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Present Status of Applied Microbiology in India

Anuradha S. Nerurkar
Department of Microbiology and Biotechnology Centre
Faculty of Science
The Maharaja Sayajirao University of Baroda
Vadodara, Gujarat. - 390 002.

13-Apr-2006 (Revised 03-Apr-2007)

Wastewater Microbiology
Self purification
Characterization of sewage
Conventional sewage treatment
Advances in wastewater treatment plants
Low cost sewage treatment plants
Water Microbiology
Drinking water microbiology
Waterborne diseases
Microbiological examination of water
Water purification
Air Microbiology
Airborne diseases
Fate and transport of microorganisms in air

Microbiological analysis of air
Control of airborne microorganisms
Food and Dairy Microbiology
Microorganisms in food
Beneficial effects of microorganism in food
Detrimental effects of microorganisms on food
Food preservation
Microbiological examination of food

Wastewater microbiology; Sewage treatment; Drinking water microbiology; Waterborne diseases; Water
purification; Airborne microorganisms; Airborne diseases; Microorganisms in food; Food preservation.

In the 1970’s a new era of Microbiology emerged and developed into the field of
Environmental Microbiology. It is closely related to Microbial Ecology, which is the study of
the interaction of microorganisms within the environment. Primary difference between these
two fields is that Environmental Microbiology is an applied field where the study of
microorganisms in the environment leading to the benefit of the society is emphasized. It is a
subset of Applied Microbiology and interfaces with water, wastewater, air, soil, food,
industrial microbiology and others. Microbial Biotechnology encompasses the field which
includes the manipulation of the microbes to increase their practical benefit wherein the
principles of Applied Microbiology are applied. Improved water and wastewater treatment,
clean environment strategies and role of microbes in food availability and quality are some of
the significant achievements of Microbial Biotechnologists who are equipped with a detailed
knowledge of environment and functioning of complex microbial community. This chapter
discusses the present status of Applied Microbiology in India in the context of world scenario
including Wastewater, Water, Air and Food & Dairy Microbiology. Some of the national
institutions engaged in research in the field of Applied Microbiology are listed in Table 1.

Table1: Institutions in India involved in environment and food related research and

S. No.
Area of Research & Development
National Environmental
Engineering Research Institute
(NEERI ), Nagpur, Maharashtra.
Environmental Engineering and
Biotechnology that concerns wastewater,
water and air
Centre for Environmental Science
and Engineering (CESE), I.I.T.,
Mumbai, Maharashtra.
Addresses needs and challenges of major
industrial sectors and international
Central Food and Technological
Research Institute (CFTRI),
Mysore, Karnataka.
Food processing, conservation and
development of related technology
Food Corporation of India (FCI)
Defense Food Research
Laboratory (DFRL), Mysore,
Food security.
DFRL, an FCI laboratory is engaged in
the field of food preservation, packaging
and analysis
National Dairy Development Board
(NDDB), Anand, Gujarat.
Promote dairy development. Operation
flood is an integrated approach to
strengthen dairy cooperative
National Dairy Research Institute
(NDRI), Karnal, Haryana.
Provides inputs for development to the
dairy industry.

Wastewater Microbiology
The new focus of environment as a whole has led to the development of new activities related
to application of principles of water, wastewater and air microbiology which forms the scope
of Environmental Biotechnology. This part addresses various aspects of wastewater
treatment. Agricultural, industrial and everyday human activities produce liquid wastewater
termed as industrial and domestic wastewater or sewage, respectively. The old practice was


to discharge the waste into the nearest water body. As the population increases, the
discharged waste remains undegraded causing oxygen depletion thereby affecting the aquatic
life. The practice of wastewater treatment started in middle of 19
century as the threat of
waterborne diseases increased. Wastewater was suitably treated before dumping it in water
body. Recently the focus of wastewater treatment has been pathogens and toxic substance
removal for water recycling, since the water supplies are limited and there is a need to reuse
the water. In India wastewater treatment plants are increasing in cities while the low cost
technologies in rural areas.

Self Purification
The fate of the discharged waste is determined by the self purification potential of the
receiving water body. Self purification is based on biogeochemical cycling activities and
inter-population relationships of the indigenous (autochthonous) microbial populations.
Organic nutrients are mineralized by the heterotrophic aquatic organisms. Ammonia is
nitrified and inorganic nutrients are utilized by higher aquatic plants. Non-indigenous
(alochthonous) population of enteric pathogens that enter through waste is eliminated by
competition and predation of the autochthonous microorganisms. The wastewater may
overwhelm the self purification capacity of the aquatic system causing pollution of receiving
water. Depending on the climatic and environmental factors the water may attain an
acceptable quality level in the stream, downstream from the sewage outfall. A well defined
profile of pollution and self purification of the receiving water is obtained by a succession of
changes in water quality that occurs on the downstream of the point source of pollution
(Figure1). Self purification is a slow process and a heavily polluted stream may have to
traverse quite a long distance for days to get purified. Whenever the wastewater is
discharged, the suspended matter either settles at the bed near the point of discharge or gets
carried away.

Figure 1: Sewage outfall and self purification

The organic matter is utilized by the aerobic microorganisms reducing the dissolved oxygen.
As the organic matter is depleted, the number of microorganisms is also reduced. The
reaeration rate of the atmosphere catches up. The water becomes clear and the stream returns
to the original condition and the self purification is accomplished. The biochemical oxygen
demand (BOD) and the dissolved oxygen (DO) of the receiving waters are parameters that
give good measure of pollution that exists in the receiving stream.


Characterization of Sewage
Domestic wastewater or sewage contains human waste like feaces, urine, and gray water.
Gray water results from washing, bathing and meal preparations. Agricultural run-off water
and waste from nearby industries may also enter the system. The important physical
characteristic of the wastewater is its total solids content. It includes floating, suspended,
colloidal and dissolved solids. Total solids are those that remain as residue upon evaporation
at 105°C.

The Total solids include suspended and filterable solids (about 1 µ size).
Settlable suspended solids are the ones that settle in Imhoff cone in 60 min., while the
remaining are non-settlable solids. Colloidal filterable solids impart turbidity to the water,
measurement of which gives an idea about the wastewater quality. Dissolved filterable solids
cannot be removed by conventional treatment and requires special treatment. Solids are
called volatile solids if they are volatile at 600°C. Minerals are non-volatile solids that form
ash when heated at 600°C. Depending on the amount of total suspended solids the sewage
can be categorized as high strength (>500 ppm ), medium strength (200-500 ppm) and weak
(< 200 ppm). The odor in sewage usually is due to presence of amines, hydrogen sulfide,
ammonia, mercaptans, skatole, organic sulfide etc. The color of fresh sewage is usually gray.
After the organics are broken down the dissolved oxygen is depleted and the color changes to
black. The chemical nature of a typical municipal sewage is primarily due to proteins,
carbohydrates and fats. Surfactants, detergents, phenols, pesticides etc. form the minor
components. Roughly the composition of the sewage can be represented in terms of its
contents (Table 2).

Table 2: Composition of average sewage

S. No.
Total solids
Dissolved solids
Total P
Settled solids
Total N
Organic P
Suspended solids
Organic N
Inorganic P

The need for characterization of sewage arises in order to evaluate its capacity to cause
pollution, decide correct type and size of the treatment plants, monitor the efficiency of the
plant, and prevent the pollution of the receiving water. It helps to establish an effective and
economical waste management system. The components of wastewater are defined under
broad categories of organic, inorganic and microbial content since exact chemical
composition cannot be determined. Each category requires specialized treatment to render it
harmless. To determine the organic content of the wastewater Biochemical oxygen demand
(BOD), Chemical oxygen demand (COD), Permangnate value (PV), Total oxygen demand
(TOD), Total organic carbon (TOC) and Theoretical oxygen demand (ThOD) tests are used.
BOD or Biochemical oxygen demand is widely used method that exploits the ability of the
microorganisms to oxidize organic matter to CO
and H
O. The indigenous microorganisms
in the wastewater are used and DO is measured initially and after a period of five days at
20°C (BOD
). The microorganisms starve after the organics are depleted and are forced to
use cellular carbon. This is called endogenous respiration. The less oxidizable organics are
utilized over a period of 28 days. This oxygen demand exerted over a period of 28 days is
called ultimate BOD (BODu). The nitrogenous organic waste exerts oxygen demand between


5 and 12 days. This is the result of autotrophic nitrification by nitrifiers called nitrogenous
oxygen demand or NOD (Figure2). Inhibitors like 1-allyl-2-thiourea (ATU), 2-chloro-6
(trichloromethyl) pyridine or nitrapyrin suppress NOD. Addition of H
followed by
neutralization with NaOH or pasteurization of seed also acheives this. Algae which releases
evolution as a result of photosynthesis may interfere with BOD. Incubation in the dark
solves this problem. BOD is a bioassay giving an idea about only biodegradable organics.
COD or Chemical oxygen demand measures the amount of oxidizing agent such as
dichromate that is utilized to completely oxidize the wastewater to CO
and H
O in presence
of concentrated H
at 100°C temperature. The unutilized dichromate is titrated with
ferrous sulfate using ferroin (1, 10-phenanthroline) indicator. Even in this strong oxidizing
environment certain aromatic compounds e.g. toluene, benzene and ammonia are not
oxidized. The biodegradable as well as some of the non-biodegradable organics also exert
oxygen demand in COD. PV or Permanganate value represents organic material that is
chemically oxidized by permanganate in presence of dilute sulfuric acid for 10 min at boiling
temperature. Upon oxidation bright pink of permanganate changes to colorless liquid serving
as a visual guide. The remaining permanganate is titrated against ammonium oxalate. This
procedure is simple and ideal for field testing. TOD or Total oxygen demand is exerted by
organic substances and minor amount of inorganic ones and measures the oxygen that is used
up in a platinum combustion chamber at 900°C. In TOC or Total organic carbon
measurement the organic matter in the wastewater is oxidized at high temperature (900°C) to
which is measured by potentiometric procedure. The biodegradable, non-biodegradable
and refractory organics that are not oxidized in COD also exert oxygen demand here. This is
fast and automated procedure giving good reproducibility. ThOD or theoretical oxygen
demand refers to calculated oxygen demand when the appropriate chemical formulae of the
contents of the waste is known. A linear relationship exists in these assay values in the order,
PV < BOD < COD < TOD <TOC< ThOD. The inorganic matter of the sewage includes
chlorides, alkalinity, nitrogen, phosphorus, sulfur, heavy metals, gases etc. Pathogens and
nonpathogens belonging to different groups like bacteria, viruses, protozoa, worms etc. are
also present in the sewage. The industry personnel use a thumb rule to assess the amenability
of a wastewater to biotreatment. Accordingly the ratio if BOD/COD > 0.6 it is easily
biodegradable, between 0.3 – 0.6 means degradable by adapted seed or inoculum, < 0.3
indicates non-amenablility to biotreatment. Generally the character of sewage is constant and
hence it can be treated by conventional scheme. This is not the case with Industrial
wastewater as it has varying character. The studies regarding the treatability of the
wastewater are needed to design a treatment scheme for it.

Conventional Sewage Treatment
The conventional method of sewage treatment attempts to maintain acceptable BOD before it
is discharged into the water body. A combination of physical unit operations and chemical
and biological processes are used. In this, the forces that favor self purification are
purposefully intensified to get the desired treatment in short time and space. Major steps in
the conventional sewage treatment are primary, secondary and tertiary or advanced treatment
(Figure 3). Primary treatment is a physical operation that separates large debris followed by
sedimentation to settle big suspended solids. 20-30% BOD that is present in the particulate
form is removed. Raw sewage passes through a metal grating that removes large debris such
as branches, tyres etc. A moving screen filters small items like bottles etc., after which a grit
tank is provided where the sewage is kept for some time for sand and gravel to settle out. The
waste then is pumped into primary settling or sedimentation tank. If dissolved solids are less,
large chunk of BOD is removed with settled sludge in the sedimentation tank. Pathogens
adsorbed to solids are removed. The effluent of primary treatment is called settled sewage.


