Microbiology of potable water

Define Potable water?

Water that is free from disease-producing organisms, poisonous substances, chemical,

biological, and radioactive contaminants which would make it unfit for human
consumption and many other uses is referred to as potable water.

Why isn’t the routine analysis of water directed towards isolation and
identification of specific pathogen?

A number of pathogens are known to be transmitted through water. So, a routine

analysis of water for the presence of these pathogens is required to prevent the spread
of waterborne diseases. This infers that the routine analysis of water would be in search
of pathogenic microorganisms, but that is not the case.

There are several reasons for this.

(i) Pathogens are likely to enter a water supply sporadically and since they may not
survive for long periods of time, they could be missed in a sample submitted to the
laboratory.

(ii) If they are present in very small numbers, pathogens are likely to escape detection
by laboratory procedures.

(iii) Bacteriological analysis of water will take a minimum of 24 hours and within this
time, if pathogens are present, many people would have consumed the water resulting
in the spread of the disease.

Because of the reasons cited above and others, the bacteriological analysis always
includes the search for certain groups of bacteria which are collectively referred to as
indicator organisms.

Define Indicator organisms? write down the criteria for an ideal indicator
organism ?

Indicator organisms: Indicator organisms are those whose presence indicates that the
water is polluted with fecal material from humans or other warm-blooded animals.

There are several criteria for an ideal indicator organism :

(i) It should be one of the intestinal microflora of warmblooded animals

(ii) It should be present when pathogens are present and absent in uncontaminated
samples.

(iii) It should be present in greater numbers than the pathogen (making detection
relatively easy)

(iv) It should be non-pathogenic.

(v) It should be detectable by simple tests.

(vi) It must survive in the environment as long as the potential pathogens.

Define coliforms , fecal coliforms and non-fecal coliforms ?

Coliforms refer to aerobic or facultatively anaerobic , gram-negative, non-spore-forming

rod-shaped bacteria that ferment lactose with the production of acid and gas within 48
hours at 35°C.

The definition includes the genera Escherichia, Citrobacter; Enterobacter and

Klebsiella.

Coliforms that found in the intestines of warm-blooded animals are referred to as fecal
coliforms , while coliforms that occur frequently on grains and plants are referred to as
non-fecal coliforms.

How can you differentiate between coliforms and fecal coliforms ?

Coliforms and fecal coliforms can be differentiated by performing ONPG test and MUG
test. These test requires 24 hours or less to complete.

Medium containing substrate o-nitrophenyl-β-D-galactopyranoside (ONPG) is inoculated

with sample containing coliforms that produce the enzyme β-galactosidase ,which acts
on ONPG and forms a yellow color , indicating their presence in the sample.

Medium containing substrate 4-methylumbelliferyl–β-D-glucoronide (MUG) is inoculated

with sample containing fecal coliforms eg E coli that produce the enzyme β-
glucuronidase ,which acts on MUG to form a fluorescent compound that glows blue when
illuminated by long-wave UV light , indicating their presence in the sample.
How can you distinguish among members of coliform group ????

Biochemical tests are performed to distinguish among members of coliform group

because they closely resemble each other in their morphological and cultural
characteristics. These tests are collectively designated as the IMViC reactions.

(I = indole , M = methyl red, Vi = Voges -Proskauer reaction , and C = citrate)

The reaction for a typical strain of Escherichia and Enterobacter are as follows:

Test Escherichia coli Enterobacter

aerogenes
I. Indole (ability to produce Indole is produced from Not produced (-)
indole) tryptophan (+)
II. Methyl red (amount of acidity Methylred is turned red, which Methylred remains
produced in a special glucose- means a lower pH (> 4.5) is yellow, which means
broth medium and detected by produced(+) less acid is produced
the PH indicator methyl-red) (-)

III. Voges-Proskauer reaction Acetylmethly carbinol is not Acetyl methly carbinol

(ability to produce produced (-) is produced (+)
acetylmethylcarbinol in a glucose
peptone medium)
IV. Citrate (utilization of sodium Citrate as the sole source of Support the growth (+)
citrate as source of carbon) carbon does not support
growth (-)

Bacteriological analysis of water

Drinking water, polluted water from natural sources and treated effluent water have to
be examined regularly for determining their quality. Examination of this sort do provide
information on the status of pollution, type of pollution and its source and type of
treatment required. In addition, it can justify whether the water can be used or not.

Tests for the detection of coliform bacteria

The standard microbiological technique involves three successive steps :

(1) Presumptive test (2) Confirmed test and (3) Completed test.

Presumptive Test

A series of lactose broth tubes are inoculated with measured amounts of water and
incubated at 35°C for 24 to 48 hours.

Gas formation in any one of the tubes within 48 hours indicates a positive presumptive
test. Absence of gas formation at the end of 48 hours constitutes a negative presumptive
test, indicates coliforms are not present.

Confirmed Test

However, it is possible that the formation of gas may not be due to the presence of
coliforms. In order to confirm the presence of coliforms, it is necessary to perform a
confirmed test. Two methods are normally employed.

In one method a drop of culture from a positive lactose broth tube is transferred to
brilliant green lactose bile broth, and is incubated for 24 to 48 hours at 35°C. The
appearance of gas within 48 hrs indicates a positive confirmed test.

In the second method a drop of culture from the positive lactose broth is streaked on a
petriplate containing eosin-methylene blue agar (EMB).

The dye methylene blue in EMB agar inhibits gram positive organisms and allows the
gram-negative coliforms to grow. Coliforms produce colonies with dark centers. E. coli
and E. aerogenes can be distinguished from one another by the size and color of the
colonies. E. coli colonies are small and have a green metallic sheen, whereas E.
aerogenes forms large pinkish colonies.

Completed Test

Isolated colonies from petri plates are transferred into lactose broth and streaked on to
an agar slant. The presence of gas in the fermentation broth and the presence of gram
negative, non-spore forming bacilli on the slant give evidence that coliforms present

Membrane-Filter Technique
Membrane-Filter Technique is widely used technique for bacteriological examination of
water to determine its potability.

In this technique, a thin membrane filter-disc is used. A measured volume of water is

filtered through filter-disc ; the bacteria are retained on the surface of the membrane
filter disk. The disc is then transferred to a Petri dish containing a thin absorbent pad
that has previously been saturated with the appropriate medium. The Petri dish
containing absorbent pad and filter-disc is incubated at 37°C for 18-24 hours. The
medium diffuses through the pores of the filter-disc and provides nutrients to the
bacteria. After incubation, colonies developed on the filter-disc are counted. The
characteristic colonies of different bacteria could now be studied to determine water
potability.

Advantages of Membrane Filtration

Permits testing of large sample volumes.

Reduces preparation time as compared to many traditional methods.

Allows isolation and enumeration of discrete colonies of bacteria.

Provides presence or absence information within 24 hours.

Effective and acceptable technique. Used to monitor drinking water in government

laboratories.

Limitation of Membrane Filtration

Samples, which contain large quantities of colloidal materials or suspended solids such
as iron, manganese, flocs, clay or algae, can clog the pores and prevent filtration.

Nuisance organisms

Aquatic microorganisms which create problems of odor, color and taste or cause
obstruction of water flow are called nuisance microorganisms.

The following are examples of nuisance microorganisms and the undesirable conditions
they cause :

Slime Forming Bacteria – Produce gummy or slimy conditions.

