Microbial, Industrial and Environmental Biotechnology

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Vivek Tripathi (answervivek@gmail.com)
Microbial, Industrial and Environmental Biotechnology
Growth Kinetics of Microorganism/Growth Curve
Growth curves are also common tools in ecological studies; they are used to track the rise and fall of
populations of plants, animals, and other multicellular organisms over time.
The increase in the cell size and cell mass during the development of an organism is termed as growth. It
is the unique characteristics of all organisms.
Growth curves are widely used in biology for quantities such as population size or biomass (in
population ecology and demography, for population growth analysis), individual body height or biomass
(in physiology, for growth analysis of individuals). Values for the measured property can be plotted on a
graph as a function of time.
Bacterial growth is the asexual reproduction, or cell division, of a bacterium into two daughter cells, in a
process called binary fission.
The growth curve has four distinct phases-
1. Lag Phase- When a microorganism is introduced into the fresh medium, it takes some time to
adjust with the new environment. This phase is termed as Lag phase, in which cellular
metabolism is accelerated, cells are increasing in size, but the bacteria are not able to replicate
and therefore no increase in cell mass. The length of the lag phase depends directly on the
previous growth condition of the organism.
2. Exponential or Logarithmic (log) Phase- During this phase, the microorganisms are in a rapidly
growing and dividing state. Their metabolic activity increases and the organism begin the DNA
replication by binary fission at a constant rate. The growth medium is exploited at the maximal
rate, the culture reaches the maximum growth rate and the number of bacteria increases
logarithmically (exponentially) and finally the single cell divide into two, which replicate into
four, eight, sixteen, thirty two and so on (That is 20, 21, 22, 23.........2n, n is the number of
generations) This will result in a balanced growth. The time taken by the bacteria to double in
number during a specified time period is known as the generation time. The generation time
tends to vary with different organisms. E.coli divides in every 20 minutes, hence its generation
time is 20 minutes, and for Staphylococcus aureus it is 30 minutes.

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3. Stationary Phase- As the bacterial population continues to grow, all the nutrients in the growth
medium are used up by the microorganism for their rapid multiplication. This result in the
accumulation of waste materials, toxic metabolites and inhibitory compounds such as antibiotics
in the medium. This shifts the conditions of the medium such as pH and temperature, thereby
creating an unfavorable environment for the bacterial growth. The reproduction rate will slow
down, the cells undergoing division is equal to the number of cell death, and finally bacterium
stops its division completely. The cell number is not increased and thus the growth rate is
established. If a cell taken from the stationary phase is introduced into a fresh medium, the cell
can easily move on the exponential phase and is able to perform its metabolic activities as usual.
4. Decline or Death Phase- The depletion of nutrients and the subsequent accumulation of
metabolic waste products and other toxic materials in the media will facilitates the bacterium
to move on to the Death phase. During this, the bacterium completely loses its ability to
reproduce. Individual bacteria begin to die due to the unfavorable conditions and the death is
rapid and at uniform rate. The number of dead cells exceeds the number of live cells. Some
organisms which can resist this condition can survive in the environment by producing
endospores.
Sterilization Techniques
Sterilization (or sterilisation) is a term referring to any process that eliminates (removes) or kills all forms
of life, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) present on a
surface, contained in a fluid, in medication, or in a compound such as biological culture media.
Sterilization can be achieved by applying heat, chemicals, irradiation, high pressure, and filtration or
combinations thereof. Sterilisation is difficult to achieve and in the case of making food safe is more
accurately described as pasteurization.
Methods of Sterilization
1. Heating
Wet Heat (Autoclaving) - The method of choice for sterilisation in most labs is autoclaving; using
pressurized steam to heat the material to be sterilized. This is a very effective method that kills
all microbes, spores and viruses, although for some specific bugs, especially high temperatures
or incubation times are required.
Autoclaving kills microbes by hydrolysis and coagulation of cellular proteins, which is efficiently
achieved by intense heat in the presence of water.
Dry Heat (Flaming, Baking) - Dry heating has one crucial difference from autoclaving. You've
guessed it – there's no water, so protein hydrolysis can't take place.
Instead, dry heat tends to kill microbes by oxidation of cellular components. This requires more
energy than protein hydrolysis so higher temperatures are required for efficient sterilization by
dry heat.
For example sterilisation can normally be achieved in 15 minutes by autoclaving at 121 °C,
whereas dry heating would generally need a temperature of 160 °C to sterilize in a similar
amount of time.

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2. Filtration
Filtration is a great way of quickly sterilizing solutions without heating. Filters, of course, work by
passing the solution through a filter with a pore diameter that is too small for microbes to pass
through.
Filters can be scintered glass funnels made from heat-fused glass particles or, more commonly
these days, membrane filters made from cellulose esters.
For removal of bacteria, filters with an average pore diameter of 0.2um is normally used. But
remember, viruses and phage can pass through these filters so filtration is not a good option if
these are a concern.
3. Chemical Sterilization
Chemicals are also used for sterilization. Although heating provides the most reliable way to rid
objects of all transmissible agents, it is not always appropriate, because it will damage heat-
sensitive materials such as biological materials, fiber optics, electronics, and many plastics.
Few chemicals as example-
Ethylene oxide (EO or EtO) gas is commonly used to sterilize objects that are sensitive to
temperatures greater than 60 °C and / or radiation such as plastics, optics and electrics.
Nitrogen dioxide (NO2) gas is a rapid and effective sterilant for use against a wide range of
microorganisms, including common bacteria, viruses, and spores.
Ozone is used in industrial settings to sterilize water and air, as well as a disinfectant for
surfaces.
Chlorine bleach is another accepted liquid sterilizing agent. Household bleach consists of 5.25%
sodium hypochlorite. It is usually diluted to 1/10 immediately before use; however to kill
Mycobacterium tuberculosis it should be diluted only 1/5, and 1/2.5 (1 part bleach and 1.5 parts
water) to inactivate prions.
Hydrogen peroxide is strong oxidant and these oxidizing properties allow it to destroy a wide
range of pathogens and it is used to sterilize heat or temperature sensitive articles such as rigid
endoscopes.
Peracetic acid a bright colorless liquid, which has a piercing odor and a pH of 2.8. Produced by
the reaction of hydrogen peroxide and acetic acid.
Glutaraldehyde a colorless, pungent liquid produced industrially by the oxidation of
cyclopentene.
Formaldehyde gas is also limited because the chemical is carcinogenic. Its use is restricted
primarily to sterilization of HEPA filters.
4. Solvents
Ethanol is commonly used as a disinfectant, although since isopropanol is a better solvent for fat
it is probably a better option. Both work by denaturing proteins through a process that requires
water, so they must be diluted to 60-90% in water to be effective.
Again, it's important to remember that although ethanol and IPA are good at killing microbial
cells, they have no effect on spores. Silver ions and silver compounds show a toxic effect on
some bacteria, viruses, algae and fungi.

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5. Radiation
UV, x-rays and gamma rays are all types of electromagnetic radiation that have profoundly
damaging effects on DNA, so make excellent tools for sterilization, the main difference between
them, in terms of their effectiveness, and is their penetration.
UV has limited penetration in air so sterilisation only occurs in a fairly small area around the
lamp. However, it is relatively safe and is quite useful for sterilizing small areas, like laminar flow
hoods.
X-rays and gamma rays are far more penetrating, which makes them more dangerous but very
effective for large scale cold sterilization of plastic items (e.g. syringes) during manufacturing.
Microbes
Microbes are single-cell organisms or a microorganism, especially a bacterium causing disease or
fermentation.
A microorganism or microbe is an organism that is so small that it is microscopic (invisible to the naked
eye).
Microorganisms are often illustrated using single-celled, or unicellular organisms; however, some
unicellular protists are visible to the naked eye, and some multicellular species are microscopic.
Discovery of microorganisms is done in 1674 by Antonie van Leeuwenhoek, using a microscope of his
own design.
Microorganisms are very diverse and include all the bacteria and archaea and almost all the protozoa.
They also include some fungi, algae, and certain animals, such as rotifers. Many macro animals and
plants have juvenile stages which are also microorganisms.
Some microbiologists also classify viruses (and viroids) as microorganisms, but others consider these as
nonliving.
Isolation of Microbes
A number of methods are available for the isolation of bacteria. The following are a few important
methods:-
1. Surface Plating
Surface plating is also called as streak culture method. is a technique used to isolate a pure
strain from a single species of microorganism, often bacteria. Samples can then be taken from
the resulting colonies and a microbiological culture can be grown on a new plate so that the
organism can be identified, studied, or tested.
2. Enrichment and Selective Media
Bacteria can be isolated by growing in enrichment or selective media. In these media,
substances which inhibit the growth of unwanted bacteria is added. So there is a growth of only
the bacteria which is wanted.
3. Aerobic and Anaerobic Conditions
Aerobic and anaerobic bacteria can be separated by cultivation under aerobic or anaerobic
conditions. The most widely used medium for anaerobic bacteria is Robertson's cooked meat
medium. It contains fat-free minced cooked meat in broth. It is covered with a layer of sterile
Vaseline.