Secondary treatment comprises the biological treatment in which remaining suspended and
dissolved organic material along with about 90-95% BOD and pathogens are removed. The
settled sewage is pumped into either trickling filter or activated sludge process for biological

Figure 2: Carbonaceous and nitrogenous BOD

Figure 3: Conventional sewage treatment

Trickling filter is a biological filter bed made of stones and is one of the oldest system. The
sewage is sprinkled by means of an overhead sprayer which is rotating at a constant speed.
The stones are 30-100 mm in size and packed at the depth of 1.8 m. Life of trickling filter is
30-50 years. As the sewage trickles down microbial biomass increases which grows in a form
of biofilm on the stones. This is called zoogleal film or schmuzedecke (German). It is
composed of bacteria, fungi, algae and protozoa.

Top 0.5 m of the filter is the heterotrophic zone and bottom 1.5 m is the autotrophic zone.
Biofilm microorganisms oxidize the organics from the trickling sewage and its thickness


increases over time. Eventually the biofilm sloughs off and new one starts building. The
trickling filter also harbour nematodes and rotifers and birds grazing on the worms. Rotifers
are minute aquatic multicellular invertebrates. The commonly found bacterial species in
trickling filter are Beggiatoa, Sphaerotilus natans, Achromobacter, Flavobacterium,
Pseudomonas and Zooglea ( Figure 4).

Figure 4: Trickling filter

Activated sludge process consists of aeration tank, clarifier and the sludge recycle tank. It
employs the microorganisms in suspended growth as opposed to attached growth in the
trickling filter. The sewage from primary settling tank is introduced in the aeration tank
equipped with aerators and mechanical stirring where digestion of organic matter of sewage
occurs. The sludge recycle reintroduces the sludge from the previous batch of the process
(Figure 5). This accelerates the development of microbial flocs teeming with actively
growing bacteria and is called activated sludge. During the holding period for 4-8h, the
heterogenous nature of organics in the sewage allows vigorous development of diverse
population predominantly belonging to species of Escherichia, Enterobacter, Pseudomonas,
Achromobacter, Flavobacterium, Zooglea, Micrococcus, Arthrobacter, cornyforms and
mycobacteria. Filamentous fungi, yeasts and protozoa occur in low numbers.

Suspended bacterial population diminishes and the bacteria associated with flocs increase in
number with time. A significant amount of dissolved organic substrate is mineralized by the
flocs. The sludge or floc can be removed by settling in the secondary sedimentation tank or
clarifier that follows. The settling character of the flocs is critical for their efficient removal.
Poor settling caused by bulking sludge is due to proliferation of filamentous bacteria like
Sphaerotilus, Beggiatoa, Thiothrix and the filamentous fungi like Geotrichum,
Cephalosporium, Cladosporium and Penicillium. The important parameters that control the
operation of the activated sludge process are organic loading rates, oxygen supply and
operation of the clarifier where thickening of the sludge is also accomplished. Sludge
settlability is determined by sludge volume index (SVI). It is determined by measuring the
sludge volume after it has settled for 30 mins. The SVI = Vx1000/MLSS where V is the
volume of settled sludge after 30 min (ml/L). A mean cell residence time of 3-4 days in the
clarifier is required for effective settling. Sudden changes in temperature, pH, absence of
nutrients and presence of toxic metals and organics results in poor settling. A high SVI of 150
ml/g indicates bulking conditions. Low dissolved oxygen, F/M ratio, nutrients (N and P) and
high sulfide, carbon : nitrogen ratio or carbon : phosphorus ratio cause filamentous bacteria to
proliferate and therefore bulking. Chlorination or H
treatment eliminate filamentous
bacteria. A portion of the settled sludge from the clarifier is recycled while the rest is wasted


and is called wasted sludge. The pathogens are reduced due to the direct effect of
competition, adsorption, predation and settling. Predation by ciliates like Vorticella, rotifers
and Bdellovibrio bacteriovorus affects all bacteria. Pathogens tend to grow poorly in these
conditions. Enteroviruses also are removed to certain degree. The content of the aeration tank
is referred to as mixed liquid suspended solids (MLSS). The organic part of the MLSS is
called MLVSS or mixed liquid volatile suspended solids, which includes non microbial
organic matter as well as dead and living microbes. A proper F/M or Food to microorganisms
ratio helps to exercise proper control over activated sludge process. F/M is expressed as (Q x
BOD) / (MLSS x V) where Q is the flow rate of the sewage in million gallons per day
(MGD) and V is the volume of the aeration tank (gallons). F/M ratio can be controlled by rate
of wasting of sludge. For conventional processes F/M ratio is 0.2 – 0.5 lb BOD
MLSS. A low F/M ratio means that the microorganisms are starved leading to increase in
efficiency of wastewater treatment.

Figure 5: Activated sludge process

Tertiary treatment comprises of a series of additional steps after secondary treatment to
further reduce organics, turbidity, nitrogen, phosphorus, metals and pathogen. Mostly some
type of physicochemical or biological treatment such as coagulation, filtration, activated
carbon, adsorption of organics, nutrient removal and disinfection is required. The tertiary
treatment is done for additional protection of wildlife after discharge in rivers, lakes etc. or
when the water is to be reused. Some of the tertiary treatment processes are given in Table 3.
Disinfection is always done before discarding the sludge. Chlorination, Ozonation, and UV
disinfection are commonly used (their mechanism is discussed in part II Water
Microbiology). They also react with other organic matter, NH
, Fe, Mn, S compounds and
also reduce BOD, color, odor and oxidize cyanides. Ozone reacts with unsaturated organics
of wastewater, also reduces foaming in addition to what chlorine does. Basically it opens the
ring and brings about partial oxidation of aromatics. Thus the aromatics become more
susceptible to conventional treatment.

Gamma irradiation to hygeinise sludge uses radioisotopes in batch or continuous mode
(Figure 6). Refractory organics (organics difficult to oxidize) and BOD can be removed by
this process. In one such plant in Gujarat a moderate dose of gamma rays of 3-5 kGy (300-
500 krads) to kill the pathogens in the sludge is provided. The sludge is exposed for a
predetermined time and dose of the source of radiation which is cobalt 60. The source and
sludge reactor is housed in a concrete cell to prevent radiations leakage. The irradiation does
not leave any residual radioactivity in the treated sludge and therefore is safe.


Table 3: Some Tertiary Treatment Processes

S. No.
Final step in sewage treatment designed to kill
Suspended solid
Microscreens, sand, anthracite or diatomaceous earth
filters employed. Coagulation with alum,
polyelectrolytes, lime and other chemicals aids the
Taste and odour removal
Activated carbons are widely used. Solutes are adsorbed
onto the carbon by means of strong Van der Waals
Ion removal
Ion exchange used wherein ions that are held to
functional groups on the surface of a solid by
electrostatic forces are exchanged for ions of different
species in solution and complete demineralization is
Nutrient removal
Removal of nitrogen and phosphorus done biologically.

Figure 6: Gamma irradiator

Nutrient removal is achieved by chemical or biological methods. However, for nitrogen
removal, biological methods are more cost effective. Activated sludge process is modified to
encourage denitrification. Chemolithotrophic nitrifiers that convert ammonia to nitrate are
slow growers and require a longer retention time in the aeration tank Anoxic conditions and
exogenous carbon are provided in separate tank to encourage denitrification to nitrogen gas
which is achieved by heterotrophic denitrifying bacteria. Phosphorus removal is also done
similarly. Under anaerobic conditions, the microbes release stored phosphorus to generate
energy. The energy liberated is used for the uptake of BOD from the wastewater. When
aerobic conditions are restored microbes exhibit phosphorus uptake level above those


normally required to support cell maintenance, synthesis and transport reactions. It is stored
as storage volutin or polyphosphate granules (storage of excess phosphate) in wasted sludge
containing excess phosphorus.

Sludge treatment is generally done anaerobically. The sludge drawn from primary settling
tank or wasted sludge of secondary clarifier or trickling filter contains putrecible organics
and 92-98% water and resists dewatering. A number of treatments including digestion are
used to stabilize the sludge (Table 4). This not only reduces the sludge volume but produces a
sludge that is odorless and ready to disperse. Sludge treatment includes all or a combination
of processes. Sludge digestion is carried out in anaerobic digester where 50% of carbon over
a period of 10-30 days is degraded (Figure 7). Methane is the byproduct. This just simplifies
the sludge organics and does not completely degrade them. Resultant stabilized sludge is a
good fertilizer and is sold so.

Table 4: Processes used in sludge treatment

S. No.
reduces the moisture in sludge, gravity thickeners and flotation
thickeners are employed.
anaerobic bacteria digest the organic contents, only biological
step in sludge treatment, carried out in anaerobic digester.
improves the drainability of the digested sludge, chemicals, heat
treatment, freezing etc. are used.
sludge is disinfected by gamma irradiation in addition to other
conventional methods.
air drying, vacuum filtration, centrifugation , heat drying etc. are
used, sand drying beds are common.

Figure 7: Anaerobic digester

The digester has facilities for mixing, gas collection, sludge addition and draw off. Sludge
has 20-100 g/L suspended organics. Bacterial counts of 10
cfu/ml is attained. It is


operated at 35-37°C at pH 6-8. Fungi and protozoa have no role. However, a complex
bacterial community is involved. The anaerobic digestion can be summarized as organic
matter  CH
+ CO
+ NH
+ H
S. Acid formation followed by methane generation are
the sequence of reactions in the digester. Complex organic polymers like proteins, fats,
carbohydrates, cellulose, lignin etc. are broken down by hydrolytic bacteria using
extracellular enzymes. The simple soluble products like sugars, fatty acids, amino acids are
fermented by acid forming or acetogenic bacteria.Volatile fatty acids like acetic, propionic
and lactic and gases like CO
and H
are the products that are converted to methane by
methanogenic bacteria. Thus the four major groups of bacteria involved in succession in
anaerobic digester are, Acid forming hydrolytic and fermentative Acetogenic, acetate and
hydrogen forming Acetoclastic, methane forming and Hydrogen utilizing methanogens.
Methanogens are slow growers as compared to the hydrolytic and acid forming bacteria. 70%
of methane is formed from acetate.The preceding bacteria provide the nitrogen source to
methanogens by reducing organic nitrogenous compounds to ammonia. Methane formation
also neutralizes the pH of the digestor slurry.Their reactions are as follows:

Acid formers and acetogenic bacteria
substrates → CO
+ H
+ acetate
substrates → propionate + butyrate + ethanol
Acetoclastic methanogens
acetate + H
O → CH
+ CO
+ energy
Hydrogen utilizing methanogens
+ H
→ CH
+ 3H
O + energy

Hydrogen utilizing methanogens reduce the partial pressure of hydrogen in the digester which
is beneficial for the activity of acetogens. Methanogens being strict anaerobes it is important
to maintain reducing environment in the digester. In the digesters where methanogenesis is
interrupted, acids accumulate. This is called stuck or sour digester. Many factors like pH,
heavy metals, toxic substances can upset the operation of the digester. Subsequently the
digested sludge is treated with other physical processes.

Finally drying is accomplished in sand drying beds. It involves air drying in shallow beds and
is the cheapest and preferred method for drying the sludge. A bed of about 250 mm of sand
over about equally thick well graded gravel layer, underlain by perforated drainage lines
spaced 2.5 – 6 m apart is prepared. The bed slopes towards the discharge and at a rate of 1 in
20. For flexibility of operation the bed area is subdivided by partitions, each approximately 6
m wide and 6-30 m long. The digested sludge is applied on to the beds in 200 mm to 300 mm
thick layers. The drying time depends on the weather and may vary from 10 days to several
weeks. The sludge cake is removed and solid as fertilizer or fuel.

Advances in Wastewater Treatment Plants
In the earlier plants mostly designed by civil engineers, the ‘bio’ component was neglected. A
detailed knowledge of Environmental Biology and more particularly of functioning of
complex microbial communities is needed to employ strategies that have a holistic approach.
Environmental Biotechnology focuses on the ‘bio’ component leading to efficient
environmental systems. The developments are concerned with the extraction of best
efficiency of microorganisms. The treatment efficiency is proportional to the amount of
biomass and contact time between the waste and the biomass. The developments in
wastewater treatment plants have been in the operation as well as design of the plant.