Iron Bacteria - Transform soluble iron compounds to insoluble compounds. Deposition

of insoluble iron compounds reduces the flow of water through pipes.
Sulfur Bacteria - Produce sulfuric acid and hydrogen sulfide ,which can make water
very acidic and impart an unpleasant odor. Sulfuric acid formation leads to corrosion of
pipes.

Algae – Produce turbidity, discoloration and unpleasant odor and taste.

Describe briefly water treatment process

Water treatment describes those processes used to make water more acceptable for a
desired end-use. The process includes

Settling :

Raw water collected from the source held in the reservoir to allow particles to settle.

Flocculation :

Water is then transferred to another tank to remove colloidal materials such as clay.
Aluminum potassium sulfate (alum) is the widely used flocculant in water treatment that
forms aggregations of fine suspended particles called floc. As these aggregations slowly
settle out ,they entrap colloidal material and carry it to the bottom.

Filtration :

After flocculation ,the water is treated by filtration that is passing it through beds of 2-4
feet of fine sand. Some protozoan cysts and oocysts are removed from water only by
such filtration treatment. The microorganisms are trapped mostly by surface adsorption
onto the sand particles. They do not penetrate the tortuous routing between the particles
,even though the openings might be larger than the microbes that are filtered out. These
filters are periodically backflushed to clear them of accumulations.

Disinfection :

Disinfection is the final process to which water is subjected prior to distribution. Common
methods of disinfection include Chlorination , UV Radiation and Ozonation

Chlorination

Chlorination is the process of adding the element chlorine to water as a method of

water purification to make it fit for human consumption as drinking water.

Disinfection is commonly accomplished by chlorination ,using either chlorine gas (Cl 2) or

hypochlorite (Ca[OCl]2) or NaOCl. Chlorine gas reacts with water to yield hypochlorous
acids
Cl2 + H2O → HOCl + HCl

Hypochlorite also reacts with water to form hypochlorous acid

Ca(OCl)2 + 2H2O → 2HOCl + Ca(OH)2 Or

NaOCl + H2O →HOCl + NaOH

The hypochlorous acid or the hypochlorite (ClO -) ion is the actual disinfectant. The total
concentration of hypochlorous acid and hypochlorite is designated as free residual
chlorine. Hypochlorite acts as a strong oxidant.

Advantages :

Inexpensive

I. Quick in action.
II. Residual Cl - can prevent further contamination

Disadvantages :

I. Produce off odor

II. Generation of chlorinated-organic compounds e.g. THM compounds that may be
carcinogenic or harmful to the environment.

Disinfection by UV Radiation

Disinfection by UV radiation involves exposing treated water to radiation from UV

light. Because no chemicals are used, the treated water has no adverse effect on
organisms that later consume it, as may be the case with other methods. UV radiation
causes damage to the genetic structure of bacteria , viruses, and other pathogens ,
making them incapable of reproduction.

Advantages :

I. Leaves no taste and odor

II. Quick in action

Has no known and toxic or significant nontoxic byproducts

Disadvantages :

I. Expensive
II. Low penetrating power of UV radiation
III. No disinfection residual
IV. Need for frequent lamp maintenance and replacement
V. Need for a highly treated effluent to ensure that the target microorganisms are
not shielded from the UV radiation (i.e., any solids present in the treated effluent
may protect microorganisms from the UV light).

Ozonation

Ozone (O3) is generated by passing oxygen (O2) through a high voltage potential
resulting in a third oxygen atom becoming attached and forming O3. Ozone is very
unstable and reactive and oxidizes most organic material it comes in contact with,
thereby destroying many pathogenic microorganisms. Ozonation destroys
Cryptosporidium, bacteria and other naturally-occurring organisms reliably.

Advantages :

I. Leaves no taste and odor

II. Reduce the formation of trihalomethanes (THMs)
III. Water treated with ozone is prone to chance recontamination due to its less
residual effect

Disadvantages :

I. Expensive
II. Skilled labor is required
Measurement of wastewater treatment efficiency

The purpose of the wastewater treatment is to

i) separate waste from water for disposal elsewhere

ii) produce an effluent which can be discharged to a receiving water body without
causing pollutions

iii) remove possible chemical and biological constituents eg – typhoid bacteria

,rotavirus ,ammonia ,cyanide ,carbohydrates etc.

Measurement of wastewater treatment efficiency is required to

i) ascertain the strength of the influent and

ii) the effluent discharged to the receiving water

Parameters for the water pollution evaluation

Parameter Pollution effect Measurement

Pathogenic Water is unsuitable for Microbiological
microorganisms human consumption or
recreation
Indicator organisms Indication for fecal Microbiological
contamination ,possible
presence of pathogen
Biodegradable organic Depletion of dissolved Microbiological ,Chemical
compounds oxygen in the receiving ,COD ,BOD ,TOC
waters
Plant nutrients (N and P) Stimulation of the growth of Microbiological ,algal growth
algae and aquatic plants assay
Toxic materials Killing of aquatic life Microbiological ,Chemical
,interference with growth or ,Biological
reproduction

Parameter evaluation :

The two objectives of waste water treatment ,separating waste from waste water and
preventing water pollution of the receiving wastes are evaluated differently.

Treatment efficiency depends on the extent to which specific waste materials are
separated from the wastewater and removal efficiencies can be calculated for no of
different components. The term efficiency is used to express information about the
fraction of the waste material originally present in the waste water that has been
separated during treatment.

Most often removal efficiency is expressed as a percentage –

Percentage efficiency = 100 ×

= Concentration of waste material in the influent & = Concentration of waste

material in the efffluent

The amount of waste material separated from the water is given by –

Waste load reduction = ( ) (Q) (f) Where

Q = waste water flow rate and

f = unit conversion factor ,ie when concentration is expressed mg/L and flow in a cubic
meters/day and f of 0.001 kg/g is used to give the waste load reduction in kg/day

Biochemical Oxygen Demand

Biochemical oxygen demand or BOD is a chemical procedure for determining the

amount of dissolved oxygen needed by aerobic biological organisms in a body of water to
break down organic material present in a given water sample at certain temperature
over a specific time period

BOD measurement involves use of special bottles with airtight stoppers. Each bottle is
first filled with test water or dilutions. The filled bottles are incubated in the dark for 5
days at 20°C, and the decrease in dissolved oxygen is determined. The more oxygen
that is used up as the bacteria degrade the organic matter in the sample, the greater the
BOD, which is usually expressed in milligrams of oxygen per liter of water.

The BOD of the undiluted waste water can be calculated by the following equation –

BOD5 of waste water = BOD5 of diluted sample × dilution factor

In order to provide safety , the residual dissolved oxygen in the BOD bottle after 5 days
of incubation should be at least 2.0 mg/L.

Chemical Oxygen demand (COD) :

Chemical oxygen demand is another means of measuring the pollutional strength of
wastewater. Organic compounds are oxidized by dichromate ions in the presence of acid
, heat or catalyst. It measures the O 2 equivalent in both waste organic matter and cells
of microbes. The chemical oxygen demand test has a major advantage over the
biochemical oxygen demand analysis because of the short time required for performance

Total organic carbon (TOC) :

Total organic carbon measurements have been used as a method for determining
pollutional levels of wastewater. It measures both biodegradable and non biodegradable
compounds. This test is a direct measure of organic content.

Algal growth test :

An algal growth test has been developed to measure the relative potential of an effluent
to support the growth of algae. The test is based on Liebig’s law of minimum ,which
states that the maximum yield is proportional to the concentration of the nutrient which
is present and biologically available in the lowest concentration in relation to the growth
requirements of the organism.