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4. Isolation by Difference in Temperature
Thermophile bacteria grow at 60° C. Some bacteria like N. magnitudes grow at 22° C. By
incubation at different temperatures, bacteria can be selectively isolated.
5. Separation of Vegetative and Spore forming Bacteria
Vegetative bacteria are killed at 80° C. But spore forming bacteria like tetanus bacilli survive at
this temperature. So by heating at 80° C vegetative bacteria can be eliminated and spore
forming bacteria can be isolated.
6. Separation of Motile and Non-motile Bacteria
This can be achieved by using Craig's tube or a U tube. In the U tube, the organisms are
introduced in one limb and the motile organism can be isolated at the other limb.
7. Animal Inoculation
Pathogenic bacteria can be isolated by inoculation into appropriate animals, e.g. Anthrax bacilli
can be isolated by inoculation into mice or guinea pigs.
8. Filtration
Bacteria of different sizes may be separated by using selective filters.
9. Micromanipulation
By means of micromanipulation, single bacterium can be separated and cultured.
Characterization of Bacteria
The Gram stain, developed in 1884 by Hans Christian Gram, characterizes bacteria based on the
structural characteristics of their cell walls.
The thick layers of peptidoglycan in the "Gram-positive" cell wall stain purple, while the thin "Gram-
negative" cell wall appears pink.
Gram Stain Procedure
1. Place slide with heat fixed smear on staining tray.
2. Gently flood smear with crystal violet and let stand for 1 minute.
3. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
4. Gently flood the smear with Gram’s iodine and let stand for 1 minute.
5. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. The
smear will appear as a purple circle on the slide.
6. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol drop
by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-decolorize.
7. Immediately rinse with water.
8. Gently flood with safranin to counter-stain and let stand for 45 seconds.
9. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
10. Blot dry the slide with bibulous paper.
11. View the smear using a light-microscope under oil-immersion.
12. "Gram-positive" cell wall stain purple, while the thin "Gram-negative" cell wall appears pink.
Classification of Microbes
Microorganisms can be found almost anywhere in the taxonomic organization of life on the planet.
Bacteria and archaea are almost always microscopic, while a number of eukaryotes are also microscopic,
including most protists, some fungi, as well as some animals and plants.

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Viruses are generally regarded as not living and therefore not considered as microorganisms, although
the field of microbiology also encompasses the study of viruses.
a) Prokaryotes
Prokaryotes are organisms that lack a cell nucleus and the other membrane bound organelles.
They are almost always unicellular.
Prokaryotes consist of two domains, bacteria and archaea.
1. Bacteria
Almost all bacteria are invisible to the naked eye, with a few extremely rare exceptions, such
as Thiomargarita namibiensis.
They lack a nucleus and other membrane-bound organelles, and can function and reproduce
as individual cells, but often aggregate in multicellular colonies.
Their genome is usually a single loop of DNA, although they can also harbor small pieces of
DNA called plasmids. These plasmids can be transferred between cells through bacterial
conjugation.
Bacteria are surrounded by a cell wall, which provides strength and rigidity to their cells.
They reproduce by binary fission or sometimes by budding.
2. Archaea
Archaea are also single-celled organisms that lack nuclei.
In the past, the differences between bacteria and archaea were not recognized and archaea
were classified with bacteria as part of the kingdom Monera, however, in 1990 the
microbiologist Carl Woese proposed the three-domain system that divided living things into
bacteria, archaea and eukaryotes.
Archaea differ from bacteria in both their genetics and biochemistry. For example, while
bacterial cell membranes are made from phosphoglycerides with ester bonds, Archaean
membranes are made of ether lipids.
b) Eukaryotes
Most living things that are visible to the naked eye in their adult form are eukaryotes, including
humans. However, a large number of eukaryotes are also microorganisms. Unlike bacteria and
archaea, eukaryotes contain organelles such as the cell nucleus, the Golgi apparatus and
mitochondria in their cells.
Unicellular eukaryotes usually reproduce asexually by mitosis under favorable conditions.
However, under stressful conditions such as nutrient limitations and other conditions associated
with DNA damage, they tend to reproduce sexually by meiosis and syngamy.
1. Protista
The protists are most commonly unicellular and microscopic. This is a highly diverse group of
organisms that are not easy to classify.
Several algae species are multicellular protists, and slime molds have unique life cycles that
involve switching between unicellular, colonial, and multicellular forms.
The number of species of protists is unknown since we may have identified only a small
portion.
2. Animals
Some micro animals are multicellular but at least one animal group, Myxozoa, is unicellular
in its adult form. Microscopic arthropods include dust mites and spider mites.

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A common group of microscopic animals are the rotifers, which are filter feeders that are
usually found in fresh water.
Some micro-animals reproduce both sexually and asexually and may reach new habitats by
producing eggs which can survive harsh environments that would kill the adult animal.
3. Fungi
The fungi have several unicellular species, such as baker's yeast (Saccharomyces cerevisiae)
and fission yeast (Schizosaccharomyces pombe).
Some fungi, such as the pathogenic yeast Candida albicans, can undergo phenotypic
switching and grow as single cells in some environments, and filamentous hyphae in others.
Fungi reproduce both asexually, by budding or binary fission, as well by producing spores,
which are called conidia when produced asexually, or basidiospores when produced
sexually.
4. Plants
The green algae are a large group of photosynthetic eukaryotes that include many
microscopic organisms. Although some green algae are classified as protists, others such as
charophyta are classified with embryophyte plants, which are the most familiar group of
land plants. Algae can grow as single cells, or in long chains of cells. The green algae include
unicellular and colonial flagellates, usually but not always with two flagella per cell, as well
as various colonial, coccoid, and filamentous forms.
Useful Microbes
1. Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of budding yeast. It is perhaps the most useful yeast
owing to its use since ancient times in baking and brewing.
It is believed that it was originally isolated from the skins of grapes.
Saccharomyces cerevisiae cells are round to ovoid, 5–10 micrometers in diameter.
It reproduces by a division process known as budding.
2. Aspergillus oryzae
Aspergillus oryzae is a filamentous fungus (a mold). It is used in Chinese and Japanese cuisine to
ferment soybeans to produce soy sauce and miso. It is also used to saccharify rice, other grains,
and potatoes in the making of alcoholic beverages such as huangjiu, sake, and shōchū. Also, the
fungus is used for the production of rice vinegars.
The protease enzymes produced by this species are marketed by the company Novozymes
under the name Flavourzyme.
3. Lactobacillus plantarum
Lactobacillus plantarum is a widespread member of the genus Lactobacillus, commonly found in
many fermented food products as well as anaerobic plant matter. It is also present in saliva
(from which it was first isolated).
Lactobacillus plantarum is commonly found in many fermented food products including
sauerkraut, pickles, brined olives, Korean kimchi, Nigerian ogi, sourdough and other fermented
plant material, and also some cheeses fermented sausages and stockfish.

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4. Thiobacillus ferrooxidans
Thiobacillus ferrooxidans is the most common type of bacteria in mine waste piles.
This organism is acidophilic (acid loving), and increases the rate of pyrite oxidation in mine
tailings piles and coal deposits.
It oxidies iron and inorganic sulfur compounds. The oxidation process can be harmful, as it
produces sulfuric acid, which is a major pollutant. However, it can also be beneficial in
recovering materials such as copper and uranium.
5. Corynebacteria
Corynebacteria are chemoorganotrophic, aerobic, or facultatively anaerobic, and they exhibit a
fermentative metabolism (carbohydrates to lactic acid) under certain conditions.
6. Acetobacter aceti
Acetobacter aceti is a Gram negative bacterium that moves using its peritrichous flagella. Louis
Pasteur proved it to be the cause of conversion of alcohol to acetic acid in 1864.
Acetobacter aceti species is used for the mass production of Acetic Acid, the main component in
vinegar. During the fermentation process of vinegar production, the bacteria, Acetobacter aceti
is used to act on wines and ciders resulting in vinegar with Acetic acid. It can be converted by a
silicone tube reactor, which aids the fermentation process with oxidation.
7. Torula
Torula, in its inactive form (usually labeled as torula yeast), is widely used as a flavouring in
processed foods and pet foods. It is often grown on wood liquor, a byproduct of paper
production, which is rich in wood sugars.
It is pasteurized and spray-dried to produce a fine, light grayish-brown powder with a slightly
yeasty odor and gentle, slightly meaty taste.
8. Lactococcus lactis
Lactococcus lactis is one of the most important micro-organisms involved in the dairy industry,
being a common leaven used in the making of many dairy products.
Lactococcus lactis is a Gram-positive bacterium used extensively in the production of buttermilk
and cheese, but has also become famous as the first genetically modified organism to be used
alive for the treatment of human disease.
9. Lactobacillus delbrueckii subsp. Bulgaricus
Lactobacillus delbrueckii subsp. bulgaricus (until 1984 known as Lactobacillus bulgaricus) is one
of several bacteria used for the production of yogurt. It is also found in other naturally
fermented products. First identified in 1905 by the Bulgarian doctor Stamen Grigorov, the
bacterium feeds on lactose to produce lactic acid, which is used to preserve milk.
It is a gram-positive rod that may appear long and filamentous. It is non-motile and does not
form spores.
10. Streptococcus thermophiles
Streptococcus thermophilus (previous name Streptococcus salivarius subsp. Thermophiles) is a
gram-positive bacteria and a homofermentative facultative anaerobe.
It is also classified as a lactic acid bacterium.
S. thermophilus is found in fermented milk products, and is generally used in the production of
yogurt.

