass in the
form of sludge granules is retained in the reactor by direct settling.
A. Fixed Film Bioreactors

Fixed film bioreactors have become conventional technology for treating biodegradable
contaminants in air and water. Principal fixed film bioreactor applications include
treatment of industrial wastewaters, leachates or ground water, and air emissions of
volatile organic compounds (VOCs).
In the reactors, biological activity usually converts contaminants to innocuous end

products such as carbon dioxide, methane, and water. Conventional fixed film reactor
approaches involve aerobic, aerobic co-metabolic (with aliphatic and aromatic organic
inducers), and anaerobic metabolism. Emerging reactor approaches also include
sequential anaerobic/aerobic metabolism.
Slurry Bioreactors
A slurry bioreactor may be defined as a containment vessel and apparatus used to

create a three phase (solid, liquid, and gas) mixing condition to hasten the
biodegradation of soil-bound and water-soluble contamination as a water slurry of the
contaminated soil, sediment, or sludge and biomass (usually indigenous bacteria)
capable of degrading targeted contaminants.
Suspended Growth Bioreactors
Suspended growth bioreactors are standard technology for treating organic contaminants in
aqueous and waste sludge systems. The reactors use microbial metabolism under aerobic,
anaerobic, or sequential anaerobic/aerobic conditions to biosorb organic compounds and
biodegrade them to innocuous residuals.
The microbial activity in the systems produces biomass that is removed by gravity
sedimentation, with a portion of the settled biomass recycled to maintain a
desired mixed liquor suspended solids concentration in the bioreactor.
The excess biomass is wasted to a

sludge disposal process. Reactor
configurations includes sequencing
batch reactors (SBRs), completely
mixed activated sludge systems, plug
flow activated sludge systems, and
aerobic and anaerobic digestors.
 The anaerobic filter reactor (AFR) has been widely applied in the beverage, food-processing,
pharmaceutical and chemical industries due to its high capability of biosolids retention. In
fact clogging by biosolids, influent suspended solids, and precipitated minerals is the main
problem for this system. Applications of both upflow and downflow packed bed processes
can be observed.
E. Completely mixed anaerobic digester
 The completely mixed anaerobic digester is the basic anaerobic treatment system with an
equal hydraulic retention time (HRT) and solids retention time (SRT) in the range of 15-40
days in order to provide sufficient retention time for both operation and process stability.
Completely mixed anaerobic digesters without recycle are more suitable for wastes with
high solids concentrations.
Anaerobic Reactor Configuratios
Anaerobic Filters
Attached growth systems
In this type of reactor, waste enters in the bottom and flows
through the rocks or plastic media used for biomass
immobilization.
Recirculation is used to dilute any toxic compound in the
influent.
The main limitations of reactor : accumulation of solids in the
packing material (plugging). So, wastes containing high amount
of suspended solids are not suitable for A.F
channeling cost of packing material
Anaerobic Reactor Configuratios
Upflow Anaerobic Sludge Blanket Reactor (UASB)
Upflow Anaerobic Sludge Blanket Reactor (UASB)
This type of reactor was developed to avoid the main problems of
the anaerobic filter.
Flow is in upward direction.
Biomass settles in the bottom usually in the forms of granule.
Advantages:
High biomass concentrations. Hence high organic loading rates can be applied.
so excellent COD removals due to high biomass concentrations.
Compared to Anaerobic filter, wastewaters with higher suspended solid
concentrations can be applied.
Disadvantages: Sludge granulation is complex and not fully understood process
Biomass escape at the effluent at higher loading rates hybrit reactors are used to avoid
this problem)
Fluidized and Expended Bed Reactors
gas In these systems microorganisms
Effluent
grow on small inert particles such as
fine sand or activated carbon
Recirculation
ORP, pH pump High Recycle ratios are used to
probes
keep the particles in suspension
Water-jacketed
glass reactor The rate of liquid flow and the
carrier resulting degree of expansion of the
bed (10-25%) determine whether
the reactor is a fluidized or an
Glass
expanded (less expansion) bed
beads reactor
wastewater
limitation: high and uniform

upflow so high pumping cost
IMPACT OF DISTILLERY EFFLUENT ON ENVIRONMENT
2. Aerobic reactors:
 Anaerobically treated distillery spent wash still contains high concentrations of
organic pollutants and as such cannot be discharged directly. Aerobic treatment
of anaerobically treated distillery spent wash has been attempted for the
decolorization of the major colorant, melanoidin and for further reduction
of the COD and BOD.
 A large number of microorganisms such as bacteria (pure and mixed culture),

cyanobacteria, yeast, fungi, etc. have been isolated in recent years that are
capable of degrading melanoidin and ultimately decolorizing the wastewater.
Molasses based alcohol distillery effluent : Problem or Opportunity!
Sugar
Fermentation + Distillation
Sugarcane Juice Molasses Ethanol
Dark colour, obnoxious

odor, very high pollutant
loading (BOD, COD, TDS)
Spent Wash
Biomethanation
Rich in potassium
Ferti-irigation,
Biocomposting
• Ground water contamination

