SEWAGE TREATMENT (WASTEWATERTREATMENT AND DISPOSAL)
The size and capacity of wastewater treatment systems are determined by the estimated volume
of sewage generated from residences, businesses, and industries connected to sewer systems as
well as the anticipated inflows and infiltration (I&I). The selection of specific on-lot, clustered,
or centralized treatment plant configurations depends upon factors such as the number of
customers being served, the geographical scenario, site constraints, sewer connections, average
and peak flows, influent wastewater characteristics, regulatory effluent limits, technological
feasibility,!energy!consumption, and the operations and maintenance costs involved.
The!predominant!method of wastewater disposal in large cities and towns is discharge into a
body of surface water. Suburban and rural areas rely more on subsurface disposal. In either case,
wastewater must be purified or treated to some degree in order to protect both!public health!and
water quality. Suspended particulates and biodegradable organics must be removed to varying
extents. Pathogenic!bacteria!must be destroyed. It may also be necessary to
remove!nitrates!and!phosphates!(plant nutrients) and to neutralize or remove industrial wastes
and toxic chemicals.
The degree to which wastewater must be treated varies, depending on local environmental
conditions and governmental standards. Two pertinent types of standards are!stream standards
and effluent standards. Stream standards, designed to prevent the deterioration of existing water
quality, set limits on the amounts of specific pollutants allowed in streams, rivers, and lakes. The
limits depend on a classification of the “maximum!beneficial!use” of the water. Water
quality!parameters!that are regulated by stream standards include dissolved oxygen, coliforms,
turbidity, acidity, and toxic substances.!Effluent standards, on the other hand, pertain directly to
the quality of the treated wastewater discharged from a sewage treatment plant. The factors
controlled under these standards usually include!biochemical oxygen demand!(BOD),
suspended solids, acidity, and!coliforms.
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Learn about primary, secondary, and tertiary wastewater treatment steps and how a Secchi disk
tests purity
STEPS IN THE MODERN TREATMENT OF WASTEWATER.
There are three levels of wastewater treatment: primary, secondary, and tertiary (or
advanced).!Primary treatment!removes about 60 percent of total suspended solids and about 35
percent of BOD; dissolved impurities are not removed. It is usually used as a first step
before!secondary treatment. Secondary!treatment!removes more than 85 percent of both
suspended solids and BOD. A minimum level of secondary treatment is usually required in
the!United States!and other developed countries. When more than 85 percent of total solids and
BOD must be removed, or when dissolved nitrate and phosphate levels must be
reduced,tertiarytreatment methods are used. Tertiary processes can remove more than 99
percent of all the impurities from sewage, producing an effluent of almost drinking-water
quality. Tertiary treatment can be very expensive, often doubling the cost of secondary treatment.
It is used only under special circumstances.
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For all levels of wastewater treatment, the last step prior to!discharge!of the sewage effluent into
a body of surface water is!disinfection, which destroys any remaining pathogens in the effluent
and protects public health. Disinfection is usually accomplished by mixing the effluent
with!chlorine!gas or with liquid solutions of hypochlorite chemicals in a contact tank for at least
15 minutes. Because chlorine residuals in the effluent may have adverse effects on aquatic life,
an additional chemical may be added to dechlorinate the effluent.!Ultraviolet radiation, which
can disinfect without leaving any residual in the effluent, is becoming more competitive with
chlorine as a wastewater disinfectant.
PRIMARY TREATMENT
Activated sludge process
Primary and secondary treatment of sewage, using the activated sludge process. (more)
Primary treatment removes material that will either float or readily settle out by!gravity. It
includes the physical processes of screening, comminution,gritremoval, and sedimentation.
Screens are made of long, closely spaced, narrowmetal!bars. They block floating debris such
as!wood, rags, and other bulky objects that could clog pipes or pumps. In modern plants the
screens are cleaned mechanically, and the material is promptly disposed of by burial on the plant
grounds. A comminutor may be used to grind and shred debris that passes through the screens.
The shredded material is removed later by sedimentation or flotation processes.