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11. Lactobacillus acidophilus
Lactobacillus acidophilus (New Latin 'acid-loving milk-bacterium') is a species of gram positive
bacteria in the genus Lactobacillus. L. acidophilus is a homofermentative, microaerophilic
species, fermenting sugars into lactic acid.
Used in the production of acidophilus buttermilk.
12. Streptococcus cremoris & lactis
Microorganism used in the production of cultured buttermilk, sour cream, cottage cheese, all
types of foreign and domestic cheeses, and starter cultures.
13. Streptococcus durans
Microorganism used in the production of soft Italian, cheddar, and some Swiss cheeses.
Single Cell Protein
The term Single Cell Protein (SCP) refers to the dried microbial cells or total protein extracted from pure
microbial cell culture (Algae, bacteria, filamentous fungi, yeasts), which can be used as food supplement
to humans (Food Grade) or animals (Feed grade).
Single-cell protein (SCP) typically refers to sources of mixed protein extracted from pure or mixed
cultures of algae, yeasts, fungi or bacteria (grown on agricultural wastes) used as a substitute for
protein-rich foods, in human and animal feeds.
The term single cell protein was coined by Carol L. Wilson during 1966.
Quorntm
is a myco-protein as a product obtained from a filamentous fungus “Fusarium venenatum A3/5”
exclusively designed for human consumption, it is only the single cell protein product which is produced
in foreign countries at present days.
Production of Single Cell Protein
The production of Single Cell Protein can be done by using waste materials as the substrate, specifically
agricultural wastes such as wood shavings, sawdust, corn cobs, and many others. Examples of other
waste material substrates are food processing wastes, residues from alcohol production, hydrocarbons,
or human and animal excreta.
The process of SCP production from any microorganism or substrate would have the following basic
steps:-
1. Amount of a carbon source; it may need physical and/or chemical pretreatments.
2. Large scale biomass fermenter.
3. Addition, to the carbon source, of sources of nitrogen, phosphorus and other nutrients
needed to support optimal growth of the selected microorganism.
4. Prevention of contamination by maintaining sterile or hygienic conditions. The medium
components may be heated or sterilized by filtration and fermentation equipment’s may be
sterilized.
5. The selected microorganism is inoculated in a pure state.
6. SCP processes are highly aerobic (except those using algae). Therefore, adequate aeration
must be provided. In addition, cooling is necessary as considerable heat is generated.
7. The microbial biomass is recovered from the medium.
8. Processing of the biomass for enhancing its usefulness and/or storability.

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Three process for single cell production based on the substrate used-
1. Symba Process
Symba process developed in Sweden utilized starchy wastes combining two yeast in
sequential mixed culture.
2. Bel Process
Bel process is largest and longest running operation using whey substrate for single cell
production.
3. Pekilo Process
Pekilo process is a continuous fermentation consuming pulp mill effluent using the
filamentous fungus for the production of single cell protein
Single Cell Protein from CO2
A species Euglena gracilis (microalgae) was selected as it has advantages such as high protein content
and high digestibility for animal feed.
The biological fixation using microalgae has been known as an effective and economical carbon dioxide
reduction technology. Carbon dioxide (CO2) fixation by microalgae has been shown to be effective and
economical.
A kinetic model was studied in order to determine the relationship between specific growth rate and
light intensity. The half-saturation constant for light intensity in the Monod model was 178.7 micromol
photons/m2/s. The most favorable initial pH, temperature, and CO2 concentration were found to be 3.5,
27 degrees C, and 5-10% (vol/vol), respectively. Light intensity and hydraulic retention time were tested
for effects on cell yield in a laboratory-scale photo-bioreactor of 100l working volume followed by semi-
continuous and continuous culture. Subsequently, an innovative pilot-scale photo-bioreactor that used
sunlight and flue gas was developed to increase production of this bioresource. The proposed pilot-scale
reactor showed improved cell yield compared with the laboratory-scale reactor.
Advantages of Single Cell Protein
1. Microorganisms have a high rate of multiplication to hence rapid succession of generation
(algae: 2-6hours, yeast: 1-3 hours, bacteria: 0.5-2 hours).
2. They can be easily genetically modified for varying the amino acid composition.
3. Microorganisms have high content of protein (A very high protein content 43-85 % in the dry
mass).
4. Microorganisms can utilize a variety of carbon sources as major energy source, and some of the
waste material can also be used as carbon source.
5. Microbial biomass production occurs in continuous cultures and the quality is consistent since
the growth is independent of seasonal and climatic variations.
6. Microbial biomass production as single cell protein is independent of seasonal as well as climatic
variations.
7. Land requirements is low and is ecologically beneficial.
8. It is not dependent on climate.
9. It is not dependent on climate.

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Disadvantages of Single Cell Protein
1. Many types of microorganisms produce some substances which are toxic to the human and also
to the animals. Therefore it has to be made sure that the produced microbial biomass does not
contain any of these toxic substances.
2. Sometimes the microbial biomass when taken as diet supplement may lead to indigestion.
3. Sometimes the microbial biomass when taken as diet supplement may lead to allergic reactions
in humans.
4. The high nucleic acid content of many types of microbial biomass products is also undesirable
for human consumption as single cell protein. Sometimes this high level of nucleic acid content
in microbial biomass will lead to kidney stone formation or gout.
5. The high nucleic acid content of many types of microbial biomass may lead to poor digestibility,
gastrointestinal problem and also some skin reactions in humans.
6. The possibility of presence of toxins or carcinogenic compounds may lead to some serious
health problems in humans as well as in animal stock.
7. Single cell protein production is a very expensive procedure as it needs high level of sterility
control in the production unit or in the laboratory.
8. Single cell protein grown as animal feed on agricultural residues will be beneficial in the future
economy of developing nations.
9. Single cell protein product may cost more than conventional food product.
Microorganism Substrate
Aspergillus fumigatus Maltose, Glucose
Aspergillus niger, A. oryzae, Cephalosporium
eichhorniae, Chaetomium cellulolyticum
Cellulose, Hemicellulose
Penicillium cyclopium Glucose, Lactose, Galactose
Rhizopus chinensis Glucose, Maltose
Scytalidium aciduphilium, Thricoderma
viridae, Thricoderma alba
Cellulose, Pentose
Paecilomyces varioti Sulphite waste liquor
Fusarium graminearum Starch, Glucose
A renewable resource is a resource which is replaced naturally and can be used again.
Examples- Oxygen, Fresh water, solar energy, Timber, and Biomass.
A non-renewable resource (also called a finite resource) is a natural resource that is used up faster than
it can be made by nature. It cannot be produced, grown or generated on a scale which can sustain how
quickly it is being consumed. Once it is used up, there is no more available for future needs. Also
considered non-renewable are resources that are consumed much faster than nature can create them.
Fossil fuels (such as coal, petroleum, and natural gas), types of nuclear power (uranium) and certain
aquifers are examples.

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Probiotics are live microorganisms (in most cases, bacteria) that are similar to beneficial microorganisms
found in the human gut. They are also called “friendly bacteria” or “good bacteria.” Probiotics are
available to consumers mainly in the form of dietary supplements and foods.
Example- Yogurt is one of the best known probiotic foods is live-cultured yogurt, especially handmade,
Miso Soup, Sauerkraut, Kefir, Kombucha, Mircoalgae, Pickles, Tempeh, Poi.
Prebiotics is a general term to refer to chemicals that induce the growth and/or activity of commensal
microorganisms (e.g., bacteria and fungi) that contribute to the well-being of their host.
The most common example is in the gastrointestinal tract, where prebiotics can alter the distribution of
organisms in the gut microbiome.
Industrial Source of Enzymes
1. Cellulases
Cellulase is any of several enzymes produced chiefly by fungi, bacteria, and protozoans that
catalyze cellulolysis, the decomposition of cellulose and of some related polysaccharides;
specifically, the hydrolysis of the 1, 4-beta-D-glycosidic linkages in cellulose, hemicellulose,
lichenin, and cereal beta-D-glucans.
Cellulases break down the cellulose molecule into monosaccharides ("simple sugars") such as
beta-glucose, or shorter polysaccharides and oligosaccharides. The name is also used for any
naturally occurring mixture or complex of various such enzymes, that act serially or
synergistically to decompose cellulosic material.
An important example is the cellulase produced mainly by symbiotic bacteria in the ruminating
chambers of herbivores that allows them to digest the cellulose from their vegetable diet.
Cellulases are also produced by a few other types of organisms, such as some termites.
Application of Cellulases
1. Cellulase is used for commercial food processing in coffee, It performs hydrolysis of
cellulose during drying of beans.
2. Cellulases are widely used in textile industry and in laundry detergents.
3. Cellulase is used in the pulp and paper industry for various purposes, and they are even
used for pharmaceutical applications.
4. Cellulase is used in the fermentation of biomass into biofuels, although this process is
relatively experimental at present.
5. Cellulase is used as a treatment for phytobezoars, a form of cellulose bezoar found in
the human stomach.
2. Xylanase
Xylanase is the name given to a class of enzymes which degrade the linear polysaccharide beta-
1, 4-xylan into xylose, thus breaking down hemicellulose, one of the major components of plant
cell walls.
Xylanases are produced by fungi, bacteria, yeast, marine algae, protozoans, snails, crustaceans,
insect, seeds, etc., (mammals do not produce xylanases). However, the principal commercial
source of xylanases is filamentous fungi.