Scope for production of • Soil degradation & loss of agri-productivity
potash fertiliser • Stress on river water quality & ecosystem
Utilisation of alcohol distillery effluent :
Integrated process for potash recovery and simultaneous environmental remediation (partial)
Pre-treatment Potash recovery (>80%)

Bio-methanated Treated spent Potash Fertiliser
spent wash (A) wash (NOP, SOP, MKP etc.)
C-rich Sludge K-depleted
spent wash (B)
Activated Carbon
Water
(suitable for recycling)
CHAPTER FIVE
VERMITECHNOLOGY:
Use of aquatic plants including

transgenics in biotechnology,
biodegradable and eco-friendly
products
Current status of Environmental

Biotechnology and future.
Vermitechnology
• Vermitechnology is a method of converting all the biodegradable
wastes such as farm wastes, kitchen wastes, market wastes, bio-
wastes of agro-based industrial wastes, livestock wastes etc. into
useful product through the action of earthworms.
• Through their digestive process, earthworms convert decaying
vegetable matter, composted green waste, animal waste and paper
into valuable nutritive products for agriculture.
• The ideal objectives of vermitechnology are to upgrade the value of
the original waste material,
– produce upgraded material in situ and to obtain a final
product free of chemical and biological pollutants.
Cont’
• Earthworms form a major component of the soil system and these
organisms have been efficiently ploughing the land for millions of
years and assist in the recycling of organic nutrients for the efficient
growth of plants.
• Compost worms are being used for recycling of organic waste and
have the potential to be used in vermitechnology waste conversion
systems for industrial or municipal applications.
• Although many species of earthworms are suitable for waste
processing; two species namely Eisenia fetida and Eudrilus
eugeniae have mainly been taken into consideration for
vermicomposting.
• The process of converting organic waste by earthworm
consumption into nutrient filled humus is called vermicomposting
Cont’
• Worm castings, the scientific name of worm manure are nature’s
finest soil conditioners that are called as “Gardeners Gold” on
account of their excellent soil amending value.
• In natural system, earthworms along with soil microorganisms
degrade organic waste materials of soil and thus maintain nutrient
flux
• The vermicompost is a product rich in organic bioremediated matter
which differ from the compost obtained from the same matrix for its
level of humification and the greater presence of microbial
metabolites responsible for the fertilizing value of casts.
• By maintaining suitable temperatures in even the coldest winter
months and creating suitable conditions for earthworms to thrive
they can process a large amounts of organic material,
– consuming, digesting and passing almost any decaying organic matter
including food waste, leaves and grass, shredded paper or cardboard
waste, and chopped straw or hay.
POTENTIAL OF VERMITECHNOLOGY
• Vermitechnology may be an ecofriendly and effective tool in

reduction of waste quantities, reuse of waste materials,
recovery materials and energy particularly relevant to
agricultural production through bioprocessing and
bioconversion.
• Vermitechnology is an appropriate technique for disposal of
biologically decomposable organic waste materials and
production of compost of high quality.
• Vermitechnology improves the composting and utilization
process of available organic and inorganic wastes in the
natural plant production cycle.
• Thus, vermis are good biological agents for recovery of
vermifertilizer and vermiprotein for wider use in
agroecosystems, aquaculture and poultry.
Cont’
• Vermicomposting has added advantage to abatement of
organic pollution by rapid reduction in the bulk density and
elimination of foul odour; production of vermifertilizer for
the use in agroecosystems and vermiprotein or vermin
from waste as feed for poultry, fish, pig and other domestic
animals
• Potential benefits of vermitechnology include; reduction of
noxious qualities of organic wastes,
– i.e., elimination of bad odours, elimination/reduction of harmful
microorganisms; production of easily handling, high value
fertilizer and protein production for food and feed.
• Certain challenging areas like enhancement of food
production, waste recycling and management of solid
wastes are targeted promising area of vermitechnology.
BIOREMEDIATION
• Huge quantities of sewage sludge; solid urban waste,
agricultural residues and agro-industrial byproducts are
produced as a result of human activities.
• Before their agronomic use, organic waste must be
subjected to appropriate treatments to both stabilize
organic matter and avoid harmful effects.
• Apart from their effect on soil fertility, the bioremediated
organic wastes have been shown effective in two important
aspects of agriculture improvement and environmental
safety.
• The role of earthworms in processing organic waste and
producing stable organic matter is a suitable alternative
way to solve at least on farm dimension; the problem of
organic waste recycles.
• Like composting, vermicomposting is aimed to recycle the
energetic resources of any kind of organic wastes.
• In nature, earthworm cast consist of excreted masses of soil,
mixed with residues of comminuted and digested plant
residues. Casts obtained by vermiculture are usually called
vermicompost.
• The vermicompost is a product rich in organic bioremediated
matter, which differs from the compost obtained, from the
same matrix for its level of humification and the greater
presence of microbial metabolites.
• These metabolites, i.e., growth regulators, polysaccharides
are strongly responsible for the fertilizing value of casts.
• Various workers all over the world have so far done
considerable work on various aspects of earthworms,
earthworm cast and vermitechnology
Cont’
• Several studies have provided in depth information on the
aspects related to earthworm ecology, relationship among
earthworms, vermiculture, waste recycling and soil fertility
• The fertilizing value of bioremediated organic wastes mainly
depends on two factors viz., starting organic matrix; that
determine the quality and the safety of the end product and
bioremediation process that furthermore determine the
level of transformation of the starting materials.
• The difference between the agronomic values of the
different bioremediated materials must be mainly found in
the different process they underwent and in the microbial
metabolites synthesized by microflora during the
composting process.
Cont’
• Vermicomposting process mineralize complex