Advances in operation of trickling filter relate to its operation in different modes and at
different rates. A scheme of alternating double filtration where the sewage is applied to two
filters in series instead of two filters in parallel is one such. Biomass accumulated in the first
filter consumes majority of organics. Order of the filter is reversed before the first filter is
clogged. The biomass in the first filter is rapidly sloughed off. A filter that drops its excess
growth continuously works efficiently. Based on the rate of operation the filters are
categorized as low rate, intermediate rate and high rate. The hydraulic loading (amount of
liquid applied) of 2-4 mgad ( million gallons per day) is used in low rate filters. 85% BOD
reduction is achieved. A well nitrified stable effluent is produced. High rate filter has
hydraulic loading of 10 mgad. Recirculation of the sewage is required to get desired lowering
in BOD. Its efficiency of organic matter removal is 65-75%. However, since the organic
matter is distributed all over, the nitrifiers do not develop. Intermediate rate filters have 4-10
mgad as hydraulic loading rate. This has more chances of clogging since the hydraulic
loading is not sufficient to push the excess growth from the stones.

Advances in operation of activated sludge process include a variety of process modifications
done to solve specific operating problems. The modifications are tapered aeration, step
aeration, high operation rate, biosorption, dispersed aeration, pure oxygen etc. (Table 5). In
the complete mix plants as opposed to plug flow the aeration tank contents are completely
mixed e.g. extended aeration, high rate plants etc. Plug flow means tubular flow where fluid
particles pass through the tank and are discharged in the same sequence in which they enter
e.g. conventional plants, step-aeration plants. The particles retain their identity and remain in
the tank for a time equal to theoretical detention time. Plug flow occurs in longer tanks with
high length : width ratio in which longitudinal dispersion is minimal. In tapered aeration the
number of diffusion tubes are increased at the head of the tank and decreased at the end of the
tank Step aeration means introduction of wastes into the aeration tank at several points rather
than all at once. The high rate plant carries 200-500 mg/L MLSS. BOD reduction is very fast
within 2-4 h. Biosorption or contact stabilization is a phenomenon observed when raw
sewage is mixed with activated sludge together in aeration tank. An instant drop in the BOD
occurs. In dispersed aeration the organisms are dispersed without any flocculation. Thus
clarifier is not required. In the Pure oxygen plants as the name suggests pure oxygen is used
for aeration.

Table 5: Modifications in operation of activated sludge process

S. No.
Operational changes
aerators are more clustered at the head of the tank than at the end
of the tank.
Step aeration
Introduction of wastes into the aeration tank at several points
High rate
The F/M is kept high to give maximum biomass.
or contact stabilization A contact time of 15-30 min is given then
the sludge is settles
The organisms are dispersed without any flocculation avoiding
the need for clarifier.
Pure oxygen
Aeration done with pure oxygen
The sludge detention time in the tank is increased where the
microorganisms go into the stationary phase and resort to
endogenous growth reducing the sludge volume.


The developments in treatment plant design are seen in biotowers, rotating biological
contactor, membrane bioreactor and upflow anaerobic sludge blanket. Biotowers are
advanced trickling filters using plastic media instead of stones to get high specific surface and
void volume. The uniform media gives better liquid distribution and is light weight allowing
construction of deep filters. High strength waters are treated satisfactorily. PVC crossflow
modules that can be fitted in a circular tank are used. The media have corrugated sheets
bonded together to prevent clear vertical openings and distribute the wastewater over the
surface of the media. Towers 20 feet high are built. BOD load distribution is vertical rather
than horizontal.

Rotating biological contactor or biodisc is also an advanced system. Closely spaced discs
usually plastic are rotated in a trough containing the sewage (Figure 8). The discs are partially
submerged and become covered with microbial slime similar to trickling filter. Continuous
rotation of the discs keeps the slime well aerated and in contact with the sewage. Biofilm on
the discs sloughs off and is removed in the subsequent settling tank. Biodisc can be used to
treat both the domestic and industrial wastewater. It requires less space than trickling filters
and is more efficient and stable in operation but needs high initial capital. The activated
sludge process generates excess sludge which is stabilized in the anaerobic digester. The
excess sludge generated in the activated sludge process creates additional burden of treating

Figure 8: Rotating biological contactor

The membrane bioreactor avoids this excess sludge production and is compact. It is a
combination of activated sludge process and membrane technology (Figure 9). It consists of
suspended growth of biomass with micro or ultrafiltration membrane system which takes
place of the clarifier in the activated sludge process.

The turbidity and suspended solid concentration of the effluent is far lower than in the
conventional treatment. All biomass is retained and is then returned as sludge The sludge age
is 30-60 days which is an advantage. A high sludge concentration is attained upto 30 g/L
which allows much larger hydraulic loading rates. Disinfection of the sewage is also not
required since the membrane with pore openings generally in the 0.1– 0.5 mm range which
retain microbes.


Figure 9: Membrane bioreactor

Upflow anaerobic sludge blanket reactor is an advanced anaerobic reactor (Figure 10). It has
changed the idea that anaerobic process is slow, unreliable, requires high temperature and
removes only 50% BOD. In UASB reactor, a granular sludge with high biomass
concentration (50 g/L) can be attained allowing high volumetric loading rates. Waste in the
range of 0.3 – 100 g BOD
/L over a temperature range of 10-35°C can be treated reliably. In
UASB reactor, the wastewater enters the reactor from the bottom via a specially designed
influent distribution system and subsequently flows upwards through the granular blanket
consisting of anaerobic bacteria. The granules settle very well at the 60-80 m/h rate. The
mixture of sludge and biogas is separated in a three phase separator situated at the top of the

Main features of the UASB reactor are effective separation of biogas, a gas solid separator
device used for this separation and development of a granular settllable sludge. Even
distribution of wastewater in the reactor helps in the fast working. The organics come in
contact with the granulated sludge blanket and are degraded anaerobically. The succession of
bacteria in the granular sludge blanket is same as in the anaerobic digester. Gas bubbles
produced also help in mixing of the contents of the reactor. Gas collected in the gas collector
is methane. A good quality treated odorless sludge leaves the UASB reactor.

Figure 10: Upflow anaerobic sludge blanket reactor


Low Cost Sewage Treatment Plants
The low cost plants are particularly relevant to developing countries like India and include
oxidation pond, septic tank and biogas plant. Oxidation ponds are also called sewage lagoons
or stabilization ponds and are the oldest of the wastewater treatment systems (Figure 11).
They cover a hectare of area and are few metres deep. They are natural stewpots where
wastewater is detained while organic matter is degraded. One to four weeks time is taken for
the decomposition of the solids. Light, heat and settling of the solids will reduce the number
of the pathogens present in the wastewater. The oxidation ponds can be of four categories
aerobic, aerated, anaerobic and facultative. These often serve as a pre-treatment for high
BOD organic wastes rich in protein and fat with heavy concentration of suspended solids.
Facultative ponds are most common for domestic waste treatment. Waste treatment is
provided by both aerobic and anaerobic processes. The depth is 1-2.5 m and there are three
zones. The upper aerated zone, middle facultative zone and a lower anaerobic zone. The
detention time varies between 5 and 80 days. Mechanically aerated ponds may be 1-2 m deep
and have a detention time of less than 10 days. In general treatment depends on aeration
time, temperature as well as type of wastewaters.

Figure 11: Oxidation pond

The biodegradable organics and turbidity is not effectively removed as compared to the
activated sludge. Given sufficient retention time the oxidation ponds can cause significant
reduction in the concentration of enteric pathogens especially helminth eggs. They are
promoted for pathogen removal from wastewater used for irrigation in the developing
countries. Major drawback of the ponds is the potential for inadequate mixing or short
circuiting because of thermal gradients. The algal photosynthesis produces oxygen. The
aerobic heterotrophs proliferate in the facultative zone and degrade organics, along with the
production of CO
. CO
serves as carbon source for algae. The anaerobic bacteria at the
bottom in anaerobic zone grow at the expense of products of heterotrophs to release CH
, H
and N
in the atmosphere.

Septic tank is used by communities where population is less in rural area and ample land is
available (Figure12). Its operation is similar to the primary treatment. Sewage is taken to a
holding tank and suspended solids are allowed to settle. The sludge in the tank is pumped out
periodically and treated. It is slowly acted upon by anaerobic bacteria to organic acids and
S, while it is in the tank. The effluent flows through a series of buried perforated tubes, the
effluent percolates into the soil where the dissolved organic compounds in the effluent
undergo biodegradation. Septic tank treatment is not reliable for destroying intestinal


pathogens. It works well when not overloaded and the drainage system is proportionate to
the load and the soil type.

Heavy clay soils require extensive drainage system because of the soil’s poor permeability.
The high porosity of sandy soils can result in chemical or bacterial pollution of nearby
drinking water supply.

Figure 12: Septic tank

Biogas technology generates biogas or marsh gas as a byproduct of anaerobic decomposition
of organic matters. It is an alternative source of energy. Biogas can be used in small family
for cooking, heating and lighting and in larger institutions for power generation. The raw
material used for biogas generation is the waste material that includes human excreta, animal
manure, sewage sludge and vegetable crop residues. All of these are rich in the nutrients
suitable for growth of anaerobes.

Biogas comprises of methane 55-65%, CO
35-45%, N
0-3%, H
0-1% and H
S 0-1%.
Methane is most desirable since it has a high calorific value (~ 9000 Kcal/m
). The heat value
of biogas is 4500-6300 Kcal/m
depending on the contents of the other gases besides
methane. Sludge produced is odorless and is good fertilizer. Pathogens are reduced, rodents
and flies are not attracted. However, there are chances of explosion. Increase in the volume of
waste, may add to water pollution. Efficient use of methane requires removal of CO
S. Floating gas holder digester is designed by Khadi and village industries commission in
India (Figure 13). It consists of a cylindrical well, made of bricks. Gas produced is trapped in
a floating cover on the surface of the digester which rises and falls on a central guide. This
cover is made of steel, ferrocement, bamboo cement, plastic, fiberglass etc. Cover is a major
cause of loss of heat. The digester may be buried under the ground to prevent heat loss. The
gas holder moves semicontinuously through a straight inlet pipe and displaces an equal
amount of slurry through an outlet pipe. The digester in India is fed with cattle dung only.
Agricultural residues if used are chopped into small pieces. The design is simple to make
except the gas holder which needs a workshop for fabrication.

Water Microbiology
Study of microorganisms and their communities in water environment is called Aquatic
microbiology, while Water Microbiology relates to the study of microorganisms in potable
water. The scope of Aquatic Microbiology is wide and includes the habitats like planktons,


benthos, microbial mats and biofilm which may be found in lakes, rivers, streams, seas,
groundwater, rain, snow and hail. Planktons are the collection of free living and drifting
microorganisms in ponds, lakes and oceans. Algae and cyanobacteria are phytoplanktons
while protozoans and microbes are zooplanktons. The transition zone between the water
column and mineral subsurface is the benthic zone inhabited by anaerobes. Microbial mat is
interfacial acquatic habitat. Microbial groups are laterally compressed into these mats.
Cyanobacteria, sulfate reducing bacteria, nitrifiers, sulfur oxidizers and photosynthetic
bacteria form the layers. Biofilms are community of microbes embedded in an organic
polymer matrix adhering to a surface which is submerged in water. The freshwater
environment study is the scope of Limnology. Lakes, ponds and bogs are called Lentic or
standing habitat and running water like streams, rivers are called Lotic habitats. Brackish
water is the term used to describe the water the salt concentration or salinity of which lies
between 0-0.5% (of freshwater) and 32-37% (of salt lakes). The salt concentration in ocean
corresponds to 3.3 – 3.7 g %. Barotolerant, barophilic and psychrophilic bacteria occur in the
deep oceans. Legionella pneumophila, Aeromonas hydrophila, Vibrio spp. and Pseudomonas
aeruginosa are indigenous aquatic bacteria that cause diseases.