Grit chambers!are long narrow tanks that are designed to slow down the flow so that solids such
as!sand, coffee grounds, and eggshells will settle out of the!water. Grit causes excessive wear and
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tear on pumps and other plant equipment. Its removal is particularly important in cities
with!combined!sewer!systems, which carry a good deal of silt, sand, and gravel that wash off
streets or land during a storm.
Suspended solids that pass-through screens and grit chambers are removed from the sewage in
sedimentation tanks. These tanks, also called!primary clarifiers, provide about two hours of
detention time for gravity settling to take place. As the sewage flows through them slowly, the
solids gradually sink to the bottom. The settled solids known as raw or primary!sludge are moved
along the tank bottom by mechanical scrapers. Sludge is collected in a hopper, where it is
pumped out for removal. Mechanical surface-skimming devices remove grease and other floating
materials.
SECONDARY TREATMENT
Secondary treatment removes the soluble organic matter that escapes primary treatment. It also
removes more of the suspended solids. Removal is usually accomplished by biological processes
in which microbes consume the organic impurities as!food, converting them into!carbon
dioxide, water, and!energyfor their own growth and reproduction. The sewage treatment plant
provides a suitable!environment,!albeit!of steel and concrete, for this natural biological process.
Removal of soluble organic matter at the treatment plant helps to protect the dissolved oxygen
balance of a receiving stream,!river, or lake.
There are three basic biological treatment methods: the trickling filter, the activated sludge
process, and the oxidation pond. A fourth, less common method is the rotating biological
contactor.
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Trickling filter
A trickling filter is simply a tank filled with a deep bed of stones. Settled sewage is sprayed
continuously over the top of the stones and trickles to the bottom, where it is collected for further
treatment. As the wastewater trickles down, bacteria gather and multiply on the stones. The
steady flow of sewage over these growths allows the microbes to absorb the dissolved organics,
thus lowering the biochemical oxygen demand (BOD) of the sewage. Air circulating upward
through the spaces among the stones provides sufficient oxygen for the metabolic processes.
Settling tanks, called!secondary clarifiers, follow the trickling filters. These clarifiers remove
microbes that are washed off the rocks by the flow of wastewater. Two or more trickling filters
may be connected in series, and sewage can be recirculated in order to increase
treatment!efficiencies.
Activated sludge
The activated!sludge!treatment system consists of an aeration tank followed by a secondary
clarifier. Settled sewage, mixed with fresh sludge that is recirculated from the secondary
clarifier, is introduced into the aeration tank. Compressed!air!is then injected into the mixture
through porous diffusers located at the bottom of the tank. As it bubbles to the surface, the
diffused air provides oxygen and a rapid mixing action. Air can also be added by the churning
action of mechanical propeller-like mixers located at the tank surface.
Under such oxygenated conditions, microorganisms thrive, forming an active,
healthy!suspension!of biological solids mostly!bacteria called activated sludge. About six hours
of detention is provided in the aeration tank. This gives the microbes enough time to absorb
dissolved organics from the sewage, reducing the!BOD. The mixture then flows from the
aeration tank into the secondary clarifier, where activated sludge settles out by gravity. Clear
water is skimmed from the surface of the clarifier, disinfected, and discharged as secondary
effluent. The sludge is pumped out from a hopper at the bottom of the tank. About 30 percent of
the sludge is recirculated back into the aeration tank, where it is mixed with the primary effluent.
This recirculation is a key feature of the activated sludge process. The recycled microbes are
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well!acclimated!to the sewage environment and readily metabolize the organic materials in the
primary effluent. The remaining 70 percent of the secondary sludge must be treated and disposed
of in an acceptable manner
Aeration treatment
Schematic diagram of a prefabricated package plant for the aeration treatment of small sewage
flows. (more)
Variations of the activated sludge process include extended aeration, contact stabilization, and
high-purity oxygen aeration.!Extended aeration and!contact stabilization systems omit the
primary settling step. They are efficient for treating small sewage flows from motels, schools,
and other relatively isolated wastewater sources. Both of these treatments are usually provided in
prefabricated steel tanks called!package plants. Oxygen aeration systems mix pure oxygen with
activated sludge. A richer concentration of oxygen allows the aeration time to be shortened from
six to two hours, reducing the required tank volume.