substances, releasing the nutrients available for
plants firstly into compost and then into soil.
• These substances, that play an important role in
the plant growth and development,
– contribute to the promotion of rooting, root growth, plant development and
improvement in crop production that are among the claimed effects
associated with the supply of bioremediated wastes.
• It is worthy to note that such substances, mainly
hormone-like compounds and metal chelating
agents have been widely used for a long time to
improve the quality of agricultural crops.
Cont’
• The chemical and biological assay carried out
on the same material submitted to the two
above reported processes shows that
hormone like substances are
– present in the vermicompost in greater amounts than in the
compost and that such compounds are present in the earthworm
cast in amounts comparable with those found in the rhizosphere of
many plants.
• It supplies the soil with plant nutrients and

organic matter that will be completely humified
over medium or long term.
• That contributes to enhance the rest effect,
responsible for the future fertility of soil.

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ARBA MINCH UNIVERSITY

COLLEGE OF NATURAL SCIENCES

ENVIRONMENTAL BIOTECHNOLOGY (Biot4142)

By:- Temam A. (Assistant Professor)

What was environmental biotechnology?

 Simple and traditional definition: use, in a controlled manner, of

 Solving environmental problems through biotechnology; e.g.

 International Society for Environmental Biotechnology, since

 Use of molecular techniques to protect the environment,

 Renewable energy and resources: engineering plants for the

 Environmental Biotechnology is the multidisciplinary

What is wastewater treatment?

Therefore, Wastewater should be

 Methods of treatment in which chemical or biological

 WWT applies any of this operations, processes or

 Removal of pollutants and the protection and

 Protection of human health by the destruction of

4. Free from materials and heat that alone, or in

 The term "flow sheet" is used to describe a particular

Presence of Fats, Oils and Greases

In the 2nd stage, nitrogenous

• The processes used

Depending upon the size of

• are the patented devices, which

Figure of Comminutor and shredder

3. Hindered/zone settling : Inter-particle forces are

 Biological treatment have obvious economic advantage, both in terms

 Micro-organisms in sewage treatment plants remove the more

 The costs of waste water treatment can be reduced by the conversion

 Micro-organisms (bacteria, fungi, protozoa etc.) are important in the

►Algae and photosynthetic bacteria utilize energy from

 Three types of processes to represent the conversion of organic

2. Anaerobic oxidation: utilize nitrates, sulphates and other hydrogen

 Methane (CH4) is a source of heat but, if released to the atmosphere

o Growth of a microbial population is defined as an increase

o Growth rate is the increase in microbial cell numbers or

o Microbial populations can grow as:

1. Batch cultures (closed systems) or

2. Continuous cultures (open systems).

The lag phase is a period of cell

2. Continuous Culture of Microorganisms

Physical and Chemical Factors Affecting Microbial

 To introduce WW contact with bacteria (cells) which feed organic

The common methods of biological wastewater treatment are:

63 Figure of Photographic view of trickling filter with its rotary distributors

Figure of Typical section of a conventional circular trickling filter

 Typically wastewater after primary treatment i.e. suspended impurities

 ACTIVATED SLUDGE PROCESS:

 Provides an excellent method of treating sewage.

Units of an Activated Sludge Plant

68 Figure 9: of a conventional AS plant giving high degree of treatment

 Combining different metabolic abilities in the same micro-organism for

 Developing new microbial properties in response to the degradation of

 Currently, some genetically modified bacteria have been approved for

 Soon, many microbiologists and molecular biologists realized the

 Genetically engineered microorganisms (GEMs) have shown potential

 Synthetic gene promoter was created

 The synthetic gene promoter contains

 The reporter gene is transfected into a

 Bacteria: coli form bacteria, salmonella, Legionella, Vibrio, etc.

 Virus: Influenza, hepatitus, polio, etc.

 Algae: dinoflagellates, diatoms, toxic algae, ciguatoxin, etc.

 Multiplex technology is being developed: one run for many

 Collection with minimal amount of samples: water, soil, or air.