Figure 13: Biogas plant

Drinking Water Microbiology
Drinking or potable water is water that is free from pathogens and chemicals that are
dangerous to human health. Any taste, odor and color must be absent from the water to be
palatable. Raw water may contain many contaminants derived from sewage and nearby
industries. Many enteric pathogens are water borne. Therefore water is treated and disinfected
to remove chemicals and pathogens respectively. The raw waste is stored in reservoirs where
the oxidizable organic materials are stabilized and discrete particles settle. The collected
water or impounded water in the reservoir irrespective of its source contain sufficient
nutrients for growth of algae which require only minerals and sunlight. Many phototrophic
and chemolithotrophic bacteria grow in the dilute environment. Heteroptrophs flourish on the
organic matter of dead autotrophs. Some organic matter is introduced by wind, rain or soil
with runoff water. Bacterial activities cause transformation of iron, pH change, CO
mineralization of organics which may lead to corrosion of pipelines and thereby fouling of
water. Various algae, protozoans and iron bacteria impart bad taste and odours. Some


produce slime causing clogging of pipes. Iron bacteria include sheathed and stalked bacteria
that are typical water organisms. They are aerobic, widely distributed in nature esp. in
stagnant water such as reservoir for potable water supply. Siderocapsa, Sphaerotilus,
Clonothrix, Leptothrix,Crenothrix, Caulobacter and Gallionella are some common
filamentous iron bacteria. Sulfur and sulfate reducing bacteria also contribute to fouling of
impounded waters.

Water-Borne Diseases
An important aspect of Water Microbiology is numerous disease causing microorganisms
spread through water. Many bacteria, viruses, fungi and protozoa are responsible for
waterborne diseases. The recurrence of waterborne illness has led to the improvement in
water purification. Some common water borne diseases are listed in the Table 6.

Table 6: Common water borne pathogens

S. No.
Diseases caused
Diseases caused
Salmonella typhi
Hepatitis A virus
Salmonella spp
Polio virus
Shigella spp.
Diseases caused
Vibrio cholerae
Giardia intestinalis
Balantidium coli
Escherichia coli
Naegleria fowleri
. ira spp. Jaundice

Microbiological Examination of Water
Heterotrophic plate count (HPC) of more than 500 / ml in tap water indicates variation in
water quality and potential for pathogen survival. They also mask the coliforms and fecal
coliforms when present in high numbers. Bottled water and charcoal filters of household taps
have high HPC. Gram negative bacteria belonging to Pseudomonas, Aeromonas, Klebsiella,
Flavobacterium, Enterobacter, Citrobacter, Serratia, Acienetobacter, Proteus, Alcaligenes,
Enteroabcter and Moroxella are detected in HPC. Monitoring and detection of indictor and
disease causing microorganisms is a major part of Sanitary Microbiology. Intestinal tract
bacteria do not survive in the aquatic environment and are under physiological stress and


loose their ability to grow on selective media. Although many pathogens can be detected
directly in water, Environmental Microbiologists have generally used indicator or index
organisms as an indirect evidence of possible water contamination by human pathogens
which are considered to be of fecal origin. The criteria for an ideal indicator organism are 1]
It should be useful for all types of water 2] It should be present whenever enteric pathogens
are present 3] The organism should have reasonably longer survival time than the hardiest of
pathogens 4] The organism should not grow in water 5] The testing method should be easy to
perform 6] The density of indictor organisms should have direct relationship to the degree of
fecal pollution 7] The organism should be always found in intestinal microflora of warm
blooded animals.

Conventionally coliforms have been used as indicator organisms of fecal pollution. Various
indicator organisms are now considered for their different attributes and usefulness as no
organism satisfies all the above criteria. Among the various indicators are coliforms, fecal
streptococci, Clostridium perfringens, Bifidobacterium and Bacteroides, phages of enteric
bacteria, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and
Aeromonas hydrophila. Coliforms are defined as facultatively anaerobic Gram negative, non-
spore forming, short rod-shaped bacteria that produce acid and gas on lactose fermentation in
prescribed culture medium within 48h at 35º C. The group includes Escherichia, Citrobacter,
Enterobacter and Klebsiella. Escherichia coli and Klebsiella pneumoniae are the important
coliforms. E. coli is a natural inhabitant of the intestine and K. pneumoniae is that of soil. The
test for coliforms involves presumptive, confirmed and completed tests (Figure 14). The
presumptive test is clubbed with the multiple tube dilution technique which gives an estimate
of most probable number (MPN) of coliforms in 100 ml of water sample and is also called
MPN test. In MPN test different sample volumes are inoculated in lactose broth or
McConkey broth at 35ºC for 48h incubation. The test is based on the principle that a single
living cell can develop into a turbid culture. By determining the average dilution at which the
tubes do not receive cells, the number of microorganisms most probably present in the
original sample can be computed using the MPN table. In the confirmed test typical greenish
metallic sheen colonies on EMB indicate fecal coliforms whereas non-fecal coliforms give
mucoid pink colonies with dark centers called atypical colonies. The completed test involves
confirmation of coliforms. Gram negative non-spore forming, lactose fermenting (within 48h,
rod shaped bacteria indicate fecal coliforms.

IMViC includes Indole production (I), Methyl red test (MR), Voges Proskauer (VP) and
Citrate utilization (C) tests and helps to distinguish and finally confirm the fecal and nonfecal
coliforms. E.coli of fecal origin gives the MR and I positive and Klebsiella pneumoniae of
non-fecal origin gives the VP and C positive

Membrane filter technique (MF) is another useful test that consists of passing the water
through a membrane filter. The filter with bacteria is transferred to the surface of a solid
medium or to an absorptive pad containing the desired liquid medium. Use of appropriate
media helps in the detection of total, fecal and non-fecal coliforms. A resuscitation medium
may be used to revive injured or stressed coliforms due to chlorination etc. The MF technique
gives good reproducibility and the results are obtained in one step, filters can be transferred
between different media, large volumes of sample can be processed, is time saving, can be
performed on site and bears low cost. While the test is not suitable for high turbidity samples,
other bacteria or metals and chemicals adsorb on the filter and may inhibit the growth of


Figure14: Scheme of microbiological examination of water

The presen/absence test (PA) is more simplified test for detecting coliforms and fecal
coliforms in which 100 ml of water sample in incubated in a single culture flask with a triple
strength broth containing lactose broth or lauryl tryptose broth. This test is based on
assumption that coliforms should be present in 100 ml of drinking water. A positive test
needs confirmation.

Colilert defined substrate test is used to differentiate the E.coli and coliforms. A 100 ml
water sample is added in a specialized medium containing o-nitropheyl β-D-
galactopyranoside (ONPG) and 4-methylumbelliferyl β-D-glucoronide (MUG) as the only
nutrients. If coliforms are present they produce β-galactoside and act on ONPG to release o-
nitrophenol, which is yellow. E. coli elaborates glucuronidase that acts on MUG the product
of which fluoresces in UV. The test is obtained in 24 h.

The regrowth of coliforms in water after disinfection is the biggest drawback of coliforms as
indicator. Fecal streptococci (FS) are group of Gram positive Lancefield group D
streptococci, grow in 6.5% NaCl, pH 9 and at 45ºC. The advantages of FS over coliforms as
indicator is that they do not regrow in water, are more resistant to environmental stress and
chlorination and generally persist longer in the environment. They suggest risk of
gastroenteritis for recreational waters and presence of enteric viruses. Streptococcus bovis
and S. equinus are found in animals while Enterococcus faecalis (S. faecalis) and E. faecium
are specific to human gut. A FC/FS i.e. fecal coliforms to fecal streptococci ratio of four


or more indicates a contamination of human origin whereas a ratio of 0.7 indicates animal
fecal pollution. MPN and MF test are also used for FS. Many other organisms are considered
as indicators for various reasons (Table 7). Phages due their presence in sewage and
similarity with enteric viruses are considered.

Table 7: Other indicator organisms

anaerobic spore former, gram
positive rod shaped and
exclusively of fecal origin. Spores
are resistant and persist for long
useful indicator of past pollution, a
tracer for less hardy indicators,
protozoans and viruses.
and Bacteroids
primarily associated with humans
they can distinguish human and
animal contamination.
B. bifidus survives for a short time
therefore its presence suggests
relatively recent pollution
F-specific RNA
phage, f2,
φx174, MS2,
Coliphages, not always seen
associated with fecal pollution
however their presence in high
numbers in wastewater and high
resistance to chlorination can be an
index of wastewater contamination
and indicators of enteric viruses.
useful for evaluation of virus
resistance to disinfectants, fate of
enteric viruses in water treatment
and surface or groundwater tracers
and presence of host.
Phages of
of human origin exclusively
An advantage over coliphage is
they help to detect human fecal
contamination.They do not
multiply in the water and have
decay rate similar to other viruses.
associated with the diseases of eye,
ear, nose and throat infections.
common opportunistic pathogen,
causes life threatening infection in
urn patients and
immunocompromised individuals.
Folliculitis, dermatitis, ear and
urinary infections are common in
ill maintained swimming pools.
this organism is of no value as
indicator of fecal pollution
however coliforms do not suit as
indictor of contamination of
swimming pool water as the
contamination is not of fecal origin.
aureus and
suggests the sanitary quality of
water because it presence is
associated with human activities
Useful for recreational waters.
occurs in uncontaminated, as well
as contaminated waters. also an
opportunistic pathogen in humans,
animals and fish.
Because of its association with
nutrient rich conditions it has been
suggested as an indicator of
nutrient rich status of the waters.


Water Purification
Water purification forms a critical link in promoting public health and safety. It involves
variety of steps which depend upon the type of impurities in the raw water source. The major
operations done are sedimentation, flocculation, filteration and disinfection. Raw water
becomes potable after this treatment (Figure 15). Impurities in raw water include suspended,
dissolved, colloidal solids; bacteria; toxic substances; color; odor and mineral or organic
matter. These can be categorized as chemical, physical and microbiological. Table 8 indicates
the drinking water standards in India. Different unit processes and operations are performed
to remove different impurities (Table 9).

Figure15: Sequence of processes in water purification

Table 8: The Bureau of Indian Standards defined levels of substances in water and their
permissible levels

S. No.
Substance / Test
Desirable limit
Maximum permissible limit*
Physical turbidity
Chemical pH
6.5 – 8.5
No relaxation
as (CaCO
) mg/l
as Cl mg/l
as Fe mg/l
as N mg/;
No relaxation
as F mg/l
Residual chlorine
0.2 – 0.5
No relaxation
as As mg/l
No relaxation
MPN/100 ml
No relaxation
E. coli
MPN/100 ml
No relaxation

* Where there is no alternative source for drinking
** Coliform should not be detected in 100 ml of any two consecutive samples


Table 9: Unit processes and operations and specific impurities removed

S. No.
Unit Processes / operations
Aeration, chemical oxidation, ion exchange,
Colour and precipitate
Chemical precipitation, (dosing, mixing,
flocculation, settling) ion exchange
Softening (Ca, Mg
Chemical coagulation, (dosing, mixing,
flocculation, settling) filtration
Turbidity removal
Aeration, chemical oxidation, adsorption
Taste and odour
Irradiation, ozonation, chlorination

Sedimentation is separation of suspended particles by natural aggregation and gravitational
settling carried out in sedimentation tank or settling basin. Some degree of sedimentation
occurs during storage. Quiescent sedimentation for a period of 30-60 days may result in
purification equivalent to filtration. Bacteria and viruses still persist. Sedimentation is
combined with coagulation. Coagulation brings about destabilization and agglomeration of
the particles. Metal salts like aluminium sulfate, ferric chloride, calcium oxide or hydroxide
are used as coagulants, metal hydroxide form precipitates in which the colloidal particles get
enmeshed and settle along with them. In flocculation the agglomeration of the destabilized
particles is induced by mechanical means into compact fast settlable particles called flocs.
The aluminium sulfate added reacts with the natural alkalinity present in water to form flocs.
Betonite, clay, activated silica etc. aid the coagulants. These agents bear negative charge
when present with positively charged metal hydroxide gives a tough dense flocs thereby
hastening flocculation. Polyelectrolytes which have ionizable carboxyl, amino, sulfonic
groups are synthetic coagulants. The flocs are allowed to settle and the supernatant is taken
for filtration.