Oxidation pond
Oxidation ponds, also called lagoons or stabilization ponds, are large, shallow ponds designed
to treat wastewater through the interaction of!sunlight, bacteria, and algae.!Algae!grow
using!energy!from the sun and!carbon dioxide!and inorganiccompounds!released
by!bacteria!in!water. During the process of!photosynthesis, the algae release oxygen needed by
aerobic bacteria. Mechanical aerators are sometimes installed to supply yet more oxygen, thereby
reducing the required size of the pond. Sludge deposits in the pond must eventually be removed
by dredging. Algae remaining in the pond effluent can be removed by filtration or by a
combination of chemical treatment and settling.
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Rotating biological contactor
In this treatment system a series of large plastic disks mounted on a horizontal shaft are partially
submerged in primary effluent. As the shaft rotates, the disks are exposed alternately to!air!and
wastewater, allowing a layer of bacteria to grow on the disks and to metabolize the organics in
the wastewater.
TERTIARY TREATMENT
When the intended receiving water is very!vulnerable!to the effects of!pollution, secondary
effluent may be treated further by several tertiary processes.
Tertiary treatment of wastewater
(Left) During the filtering step, wastewater from secondary treatment, still containing suspended
solids, pours from a trough and percolates through a filter bed made of porous media such as
sand, gravel, and anthracite. The filtered water is then piped away for disposal. (Right) In the
backwashing step, entrained solids are periodically flushed from the filter media by pumping
filtered water back through the assembly. The backwash water, carrying suspended solids, is
returned to the beginning of the wastewater treatment process. (more)
For the removal of additional suspended solids and!BODfrom secondary effluent, effluent
polishing is an effective treatment. It is most often accomplished using granular media filters,
much like the filters used to purify drinking water. Polishing filters are usually built as
prefabricated units, with tanks placed directly above the filters for storing backwash water.
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ANALYSIS OF BACTERIA IN FOOD
Analyzing bacteria in food is crucial for ensuring food safety and preventing foodborne illnesses.
Microbiological analysis of food involves the detection, enumeration, and identification of
bacteria present in food products. Here's an overview of the steps involved in analyzing bacteria
in food:
1. Sampling
Representative Sampling: Collecting food samples from different batches, lots, or points in the
production process to ensure representativeness.
Aseptic Techniques: Using sterile sampling equipment and procedures to prevent contamination
during sample collection.
2. Sample Preparation
Homogenization: Mixing or blending food samples to ensure uniform distribution of bacteria
throughout the sample.
Dilution: Diluting samples with sterile diluents to achieve appropriate bacterial counts for
enumeration.
3. Microbiological Analysis
Total Viable Count (TVC):
Description: Determination of the total number of viable bacteria in a food sample.
Method: Spread plate or pour plate method using culture media such as Plate Count Agar (PCA)
or Standard Methods Agar (SMA).
Incubation: Incubating plates at appropriate temperatures (usually 30-37°C) for 24-48 hours.
Enumeration: Counting colonies and expressing results as colony-forming units per gram
(CFU/g) or milliliter (CFU/mL) of food.
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Specific Pathogen Detection:
Description: Identification and enumeration of specific pathogenic bacteria such as Salmonella,
Escherichia coli (E. coli), Listeria monocytogenes, and Staphylococcus aureus.
Methods: Culture-based methods (enrichment, selective media) or molecular techniques (PCR,
qPCR) for targeted detection and identification.
Indicator Organisms:
Description: Detection of indicator organisms such as coliforms, fecal coliforms, and
Enterobacteriaceae, which serve as indicators of fecal contamination and hygienic conditions.
Methods: Membrane filtration, multiple-tube fermentation, or most probable number (MPN)
techniques using selective and differential media.