The EPA employs – Selenastrum capricornutum

i) In this test ,simply algal growth is measured. So ,the sample must be filtered to
remove indigenous algae. For nutrient study ,the sample is autoclaved and then filtered.

ii) Disodium EDTA is added as chelating agent (1 mg/L).It removes Fe ,Ca ,Mg and other
heavy metals that are toxic to the algae.

iii) Then N,P or wastewater is added to the sample

iv) Incubation period is 14 days.

v) Biologically available Na and P are determined from the dry weight of the algal mass.

vi) Each µg of P will support 0.430 mg of S capricornutum.Each µg of N will support

0.038 mg dry weight of S capricornutum

Those water containing greater than 0.015 mg of biologically available.P and 0.165 mg
of biologically available N per liter are considered to be eutrophic.

Uses :

i) To determine the reduction of N and P that is necessary to protect the water quality.
ii) To measure the biologically available N and P in the plant effluent as well as assess
the trophic status of the receiving water body.

Bioassays using bacteria :

Several bacterial assays available for determining the toxicity of environmental sample.
Toxicity test using bacteria have been developed based on –

- growth inhibition

- oxygen uptake

- colony formation

- motility

-bioluminescence

The most widely used test using microorganisms are

(a) The Microtox test

(b) Met PAD test

(c) Met PLATE test

The Microtox tests : There are three versions of the Microtox assays –

(i) An acute test

(ii) A chronic test

(iii) A mutagenicity test

Based on bioluminescence :

The acute Microtox assay :

This assay uses freeze dried cultures of the bioluminescent marine bacterium Vibrio
fischeri which emits light as an end product of its respiration.

The test is based on the inhibition of Vibrio fischeri by toxic chemicals. It has been
used for determining the toxicity of wastewater effluent
The lyophilized bacteria are rehydrated with microtox reconstitution solution to produce
a ready-to-use cell suspension.

In the assay ,this test organisms are exposed to a test sample to measure the toxic
effect of the sample.

The light output of the luminescent bacteria is measured to before and the after they are
exposed to a toxic sample.

The light of a control that contains no sample is used to determine lost light. The
luminescent bacteria is exposed wastewater sample for 30 min to perform this assay.

Met PAD :

MetPAD heavy metal toxicity testing kit is specific for heavy metal toxicity and is based
on enzyme inhibition in a bacterial strain by bioavailable heavy metals in aqueous
samples. Bacterial response to a toxic sample is conveniently observed on the test pad.
The intensity of the purple color on the pad is inversely proportional to the sample
toxicity.

The MetPAD kit includes freeze dried E coli ,diluent ,phosphate buffer and three petri
dishes – each containing a yellow filter pad saturated with substrate for the enzyme β
galactosidase.

To perform this assay ,the bacterial reagent is rehydrated and mixed with the sample.

At the end of a 90 min exposure period ,a drop of the suspension is placed on the assay
pad to measure enzyme activity.

A purple color gives indication of in galactosidase activity and the intensity of the other is
inversely proportional to the sample toxicity.

Color intensity for a given sample is compared to that of a control prepared by

suspending E coli in the diluent.

Met PLATE :

The MetPLATE kit is specific for heavy metal toxicity. The MetPLATE kit is based on
enzyme inhibition in a bacterial strain by bioavailable heavy metals in aqueous samples.
Bacterial response to a toxic sample is conveniently observed on the 96-well microplate.
The intensity of the purple color is inversely proportional to the sample toxicity.
Absorbance is measured at 575 nm using a microplate reader.
There are also specific biosensors that can detect the presence of specific metals in
environmental samples.These biosensors are constructed by fusing an inducible
promoter from a metal resistance operon to a reporter gene that codes for the
production of bioluminescence or a specific enzyme. Mercury and arsenite can be
detected by such biosensors.

The ATP-TOX assay :

This assay is based on both the growth inhibition and ATP measurement of E coli and
inhibition of luciferase activity.

Toxi-ChromoTest

The Toxi-ChromoTest procedure exposes the bacteria to the toxicants in the sample for a
short (90-minute) incubation period. After the incubation period, a chromogenic
substrate is added. If the sample is toxic, no color will develop and if the sample is non-
toxic, a distinctive blue colour quickly develops.

Toxicity testing based on growth inhibition

Toxicity testing may also be based on growth inhibition of pure or mixed cultures or
waste water microorganisms. The assays consist of determining changes in bacterial
densities via measurement of the optical density of the bacterial suspension ,determining
inhibition zones on solid growth media or by ATP measurement.

Toxicity testing based on effect on Nutrient cycle :

Toxic chemicals may exert an adverse effect on Nutrient cycling by microorganism.

Nitrification is probably the most sensitive step to environmental toxicants. Toxcity
assays based on the inhibition of both Nitrosomonas and Nitrobacter are developed to
determine the toxicity in waste water samples.

The impact of toxic chemicals on the carbon is conveniently determined by measuring

the inhibition of microbial respiration ,which can be measured with oxygen electrodes
,manometers or electrolytic respirometers.

Use of algae :

Owing to their role as primary producers and crucial components of the food chain ,micro
algae (eg selenastrum capricornutrum.) are useful indicator microorganism to assess the
eutrophication potential of surface waters.
Microorganisms and some novel pollution problem

Biodegradation is the process of chemical breakdown of a substance to smaller

products by the act of microorganisms or their enzymes. Biodegradation is often used
interchangeably with “mineralization”, but, in fact, mineralization represents the
breakdown of organic materials into inorganic forms brought about mainly by
microorganisms.

Bioremediation is the removal or detoxification of man-made xenobiotic compounds

using microorganisms.

Principle of microbial infallibility

Microorganism has the capability of degrading all naturally occurring compounds; this is
known as the principle of microbial infallibility proposed by Alexander in 1965.
Microorganisms are also able to degrade many of the xenobiotic compounds, but they
are unable to degrade many others.

Xenobiotics are synthetic (man-made) organic compounds that are foreign to existing
biological system.

The compounds that resist biodegradation and thereby persist in the environment are
called recalcitrant.

The xenobiotic compounds may be recalcitrant due to one or more of the following
reasons:

(i) they are not recognised as substrate by the existing degradative enzymes,

(ii) they are highly stable, i.e., chemically and biologically inert due to the presence of
substitution groups like halogens, nitro-, sulphonate, amino-, methoxy- and carbamyl
groups,

(iii) they are insoluble in water, or are adsorbed to external matrices like soil,

(iv) they are highly toxic or give rise to toxic products due to microbial activity,
(v) their large molecular size prevents entry into microbial cells,

(vi) inability of the compounds to induce the synthesis of degrading enzymes, and

(vii) lack of the permease needed for their transport into the microbial cells.

The structural features that make these compounds resistant to microbial degradation
include the following:

(i) Presence of halogens in the place of hydrogen in the molecule; the carbon-halogen
bond is highly stable and its cleavage requires considerable energy.

(ii) Substitution of -H by other groups like nitro-, sulphonate, methoxy-, amino- and
carbamyl groups

(iii) Cyclic structures, aromatic compounds, cycloalkanes and heterocyclic compounds

are more recalcitrant than linear chain or aliphatic compounds

(iv) Branched linear chains resist biodegradation etc. In general, the more complex is
the structure of axenobiotic compound, the more resistant it is to biodegradation. Many
other xenobiotics resist biodegradation due to their large molecular size and insolubility
in water.