Filtration is a process of separation of suspended matter from water by passing it through
porous medium in this case a filter. The filter is filled with media which may be sand,
crushed anthracite, coal, diatomaceous earth, activated carbon, plastic spheres, rings or metals
fabrics. The gravity filters namely slow sand and rapid sand filters are currently used. In slow
sand filters there is a slow passage of water through a bed of sand in which a microbial layers
covers the surface of each sand grain (Figure 16). It is a biological process. Wastewater-borne
microbes are removed by adhesion to the slimy microbial layer or biofilm Cryptosporidium,
Giardia and Cyclospora are protozoans which have cysts that are removed by slow sand
filter. Virus levels after filtration goes to 90-99% aided by chemical oxidants, high pH,
photooxidation. However none of the processes remove the viruses completely. The slow
sand filter is a large sand and gravel bed on acre or more in area of land build up over drain
pipes. Coarse gravel (5 mm dia) is at the bottom graduating in size to the top layer of fine
sand (0.25 – 0.35 mm dia). About 5 million gallons per day of water is filtered. The organic
chemical content is reduced due to biochemical oxidation by the biofilm bacteria. When the
biofilm thickens the rate of filtration goes down. It is then stopped and the biomass removed
normally before refilling the filter.


Figure 16: Slow sand filtration

About 40% color reduction is observed. Slow sand filter is simple to construct and operate. It
gives uniform water quality and effectively removes bacteria. The drawbacks are that it
requires large area and turbid waters more than 30-50 ppm cannot be filtered. Since it is
biological process, seasonal variation in operation is observed. The rapid sand filter depends
on physical trapping of particles and flocs and operates on mechanical basis (Figure 17). It
takes less area and no biofilm formation occurs.

Figure 17: Rapid sand filter

The water after coagulation and flocculation is passed through rapid sand filter. As the name
suggests it gives about 200 million gallons water per day. Therefore it is more commonly
used. The size of sand is 0.35 – 0.5 mm dia. which is larger than the sand grains in slow sand
filter. There is an underdrainage over which sand bed is laid. This collects the filtered water.
The arrangement of backwash by reversing the flow of cleaned water and bubbling air
through the sand bed helps to clean the sand particles when the filtration rate goes down. The
wash water is wasted. The rapid sand filter therefore can accommodate relatively more turbid
water than slow sand filter. It can also be operated continuously and requires less
maintenance. Though requirement of skilled personnel, less effective bacteria removal and


more operational troubles are its negative aspects. Disinfection is imperative after all
filtration processes.

Modern water purification facilities make use of membrane filtration. This is an alternative to
distillation. It recovers large quantity of water from dilute solutions of minerals or pollutants
as is found in sea or brackish water. This assumes importance in reclamation of domestic or
industrial effluents in the event of water scarcity. The reverse osmosis process provides a
solution to this problem. It requires low energy and is highly selective in the removal of
dissolved solids. Osmosis is a process of selective transport of aqueous solutions through
semipermeable membrane. The principle of desalination by reverse osmosis is essentially the
same as that of the osmotic process except that the process is reversed. The influent water is
forced through a semipermeable cellulose acetate membrane under high pressure upto or
greater than 1500 psi. Usually 40 psi for tap water and 1500 psi for sea water is applied. The
membrane allows the water to pass through excluding majority of the dissolved solids. It may
exclude 90-95% of sodium chloride in salt water while most other salts are excluded to a
greater extent e.g. 99-99.7% CaSO
. Discrete and particulate organic material, proteins,
bacteria and viruses are excluded to an even greater extent. The process is also used in
tertiary sewage treatment. Softening of water is needed when the water has hardness.
Sedimentation and filtration remove suspended and colloidal solids from raw water. The
dissolved alkaline minerals like carbonates and bicarbonate when associated with sodium and
potassium cause hardness. Hardness in water comes from nearby mining operations or acidic
industrial wastes. Removal of minerals from water is known as demineralization process.
Treatment with lime, lime-soda, boiling and ion exchange are methods used to remove
minerals. Disinfection refers to reduction of bacterial population to a safe level as opposed to
sterilization. As a result of disinfection bacterial, protozoal and viral diseases have been
curbed. Chlorination is the least expensive method and easy to administer. Chlorine is a
strong oxidizing agent which when added to water as a gas forms hypochlorous and
hydrochloric acid. Cl
OHCl+HOCl (hydrochlorous acid). The HOCL is very unstable
and decomposes quickly by releasing nascent oxygen which is a strong oxidizing agent HOCl
 HCL + (O). The action of this nascent oxygen on cellular components is indiscriminate. It
oxidizes protein and reversibly binds to –SH groups and denatures essential cellular enzymes,
alters permeability of cell membrane, interferes with membrane function, and denatures
nucleic acids. Chlorine in the form of HOCl is called the free available chlorine while when it
is combined with ammonia and nitrogen containing organic substances it forms combined
chlorine. The reactions of chlorine and ammonia are of importance in water disinfection.
These form monochlramines ( NH
+ HOCl  NH
Cl + H
O ), dichloramines (NH
Cl +
+ H
) and trichloramines (NHCl
+ HOCl  NCl
+ H
O). These products
have disinfecting power of HOCl but much less at a given concentration than chlorine. 1
mg/L (1 ppm) chlorine for 30 min. significantly reduces the bacterial numbers. Presence of
interfering substances like NH
increase the required chlorine dose due to formation of
compounds with lesser disinfection power. 30 ppm chlorine inactivate enteric viruses and
destroy protozoans. Chloramines are much less effective in the inactivation of viruses.
Chlorine affects the protein capsid and interacts with nucleic acid, 0.8 ppm chlorine
inactivates the poliovirus RNA. For the double stranded RNA rotavirus the coat is the target.
A concern with chlorination is the formation of disinfection byproducts (DBPs) such as
halomethanes, a group of compounds that may be carcinogenic are formed when chlorine
reacts with organic matter. Another factor that affects chlorination is temperature of water
(higher the temperature more the dissolved chlorine). Presence of reduced ions or compounds
such as nitrite, H
S, Mn, Fe etc. lessen the disinfecting power of chlorine by getting oxidized
themselves. Taking this into consideration, enough chlorine must be added to leave a residual
of 0.2 – 1 ppm of free chlorine after all microorganisms and extraneous organic matter have


been saturated with chlorine called breakpoint chlorination (Figure 18). Formation of
chloramines is dependent upon the ratio of chlorine to ammonia, chlorine dose, temperature
and pH. Upto chlorine to ammonia mass ratio of 5 the predominant product is
monochlramine which has the best disinfection power out of the three mono, di and
trichloramines. Since they are slow acting they have been used as secondary disinfectants
when a residual in the distribution system is desired. Usually chloramines are added after
ozonation since the latter does not leave any residuals. Otherwise the bacteria grow in the
pipelines and develop into biofilms. Chloramines inactivate the –SH containing enzymes and
to a lesser extent react with nucleic acid to kill the cells. Viruses are inactivated by
chloramines by reacting with nucleic acid.

Figure 18: Breakpoint chlorination

Ozonation is a powerful oxidizing process in water disinfection. Ozone (O
) is produced by
passing electric discharge through a stream of air or oxygen. It is more expensive than
chlorination. The advantage is that it does not produce trihalomethane or DBPs. Aldehydes or
bromates formed by ozone however may have adverse health effects. Its effectiveness is not
influenced by pH or NH
. It is more powerful than chlorine. The c.t value for ozone for 99%
inactivation of bacteria is 0.0011-0.2 and for viruses it is 0.04-0.42. c.t value is the product of
the concentration of the disinfectant and the time required to inactivate a certain percentage
of population under certain conditions of pH and temperature. Mechanism of disinfection of
ozone is similar to chlorine.

Heavy metal ions such as Cu, Ag, Zn, Pb, Cd, Ni and Co exhibit what is called their
oligodynamic action against bacteria. Cu and Ag are used in water treatment. Cu helps to
control Legionella in hospital disinfection system. Silver is used as a bacteriostatic agent. It is
added to activated carbon in faucet mounted devices for home use. The antimicrobial range
for copper is 200-400 g/L while that of silver is 40-90 µg/L. Unlike other disinfectants metals
remain active for long time in water. Their action is enhanced in the presence of low
concentration of oxidizing agents due to synergistic action. The inactivation of
macromolecules (protein and nucleic acids) is due to site specific Fenton mechanism. The
metal ion binds to the biological target and is reduced by superoxide radicals or other
reductants and subsequently reoxidized by H
generating hydroxide radicals. Repeated


cyclic redox reaction may result in multihit damage as radical formation occurs near the
target site. Cu and Ag may also bind to enzymes interfering in their normal function.

UV disinfection is now increasingly used in water and wastewater because it does not produce
any toxic byproducts (potential carcinogens), taste or odor. The need of storage is avoided.
But the high cost, no residual disinfectant, difficulty in standardizing UV dose, maintenance
and cleaning of UV lamp and potential reactivation of some enteric bacteria after UV
treatment are some of the disadvantages of this method. Nevertheless, low cost, more
efficient lamps and reliable equipments available makes UV more attractive. It is used in
pharmaceuticals, cosmetic, beverage and electronics industries. Cell clumping, suspended
solids, turbidity, presence of humic substances, phenolic compounds, lignin sulfonates and
ferric ion affect UV transmission in water. Thus filtration is necessary before UV
disinfection. The UV resistance of double stranded DNA viruses > MS2 phage> coliphage >
bacterial spores > double stranded RNA enteric viruses > single stranded RNA enteric viruses
> vegetative bacteria. The radiation of 260 nm is the most lethal for microbes since nucleic
acids absorbs at this wavelength. Figure 19 shows a typical UV germicidal lamp. Total and
fecal coliforms are capable of photoactivation but fecal streptococci are not. Ionising
radiations viz. gamma radiations are also used as in wastewater treatment for disinfection.

Figure 19: Ultraviolet germicidal lamp

Air Microbiology
Aerobiology is defined as the study of life present in the air. Aeromicrobiology relates to the
study of environmentally relevant microorganisms. Intramural Aerobiology deals with indoor
environment while Extramural Aerobiology deals with outdoor environment. No organism is
indigenous to the atmosphere. Microorganisms exist within 300-1000 feet of earth’s surface
that have become attached to fragments of dried leaves, straw or dust particles light enough to
be blown by wind. In dry whether the microbial load of air is high while in wet weather the
rain washes the microorganisms from the air. Air is not a medium in which microorganisms
grow, but it is a carrier of dust and droplets that may be laden with microorganisms. Large
droplets settle out quickly while the droplet nuclei remain afloat. The spore formers and cyst
formers are likely to survive better in the atmosphere for longer period. Depending upon the
type and the climatic conditions the persistence of microorganisms is observed. The
microorganisms come into the air via both land and water. Wind creates dust laden with
microbes. From the ocean surface water droplets laden with microbes arise. Various
agricultural, industrial and municipal processing facilities have the potential for generating


microbe laden aerosols. The irrigation sprinkler, grain thrashing, sewage treatment facility,
abattoirs etc. can serve as sources.

Airborne Diseases
Airborne diseases are caused by hardy microorganisms and include diseases of plants,
animals and human. The impact of plant pathogens esp. fungi on agricultural economy is
enormous. Infections of pet and livestock by airborne pathogens is also significant as are the
diseases in humans . The kinds of pathogenic microorganisms present in the atmosphere
associated with humans are viruses, protozoa, molds and bacteria (Table10). Table 11
summarises the wide adverse effects on humans by contaminants present in the air which also
includes the bacterial and fungal toxins. Air pollution by exhausts from industries is another
matter. The consequences of industrial air pollution are irritation of skin, eyes and respiratory
tract, thereby posing health hazards. Toxins of Clostridium botulinum is a potential biological
warfare agent. LPS, endotoxins derived from gram negative bacteria are another example of
air borne toxins. Bacillus anthracis has been used effectively in germ or biological warfare. A
sophisticated training and machinery is required to tackle these agents.