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Commercial applications for xylanase include the chlorine-free bleaching of wood pulp prior to
the papermaking process, and the increased digestibility of silage (in this aspect, it is also used
for fermentative composting).
3. Pectinases
Pectinase is an enzyme that breaks down pectin, a polysaccharide found in plant cell walls.
Pectinases are one of the upcoming enzymes of fruit and textile industries. These enzymes
break down complex polysaccharides of plant tissues into simpler molecules like galacturonic
acids. The role of acidic pectinases in bringing down the cloudiness and bitterness of fruit juices
is well established.
They can be extracted from fungi such as Aspergillus niger. The fungus produces these enzymes
to break down the middle lamella in plants so that it can extract nutrients from the plant tissues
and insert fungal hyphae. If pectinase is boiled it is denatured (unfolded) making it harder to
connect with the pectin at the active site, and produce as much juice.
Pectinase enzymes are used for extracting juice from purée. This is done when the enzyme
pectinase breaks down the substrate pectin and the juice is extracted. The enzyme pectinase
lowers the activation energy needed for the juice to be produced and catalyzes the reaction.
4. Amylase
Amylase is an enzyme that catalyses the hydrolysis of starch into sugars.
Amylase is present in the saliva of humans and some other mammals, where it begins the
chemical process of digestion.
Foods that contain large amounts of starch but little sugar, such as rice and potatoes, may
acquire a slightly sweet taste as they are chewed because amylase degrades some of their
starch into sugar.
Amylase is used in fermentation, also in bread making and to break down complex sugars, such
as starch (found in flour), into simple sugars.
5. Lipase
A lipase is an enzyme that catalyzes the hydrolysis of fats (lipids). Lipases are a subclass of the
esterases.
Lipase is an enzyme that the body uses to break down fats in food so they can be absorbed in
the intestines. Lipase is primarily produced in the pancreas but is also in the mouth and
stomach.
Lipases perform essential roles in the digestion, transport and processing of dietary lipids (e.g.
triglycerides, fats, oils) in most, if not all, living organisms. Genes encoding lipases are even
present in certain viruses.
Uses
1. Lipases serve important roles in human practices as ancient as yogurt and cheese
fermentation.
2. Lipases are also being exploited as cheap and versatile catalysts to degrade lipids in more
modern applications.
3. Recombinant lipase enzymes used in baking, laundry detergents and even as biocatalysts in
alternative energy strategies to convert vegetable oil into fuel.

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6. Proteases
A protease (also called peptidase or proteinase) is any enzyme that performs proteolysis, that is,
begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in
the polypeptide chain forming the protein.
Proteases have evolved multiple times, and different classes of protease can perform the same
reaction by completely different catalytic mechanisms.
Proteases can be found in animals, plants, bacteria, archaea and viruses.
Uses
1. Proteases are used in industry, medicine and as a basic biological research tool.
2. Digestive proteases are part of many laundry detergents and are also used extensively in the
bread industry in bread improver.
3. A variety of proteases are used medically both for their natural function (e.g. controlling
blood clotting) and for completely artificial functions (e.g. for the targeted degradation of
pathogenic proteins).
4. Highly specific proteases such as TEV protease and thrombin are commonly used to cleave
fusion proteins and affinity tags in a controlled fashion.
Commercial Production
Antibiotics
Antibiotics or antibacterials are a type of antimicrobial used specifically against bacteria, and are often
used in medical treatment of bacterial infections, they may either kill or inhibit the growth of bacteria.
Several antibiotic agents are also effective against a number of fungi, protozoans and some are toxic to
humans and animals, even when given in therapeutic dosage.
Antibiotics are not effective against viruses.
Antibiotics revolutionized medicine in the 20th century, and have together with vaccination lead to the
near eradication of diseases such as tuberculosis in the developed world.
Example of Antibiotics
1. Penicillin
Penicillin (sometimes abbreviated PCN or pen) is a group of antibiotics derived from Penicillium
fungi, including penicillin G ‘Penicillium chrysogenum’ (intravenous use), penicillin V (oral use),
procaine penicillin, and benzathine penicillin (intramuscular use).
Penicillin antibiotics were among the first drugs to be effective against many previously serious
diseases, such as bacterial infections caused by staphylococci and streptococci.
All penicillins are β-lactam antibiotics and are used in the treatment of bacterial infections
caused by susceptible, usually Gram-positive, organisms.
Production
Penicillin is a secondary metabolite of certain species of Penicillium and is produced when
growth of the fungus is inhibited by stress. It is not produced during active growth. Production is
also limited by feedback in the synthesis pathway of penicillin.
α-ketoglutarate + AcCoA → homocitrate → L-α-aminoadipic acid → L-lysine + β-lactam

15
The by-product, l-lysine, inhibits the production of homocitrate, so the presence of exogenous
lysine should be avoided in penicillin production.
The Penicillium cells are grown using a technique called fed-batch culture, in which the cells are
constantly subject to stress, which is required for induction of penicillin production. The
available carbon sources are also important: Glucose inhibits penicillin production, whereas
lactose does not. The pH and the levels of nitrogen, lysine, phosphate, and oxygen of the
batches must also be carefully controlled.
2. Streptomycin
Streptomycin is an antibiotic (antimycobacterial) drug, the first of a class of drugs called
aminoglycosides to be discovered, and it was the first effective treatment for tuberculosis.
It is derived from the actinobacterium Streptomyces griseus.
Streptomycin is a bactericidal antibiotic.
Adverse effects of this medicine are ototoxicity, nephrotoxicity, fetal auditory toxicity, and
neuromuscular paralysis.
Streptomycin, in combination with penicillin, is used in a standard antibiotic cocktail to prevent
bacterial infection in cell culture.
Production of streptomycin is done by the surface culture of Streptomyces griseus in pint milk
bottles on a papain digest of beef + meat extract + glucose + mineral salt medium.
Amino acids
Amino acids are biologically important organic compounds composed of amine (-NH2) and carboxylic
acid (-COOH) functional groups, along with a side-chain specific to each amino acid.
The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen, though other elements
are found in the side-chains of certain amino acids.
An essential amino acid or indispensable amino acid is an amino acid that cannot be synthesized de
novo (from scratch) by the organism being considered, and therefore must be supplied in its diet. The
nine amino acids humans cannot synthesize are phenylalanine, valine, threonine, tryptophan,
methionine, leucine, isoleucine, lysine, and histidine.
Six amino acids are considered conditionally essential in the human diet, meaning their synthesis can be
limited under special pathophysiological conditions, such as prematurity in the infant or individuals in
severe catabolic distress. These six are arginine, cysteine, glycine, glutamine, proline and tyrosine.
Five amino acids are dispensable in humans, meaning they can be synthesized in the body. These five
are alanine, aspartic acid, asparagine, glutamic acid and serine.
Class Name of the amino acids
Aliphatic Glycine, Alanine, Valine, Leucine, Isoleucine
Hydroxyl or Sulfur/Selenium-
containing
Serine, Cysteine, Selenocysteine, Threonine,
Methionine
Cyclic Proline
Aromatic Phenylalanine, Tyrosine, Tryptophan
Basic Histidine, Lysine, Arginine
Acidic and their Amide Aspartate, Glutamate, Asparagine, Glutamine

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Insulin
Insulin is a peptide hormone produced by beta cells in the pancreas. It regulates the metabolism of
carbohydrates and fats by promoting the absorption of glucose from the blood to skeletal muscles and
fat tissue and by causing fat to be stored rather than used for energy. Except in the presence of the
metabolic disorder diabetes mellitus and metabolic syndrome, insulin is provided within the body in a
constant proportion to remove excess glucose from the blood, which otherwise would be toxic. When
blood glucose levels fall below a certain level, the body begins to use stored glucose as an energy source
through glycogenolysis, which breaks down the glycogen stored in the liver and muscles into glucose,
which can then be utilized as an energy source. As a central metabolic control mechanism, its status is
also used as a control signal to other body systems (such as amino acid uptake by body cells). In
addition, it has several other anabolic effects throughout the body.
When control of insulin levels fails, diabetes mellitus can result.
Production

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Steroids
Steroids comprise a group of cyclical organic compounds whose basis is a characteristic arrangement of
seventeen carbon atoms in a four-ring structure linked together from three 6-carbon rings followed by a
5-carbon ring and an eight-carbon side chain on carbon 17 (illustration on right). These rings are
synthesized by biochemical processes from cyclization of a thirty-carbon chain, squalene, into lanosterol
or cycloartenol.
Hundreds of distinct steroids are found in animals, fungi, plants, and elsewhere and many steroids are
necessary to life at all levels.
Steroid and their metabolites are frequently used signalling molecules. The most notable examples are
the steroid hormones.
Steroids along with phospholipids function as components of cell membranes. Steroids such as
cholesterol decrease membrane fluidity.
Production
Ethanol
Commonly referred to simply as alcohol or spirits, ethanol is also called ethyl alcohol, and drinking
alcohol. It is the principal type of alcohol found in alcoholic beverages, produced by the fermentation of
sugars by yeasts.
It is a neurotoxic psychoactive drug and one of the oldest recreational drugs used by humans. It can
cause alcohol intoxication when consumed in sufficient quantity.
Ethanol is used as a solvent, an antiseptic, a fuel and the active fluid in modern (post-mercury)
thermometers. It is a volatile, flammable, colorless liquid with a strong chemical odor.
Production