Biomagnification :

Biomagnification is a process in which chemical substances become more concentrated

at each higher trophic level. The figure shows how DDT becomes concentrated in the
tissues of organisms representing four successive trophic levels in a food chain.

Spraying a marsh to control mosquitoes will cause trace amounts of DDT to accumulate
in the cells of microscopic aquatic organisms, the plankton, in the marsh.

In feeding on the plankton, filter-feeders, like clams and some fish, harvest DDT as well
as food. (Concentrations of DDT 10 times greater than those in the plankton have been
measured in clams.)

The process of concentration goes right on up the food chain from one trophic level to
the next. Gulls, which feed on clams, may accumulate DDT to 40 or more times the
concentration in their prey. This represents a 400-fold increase in concentration along
the length of this short food chain.

Some carnivores at the ends of longer food chains (ospreys, pelicans, falcons, and
eagles) suffered serious declines in reproduction because of biomagnification.
Transformation of DDT :

Recalcitrant halocarbons :

Many xenobiotic pollutants that have proven recalcitrant to microbial attack are
halocarbons. The carbon halogen bond is highly stable and its cleavage requires
considerable energy , hence , halocarbons are chemically and biologically inert.

Important groups of halocarbons include the C1-C2 haloalkyl propellants ,solvents and
refrigerants ,haloaromatics such as chlorobenzenes ,chlorophenols and
chlorobenzoates ,polychlorinated or polybrominated biphenyls and triphenyls
,chlorodibenzodioxins and chlorodibenzofurans.Some organochlorine insecticides are also
highly recalcitrant.

Haloalkyl Propellants and Solvents :

Haloalkyl propellants and solvents are C1-C2 alkanes in which all or nearly all hydrogen
atoms have been replaced by fluorine-chlorine combinations. They serve as solvents and
aerosol propellants in spray cans for such things as cosmetics ,paint ,and insecticides
and as the working fluid in the condenser units of air conditioners and refrigerators. Most
commonly used have been the CCl 3F and CCl2F2 freons ,designated by the codes F-11
and F-12 ,respectively. Dichloromethane ,chloroform ,carbon tetrachloride ,dichloro
,trichloro and tetrachloroethenes are important industrial and cleaning solvents. Spilled
halocarbon propellants and solvents have seriously contaminated groundwater. Being
inert and quite volatile ,some of these components rise to the stratosphere upon
release ,depleting Earths protective ozone layer by photo chemical interactions. Partial
destruction of the ozone layer would result in increased UV radiation on the surface of
Earth ,causing an increased incidence of skin cancer and mutagenesis.

Intensive research efforts are under way to find a suitable substitute for halocarbons as
refrigerators. A new generation of refrigerants is gradually replacing the freons in
refrigeration and air conditioning units. These C2 based halocarbons are not
biodegradable but they contain chlorine rather than fluorine substituents. They are
photochemically less destructive for the ozone layer than the classic freons.

Dehalogenation of organic compounds is favored under anaerobic conditions ;

microorganisms have been found that produce dehalogenases and carry out reductive
dehalogenation.

Anaerobic dechlorination of PCE :

Sulfate-reducing bacteria transform tetrachloroethene (perchloroethene ,PCE) to

trichloroethene and dichloroethene by anaerobic dehalogenation. Dichloroethene is
further dechlorinated to the toxic product vinyl chloride which then yields ethene. Such
PCE degradation has been demonstrated in a methanogenic bacterial consortium
growing on acetate.
Fig : Anaerobic dechlorination of PCE by methanogenic consortia in the presence of
acetate

Aerobic degradation of TCE :

Aerobic degradation of trichloroethene (TCE) ,a widely distributed halocarbon

pollutant ,by a methane-utlizing microbial consortium has been demonstrated. TCE
converts to TCE epoxide by methane monooxygenase ,which subsequently hydrolyzes to
polar products (formate ,glyoxylate ,and dichloroacetate ) utilizable by
microorganisms.As a curious side product ,small amounts of chloral (Cl 3C . CHO) are also
formed.

Fig : Aerobic cometabolism of TCE by methanotrophic microorganisms

TCE is also attacked by toluene dioxygenase of Pseudomonas. The described two mono
and dioxygenase systems fail to cometabolize PCE. Methylcoccus capsulatus was
reported to convert chloro and bromomethane to formaldehyde ,dichloromethane to CO
and trichloromethane to CO2 while growing on methane.

Thus , methanotrophic bacteria show some promise for bioremediation of halocarbon-

polluted aquifers.

Halobenzenes ,Halophenolsand Halobenzoates :

Chlorobenzenes are extensively used as industrial solvents. With chlorobenzenes and

other haloaromatics ,it is a common feature that their aerobic biodegradability decreases
with the number of halosubstituents ,but the same halogenated aromatics are
dechlorinated under anaerobic conditions with relative ease (easily). As the
halosubstituents are sequentially removed ,the products are less and less likely to be
dehalogenated further and monochlorobenzene is not dechlorinated anaerobically.
Hexachlorobenzene is dechlorinated rapidly to 1,3,5-trichlorobenzene in anaerobic
sewage sludge ,but further dechlorinaiton to 1,3-dichloro- and monochlorobenzenes is
slow and incomplete. In contrast ,mono and dichlorobenzenes are degraded aerobically
with relative ease of by various Pseudomonas and Alcaligenes strains .The aerobic
biodegradation of trichloro – and tetrachlorobenzenes is more difficult ,but some
Pesudomonas strains with such capabilities were isolated.

Chlorophenols ,particularly the highly chlorinated pentachlorophenol (PCP) ,are used as

preservatives for wood and canvas. Other chlorophenols are used in the synthesis of
pesticides, resins and dyes. These products are toxic to microorganisms due to difficulty
to degrade. Even ,in appropriately low concentrations ,various microorganisms are
capable of degrading chlorophenols under both aerobic and anaerobic conditions.

Under aerobic condition , PCP is converted to tetrachlorohydroquinone by a

monooxygenase. A Flavaobacterium sp and Mycobacterium chlorophenolicum
were described to catalyze this reaction. Tetrachlorohydroquinone is stepwise
dechlorinated to 2,6-dichlorohydroquinone which then yields CO2 and H2O.

Under anaerobic condition ,PCP is reductively dechlorinated stepwise to phenol. Phenol

can be metabolized further to methane and CO2.

Recalcitrant Nitroaromatic Compounds :

Nitaromatics are used as explosives such as trinitrotoluene (TNT) , solvents such as

nitrobenzene and pesticides ,such as nitropehnols. Their manufacture and disposal left
many sites polluted. The toxicity of these residues are high. Similar to halogen
substituents ,nitro substitution of aromatic rings are has a strong negative effect on
biodegradability. Both halogen and nitro substituents are electron withdrawing and tend
to deactivate the aromatic ring. Compounds with several nitro-susbtituents tend to be
recalcitrant. The biodegradation of nitroaromatic compounds tends to be slow and often
leads to bound residues in soils and sediments. Similarly to chloroaromatics ,extensively
nitro-substituted aromatics are more easily transformed under reductive than under
oxidative conditions. As an example of a monoaromatic compound ,the oxidative and the
reductive pathways of nitrobenzene biodegradation are shown in figure.In the oxidative
pathway ,nitrobenzene converts to nitrobenezenediol by a dioxygenase and NO2 - is
spontaneously released during arrangement to catechol. Subsequently ,the catechol ring
is opened by meta cleavage. In the reductive pathway ,the nitro group is stepwise
reduced through nitrosobenzene to phenylhydroxylamine. The latter undergoes
rearrangement by a mutase to 2-aminophenol.The 2-aminopehnol ring is subsequently
oxidatively opened and the nitrogen is eliminated as ammonium during conversion to
oxalocrotonate.
Fig : Oxidative and reductive pathways of nitrobenzene biodegradation ,catalyzed by
Comamonas sp and Pseudomoans pseudocaligenes ,respectively.

