Microbial, Industrial and Environmental Biotechnology

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Vivek Tripathi ([email protected])

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

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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.

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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.

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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.

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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:

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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.

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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).

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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

Education

Microbial, Industrial and Environmental Biotechnology