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Butanol
Butanol (also butyl alcohol) refers to a four-carbon alcohol with a formula of C4H9OH. There are four
possible isomeric structures for butanol, from a straight-chain primary alcohol to a branched-chain
tertiary alcohol.
It is primarily used as a solvent, as an intermediate in chemical synthesis, and as a fuel. It is sometimes
also called biobutanol when produced biologically.
Butanol is considered as a potential biofuel (butanol fuel). Butanol at 85 percent strength can be used in
cars designed for gasoline (petrol) without any change to the engine (unlike 85% ethanol), and it
contains more energy for a given volume than ethanol and almost as much as gasoline, so a vehicle
using butanol would return fuel consumption more comparable to gasoline than ethanol. Butanol can
also be used as a blended additive to diesel fuel to reduce soot emissions.
Production
Acetone
Acetone (systematically named propan-2-one) is the organic compound with the formula (CH3)2CO. It is
a colorless, volatile, flammable liquid, and is the simplest ketone.
Acetone is miscible with water and serves as an important solvent in its own right, typically for cleaning
purposes in the laboratory.
Acetone is produced and disposed of in the human body through normal metabolic processes. It is
normally present in blood and urine. People with diabetes produce it in larger amounts.
About a third of the world's acetone is used as a solvent, and a quarter is consumed as acetone
cyanohydrin a precursor to methyl methacrylate.
Production

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Glycerol
Glycerol (also called glycerine or glycerin) is a simple polyol (sugar alcohol) compound.
It is a colorless, odorless, viscous liquid that is widely used in pharmaceutical formulations.
Glycerol has three hydroxyl groups that are responsible for its solubility in water and its hygroscopic
nature. The glycerol backbone is central to all lipids known as triglycerides.
Glycerol is sweet-tasting and generally considered non-toxic.
Glycerol is a byproduct in the production of biodiesel.
In food and beverages, glycerol serves as a humectant, solvent, and sweetener, and may help preserve
foods.
Glycerol is used in medical and pharmaceutical and personal care preparations, mainly as a means of
improving smoothness, providing lubrication and as a humectant.
Glycerol is used to produce nitroglycerin, which is an essential ingredient of various explosives such as
dynamite, gelignite, and propellants like cordite.
Alkaloid
Alkaloids are a group of naturally occurring chemical compounds (natural products) that contain mostly
basic nitrogen atoms.
This group also includes some related compounds with neutral and even weakly acidic properties.
In addition to carbon, hydrogen and nitrogen, alkaloids may also contain oxygen, sulfur and more rarely
other elements such as chlorine, bromine, and phosphorus.
Alkaloids are produced by a large variety of organisms including bacteria, fungi, plants, and animals.
They can be purified from crude extracts of these organisms by acid-base extraction.
Many alkaloids are toxic to other organisms.
They often have pharmacological effects and are used as medications, as recreational drugs, or in
entheogenic rituals.
Examples are Quinine, Colchicine, Nicotine, Morphine, Caffeine, Cocaine etc.

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Vitamins
A vitamin is an organic compound and a vital nutrient that an organism requires in limited amounts.
An organic chemical compound (or related set of compounds) is called a vitamin when the organism
cannot synthesize the compound in sufficient quantities, and must be obtained through the diet; thus,
the term "vitamin" is conditional upon the circumstances and the particular organism.
For example, ascorbic acid (vitamin C) is a vitamin for humans, but not for most other animal organisms.
Supplementation is important for the treatment of certain health problems, but there is little evidence
of nutritional benefit when used by otherwise healthy people.
Anti-vitamins are chemical compounds that inhibit the absorption or actions of vitamins. For example,
avidin is a protein in egg whites that inhibits the absorption of biotin.

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Production
Pollution
Pollution is the introduction of contaminants into the natural environment that cause adverse change or
the presence in or introduction into the environment of a substance which has harmful or poisonous
effects.
Pollution can take the form of chemical substances or energy, such as noise, heat or light.
Pollutants, the components of pollution, can be either foreign substances/energies or naturally
occurring contaminants.
Pollution is often classed as point source or nonpoint source pollution.
Types of pollution
1. Air pollution
The release of chemicals and particulates into the atmosphere. Common gaseous pollutants
include carbon monoxide, sulfur dioxide, chlorofluorocarbons (CFCs) and nitrogen oxides
produced by industry and motor vehicles. Photochemical ozone and smog are created as
nitrogen oxides and hydrocarbons react to sunlight. Particulate matter, or fine dust is
characterized by their micrometer size PM10 to PM2.5.
2. Light pollution
Includes light trespass, over-illumination and astronomical interference.
3. Noise pollution
It encompasses roadway noise, aircraft noise, industrial noise as well as high-intensity sonar.
4. Soil Contamination
Occurs when chemicals are released by spill or underground leakage. Among the most
significant soil contaminants are hydrocarbons, heavy metals, MTBE, herbicides, pesticides
and chlorinated hydrocarbons.
5. Radioactive contamination
Resulting from 20th century activities in atomic physics, such as nuclear power generation
and nuclear weapons research, manufacture and deployment.

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6. Water pollution
By the discharge of wastewater from commercial and industrial waste (intentionally or
through spills) into surface waters; discharges of untreated domestic sewage, and chemical
contaminants, such as chlorine, from treated sewage; release of waste and contaminants
into surface runoff flowing to surface waters (including urban runoff and agricultural runoff,
which may contain chemical fertilizers and pesticides); waste disposal and leaching into
groundwater; eutrophication and littering.
Pollution control
Pollution control is a term used in environmental management. It means the control of emissions and
effluents into air, water or soil.
1. Recycling
Recycling is a process to change waste materials into new products to prevent waste of
potentially useful materials, reduce the consumption of fresh raw materials, reduce energy
usage, reduce air pollution (from incineration) and water pollution (from landfilling) by reducing
the need for "conventional" waste disposal, and lower greenhouse gas emissions as compared
to plastic production.
2. Waste minimization
Waste minimization is a process of elimination that involves reducing the amount of waste
produced in society and helps eliminate the generation of harmful and persistent wastes,
supporting the efforts to promote a more sustainable society.
3. Compost
Compost is organic matter that has been decomposed and recycled as a fertilizer and soil
amendment. Compost is a key ingredient in organic farming.
4. Dust Collector
A dust collector is a system used to enhance the quality of air released from industrial and
commercial processes by collecting dust and other impurities from air or gas. Designed to
handle high-volume dust loads, a dust collector system consists of a blower, dust filter, a filter-
cleaning system, and a dust receptacle or dust removal system. It is distinguished from air
cleaners, which use disposable filters to remove dust.
5. Sewage Treatment
Sewage treatment is the process of removing contaminants from wastewater, including
household sewage and runoff (effluents). It includes physical, chemical, and biological processes
to remove physical, chemical and biological contaminants. Its objective is to produce an
environmentally safe fluid waste stream (or treated effluent) and a solid waste (or treated
sludge) suitable for disposal or reuse (usually as farm fertilizer).
Reducing Chemicals Environmental Impact
I. Pesticides, fungicides, weedicides are not used in the garden.
II. Natural air fresheners – such as natural oils – are used instead of chemical air fresheners.
III. We should use bio fertilizer.
IV. Waste chemicals should not be drain into the water.
V. Chemically synthesized fertilizer should not be used in the soil.
VI. Release of toxic chemical gases in environmental should not be done.

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Weedicides
A weedicide is usually a chemical or organic substance which is used to remove unwanted plants mainly
like weeds which effect the healthy growth if the plant. But excessive use of weedicides can also effect
the growth of plants and cause them to become harmful when consumed.
Weedicide has direct effect on cropes.
Some common weedicides are 2, 4-D, Metachlor, Nitofen.
Pesticides
Pesticides a substance used for destroying insects or other organisms harmful to cultivated plants or to
animals.
Pesticides meant for attracting, seducing, and then destroying, or mitigating any pest.
They are a class of biocide.
The most common use of pesticides is as plant protection products (also known as crop protection
products), which in general protect plants from damaging influences such as weeds, plant diseases or
insects.
Example- benomyl, carboxin, fenac, 2, 3, 6-TBA, 2, 4, 5-T
Bio pesticides
Bio pesticides are certain types of pesticides derived from such natural materials as animals, plants,
bacteria, and certain minerals.
For example, canola oil and baking soda have pesticidal applications and are considered bio pesticides.
Advantage
1. Environment Friendly
2. Cost is low
3. Do not leave any residue to food material
4. Insect do not develop resistance towards it.
5. Highly specific
6. Not harmful.
Disadvantage
1. Slow speed of action
2. Highly specific
Fertilizer
Fertilizer (or fertiliser) is any material of natural or synthetic origin (other than liming materials) that is
applied to soils or to plant tissues (usually leaves) to supply one or more plant nutrients essential to the
growth of plants.
Conservative estimates report 30 to 50% of crop yields are attributed to natural or synthetic commercial
fertilizer. Global market value is likely to rise to more than US$185 billion until 2019.
Classification
1. Single nutrient ("straight") fertilizers
The main nitrogen-based straight fertilizer is ammonia or its solutions.
Ammonium nitrate (NH4NO3) is also widely used.
Urea is another popular source of nitrogen, having the advantage that it is a solid and non-
explosive, unlike ammonia and ammonium nitrate, respectively.