Under anaerobic and microaerophilic conditions , the nitro groups of TNT are reduced
one by one to amino groups ,but each subsequent reduction is slower and less complete.
The first reduction may be catalyzed even by aerobic microbes , second by facultative
anaerobes and the last reduction by strict anaerobes. The reduction sequence does not
lead to mineralization but rather to polymerization or binding of the residues.

Polychlorinated biphenyls (PCBs)

Polychlorinated biphenyls are a group of manufactured organic compounds with 1 to 10

chlorine atoms attached to biphenyl, which is a molecule composed of two benzene
rings. Chlorine atoms may be present at some or all of the 10 possible positions which
are numbered 2–6 on one ring, and 2'–6' on the other ring
Fig : Chemical structure of PCBs.

Properties

Polychlorinated biphenyls (PCBs) are oily liquids, clear to light-yellow in color, with no
smell or taste. They have low vapor pressure , high boiling point , low solubilities in
water ,good solubilities in most organic solvents , oils and fats , low electrical
conductivity etc. Because of these properties ,they have been used as plasticizers in
polyvinyl polymers , as insulators-coolants in transformers and capacitors and as heat-
exchange fluids in general. Minor uses involve adhesives , flame retardants , paints ,
sealants , fixatives in microscopy (such as oil immersion) , inks , surface coatings etc.

As the degree of chlorination increases, melting point and lipophilicity increase, but
vapor pressure and water solubility decrease.

Production

The commercial production of PCBs started in 1929 by Montesano (Company) in the

United States but their use has been banned or severely restricted in many countries
since the 1970s and 80s because of the possible risks to human health and the
environment.

Sources of PCB pollution:

- Landfills containing transformers, capacitors, and other PCB waste can release PCBs
into the air.

- The incineration of municipal waste may lead to PCB pollution and produce dangerous
by-products, such as hydrogen chloride and dioxins (PCDDs and PCDFs).

- PCBs can also evaporate from contaminated water bodies, such as the North
American Great Lakes.

Effects :

Concentrations of PCB over 1 ppm have been detected in one-third of the sampled U.S
population. In some fresh water habitats ,older individuals of predatory fish ,such as
trout and salmon ,accumulate 20-30 ppm of PCBs and predatory and fish eating birds
were found to contain several hundred ppm of PCBs. In 1968 , PCB contaminated
cooking oil poisoned nearly a thousand people in japan ; the main symptoms liver
damage and a severe skin condition called chloracne . In this case , the victims had
consumed several grams of the substance. Low PCB intakes from general environmental
contaminations are not known to harm humans or most other mammals ,but along with
chlorinated hydrocarbon insecticides ,PCBs were implicated in eggshell thinning and
reproductive failure of some predatory and fish eating birds. Besides having acute effects
,PCBs and some other chlorinated hydrocarbons ,including DDT – are under suspicion of
being potential human carcinogens ,although this point is still controversial.

Although PCBs are relatively resistant to biodegradation ,a number of microorganisms

have been isolated that transform them. Aerobic degradation of PCBs is carried out by
the white rot fungus Phanerochaete ,by Acinetobacter and by Alcaligenes and
anaerobically by reductive dechlorination. Degradation of PCBs typically is by
cometabolism and is enhanced by the addition of less chlorinated analogs such as
dichlorobiphenyl. The more extensively chlorinated PCBs are more likely to be
chlorinated than the less chlorinated ones. Meta and para positions are dechlorinated in
preference to ortho positions.

Frequent contaminants of PCBs are 2,3,7,8-tetrachlorodibendioxin (TCDD) and 2,3,7,8-

tetrachlorodibenofuran . Some of these contaminants arise during manufacture ,others
are formed through thermal degradation of the chlorophenols and chlorobiphenyls. These
and related chlorodibenzodioxins and chlorodibenzofurans are extremely toxic and highly
resistant to biodegradation.

2,3,7,8 TCDF 2,3,7,8 TCDD

Polybrominated biphenyls (PBBs) ,which are used as flame retardants and

polychlorinated terphenyls (PCTs) ,which have three attached phenyl rings and are
employed as plasticizers ,have pollution potentials similar to PCBs.They have been
manufactured on a much smaller scale than PCBs.

Decabromobiphenyl Tetradecachloroterphenyl

Synthetic Polymers :
Synthetic polymers are often referred to as "plastics" . They are easily molded into
complex shapes, have high chemical resistance, and are more or less elastic. These
properties have made them popular in the manufacture of garments, durable and
disposable goods, and packaging materials. It is estimated that over 90% of the plastic
materials, mainly disposable goods and packaging materials, consist mainly of
polyethylene, polyvinyl chloride and polystyrene.These materials have molecular weights
ranging from several thousand to 150,000 and appear to resist biodegradation
indefinitely.

Some reports were made earlier that the plastics are biodegradable but slowly. This was
based primarily on the fact that the plastics become brittle after some time. But, the
closer scrutiny reveals that this is because of the degradation of the plasticizers not the
basic polymer. Plasticizers are esters of long chain fatty acids and alcohols and are, in
fact, the additives rendering flexibility to plastics. Plasticizers biodegradation makes the
plastic brittle, but the polymer structure of the plastic remains unaffected.

Resistance to biodegradation seems to be associated with their excessive molecular size. If their
molecular size is reduced considerably in short polymer chain fragments. e.g., by pyrolysis , the
fragments will become biodegradable.

Polyethylene

Polyvenyl Chloride

Polystyrene

A new agricultural practice involving plastics ,the so called film-mulching technique

lends special urgency to the development of biodegradable plastic film material. This
practice involves covering fields with plastic film in order to control weeds and conserve
moisture. It is impractical to re-use or even to collect the film at the end of the growing
season ; a large accumulation of plastic fragments in the field is equally undesirable.
Because polyethylene , polybutene and other polyolefins are susceptible to
photochemical degradation ,it is possible to devise (prepare) materials that sustain (add)
sufficient photochemical damage during a growing season to become susceptible to
subsequent microbial degradation.
Truly biodegradable poly-β-hydroxyalkanoates are produced by Alcaligenes eutrophus
,Bacillus ceresus and various Pseudomonads. These intracellular storage products have
thermoplastic properties and can be molded. Their properties can be influenced by the
substrate fed to the bacteria. A β-hydroxybutyric and β-hydroxyvaleric acid copolymer
gives the best properties. Up to 50% of the cell dry weight is β-hydroxyalkanoate under
the proper growth conditions. But the price of this product is 5 to 7 times higher than
that of petrochemical-based polyethylene. Recently ,some shampoo bottles and
disposable diapers were also manufactured from this material. The synthetic
caprolactone polyester that contains hydrolyzable bonds is also a good candidate as
biodegradable plastic.