Table 10: Air-borne human diseases of importance and their causative agent

Streptococcus pyogenes
Sore throat
Influenza virus
Hepatitis virus
Streptococcus pneumoniae
Herpes virus
Chicken pox
Klebsiella pneumoniae
atypical pneumonia
Picorna virus
Neisseria meningitidis
Dengue fever
Yersinia pestis
Bubonic plaque
Rubella virus
Bordetella pertussis
Whooping cough
Measles virus
Haemophilus influenzae
Influenza virus
Nocardia asteroids

Aspergillus fumigatus

Blastomyces dermatiridi

Coccidioides immitis

Cryptococcus neoformans

Histoplasma capsulatum

Pneumocystis carinii


Table 11: Adverse effects on humans and environment associated with exposure to
airborne microorganisms

S. No.
Hypersensitivity, respiratory
Plant diseases, odour
Allergy, skin problems,
respiratory infections
Deterioration of building
materials, odour, plant diseases
Cough, headache, respiratory
Cough, headache, respiratory
Disease in livestock
Encephalitis, hypersensitivity
Protect other bacteria, disease to
Crop and livestock disease

Fate and Transport of Microorganisms in Air
The atmosphere is inhospitable place for microbes because of stress due to environmental
factors like dessication, temperature, relative humidity, radiation and oxygen. The majority
of airborne microorganisms are immediately inactivated as the result of this. Those hardy
microbes that can survive these factors are able to cause the diseases. Oxygen stress relates to
the reactive oxygen species. Another term open air factor (OAF) in this connection describes
the environmental effect that cannot be replicated in the laboratory settings. When ozone and
hydrocarbons (usually ethylene) alone with the oxygen act together the inactivation rates of
microbes is greatly affected. Microorganisms like fungi cannot survive and complete their
life cycles by staying afloat in air for an indefinite period of time. Bioaerosols in the air
harbor the microbes. They vary in size. Large droplets in the air are of 100 µ and like dust
settle rapidly in quiet air. Small droplets on the other hand in warm, dry atmosphere dry
quickly to form droplet nuclei. These are of 2-5 µm size, light and may float about for many
minutes or even hours. The microorganisms in them are protected by dried mucus which
coats them. Being small these escape the mechanical traps of the upper respiratory tract and
enter the lungs. They may settle on the alveolar tissue. Transmission of airborne pathogens
usually occurs via droplet nuclei. The aeromicrobiological pathway (AMB) describes the
launching of the aerosols in the air, the subsequent transport via diffusion and dispersion, and
finally their deposition e.g. droplet nuclei having influenza virus which is launched via
coughing, sneezing or taking in the air.

Microbological Analysis of Air
Many sampling devises are available for air sampling. The impingement, bubbling,
atomizing, electrostatic membrane filter devices etc. are some of the devices used for
collection of air. The choice rests on the availability, cost, air volume, mobility, sampling
efficiency and overall efficacy of the device for sample microorganisms. On the basis of their


sampling method, several type of samplers using impingement, centrifugation, filtration and
deposition are available. Deposition is easiest and most cost effective method of sampling. It
can be accomplished by merely opening an agar plate and exposing it to air which results in
direct impaction, gravity settling and other depositional forces. This has quite low sampling

Impingement is the trapping of airborne particles in liquid matrix. Impingement device
operates by drawing air through an inlet that is similar in shape to the human nasal passage.
Anderson sampler is multilevel, multiorifice, cascade sampler which is commonly used
(Figure 20). The air that is sucked through the sampling port strikes agar plates. Larger
particles are collected on the first layer and each successive stage collects smaller and smaller
sized particles.

Figure 20: Anderson sampler

Filtration is the trapping of airborne particles by size exclusion and deposition is the
collection of airborne particles using only naturally occurring deposition forces. Filtration and
deposition are widely used for cost and portability reasons. The filtration is used for
lipopolysaccharide airborne levels. Limulus amoebocyte lysate assay involves the
amoebocyte lysate obtained from blood cells of Limulus a horseshoe crab. It contains an
enzyme linked coagulation system which is activated by the presence of LPS. Centrifugal
sampler uses circular flow patterns to increase the gravitational pull within the sampling
device in order to deposit particles (Figure 21). The cyclone, a tangential inlet and return flow
sampling device is the most common type. As a result of increased centrifugal forces
imposed on particles in the airstream the particles are sedimented out. Liquid medium is used
to trap the microbes. Its efficiency is low, but it is inexpensive, easily sterilized and portable.
Traditionally culturable and microscopic methods have been used. Now immunoassay
method is used extensively in biomedical research for bioaerosol analysis. Fluorescence
immunoassay and radioimmunoassay are used for allergens such as dust mite allergen and
animal dander. Biochemical assays are used to measure endotoxins or mycotoxins. Molecular
techniques like PCR has enhanced the detection of microorganisms in variety of matrices.


Figure 21: Centrifugal sampler

Control of Airborne Microorganisms
Thorough dilution of contaminated air by ventilation is a very effective means of controlling
airborne diseases indoors. However, it is expensive since special ducts or blowers need to be
installed. Disinfection or more rarely sterilization of air is desirable. Three general methods
used for control of microorganisms of indoor air are radiation, bactericidal vapors and dust
control. Irradiation with ultraviolet light of the 254 nm wavelength is sufficiently
microbicidal at the same time not irritating. UV lamps are attached at strategic points
overhead. Deflectors are provided to prevent direct exposure of persons to UV rays which
may be permanently injured. Many substances are lethal to microorganisms in the vapor
phase. These are formaldehyde, ethylene oxide, β propiolactone which are used as
bactericidal vapor. Probably the most effective are propylene glycol and triethylene glycol.
These are odorless, tasteless, nonirritating, nontoxic, noncorrosive and nonexplosive. They
are highly effective in killing bacteria in the air although ineffective in the form of
concentrated aqueous solutions. As little as 0.5 mg/L of propylene glycol vapor for 15
seconds can sterilize heavily contaminated air. Other agents like orthophenylphenol etc are
used for surface applications. Dust control is also good means of keeping microorganisms out
of the air especially indoors or intramural air, as droplet nuclei remain afloat for long time in
the air. In hospital wards dust from clothing or bedding can be a means of disease
transmission. Floors and sweepings are oiled to control dust. Microbiological laboratories
carrying out research with pathogens and recombinant microbes, generate aerosols via
centrifuges, vortex etc. Special equipments e.g. the biosafety cabinets and other containment
devices are designed to control spread of airborne microbes. Four biosafety levels (1-4) as
described in Table 12 are generally available depending on the type of research conducted
and the laboratories are called biosafety laboratories. The levels show increasing stringency
as the nature of work becomes increasingly dangerous.


Table 12: Levels of biohazard control

Biosafety level 1
work with well characterized agents with standard microbiological
techniques not associated with disease in healthy adult humans are
handled, only general restrictions are placed on public access e.g. teaching
Biosafety level 2

work with agents that are of moderate hazard to humans and the
environment , personnel have specialized training in the handling of
pathogens access to the work areas is limited.
Biosafety level 3
laboratories where agents cause serious or fatal disease due to AMB
exposure are handled, personnel are specifically trained to handle
pathogens, procedures conducted in biological safety cabinets and other
containment devices, facilities have permanent locks to control access,
negative airflow, and filtered ventilation in order to protect the public and
the environments, safety hoods used and clothes must be changed before
leaving the premises.
Biosafety level 4
highest level, for organisms causing life threatening disease in association
with aerosolization, personnel with highly specialized training, the
laboratories are 100% isolated from other buildings, positive pressure
ventilation provided to prevent spread of microorganisms into the
environment, laboratories with complete containment, personnel required
to wear specialized clothing which is removed and sterilized before
leaving the containment areas, personnel are required to shower before
leaving , all air in and out of these laboratories is sterilized by filtration
and germicidal treatment.

Food and Dairy Microbiology
The modern human diet includes a wide variety of foods including fruits, vegetables, meat,
milk etc. Raw food if properly handled and processed should contain low levels of
microorganisms. Different foods provide different conditions for microbial growth and thus
differ in their microbial contents. Food Microbiology encompasses the study of
microorganisms causing detrimental as well as beneficial effects in food. Microbial
Biotechnology is a modern adjunct of the traditional Food and Industrial Microbiology.

Microorganisms in Food
Various kinds of microorganisms are associated with food. Microorganisms in fruits and
vegetables depends on the post harvest handling. Fungi are predominant organisms associated
with low acid food while the medium acid kinds have bacteria associated with them. Milk as
soon as it is drawn from the cow has low microbial count. The milking equipment, the cow,
personnel, air, lack of sanitary practices contaminate the milk. Refrigerated meat has low
counts. Chopping and grinding increases the numbers as new surfaces are introduced.
Microflora in the poultry are introduced during killing, defeathering and evisceration. Eggs
are free of microorganisms due to the hard shell and the underlying thin membrane. Cracks in
the shell however will result in contamination.


Beneficial Effects of Microorganisms in Food
The effect of microorganisms on food can be classified as Beneficial effects e.g. fermented
foods, microbes as food and genetically modified foods; Neutral effects and Detrimental
effects e.g. food spoilages and food poisonings & infections. Food fermentations generate
organic compounds and transform foods into those with more desirable characteristics like
enhancement of flavor, texture, digestibility etc. Cheese is an ancient fermented food
involving a significant role of microorganisms in its preparation. Different starter cultures
with variable conditions result in unique flavor and texture characteristic of cheese. Lactic
acid bacteria are used as starter culture. Casein, the milk protein is destabilized by enzymes
and heat in presence of calcium in the process of curdling. The κ-casein portion of casein has
stabilizing effect. It is converted to para-casein by the action of rennet which cleaves phe-met
(105-106) peptide bond. Rennet has the desirable high clotting to proteolysis ratio as
compared to other similar enzymes. The C:P of rennet (chymosin) > Mucor rennet>
endothecia rennet > bovine pepsin. The excessive proteolysis of curd causes bitterness in
cheese. Solids after curdling are separated and formed into cubes for unripened cottage
cheese or paneer. In cream cheese preparation the starting material is cream. In the ripened
cheeses selected bacterial and fungal species under specific conditions of time, temperature
and humidity are encouraged to grow.

Ripening gives the characteristic flavor that is end result of release of many compounds due
to the enzymes released by the ripening agents. Thus every cheese uniquely has a typical
taste, flavor, texture and appearance. Another classification of cheese is based on the specific
amount of moisture removed from the curd with the help of salt. The soft cheese has 50-58%,
semi-hard 45% and hard cheeses have < 40% moisture (Table 13).

Apart from cheese many fermented dairy products with enhanced nutritive and probiotic
values are consumed world over (Table 14) The probiotic effects of these products include
many beneficial effect. It modifies microbial flora in lower intestine, have antimicrobial
activity, minimizes lactose tolerance, lowers serum cholesterol, have anticancer activity,
promote calcium absorption and synthezise B complex vitamins.

Bread is also an ancient food. Baker’s yeast Saccharomyces cerevisiae is added to dough for
leavening which causes the dough to rise. The maltase, invertase and amylase of yeast acts on
starch to produce sugars. Further fermentation of sugars is done to produce minimum alcohol
and majorly CO
. Alcohol imparts flavor while CO
causes the rise of dough giving it a
texture. The dough is conditioned during fermentation by protease action on the flour
producing gluten. Gluten makes the dough elastic and retains the CO
. The leavening is done
for 2h at 27°C. This follows baking at 100°C which kills the yeast, inactivates the enzymes,
evaporates alcohol and expands the gas. Gelatinisation of starch due to action of enzymes
earlier results in setting of the bread. The structural support to dough is given by the gluten
while in the baked bread it is given by gelatinized starch. In rye bread, yeast like Torulopsis
holmii, hetero-fermentative (those producing along with lactic acid other fermentation
products) Lactobacillus spp. L. plantarum, L. brevis, L. bulgaricus, Leuconostoc
mesenteroides and Streptococcus thermophilus are used.