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2. Multinutrient fertilizers
These fertilizers are the most common. They consist of more than two or more nutrient
components.
3. Binary (NP, NK, PK) fertilizers
Major two-component fertilizers provide both nitrogen and phosphorus to the plants. These are
called NP fertilizers. The main NP fertilizer are monoammonium phosphate (MAP) and
diammonium phosphate (DAP). The active ingredient in MAP is NH4H2PO4. The active
ingredient in DAP is (NH4)2HPO4. About 85% of MAP and DAP fertilizers are soluble in water.
4. NPK fertilizers
NPK fertilizers are three-component fertilizers providing nitrogen, phosphorus, and potassium.
The Global Environment Monitoring System (GEMS)
The Global Environment Monitoring System (GEMS) is a collective effort of the world community to
acquire, through monitoring, the data which are needed for the rational management of the
environment.
The Global Environment Monitoring System (GEMS) arose from recommendations of the United Nations
Conference on the Human Environment which was held in Stockholm in 1972.
The GEMS Programme Activity Centre (PAC) at UNEP headquarters in Nairobi, Kenya, coordinates all
that it can of the various environmental monitoring activities which are carried on throughout the
world—particularly those within the United Nations System.
Programme Activity Centre (PAC), in the manner of UNEP itself, is not operational but works mainly
through the intermediary of the Specialized Agencies of the United Nations System—most notably FAO,
ILO, UNESCO, WHO, and WMO—together with appropriate intergovernmental organizations such as
IUCN.
The GEMS monitoring system consists of five closely interrelated programmes which have built-in
provision for training and for rendering technical assistance to ensure the participation of countries that
are inadequately provided with personnel and equipment.
The five are:
1. Climate-related monitoring;
2. Monitoring of long-range transport of pollutants;
3. Health-related monitoring (concerned with pollutional effects)
4. Ocean monitoring; and
5. Terrestrial renewable-resource monitoring.
Monitored data are gathered at suitable coordinating centers for each network at which appropriate
data-bases have been, or are being, established. Data are analyzed to produce periodic regional and
global assessments which are reported at intervals that are appropriate to the variable which is being
considered.

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Sewage treatment
Sewage treatment is the process of removing contaminants from wastewater, including household
sewage and runoff (effluents). It includes physical, chemical, and biological processes to remove
physical, chemical and biological contaminants. Its objective is to produce an environmentally safe fluid
waste stream (or treated effluent) and a solid waste (or treated sludge) suitable for disposal or reuse
(usually as farm fertilizer).
Process
Sewage can be treated close to where the sewage is created, a decentralized system (in septic tanks,
biofilters or aerobic treatment systems), or be collected and transported by a network of pipes and
pump stations to a municipal treatment plant, a centralized system (see sewerage and pipes and
infrastructure). Sewage collection and treatment is typically subject to local, state and federal
regulations and standards. Industrial sources of sewage often require specialized treatment processes.
Sewage treatment generally involves three stages, called primary, secondary and tertiary treatment.
1. Primary Treatment
Primary treatment consists of temporarily holding the sewage in a quiescent basin where heavy
solids can settle to the bottom while oil, grease and lighter solids float to the surface. The
settled and floating materials are removed and the remaining liquid may be discharged or
subjected to secondary treatment.
2. Secondary Treatment
Secondary treatment removes dissolved and suspended biological matter. Secondary treatment
is typically performed by indigenous, water-borne micro-organisms in a managed habitat.
Secondary treatment may require a separation process to remove the micro-organisms from the
treated water prior to discharge or tertiary treatment.
3. Tertiary Treatment
Tertiary treatment is sometimes defined as anything more than primary and secondary
treatment in order to allow rejection into a highly sensitive or fragile ecosystem (estuaries, low-
flow Rivers, coral reefs). Treated water is sometimes disinfected chemically or physically (for
example, by lagoons and microfiltration) prior to discharge into a stream, river, bay, lagoon or
wetland, or it can be used for the irrigation of a golf course, green way or park. If it is sufficiently
clean, it can also be used for groundwater recharge or agricultural purposes.

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Solid Waste Treatment
Solid waste are wastes that are not liquid or gaseous the term solid waste means: Material such as
household garbage, food wastes, yard wastes, and demolition or construction debris.
Solid wastes are all the discarded solid materials from municipal, industrial, and agricultural activities
The objective of solid wastes treatment to control, collect, process, dispose of solid wastes in an
economical way consistent with the public health protection.
Arbiogaz is one of the leading firms in Turkey for solid waste processing plants. Biogas is produced as a
result of anaerobic fermentation of organic materials collected from municipal, agricultural and
industrial wastes, including treatment plant sludge.
Three types of characteristics:
 Physical
 Chemical and
 Biological
Solid Waste Treatment Method
Several methods are used for treatment and disposal of solid waste. These are:
1. Composting
It is a process in which organic matter of solid waste is decomposed and converted to humus
and mineral compounds.
Compost is the end product of composting, which used as fertilizer.
2. Landfilling
A landfill site is a site for the disposal of waste materials by burial and is the oldest form
of waste treatment.
3. Pyrolysis
Pyrolysis heating of the solid waste at very high temperature in absence of air.
Carried out at temp. Between 500 ˚C – 1000 ˚C.
Gas, liquid and chars are the byproducts.
4. Recycling
Recycling is processing used materials into new products.
It reduce the consumption of fresh raw materials, reduce energy usage, reduce air pollution
(from incineration) and water pollution (from landfilling).
Recycling is a key component of modern waste reduction and is the third component of the
"Reduce, Reuse, and Recycle" waste hierarchy.
5. Incineration
Incineration is a waste treatment process that involves the combustion of organic substances
contained in waste materials.
Incineration and other high temperature waste treatment systems are described as "thermal
treatment".
Incineration of waste materials converts the waste into ash, flue gas, and heat.
Incinerators are used for this process. Important points regarding incineration are:
a) Supplying of solid waste should be continuous.
b) Waste should be proper mixed with fuel for complete combustion.
c) Temperature should not less than 670 ˚C.

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Advantages
a) Most hygienic method.
b) Complete destruction of pathogens.
c) No odor trouble.
d) Heat generated may be used for steam power.
e) Clinkers produced may be used for road construction.
f) Less space required.
g) Adverse weather condition has no effect.
Disadvantages
a) Large initial expense.
b) Care and attention required otherwise incomplete combustion will increase air
pollution.
c) Residues required to be disposed which require money.
d) Large no of vehicles required for transportation.
Biofuel
A biofuel is a fuel whose energy is derived from biological carbon fixation, such as plants.
Carbon fixation or carbon assimilation refers to the conversion process of inorganic carbon (carbon
dioxide) to organic compounds by living organisms.
Biofuels are made by a biomass conversion (biomass refers to recently living organisms, most often
referring to plants or plant-derived materials), this biomass can be converted to convenient energy
containing substances in three different ways: thermal conversion, chemical conversion, and
biochemical conversion. This biomass conversion can result in fuel in solid, liquid, or gas form. This new
biomass can be used for biofuels. Biofuels have increased in popularity because of rising oil prices and
the need for energy security.
Bioethanol is an alcohol made by fermentation, mostly from carbohydrates produced in sugar or starch
crops such as corn, sugarcane, or sweet sorghum.
Biodiesel can be used as a fuel for vehicles in its pure form, but it is usually used as a diesel additive to
reduce levels of particulates, carbon monoxide, and hydrocarbons from diesel-powered vehicles.
Biodiesel is produced from oils or fats using transesterification and is the most common biofuel in
Europe.
First-generation biofuels
'First-generation' or conventional biofuels are made from sugar, starch, or vegetable oil.
1. Ethanol
Biologically produced alcohols, most commonly ethanol, and less commonly propanol and
butanol, are produced by the action of microorganisms and enzymes through the fermentation
of sugars or starches (easiest), or cellulose (which is more difficult).
2. Biodiesel
Biodiesel is the most common biofuel in Europe. It is produced from oils or fats using
transesterification and is a liquid similar in composition to fossil/mineral diesel. Chemically, it
consists mostly of fatty acid methyl (or ethyl) esters (FAMEs).

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3. Bioethers
Bioethers (also referred to as fuel ethers or oxygenated fuels) are cost-effective compounds that
act as octane rating enhancers."Bioethers are produced by the reaction of reactive iso-olefins,
such as iso-butylene, with bioethanol."Bioethers are created by wheat or sugar beet. They also
enhance engine performance, whilst significantly reducing engine wear and toxic exhaust
emissions.
4. Biogas
Biogas is methane produced by the process of anaerobic digestion of organic material by
anaerobes. It can be produced either from biodegradable waste materials or by the use of
energy crops fed into anaerobic digesters to supplement gas yields. The solid byproduct,
digestate, can be used as a biofuel or a fertilizer.
5. Solid Biofuels
Examples include wood, sawdust, grass trimmings, domestic refuse, charcoal, agricultural waste,
nonfood energy crops, and dried manure.
When raw biomass is already in a suitable form (such as firewood), it can burn directly in a stove
or furnace to provide heat or raise steam.
Second-generation (advanced) biofuels
Second generation biofuels, also known as advanced biofuels, are fuels that can be manufactured from
various types of biomass. Biomass is a wide-ranging term meaning any source of organic carbon that is
renewed rapidly as part of the carbon cycle. Biomass is derived from plant materials but can also include
animal materials.
First generation biofuels are made from the sugars and vegetable oils found in arable crops, which can
be easily extracted using conventional technology. In comparison, second generation biofuels are made
from lignocellulosic biomass or woody crops, agricultural residues or waste, which makes it harder to
extract the required fuel.