Alkyl benzyl sulfonates :

Alkyl Benzyl sulfonates are the major components of laundry detergents. Like other
surface active agents ,the molecule has a polar (sulfonate) and a nonpolar (alkyl) end.
Cleaning ,occurs when these molecules form a monolayer around lipophilic droplets or
particles. The molecules orient with their non polar end toward the lipophilic substance
and their sulfonate end toward the surrounding water. Non linear alkyl benzyl sulfonates
(ABS) are resistant to biodegradation and cause extensive foaming of rivers that receive
ABS-containing wastes. The methyl branching of the alkyl chain interferes with
biodegradation by blocking the normal  -oxidation sequence ..The ABS-LAS story is
particularly important because it is the first instance in which a synthetic compound's
structure is altered to avoid recalcitrance while essentially preserving the other useful
characteristics of the compound.
 Hazardous wastes

A hazardous waste is waste that poses substantial or potential threats to public health
or the environment

Hazardous wastes include:

I. Household hazardous waste

II. Agricultural wastes which are returned to the ground as fertilizer;
III. Mining overburden returned to the mine site;
IV. Utility wastes from [coal] combustion to produce electricity;
V. Oil and natural gas exploration drilling waste;
VI. Wastes from the extraction of beneficiation, and processing of ores and minerals,
including coal
VII. Cement kiln wastes;
VIII. Wood treated with arsenic preservatives.
IX. Certain chromium-containing wastes
X. Recycled hazardous wastes
 Household Hazardous Waste (HHW) is waste that is generated from residential
households.

Household Hazardous Waste (HHW) includes

I. Paints and solvents

II. Automotive wastes (used motor oil, antifreeze, etc.)

III. Pesticides (insecticides, herbicides, fungicides, etc.)

IV. Mercury-containing wastes (thermometers, switches, fluorescent lighting, etc)

V. Electronics (computers, televisions, cell phones)

VI. Aerosols / Propane cylinders

VII. Caustics / Cleaning agents

VIII. Refrigerant-containing appliances

IX. Batteries

X. Ammunition

XI. Radioactive waste

Properties of hazardous waste :

1. Explosive : Substances and preparations which may explode under the effect of
flame or which are more sensitive to shocks or friction than dinitrobenzene.

2. Oxidizing : Substances and preparations which exhibit highly exothermic reactions

when in contact with other substances ,particularly flammable substances.

3. Highly flammable : Liquid substances and preparations having a flash point below
21 C.

4. Flammable : Liquid substances and preparations having a flash point equal to or

greater than 21 C and less than or equal to 55 C.

5. Irritant : Non corrosive substances that upon contact can cause inflammation with
the skin.

6. Harmful : Substances and preparations which , if they are inhaled ,ingested or

penetrate the skin cause limited health hazard.
7. Toxic : : Substances and preparations which , if they are inhaled , ingested or
penetrated can lead serious , acute or chronic health risk and even death.

8. Carcinogenic : Substances that may induce cancer or increase its incidence.

9. Corrosive : Substances that may destroy any living tissue on contact.

10. Infectious : Substances containing viable microorganism or their toxin which may
cause disease in man or other living organisms.

11. Tetratogenic : Substances that may induce non hereditary congenital

malformations or increase their incidence.

12. Mutagenic : Substances that may induce hereditary genetic defects or increase
their incidence.

13. Substances which release toxic or very toxic gases in contact with water ,air or an
acid.

14. Ecotoxic : Substances that present or may present immediate or delayed risk for
one or more sectors of the environment.

15. Substances capable by any means after disposal of yielding another substance eg –
a leachate

Solid waste treatment technologies

There are two principle ways to treat the solid wastes : Landfills and Composting

Landfills :

Landfills represent a simple means of solid waste disposal that relies on anaerobic
microbial processes to decompose the wastes. In this procedure solid wastes ,both
organic and inorganic ,are deposited in low lying and hence low-value land.

To avoid, foul odor and attraction of insects and rodents, each day's waste deposit is
covered over with a layer of soil, creating a sanitary landfill.

For 30-50 years, the organic matter undergoes slow, anaerobic microbial decomposition
and the products which result include CO 2, H2O, CH4, low mol. wt. alcohols, and acids
which diffuse in the surrounding air and water.
In the construction of a landfill ,design is important to prevent anaerobic decomposition
products ,heavy metals and a variety of hazardous pollutants from seeping out of the
landfill site into underground aquifers , polluting much-needed urban water resources.
Some modern landfills are designed to collect the methane from anaerobic microbial
decomposition so that it can be used as a fuel. Porous pipes extend into the landfill to
the methane gas. A plastic liner and sealed layers prevent diffusion of methane out of
the landfill. Another way is to place an impermeable clay cap on the landfill ,thus
preventing water infiltration and the leaching of pollutants.

Advantages :

i. Safe way for the disposal of solids wastes include diapers and others materials
contaminated with human fecal material

ii. Developed landfill can be used for construction and recreation purposes

iii. Formation of methane which can be used as a fuel.

Disadvantages :

i. The limited number of suitable disposal sites available in urban areas are rapidly
becoming filled , necessitating longer hauling of solid waste to more distant sites

ii. Premature construction of landfills may result in structural damage to the buildings
due to subsidence and in an explosion hazard due to methane seeping into basements.

iii. Methane seepage may also damage plantings on the disposal site

iv. Gas collected from landfills contains high amounts of N 2 ,CO2 and H2S impurities
along with CH4 and so requires purification prior to use.

Composting :

Composting is a microbial process that converts putrefiable organic waste materials into
a stable ,sanitary ,humus like product. The various composting methods are used. These
include the following:

(i) Static piles :

The static pile process is simple but relatively slow ,typically requiring many months for
stabilization. Under favorable conditions ,self heating in static piles typically raises the
temp inside a compost pile to 55-60 C or above in 2-3 days. O 2 concentrations in the
compost are usually 5 times lower than in ambient air.
(ii) Aerated pile :

The aerated pile process achieve substantially faster composting rates through improved
aeration. Inside the pile the temperatures rise to self-limiting levels of 70-80 C .The
heat generated by the biodegradation process is effectively used in evaporating water
and results in a dryer and more stable compost. The aerated pile process goes to
completion in about 3 weeks.

(iii) Continuous feed reactors :

It uses a reactor that permits control of the environmental parameters. The reactor is
maintained at thermophilic temperature .The heat generates due to higher respiration
activity and may rise up to 80 C. Here composting is complete in 2-4 days. But this
method is expensive.

Fig : Temperature profile of a static in compost pile

Fig : Concentration of O2 in a compost pile

The composting process initiated by mesophiles. As the temp rises these are replaced by
thermophiles Thermophilic bacteria prominent in the composting process are Bacillus
stearothermophilus , Thermomonospora , Thermoactinomycetes , Clostridium
thermocellum . Important fungi are Geotrichum candidum , Aspergillus fumigatus ,
Mucor pusilus and Torula thermophila

For optimal composting ,several conditions are critical. Adequate moisture (50-60%
water content) must be present ,but excess moisture (70 %or above) should be avoided
as it interferes with aeration and lowers self-heating. Carbon to nitrogen ratios should
not be greater than 40 : 1 .Lower nitrogen content does not permit the formation of a
sufficient microbial biomass. Excessive nitrogen C :N = 25 : 1) leads to volatilization of
ammonia causing odor problems and lowers the fertilizer value of the resulting compost.

Compost is a good soil conditioner and supplies some plant nutrients. But before using
as fertilizer the compost must be tested for the presence of heavy metals.