Table13: Major types of cheeses and microorganisms involved

Type of cheese
Microorganisms in
early stage
Microorganisms in late
Soft, unripened
Cottage cheese

Mozzarella (Italy)

Paneer (India)

Lactococcus lactis ,
Leuconostoc cremoris

All are unripened
Soft, ripened
Brie (France)

Camembert (France)

Lactococcus lactis,

L . lactis, L .cremoris

Penicillium camemberti
P. candidum,
Brevibacterium linens
P.camemberti, B.linens
Limburger (Belgium)
Monterey (US)
Muenster (US)
Roquefort (France)

L.lactis, L. cremoris
L.lactis, L. cremoris
L.lactis, L. cremoris
L.lactis, L. cremoris
L.lactis, L. cremoris
L.lactis, L. cremoris

B linens
B linens
B linens
Hard, ripened
Cheddar (UK)

Colby (US)

Edam (Netherlands)
Gouda (Netherlands)
Swiss (Switzerland)

Enterobacter durans
Enterobacter durans
L.cremoris, L.lactis
L.cremoris, L.
L. lactis, L.helveticus

L.casei ,L. plantarum


Very hard, ripened
Parmesan (Italy)

L.lactis, L.cremoris
S. thermophilus

Lactobacillus bulgaricus


Table 14: Fermented milk products

Starter culture
Streptococcus thermophilus, Lactobacillus bulgaricus
Cultured buttermilk
S.lactis, S.crenoris, Leuconostoc citrovorous or L.dextranicurn
Bulgarian milk
Acidophilus milk
Kefir and Kumiss
S.lactis, L.bulgaricus, lactose fermenting yeast

Alcoholic beverages are produced by special strains of Saccharomyces cerevisiae carrying
out alcoholic fermentation in anaerobic conditions. Wine, Beer and distilled liquor are
included in alcoholic beverages. Wine is fermented grape juice or juice from other fruits. The
grapes are crushed to form juice called must. Special strains of Saccharomyces cerevisiae
called wine yeast is used as inoculum. The conditions for fermentation include 24-27°C
temperature for 3-5 days for red wine (from blue grapes) and 10-21°C for 7-14 days for
white wines (from green grapes). Wines are called dry when no residual sugar is present.
Sweet wines have residual sugar while sparkling wine has CO
imbibed in it.. Anthocyanin in
the skin of grapes gives it a red color. The wine is racked subsequent to fermentation.
Racking filters the wine. Generally wines are not distilled. Their spoilage is controlled by
pasteurization. Enzymes like pectinases are used to clarify the wine before pasteurization.
Beer and ale are called malt beverages because the substrate involves barley malt. Barley is
malted by germination which releases mixture of amylases and proteinases. Malt adjuncts
like corn, rice and wheat are added followed by mashing. The amylases act on carbohydrates
and proteins of the adjunct. The mash is cooked at 70°C facilitating rapid starch hydrolysis.
The insoluble material settles giving a clear liquid called wort. Hops that are dried flowers of
Humulus lupulus or hop plant are added and the mixture is cooked. This concentrates the
wort, inactivates the enzymes, kills microorganisms and extracts soluble flavoring
compounds from the hops. Resins and humulone have antibacterial property that protects the
wort against Gram positive bacteria. The next step is the actual batch fermentation using
special strains of Saccharomyces cereivisae called Brewer’s yeast. The S. cerevisiae is top
fermenting yeast since it flocculates while S. carlsbergensis is called bottom fermenting
since it settles down at appropriate time, partially clarifying the beer. Inoculation of wort is
called pitching. After initial aeration the fermentation is allowed to become anaerobic. Small
amount of glycerol and acetic acid are formed. Higher alcohols, acids and esters also
contribute to flavor. The resulting product is called green beer. This needs aging to achieve
the flavor, taste and aroma of the finished beer. During aging precipitation of proteins, yeast
and resins occur mellowing the beer. Mature beer is filtered and carbonated to achieve a CO

content of 0.42-0.52%. In commercial process the CO
is collected during fermentation and
reinjected. The alcohol contents of beer ranges from 3.6-8%. Light beer (low calorie), ale and
sake (rice beer) are the variations of beer. Like wine, pasteurization prevents spoilage of beer.
Distilled liquors or spirits are high alcohol containing hard liquors. The general types of
distilled liquors include brandy from fermented fruit juices, rum from fermented molasses
and whisky from fermented mash of mixed grains. Distiller’s strains of S. cereivisiae
are used. Microorganisms contribute significantly to improve fruits vegetables and beans
after fermentation (Table 15).


Table 15: Fermented fruits, vegetables and beans

Process / Microorganisms
Sufu (Chinese)
Tofu-coagulated soybean fermented by Actinomucor elegans and
Mucor spp.
Soybean mash is fermented by Rhizopus oligosporus and R. oryzae

Shredded cabbage containing 2.2 – 2.8% NaCl is fermented by
Leuconostoc mesenteroides, Lactobacillus plantarum and
Lactobacillus brevis successively

Cucumber + dill seeds in brine at 50% NaCl is fermented by
Leuconostoc mesenteroides, Enterococcus faecalis, Pediococcus
cerevisiae, Lactobacillus brevis, L. plantarum (dominant)
Grass, chopped corn and animal feed is stored under moist
anaerobic conditions, to undergo lactic acid mixed fermentation
Lactic acid fermentation by Leuconostoc spp. followed by L.
L. brevis, yeasts and various bacteria.

Many fermented grain products are used world over. One such is soy sauce, a brown, salty,
tangy sauce used as condiment by Japanese and Chinese. A mash of soy beans, wheat and
wheat bran is fermented by Aspergillus oryzae in solid substrate fermentation. The mouldy
substrate obtained at the end of the fermentation is called koji, which is extracted after
drying. Proteases, amylases and lactic acid of the bacteria act on soybean and wheat. The
extract, autoclaved soybean and crushed, steamed wheat are mixed. The resulting mixture is
called maromi. This is incubated at 30°C for 10 weeks to an year. During this period a
succession of microorganism Pediococcus soyae, Yeasts such as Saccharomyces rouxii,
Zygosaccharomyces soyae, Torulopsis carry out alcoholic fermentation.

Aspergillus oryzae is the most important organism. Lactobacillus spp. produce lactic acid
thereby preventing spoilage. Miso is also produced by koji fermentation of rice by A. oryzae.
It is ground to paste and combined with other food. Natto is produced from boiled soybeans
by Bacillus subtilis. Proteinase softens and increase flavour of soybeans. Poi is a Polynesian
fermented product. Stems of Toro plant are steamed, ground and subjected to fermentation by
succession of coliforms, Pseudomonas, Lactobacillus, Streptococcus and Leuconostoc.
Finally yeasts and Geotrichum candidum flourish. The fermentation product are lactic acid,
acetic acid, formic acid and CO

Microorganisms as products include Baker’s yeast and Single cell protein. The Baker’s yeast
is produced by growing special Saccharomyces cerevisiae strains with high growth rate and
white color, for their biomass in molasses and mineral medium. 0.5 – 1.5% sugar is used
since too high a concentration represses respiratory enzymes. The yeast cream which is white
in color is collected, centrifuged and dewatered. The processing steps decide whether a high
moisture containing compressed yeast is final product or granulated active dry yeast (8%
moisture). The compressed yeast requires refrigeration while the active dry yeast does not.
The wine, brewer’s, distiller’s and baker’s yeast can also be used as food or feed supplement


called single cell protein (SCP). In fact a variety of bacteria, yeasts and fungi have been
cultivated as SCP, so named because they are derived from single celled organisms (Table
16). The protein is extracted from cultivated microbial biomass.

Table16: Comparison of various parameters of SCP production from algae, fungi and

Growth rate
Quite high
Lower than
Light, CO
Wide range
Agricultural waste,
, C
molasses etc.
Wide range,
molasses, wheat,
molasses, whey
Upto 11

Precautions needed

amino acids
Nucleic acid
Endotoxin from
Gm negative
40-60 %
50-83 %
30-70 %
30-70 %

It can replace costly protein supplements like soymeal and fishmeal. Moreover agricultural
waste and inexpensive substrate can be used as raw material for SCP production giving
value-added products. The proteins from microbial sources have all the essential amino acids.
Algae are rich in vitamins and low nucleic acids. Fungi are rich in B-complex vitamins.
Yeasts also contain vitamins. Bacterial SCP has highest proteins (80%) but nucleic acids
especially RNA is high. The limitations of SCP are presence of undigestible cellulosic cell
wall in algae, mycotoxins of fungi, high cost of protein extraction and nucleic acid contents
in bacteria. Yeast have acceptability due to familiarity while with others there is a
psychological barrier to use the bacteria as major food source. The different microorganisms
as SCP are grown on different inexpensive substrates (Table 16 ).

Food additives enhance the quality of food nutrition and flavour. Many vitamins, amino
acids, enzymes, nucleotides and organic acids used in food industry are obtained from
microbial cultures. The metabolic control mechanisms that prevent it from overproducing
these compounds must be circumvented before large quantities of the desired byproduct can
be synthesized and harvested. Nucleotides are used to enhance taste of food. Enzymes are


used in various roles in number of processes related to food processing (Table 17). Vitamins
are essential nutritional factors used as dietary supplements. Phenylalanine and aspartic acid
are ingredients of aspartame, artificial sweetener. Amino acids from microbial fermentation is
advantageous since products include only L-amino acids while chemical synthesis gives a
racemic mixture. Economic production of amino acids is possible due to strains defective in
regulation. Several organic acids including acetic, gluconic, citric, itaconic, gibberellic and
lactic acid are produced by microbial fermentations (Table 18).

Table17: Common microbial food enzymes

Reaction & Applications
Starch  sugar, brewing, syrup production
Saccharomyces cerevisiae
Sucrose  glucose + fructose manufacture
of candies
Pectin  oligosaccharides + galacturonic
acid. fruit juice clarification and preparation
of concentrates
Endothocia, Mucor
Coagulation of casein, curdling in cheese
Bacillus, Aspergillus
Protein hydrolysis, meat tenderization
Diacetyl reductase
Enterobacter aerogenes
Diacetyl removal, prevention of certain off
flavors in beer and fruit juices
Kluveromyces fragilis
Lactose  galactose + glucose, digestion of
lactose in milk,prevention of lactose
crystallization in ice cream
Aspergillus niger
Elimination of naringin, removal of bitter
taste from orange juice
Glucose oxidase
Aspergillus niger
Glucose  gluconic acid, prevention of
browning in dried eggs
Glucose isomerase
Glucose  fructose, preparation of very
sweet syrups

Vinegar production is a two step process, first is the anaerobic alcoholic fermentation by S.
cerevisiae followed by oxidative transformation of alcohol to give acetic acid by Acetobacter.
The starting material may be fruits such as grapes, oranges, apples, pears, vegetables such as
potatoes, malted cereals such as barley, rye, wheat and corn and sugary syrups such as
molasses, honey etc. The resulting vinegar is called wine vinegar from wine, cider vinegar
from apple and so on. The Acetobacter process has evolved progressively in fermenter design
to accomplish optimum oxygen transfer to bacteria. In Orleans process the acetic acid
bacteria grow as a film on the top of shallow static pans. Next evolved the Fringe’s vinegar
generators with wood shavings on which the bacteria grow. Alcohol trickle down and is
oxidized to acetic acid. Now submerged culture reactors are employed. These are called
acetator and cavitator. Forced aeration maximizes the rate of acetic acid production. The
product is filtered and allowed to age to achieve the desired flavour.


Table 18: Some vitamins, amino acid and organic acid produced by fermentation

Vitamin B12
Propionibacterium shermanii
Ashbya gosypii
Vitamin C precursor
(5 ketogluconic acid)
Gluconobacter oxidans
Homoserine requiring auxotroph of
Corynebacterium glutamicum
Glutamic acid
(used in aginomoto or mono-sodium
Corynebacterium, Brevibacterium or
Arthrobacter spp.
Citric acid
Aspergillus niger
Lactic acid
Lactobacillus delbruckii
Fumaric acid
Gluconic acid
Aspergillus niger

Genetically modified food also called genetically engineered food, Frankenfood or genetically
altered food. Genetically modified organisms are those whose genetic material is modified to
create new life forms that would never occur in nature under natural conditions of crossbreed
or natural recombination. To create genetically engineered crops genes from bacteria, viruses,
plants, animals have been inserted into plants like soybean, corn, canola and cotton. Thus
genetic manipulation is directed to have food with long shelf life, enhanced disease
resistance, enhanced taste, improved appearance and high nutritive value. A concern
expressed is about the safety of the products since the regulations and laws are same as for
the natural foods. However, the manufacturer must show that it does not contain allergic or
toxic substances. Additionally changes in the levels of important nutrients have to be
reported. Many products are on the shelves in the market. Some of the products on the way
are listed in the Table 19. Rot-resistant tomatoes are among the first commercially available
GM food. It is a product of antisense technology. Rotting is the effect of softening enzymes,
fungal growth, or over ripening of the fruit. To prevent premature ripening (before arrival in
the market) tomatoes that cannot express a ripening associated gene have been produced.
This is accomplished by inserting another gene into the tomato plant, a gene with a nucleotide
sequence complementary to that of the gene that produces an ethylene generating enzyme
(ethylene is the plant hormone that triggers natural ripening). The single-stranded messenger
RNAs transcribed from these genes are complementary and form a double-stranded RNA
within the cell that cannot be translated. When ripening is desired it can be induced by
exposing the tomatoes to ethylene. The antisense gene is introduced via Agrobacterium
tumifaciens Ti plasmid.