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Bioremediation
Bioremediation is a waste management technique that involves the use of organisms to remove or
neutralize pollutants from a contaminated site.
According to the EPA (Environmental Protection Agency), bioremediation is a “treatment that uses
naturally occurring organisms to break down hazardous substances into less toxic or non-toxic
substances”.
Bioremediation can be generally classified as in situ or ex situ.
In situ bioremediation involves treating the contaminated material at the site, while ex situ involves the
removal of the contaminated material to be treated elsewhere.
Some examples of bioremediation related technologies are phytoremediation, bioventing, bioleaching,
landfarming, bioreactor, composting, bioaugmentation, rhizofiltration, and biostimulation.
Bioremediation may occur on its own (natural attenuation or intrinsic bioremediation) or may only
effectively occur through the addition of fertilizers, oxygen, etc., that help encourage the growth of the
pollution-eating microbes within the medium (biostimulation).
Microorganisms used to perform the function of bioremediation are known as bioremediators.
The idea of bioremediation has become popular with the onset of the twenty-first century. In principle,
genetically engineered plants and microorganisms can greatly enhance the potential range of
bioremediation. For example, bacterial enzymes engineered into plants can speed up the breakdown of
TNT and other explosives. With transgenic poplar trees carrying a bacterial gene, methyl mercury may
be converted to elemental mercury, which is released to the atmosphere at extreme dilution. However,
concern about release of such organisms into the environment has limited actual field applications.
Essential Factors for Microbial Bioremediation
Factor Desired Conditions
Microbial
Population
Suitable kinds of organisms that can biodegrade all of the contaminants
Oxygen Enough to support aerobic biodegradation (about 2% oxygen in the gas phase or 0.4
mg/liter in the soil water)
Water Soil moisture should be from 50–70% of the water holding capacity of the soil
Nutrients Nitrogen, phosphorus, sulfur, and other nutrients to support good microbial growth
Temperature Appropriate temperatures for microbial growth (0–40˚C)
pH Best range is from 6.5 to 7.5
In Situ Bioremediation
In situ processes (degrading the contaminants in place) are often recommended because less material
has to be moved.
These processes can be designed with or without plants.
Plants have been used because they take up large quantities of water, this helps to control
contaminated water, such as a groundwater contaminant plume, in the soil.
Aerobic (oxygen-using) processes may occur in the unsaturated layer of soil, the vadose zone, which is
found above the water table.
The vadose zone is defined as the layer of soil having continuously connected passages filled with air,
while the saturated zone is the deeper part where the pores are filled with water. Oxygen moves in the
unsaturated zone by diffusion through pores in the soil. Some plants also provide pathways to move
oxygen into the soil. This can be very important to increase the aerobic degradation of organic
compounds.

30
Algae Bioremediation
Algae bioremediation is unique because it is a self-sustaining cycle (Figure 1). To oxidize contaminants
into less-harmful metabolites, algae extract and utilize oxygen from its surrounding environment; these
metabolites include CO2 and H2O. For growth, algae use photosynthesis, which requires CO2 and H2O.
Photosynthesis, in turn, releases oxygen that algae can employ for further contaminant oxidation, thus
repeating the cycle.
Figure 1: Algae Bioremediation. Metabolism of the contaminants through algae produces CO2, H2O, and
less-harmful components. In turn the algae are able to use the CO2, H2O, and less-harmful components
as nutrition necessary for photosynthesis and sustainability.
Mycoremediation is a form of bioremediation in which fungi are used to decontaminate the area. The
term mycoremediation refers specifically to the use of fungal mycelia in bioremediation.
Advantages and disadvantages of Bioremediation
 Advantages
1) Bioremediation is a natural process.
2) It is cost effective.
3) Toxic chemicals are destroyed or removed from environment and not just merely separated.
4) Low capital expenditure.
5) Less energy is required as compared to other technologies
6) Less manual supervision.
 Disadvantages
1) The process of bioremediation is slow. Time required is in day to months.
2) Heavy metals are not removed.
3) For in-situ bioremediation site must have soil with high permeability.
4) It does not remove all quantities of contaminants.

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Biodegradation
Biodegradation is the chemical dissolution or degradation of materials by bacteria or other biological
means, degradation caused by enzymatic process resulting from the action of cells.
Biodegradable matter is generally organic material such as plant and animal matter and other
substances originating from living organisms, or artificial materials that are similar enough to plant and
animal matter to be put to use by microorganisms.
In nature, different materials biodegrade at different rates, and a number of factors are important in the
rate of degradation of organic compounds.
To be able to work effectively, most microorganisms that assist the biodegradability need light, water
and oxygen. Temperature is also an important factor in determining the rate of biodegradability.
Biodegradation of Natural Product (For example Oil)
Biodegradation is the process of breaking down material in simpler components by living organisms,
most often microorganisms.
Oil spills occur due to accidents in the industry as a result of extraction or transportation. Since such
spills spread over great areas and have deleterious effects on living organisms. It is important to use
environmentally friendly mechanisms for their cleanup.
There are many microorganisms that can break down petroleum, the most prominent being
hydrocarbonoclastic bacteria. A representative of this group is Alcanivorax borkumensis, and its genome
contains genes that code for the degradation of alkanes.
Petroleum (crude oil) is a liquid fossil fuel. It is a product of decaying organic matter, such as algae and
zooplankton. It is one of the major energy sources in the world, and is also used by the chemical industry
to manufacture a large number of consumer products.
Oil spills in marine environments are especially damaging because they cannot be contained and can
spread over huge areas.
In the environment, such spills are naturally cleaned by microorganisms that can break down the oil,
some microorganisms produce enzymes that can degrade a variety of chemical compounds, including
hydrocarbons like oil.
The dominant group of such bacteria are the hydrocarbonoclastic bacteria (HCB). The concentration of
these bacteria increases significantly in areas of oil spill. One of the best studied representative of this
group is Alcanivorax borkumensis; it's also the only one to have its genome sequenced. This species
contains individual genes responsible for breaking down certain alkanes into harmless products. It also
possesses genes to direct the production of a layer of biosurfactant around the cell to enhance the oil
emulsification. The addition of nitrogen and phosphorus to the Alcanivorax environment increases its
growth rate. However, the addition of these nutrients in natural environments to improve the cleanup
of oil spills is not desirable, since it can have an overall negative impact on the ecosystem.
Aside from hydrocarbons, crude oil contains additional toxic compounds, such as pyridine. These are
degraded by representatives of other genera such as Micrococcus and Rhodococcus. Oil tarballs are
biodegraded slowly by species from the genera Chromobacterium, Micrococcus, Bacillus, Pseudomonas,
Candida, Saccharomyces and others. In the cleanup of the Deepwater Horizon oil spill, genetically
modified microorganisms were used, but some scientists suspect they might have caused health issues
for people in the affected areas.

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Biodegradation of Xenobiotics
Xenobiotics are that compound which have been produced artificially by chemical synthesis for
industrial or agricultural purposes e.g. DDT, halogenated H.C., aromatics, pesticides, PCB, PAH, lignin.
Use of pesticides has benefited the modem society by improving the quantity and quality of the worlds'
food production. Gradually, pesticide usage has become an integral part of modern agriculture system.
Many of the artificially made complex compounds i.e. xenobiotics persist in environment and do not
undergo biological transformation.
Microorganisms play an important role in degradation of xenobiotics, and maintaining of steady state
concentrations of chemicals in the environment. The complete degradation of a pesticide molecule to its
inorganic components that can be eventually used in an oxidative cycle removes its potential toxicity
from the environment.
Microbial Degradation of Xenobiotics
Biodegradation of pesticides occurs by aerobic soil microbes. Pesticides are of wide varieties of
chemicals e.g. chlorophenoxyalkyl caboxylic acid, substituted ureas, nitrophenols, tri-azines, phenyl
carbamates, orga-nochlorines, organophosphates, etc. Duration of persistence of her-bicides and
insecticides in soil is given in Table 21.2. Otganophos-phates (e.g. diazion, methyl par-athion and
parathion) are perhaps the most extensively used insecti-cides under many agricultural sys-tems.
Biodegradation through hy-drolysis of p-o-aryl bonds by Pseudomonas diminuta and Flavobacterium are
considered as the most significant steps in the detoxification of organophospho-rus compounds.
Organomercurials (e.g. Semesan, Panodrench, Panogen) have been practiced in agriculture since the
birth of fun-gicides. Several species of Aspergillus, Penicillium and Trichoderma have been isolated from
Semesan-treated soil. More-over, they have shown ability to grow over 100 ppm of fungicide in vitro.
The major fungicides used in agriculture are water soluble derivatives such as Ziram, Ferbam, Thiram,
etc. All these are de-graded by microorganisms.