Composting can also be accomplished anaerobically using the DRANCO (DRy ANaerobic
Conversion) process. The DRANCO process is carried out at thermophilic temp in
specially constructed airtight reactors. The reactor is charged from the top with 35-40 %
solid organic wastes is preheated to 50 C by steam and moves passively through the
verticle cylinder of the reactor. Time required for total mineralization is 18-21 days.
Steam is injected to increase the temp. About 30% of the generated biogas is used to
heat the DRANCO vessel and 70 % is used to produce electricity. The discharged and
stabilized waste is called humotate used for gardening purpose.

Sewage treatment

Sewage treatment is the process of removing contaminants from wastewater and

household sewage.

Its objective is to produce a treated effluent and a solid waste or sludge suitable for
discharge or reuse back into the environment.

Sewage treatment generally involves three stages, called primary, secondary and
tertiary treatment.

Primary Treatment

This removes only suspended solids in settling tanks or basins. Solids are drawn off from
the bottom. They may be subjected to anaerobic digestion or composting prior to final
disposal in landfills or as soil conditioner

The liquid portion of the sewage containing dissolved organic matter is subjected to
secondary treatment or is discharged after primary treatment only. Liquid wastes vary in
composition and if they contain mainly solids and little dissolved organic matter ,primary
treatment may remove 70-80 % of the BOD and may be considered adequate. For
typical domestic sewage (table) , however , primary treatment removes only 30-40 % of
the BOD and secondary treatment is necessary for acceptable BOD reduction.

Table : Characteristics of typical municipal wastewater

Particles Concentration (mg/L)

Solids ,total 700
Dissolved 500
i. Fixed 300
ii. Volatile 200
Suspended 200
i. Fixed 50
ii. Volatile 150
Ultimate biochemical O2 300
demand
Total organic carbon 200
(TOC)
Chemical Oxygen 400
demand
N2 40
Organic 15
Free ammonia 25
Nitrites 0
Nitrates 0
Phosphorous 10
i. Organic 3
ii .Inorganic 7
Grease 100

Secondary treatment

Secondary treatment is designed to substantially degrade the biological content of the

sewage which are derived from human waste, food waste, soaps and detergent.
Secondary treatment systems are classified as fixed-film or suspended-growth systems.

Fixed-film systems include trickling filter system and rotating biological contactor
,where the biomass grows on media and the sewage passes over its surface.

Suspended-growth systems include activated sludge system ,where the biomass is

mixed with the sewage and can be operated in a smaller space than fixed-film systems
that treat the same amount of water.

Trickling filter system :

Trickling filter system is a wastewater treatment system that biodegrades organic

matter. It consists of a circular or rectangular bed of stone, gravel or synthetic material.
There is a rotating arm above the surface of filter bed. The rotating arm sprays the
settled sewage over the bed either continuously or intermittently that slowly percolates
through this porous bed. The spraying saturates the liquid with oxygen.

Dense , slimy bacterial growth coats the porous material of the filter bed. Zooglea
ramigera and similar bacteria play a key role in generating the slime matrix which
accomodates a heterogenous microbial community including bacteria , fungi ,
protozoa ,nematodes and rotifers. This microbial community absorbs and mineralizes the
dissolved organic nutrients in the sewage ,thus reducing the BOD of the effluent.

Fig : Tricking filter system

A food web is established based upon the microbial biofilm. Insects , mainly fly larvae
,consume the excess microbial biomass generated but constitute a nuisance to nearby
residential areas. They can be controlled by the continuous rather than intermittent
operation of the trickling filters ,because continuous spreading suppresses the fly
larvae ,which successfully feed on the exposed microbial biomass only when the
sprinkling is shut off.
Fig : Main pathways of energy and materials transfer during treatment of dissolved
organic wastes by tricking filter

Advantage :

Simple and inexpensive

Disadvantages :

Nutrient overload may lead to excess microbial slime ,reducing percolation rates and
necessitating a renewal of the trickling filter bed.

Cold winter temeperatures strongly reduce the effectiveness

Rotating biological contactor :

Rotating biological contactor is a more advanced type of aerobic film flow type
treatment system. Here, closely spaced discs, usually made of plastic are rotated in a
trough containing the sewage effluent. The discs are partially submerged and become
coated with a microbial slime similar to that developing in trickling filters. Continuous
rotation of the discs keeps the slime well aerated and in contact with the sewage.

Biofilms developing on an RBC comprise a complex and diverse microbial community of

diverse bacetrial ,protozoan and metazoan populations. Among the filamentous
microorganisms are populations of Sphaerotilus , Beggiatoa , Nocardia and Oscillatoria.
When the film becomes so thick that 0 2 and nutrients fail to reach the inner portion of
film, the innermost microbes die, causing detachment of the film. The system is used for
both the treatment of domestic sewage and industrial effluents.

Fig : Rotating biological contactor

Advantages :

Requires less space

More efficient and stable in operation

Produce no aerosols

Disadvantage :

High installation cost

Fluidized bed reactors :

In fluidized bed reactors the support materials – pumice ,sand or plastic are fluidized by
pumping waste water up through the medium (anaerobic ) or by injecting air or oxygen
(aerobic).In the first instance ,the upflow velocity is high enough to fluidize the support
particles so that they are not in direct contact with each other but not so high as to
shear off the biofilm.

Advantage :

Provides greater surface area available for biomass support because the particles are not
in direct contact with each other

Quick method

Designed for the removal of nitrate as well as the reduction of BOD

Disadvantage :

Expensive

Activated sludge system :

Activated sludge system is a highly efficient system for the aerobic biological treatment
of industrial or municipal wastes. After primary treatment ,the sewage is introduced into
an aeration tank. Aeration is provided by air injection or mechanical stirring. Microbial
activity is maintained at high levels by reintroduction of most of the settled activated
sludge ,hence the name of the process. During the holding period in the aeration tank
,vigorous development of heterotrophic microorganisms takes place.

The heterogenous nature of the substrate allows the development of diverse

heterotrophic bacterial populations. Gram negative rods predominate with Escherichia ,
Enterobacter ,Pseudomonads Achromobacter ,Zooglea and Flavobacterium. Micrococcus ,
Arthrobacter ,various coryneforms and mycobacteria also occur along with Sphaerotilus
and other filamentous bacteria. Filamentous fungi and yeasts normally occur in low
numbers and play a subordinate role The protozoa are mainly represented by the
ciliates. Along with rotifers ,these protozoa are important predators of bacteria. The
bacteria occur individually in free suspension and also aggregate as floc. The floc
consists predominantly of microbial biomass cemented by bacterial slimes ,such as those
produced by Zooglea ramigera and similar organisms. Most of the ciliate protozoa ,such
as vorticella are of the attached filter-feeding type ; they adhere to the floc but feed
predominantly on the bacteria suspension.

During the holding period ,a portion of the dissolved organic matter is mineralized.
another portion is converted to microbial biomass. Most of the microbial biomass are
associated with the floc that can be removed by settling. Poor settling characteristics are
associated with bulking of the sewage sludge ,caused by the proliferation of filamentous
bacteria such as Beggiatoa ,Sphaerotilus , Thiothrix and Bacillus and with filamentous
fungi ,such as Geotrichum ,Cephalosporium ,Cladosporium and Penicillium. The causes of
bulking may be associated with high C : N and C :P ratios or low dissolved O 2
concentrations.