BT cotton has an advantage of enhanced protection against lepidopteran insect the spp. that
causes disease in cotton. The cotton is genetically engineered to produce Bacillus
thuringensis var kurstaki insecticidal crystal protein and have inbuilt protection against these
insect pests.


Table 19: Genetically modified products and modification

Rapeseed, tobacco, Soybean, Corn
Herbicide resistance
Cotton, Potato
Insect resistance
Baker’s yeast
Increased fermentation speed
Tomatoes (Flavr savr)
rot resistant
Rice (Golden rice)
Vitamin A gene introduced
Soybean glycinin gene has 20% more protein
Withstand high glyphosphate
Herbicide resistance
has α-amylase inhibitor
Pharmaceutical crops
Vaccine producing
Growth faster

Detrimental Effects of Microorganisms on Food
Food being perishable commodity is liable to microbial spoilage by intrinsic factors that
include pH, moisture content, water activity or availability, oxidation-reduction potential and
presence of antimicrobial substances. Substances like aldehydes, phenols, coumarines,
lyzozyme in various foods prevent spoilage to certain extent. Moulds in moist conditions
spoil corn and grains. Claviceps purpura produces hallucinogenic alkaloids that causes
ergotism, a toxic condition. Aflatoxins and fumonisins of fungalorigin are carcinogenic.
Aflatoxins are produced by Aspergillus flavus in infected grains and nuts. Fumonisins are
produced by Fusarium moniliforme and is implicated in oesophageal cancer in humans.
Meats and dairy products are easily spoilt. Microbial spoilage causes putrefaction
(degradation of proteins) since they are high protein containing foods. Food may be spoilt
before canning or due to improper canning or leakage of cans during cooling. Spoiled canned
food can alter the color, texture, odour and taste. Organic acids, sulfides and gases like CO

and H
S spoilage in canned food can be classified according to the can’s appearance
(Figure 22).

Raw milk undergoes a predictable four step succession of microorganisms during spoilage.
Acid production by Lactobacillus lactis is followed by additional acid production associated
with the growth of more acid tolerant Lactobacillus. At this point yeasts and moulds become
dominant and degrade the lactic acid gradually decreasing the acidity. Eventually protein
digesting bacteria become active resulting in putrid odor and bitter flavor making the milk
clear (Figure 23)

Raw and pasturised milk are also spoilt by Streptococcus lactis (souring), coliforms (gas
production), Micrococcus, S. faecalis, Pseudomonas spp., Flavobacterium spp.,
Chromobacterium spp. (proteolysis), Alcaligenes viscolactis, Klebsiella pneumoniae,
Enterobacter aerogenes (ropines). Moulds like Geotrichum, Cladosporium and Penicillium
spp. also attack fermented milk. Fruits and vegetables have much lower protein and fat
contents. Therefore, they undergo spoilage by bacteria that degrade the carbohydrates. Soft
rot is caused by Erwinia carotovora that produces hydrolytic enzymes like pectinases.


Initially the fruits are attacked by moulds damage the outer layer leaving it open to bacterial
attack. Lactobacillus and Leuconostoc spp. spoil the frozen citrus products by virtue of
production of diacetyl-butter flavours. Saccharomyces and Candida are predominant spoilage
organisms in juices. Pseudomonas spp. causes green rot, Alcaligenes, coliforms,
Acinetobacter, and Pseudomonas cause colorless rot and Proteus causes black rot in eggs.
Contaminated eggs with Salmonella is a common source of food poisoning.

Can of spoiled food

Gas (swollen can) No gas (can not swollen)

+ CO
hydrogen yeast
swell non- Bacillus sp.
biological Rotten egg pH Mouldy
sulfide spoilage drop can
C. nigrificans leak

Thermophilic anaerobic Mesophilic
sporeformers bacteria
“TA” spoilage

Thermophilic Mesophilic
sporeformens organisms

Sour or rancid odour Putrid odour
Clostridium sporogens
other spp.
“Flat sour Mixed
spoilage” non-sporeform
B thermoacidurans. can leak

Rancid or Mixed Facultative
butyric odour flora anaerobic Lactobacillus spp.
C. butyricum can sporeforms spoilage of canned food

leak Bacillus sp.

Figure 22: Spoilage of canned food


Figure 23: Succession of microorganisms in raw milk

Food borne diseases or Food poisoning is a general term used meaning foodborne illnesses.
It may be caused by plant, animal or chemical contaminations in food apart from microbial
ones. There are two general categories of microbial food poisonings. Firstly, food
intoxication results from the ingestion of exotoxin secreted by bacterial cells growing in food.
Vomiting, diarrhoea or severe muscle spasm are the symptoms. The second category is food
infection, which is associated with the ingestion of the microorganism that cause
gastrointestinal disorders. The major forms of bacterial food poisonings are summarized in
Table 20.

Table 20: Major forms of bacterial food poisonings

I) Intoxications
Food (s)
Staphylococcus enteritis by S. aureus
custards, salad, dressings, pastries
Botulism by Clostridium botulinum
poorly canned low acid food
Enterotoxaemia by C. perfringenes
inadequately cooked meat
Enterotoxaemia by Bacillus cereus
reheated rice, potatoes, puddings
II) Infections

typhoid by Salmonella typhi,Salmonellosis by
Poultry, eggs, dairy products, mango
Shigellosis by Shigella spp.
Unsanitary canned food
Enteritis by Vibrio parahaemolyticus
poorly cooked seafood
Listeriosis by Listeria monocytogenes
poorly pasteurized milk, cheese
Enteritis by E. coli
contaminated raw vegetables, cheese

Food Preservation
Preservation methods are aimed at preventing the incorporation of microbes into food,
removing or destroying microbes in food and keeping microbes from multiplying. Modern
food preservation uses refrigeration, freezing, dehydration and canning. Aseptic handling and
processing prevents the entry of microbes in the food. Fruits, vegetables, grains are
vigorously washed to reduce contaminants level. Meat, milk and eggs are taken as aseptically


as possible to reduce contamination during handling. Flies and insects are stopped by
covering food or eliminating pests from canning area. Utensils are cleaned properly and care
is taken to avoid cross contaminating cutting boards. Refrigeration retards microbial growth,
however, on extended storage psychrophiles produce spoilage. High temperature is the safest
and reliable method. Moist heat is generally used. Canning introduced by Appert is called
Appertization or commercial sterilization intended to destroy the C.botulinum spores and not
complete sterilization. Enough heat is supplied for 12D treatment which means 12 decimal
reduction or reduction in microbial population by 12 log cycles. Canned food is treated in
special containers called retorts (which work on the principle of autoclave) at 115°C for 25-
100 min. depending on the nature of the food. Pasteurization, another widely used process
for milk, beer, wines etc. kills most of the disease causing bacteria esp. Mycobacterium
tuberculosis in milk. The LTH, low temperature holding process or vat pasteurization is
heating each and every particle of milk at 145°F (62.8°C) for 30 min. The HTST or high
temperature short time process or flash pasteurization uses 161°F (71.7°C) for 15 sec and the
UHT, ultra high temperature is treated at 141°C for 2 sec. Irradiation both ionizing and
nonionizing are used to target opportunistic pathogens like E.coli 0157H7. Herbs, spices and
seasonings are irradiated to keep the microbial count low. It is also used to kill the insects
from wheat products, prevent sprouting of potatoes during storage and delay fruit ripening.
People need to be educated as regards the safety of irradiated food. A more familiar word like
cold pasteurization should be substituted to facilitate that. The irradiated foods carry the
international irradiation logo. For deeper penetration gamma rays dose of 4.5 – 5.6 megarads
produced by Cobalt 60 are used. Alternatively high energy electron accelerator generate
electrons that are faster than γ rays but have low penetration. Dehydration is an ageold
process. Sun drying is used for grains, meat, fish and fruits. Microwaves are high frequency
electromagnetic microwaves that create vibrations among the molecules of food creating heat
that kills the microorganisms. High osmotic process is used to withdraw water from microbial
metabolism. The effect is similar to dehydration. Chemicals that are GRAS, Generally
regarded as safe include organic acids, sulfite, ethylene oxide as sterilents. Sodium nitrite and
ethyl formate are solid preservatives. The Food and Drug administration of US gives the
GRAS status to harmless chemicals. These chemicals damage the plasma membrane,
denature various cell proteins, interfere with the functioning of nucleic acids and kill
microorganisms. The concentrations used are 0.1 –0.3% or 15–700 ppm. Antibiotics like nisin
a polypeptide agent are used in food preservation. Nisin is produced by Lactococcus lactis
and affects gram positive bacteria

Microbiological Examination of Food
A problem in maintaining the food safety is the need to detect microorganisms in order to
curb outbreaks. Microbiological examination provides information on quality of raw food,
cleanliness of food handling operations and efficacy of preservation method. Spoilage
organisms can be detected making way to prevent it. The Figure 24 gives a generalized
scheme for microbiological examination of food. Direct isolation have several problems.
Instead rapid techniques like fluorescent antibody technique, ELISA and radioimmunoassay
are valuable for the detection of small amounts of pathogen specific antigens. In one such
advanced technique immunomagnetic Dyna beads coated with antibodies are used to fish
microorganisms from food and grow on selective media. DNA/RNA probes are tagged with
enzymatic, isotopic, chromogenic, luminescent or fluorescent markers. When clubbed with
PCR even a few cells can be easily detected.


Food sample

-Direct microscopic - Standard plate count Identification of specific
examination microorganisms based
-Howard mould -Differential examination on guide for tests to
counting for different bacteria, moulds, perform the examination
-Wet mounts yeasts spoilage. of foods according to the
-Metabolic types -Categories of microorganisms
lipolytic, proteolytic,
-Physiological types
aerobic, anaerobic
facultative, microaerophilic
mesophilic, psychrophilic

Figure 24: Generalised scheme for microbiological examination of food

WHO has formulated Hazard Analysis and Critical Control Point (HACCP) program. It is a
management system in which food safety is addressed through the analysis and physical
hazards from raw material production, procurement and handling to manufacturing
distribution and consumption of the finished product.

Suggested Reading
1. Atlas R.M., [1997] Principles of Microbiology, 2
edn. Wm.C.Brown Publishers, USA.
2. Tchobanoglous G.and F.L.Franklin[1995] Wastewater Engineering Treatment, Disposal and Reuse 3

edn. Metcalf and Eddy Inc. Tata McGraw-Hill Publishing Co. Ltd., N. Delhi.
3. Frobisher M., R.D.Hinsdill , R.T.Crabtee and C.R.Goodheart [1974] Fundamentals of Microbiology,
edn. W.B.Saunders Co.,London.
4. Hammer M.J. and M.J. Hammer Jr. [1996] Water and Wastewater technology 3
edn. Prentice Hall,
5. Prescott L.M.,J.P.Harley and D.A.Klein [2002] Microbiology, 5
edn. McGraw Hill, N.York.
6. Hurst C.J., G.R.Knudson, M.J.McInerney, L.D.Stetzenbach and M.V.Walt [1997] Manual of
Environmental Microbiology ASM Press, USA.
7. Peleczar Jr.M.J., E.C.S.Chan and N.R. Krieg, [1993] Microbiology : concepts and applications ,
International edn. McGraw Hill Inc., N.York.
8. Maier R.M., I.L.Pepper and C.P.Gerba [2000] Environmental Microbiology, Academic Press, N.York.