33
Microbial Desulphurization
Desulphurize means to free or become free from sulphur.
Phototropic bacterium (PCB) is one of the soil microorganisms widely distributed in the environment,
playing an important role in food chain among all animals and plants. Phototropic bacterium, a nitrogen-
fixing organism, also provides nutrients for animals and plants-the bacterium is rich in amino acid,
nucleic acids, hormones, coenzymes and vitamins produced within the cell.
Phototropic bacterium converts toxic hydrogen sulfide to non-toxic form, thus reducing the growth of
sulfate reducing bacteria.
Microbial Desulphurization of Coal
The sulfur found in coal is generally divided into three forms: pyritic, sulfate, and organic sulfur.
Originating from in-situ oxidation of metal sulfides, sulfates axe present in coal at low concentrations.
Being soluble in water, they are relatively easy to be leached from the coal. Similarly, microbial
depyritization promotes the oxidative conversion of inorganic sulfur compounds to water-soluble
products. The pyrite removal results from the combined effects of direct bacterial attack and indirect
chemical solubilization. In the former, pyrite (FeS2) is oxidized by bacteria into Fe2 (SO4) 3; in the latter,
ferric iron is the actual oxidizing agent and microorganisms serve to regenerate the ferric iron from
ferrous iron.
Fermentation
Fermentation is a metabolic process that converts sugar to acids, gases, and/or alcohol.
Fermentation occurs in yeast and bacteria, but also in oxygen-starved muscle cells, as in the case of
lactic acid fermentation.
Fermentation is also used more broadly to refer to the bulk growth of microorganisms on a growth
medium, often with the goal of producing a specific chemical product.
The science of fermentation is known as zymology.
Fermentation takes place in the lack of oxygen (when the electron transport chain is unusable) and
becomes the cell’s primary means of ATP (energy) production. It turns NADH and pyruvate produced in
the glycolysis step into NAD+ and various small molecules depending on the type of fermentation (see
examples below). In the presence of O2, NADH and pyruvate are used to generate ATP in respiration.
This is called oxidative phosphorylation, and it generates much more ATP than glycolysis alone.
Fermentation has been used by humans for the production of food and beverages since the Neolithic
age.
For example, fermentation is employed for preservation in a process that produces lactic acid as found
in such sour foods as pickled cucumbers, kimchi and yogurt, as well as for producing alcoholic beverages
such as wine and beer. Fermentation can even occur within the stomachs of animals, such as humans.
Auto-brewery syndrome is a rare medical condition where the stomach contains brewer’s yeast that
break down starches into ethanol; which enters the blood stream.
Fermentation does not necessarily have to be carried out in an anaerobic environment. For example,
even in the presence of abundant oxygen, yeast cells greatly prefer fermentation to aerobic respiration,
as long as sugars are readily available for consumption.
Lactic acid fermentation refers to two means of producing lactic acid:
 Homolactic fermentation is the production of lactic acid exclusively
 Heterolactic fermentation is the production of lactic acid as well as other acids and alcohols.

34
Methods and Types of Fermentation
Ethanol Fermentation
The chemical equation below shows the alcoholic fermentation of glucose, whose chemical formula is
C6H12O6. One glucose molecule is converted into two ethanol molecules and two carbon dioxide
molecules:
C6H12O6 → 2 C2H5OH + 2 CO2
C2H5OH is the chemical formula for ethanol.
Before fermentation takes place, one glucose molecule is broken down into two pyruvate molecules.
This is known as glycolysis.
Types of Fermentation Processes
1. Batch Fermentations
The "batch culture" fermentation is also known as "closed culture" system, in this process
fermenter is filled with the prepared mash of raw mate-rials to be fermented. The temperature
and pH for microbial fermen-tation is properly adjusted, and occasionally nutritive supplements
are added to the prepared mash.
There is no refill of nutrients once the fermentation process has started and the product is
recovered at the end of the process.
As time pass, they increase in number with rapid use of the nutrients and simultaneously
produce toxic metabolites, due to production of toxic metabolites the growth of organisms slow
down during the later stages of the fermentation process.
Once the process is completed, then, the fermentation vessel is cleaned properly, sterilized
before it use for another batch process.
Growth curve of microorganism:

35
2. Fed-batch Fermentations
Fed-batch fermentation defined as an operational technique in biotechnological processes
where one or more nutrients (substrates) are fed (supplied) to the bioreactor during microbial
growth and in which the product(s) remain in the bioreactor until the end of the run.
An alternative description of the method is that of a fermentation in which "a base medium
supports initial cell culture and a feed medium is added to prevent nutrient depletion".
It is also a type of semi-batch culture. In some cases, all the nutrients are fed into the bioreactor.
The advantage of the fed-batch culture is that one can control concentration of fed-substrate in
the culture liquid at arbitrarily desired levels (in many cases, at low levels).
Generally speaking, fed-batch culture is superior to conventional batch culture when controlling
concentrations of a nutrient (or nutrients) affect the yield or productivity of the desired
metabolite.
3. Continuous Fermentation
In batch cultures, nutrients are not renewed and so growth remains exponential for only a few
generations.
Microbial population can be maintained in a state of exponential growth for a long time by using
a system of continuous culture. This is known as steady state or balanced growth.
Balanced growth is maintained by supplying medium continuously. Medium is designed in such
a way that growth is restricted by substrate and not by toxin accumulation. Thus exponential
growth will continue by addition of new fresh medium.
Exponential growth is continued till the fermentor is completely filled with media. However if
the overflow device is attached to the fermentor then fresh medium can be added continuously
by transferring same volume of culture form the fermentor then cells can be produced
continuously.
At steady state the specific growth rate (μ) of the micro-organism is equal to the dilution rate
(D). The dilution rate is defined as the rate of flow of medium over the volume of culture in the
bioreactor.
Late nutrient addition in continuous fermentation process-
During fermentation amino acids are not taken up equally by the yeast cell - some are utilized at
beginning of the growth cycle, some later, and some not at all. Ammonia, on the other hand is
consumed preferentially to amino acids. Therefore, timing of DAP (diammonium phosphate)
(25.8% ammonia, 74.2% phosphate) addition is important. One large addition of DAP at the
beginning may delay/inhibit uptake of amino acids.
Multiple additions are preferred.
Adding nutrient supplements all at once can lead to too fast of a fermentation rate and an
imbalance in uptake and usage of nitrogen compounds. Supplements added too late (after half
the fermentation) may not be used by the yeasts, in part because the alcohol prevents their
uptake. For the same reason, adding nutrients to a stuck fermentation seldom does any good at
all. Do not wait until you have a sluggish or stuck fermentation to add nutrients.

36
4. Aerobic fermentation
Aerobic fermentation means that oxygen is present. Wine, beer and acetic acid vinegar (such as
apple cider vinegar), need oxygen in the “primary” or first stage of fermentation.
When creating acetic vinegar, for example, exposing the surface of the vinegar to as much
oxygen as possible, creates a healthy, flavorful vinegar with the correct pH.
5. Anaerobic fermentation
Anaerobic fermentation is a method cells use to extract energy from carbohydrates when
oxygen or other electron acceptors are not available in the surrounding environment. This
differentiates it from anaerobic respiration, which doesn’t use oxygen but does use electron-
accepting molecules that come from outside of the cell. The process can follow glycolysis as the
next step in the breakdown of glucose and other sugars to produce molecules of adenosine
triphosphate (ATP) that create an energy source for the cell.
Dual/Multiple Fermentation
A method of continuous product formation using at least two continuous fermentation units and a
microorganism capable of being induced, in response to environmental conditions, to undergo a genetic
alteration from a state favoring microorganism growth to a state favoring product production by the
microorganism.
The first continuous fermentation unit is maintained at environmental conditions selected to favor
growth of the microorganism and to be non-permissive for the genetic alteration. The microorganism is
grown continuously in the first unit, and a portion of the growing microorganism cell mass is transferred
via connecting means to the second continuous fermentation unit. Either the connecting means or the
second unit is maintained at second environmental conditions selected to effect the genetic alteration.
The altered microorganism is cultured in the second unit. Exudate from this second fermenter
(containing microorganism mass and medium) is continually removed and the product which is present,
either in the microorganisms themselves or the medium surrounding them, is extracted.
Fermenter
A fermenter is an apparatus that maintains required optimal environmental conditions for the growth of
industrially important microorganisms, used in large scale fermentation process and in the commercial
production of a range of fermentation products like Antibiotics, Enzymes, Organic acids, Alcoholic
beverages etc.
An ideal fermenter maintains optimal environmental conditions throughout the process for the process
organisms, added substrates and additives for a quality end product.
Many times, the terms “Bioreactor” and “Fermenter” are used synonymously. There is a very minor
difference between these two-
The bioreactor is used for the mass culture of plant and animal cells, while fermenter is mainly used for
microbial culture.
The operational parameters and design engineering of fermenters and bioreactors are identical.
1. A bioreactor should provide for the following:
2. Agitation (for mixing of cells and medium).
3. Aeration (aerobic fermenters; for O2 supply).
4. Regulation of factors like temperature, pH, pressure, aeration, nutrient feeding etc.
5. Sterilization and maintenance of sterility.
6. Withdrawal of cells/medium (for continuous fermenters).

37
Modern fermenters are usually integrated with computers for efficient process monitoring, data
acquisition etc.
S.No. Parts of Fermenter Function
1. Impellor (agitator) To stir the media continuously and hence prevent cells from settling
down, and distribute oxygen throughout the medium.
2. Sparger (Aerator) Introduce sterile oxygen to the media in case of aerobic
fermentation process.
3. Baffles (vortex
breaker)
Disrupt vortex and provide better mixing.
4. Inlet Air filter Filter air before it enter the fermenter.
5. Exhaust Air filter Trap and prevent contaminants from escaping.
6. Rotameter Measure flow rate of Air or liquid.
7. Pressure gauge Measure pressure inside the fermenter.
8. Temperature probe Measure and monitor change in temperature of the medium during
the process.
9. Cooling Jacket To maintain the temperature of the medium throughout the
process.
10. pH probe Measure and monitor pH of the medium.
11. Dissolve Oxygen
Probe
Measure dissolve oxygen in the fermenter.
12. Antifoam Breakdown and prevent foams.
13. Valves Regulation and control the flow liquids and gases.
14. Control panel Monitor over all parameters.
15. Sampling pint To obtain samples during the process.
16. Foam probe Detect the presence of the foam.
17. Level probe Measure the level of medium
18. Acid Maintain the required pH of the medium by neutralizing the basic
environment
19. Base Maintain the required pH of the medium by neutralizing the acidic
environment

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