The activity of these aerobic microorganisms oxidizes much of the sewage organic
matter into carbon dioxide and water. Soluble organic matter in the sewage is
incorporated into the floc and its microorganisms. Aeration is discontinued after 4 to 8
hours, and the contents of the tank are transferred to a settling tank, where the floc
settles out, removing much of the organic matter. These solids are subsequently treated
in an anaerobic sludge digester. The clear effluent is disinfected and discharged
A portion of the settled sludge is recycled for inoculation of the incoming raw sewage
referred to as activated sludge ,the excess sludge requires incineration or additional
treatment by anaerobic digestion or composting or disposal in landfills.

It is widely used for the treatment of domestic sewage and industrial effluents.

Fig : Diagram of an activated sludge system.

Advantages :

Reduces BOD

Removes inorganic nitrogen and phosphate

Reduces the numbers of intestinal pathogens in the sewage ,even prior to final
disinfection by chlorinaiton.

Predation by ciliates ,rotifers and Bdellovibrio is probably indiscriminate and affects

pathogen as well as nonpathogenic organisms , however ,pathogens tend to grow poorly
or not at all under the conditions.

Variation of activated sludge :

Two variations of the activated sludge process have been developed for treatment of
high BOD industrial wastes. These are designed to improve the O 2 transfer rate and thus
achieve a higher BOD reduction rate per unit volume of treatment system.

The UNOX process uses oxygen instead of air. Closed tanks and a specially designed
stirring system prevent the wastage of O2.

The ICI deep shaft process uses air injection and achieves high O 2 dissolution by
increasing hydrostatic pressure through an ingenious circulation pattern.
Fig : UNOX process

Fig : ICI deep shaft process

Why it is important to remove inorganic nitrogen and phosphate from

wastewater ?

Wastewater may contain high levels of the nutrients nitrogen and phosphorus. Excessive
release to the environment can lead to a build up of nutrients, called eutrophication,
which can in turn encourage the overgrowth of weeds, algae, and cyanobacteria (blue-
green algae). This may cause an algal bloom, a rapid growth in the population of algae.
Owing to various known or unknown causes ,such as a period of cloudy weather that
intensifies mutual sheding , exhaustion of micronutrients ,toxic products or disease ,the
algal population eventually crashes. The decomposition of the algae by bacteria uses up
so much of oxygen in the water that most or all of the animals die, which creates more
organic matter for the bacteria to decompose. In addition to causing deoxygenation,
some algal species produce toxins that contaminate drinking water supplies. So it is
important to remove inorganic nitrogen and phosphate from wastewater

Nitrogen removal:

The removal of nitrogen is accomplished through the biological oxidation of nitrogen

from ammonia (nitrification) to nitrate, followed by denitrification, the reduction of
nitrate to nitrogen gas. Nitrogen gas is released to the atmosphere and thus removed
from the water.

Nitrification itself is a two-step aerobic process, each step facilitated by a different type
of bacteria. The oxidation of ammonia (NH 3) to nitrite (NO2−) is most often facilitated by
Nitrosomonas spp. Nitrite oxidation to nitrate (NO 3−), though traditionally believed to be
facilitated by Nitrobacter spp.

Denitrification requires anoxic conditions to encourage the appropriate biological

communities to form. It is facilitated by a wide diversity of bacteria.The activated sludge
process (if designed well) can do the job the most easily.Since denitrification is the
reduction of nitrate to nitrogen gas, an electron donor is needed. This can be depending
on the wastewater, organic matter or methanol.

Phosphorous removal:

Phosphorus can be removed biologically in a process called enhanced biological

phosphorus removal. In this process, specific bacteria, called polyphosphate
accumulating organisms (PAOs) incorporate large amounts of fatty acids and store them
in the form of poly-β-hydroxybutyrate (PHB) under anaerobic conditions. Phosphate is
released into soluble form during this period. When the sewage is subsequently aerated
the poly-p bacteria rapidly oxidize their intracellular PHB reserves while engaging in
`luxury phosphate uptake’. The incorporated phosphate is stored as energy rich
polyphosphate within the cell , thus removing phosphorous from liquid effluent.

Several biological phosphorous removal systems based on the described mechanisms

have been designed and incorporated into the activated sludge treatment system.

The A/O process consists of a modified activated sludge system that involves anaerobic
and aerobic treatment stages. During the anaerobic phase , inorganic phosphorous is
released from the cells as a result of polyphosphate hydrolysis. The energy liberated is
used for the uptake of BOD from waste water. During the aerobic phase ,soluble
phosphorous is taken up by bacteria ,which synthesize polyphosphates ,using the energy
released from BOD oxidation. The A/O process results in the removal of phosphorous
and BOD from effluents and produces a phosphorous-rich sludge.

Fig : A/O process for the removal of Phosphorous

Anaerobic sludge digestors:

The solids which accumulate from the sedimentation tanks and the primary and
secondary sludge are treated further in anaerobic (sludge) digestion tank. It is a
specially designed tank which is maintained free of oxygen. It may be a completely
closed tank or may be provided with a lid at the top.

The contents are introduced into the tank through an inlet near the top. There is an
outlet at the bottom to remove the digested sludge. There is also a vent in the lid
through which any gas produced during the digestion escapes.

Both anaerobic and facultative bacteria degrade the organic matters by converting them
into soluble substances and gaseous products. The gases consist mainly of methane (60-
70%) and carbon dioxide (20 -30%). In addition hydrogen and nitrogen are also
produced during sludge digestion in small amounts.

The anaerobic digestion of wastes can be considered as a two-step process. First

complex organic materials ,including microbial biomass ,are depolymerized and
converted fermentatively to fatty acids ,CO 2 and H2. A large variety of nonpathogenic
obligately or facultatively anaerobic participate in these processes. In the next step ,
methane is generated ,either by the direct reduction of methyl groups to methane or by
the reduction of CO2 to CH4 by molecular hydrogen or by other reduced fermentation
products ,such as fatty acid ,methanol or even carbon monoxide.

The operation of anaerobic digester requires the close control of several parameters such
as retention time ,temp , PH and C : N and C : P ratios. The reactors are usually heated
to 35-37 C for optimal performance. Control is necessary for PH to stay within the 6-8
values ,with PH 7 being optimal.

Fig : Anaerobic digester

Septic tank :

Septic tank is a kind of sewage settling tank. In rural areas, individual houses treat their
sewage wastes in septic tanks built at ground levels near the house. As sewage enters
this type of tank, sedimentation occurs at the bottom leaving the liquid with fewer
suspended solids to be discharged from the tank.

The sedimented solids are subjected to degradation by anaerobic bacteria. As a result

hydrogen sulfide and other organic acids are produced. These products along with the
effluent water on the upper portion are discharged into the adjacent ground.

Sedimented sludge accumulated at the bottom of septic tank is removed and may be
dried, ground and spread on soil as a fertilizer.

Tertiary Treatment :
The purpose of tertiary treatment is to provide a final treatment stage to raise the
effluent quality before it is discharged to the receiving environment (sea, river, lake,
ground, etc).This is designed to remove non-biodegradable organic pollutants such as
chlorophenols , polychlorinated biphenyls and other synthetic pollutants and mineral
nutrients, especially N2 and P salts. Activated carbon filters are normally used in their
removal from secondary treated effluents.

To prevent eutrophication, phosphate is removed from sewage by precipitation as

calcium, aluminium or iron phosphate. Breakpoint chlorination is a process to remove
NH3.

[ Sewage is the used water supply containing domestic waste together with human
excrement and wash water and industrial waste ,including acids ,greases ,oils ,animal
matter ,vegetable matter and storm waters.]