4. Identification
Biochemical Tests: Performing biochemical assays (e.g., catalase, oxidase, fermentation tests)
to identify bacterial species based on metabolic characteristics.
Molecular Methods: Using molecular techniques such as polymerase chain reaction (PCR),
DNA sequencing, or mass spectrometry for rapid and accurate identification of bacterial species.
5. Data Analysis and Interpretation
Quantitative Analysis: Calculating bacterial counts and concentrations based on enumeration
results.
Qualitative Analysis: Identifying specific bacterial species and assessing their significance in
terms of food safety and quality.
Comparison with Regulatory Standards: Evaluating results against established
microbiological criteria and regulatory limits for various food products.
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6. Reporting
Documentation: Recording and documenting all analysis procedures, results, and
interpretations.
Communication: Reporting findings to relevant stakeholders, including food producers,
regulatory agencies, and consumers.
7. Corrective Actions
Risk Assessment: Assessing the potential risks associated with detected bacterial contamination
and determining appropriate corrective actions.
Hazard Control: Implementing control measures to mitigate bacterial hazards, such as
improving sanitation practices, adjusting processing parameters, or recalling contaminated
products if necessary.
ANALYSIS OF BACTERIA IN WATER
Analyzing bacteria in water is crucial for assessing water quality, identifying potential health
risks, and ensuring the safety of drinking water and recreational water bodies. Here's an overview
of the steps involved in analyzing bacteria in water:
1. Sampling
Sampling Locations: Collecting water samples from different points in the water distribution
system, such as source water, treatment plants, distribution networks, and recreational water
bodies.
Sampling Frequency: Conducting regular monitoring and sampling according to regulatory
requirements and risk assessments.
Sampling Techniques: Using appropriate sampling techniques and equipment to ensure
representative samples and minimize contamination.
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2. Sample Preservation and Handling
Cold Chain Management: Maintaining proper temperature control (usually refrigeration or ice)
during sample transport and storage to preserve bacterial viability.
Sterile Containers: Using sterile sampling bottles or containers to prevent contamination during
sample collection and transportation.
3. Microbiological Analysis
Total Coliforms and Escherichia coli (E. coli):
Description: Detection and enumeration of indicator bacteria such as total coliforms and E. coli,
which serve as indicators of fecal contamination and water quality.
Methods: Membrane filtration or multiple-tube fermentation techniques using selective and
differential media such as m-Endo Agar or Colilert.
Incubation: Incubating samples at appropriate temperatures (usually 35-37°C) for 24-48 hours.
Interpretation: Presence of coliforms indicates potential fecal contamination, while the
presence of E. coli suggests recent fecal pollution and increased health risks.
Heterotrophic Plate Count (HPC):
Description: Enumeration of heterotrophic bacteria that can grow on standard culture media
under aerobic conditions.
Method: Spread plate or pour plate method using non-selective media such as R2A Agar.
Incubation: Incubating plates at low temperatures (20-25°C) for 48-72 hours to encourage the
growth of a wide range of heterotrophic bacteria.
Interpretation: Elevated HPC counts may indicate deteriorating water quality or inadequate
disinfection.
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Other Pathogenic Bacteria:
Description: Detection and identification of specific pathogenic bacteria such as Salmonella,
Vibrio, Campylobacter, and Legionella.
Methods: Culture-based methods (enrichment, selective media) or molecular techniques (PCR,
qPCR) for targeted detection and identification.
Interpretation: Presence of pathogenic bacteria indicates potential health risks and requires
immediate action.
4. Data Analysis and Interpretation
Quantitative Analysis: Calculating bacterial counts and concentrations based on enumeration
results.
Qualitative Analysis: Assessing the significance of bacterial indicators and pathogens in terms
of water quality, health risks, and regulatory compliance.
Comparison with Regulatory Standards: Evaluating results against established
microbiological criteria and regulatory limits for various types of water (e.g., drinking water,
recreational water).
5. Reporting and Action
Documentation: Recording and documenting all analysis procedures, results, and
interpretations.
Communication: Reporting findings to relevant stakeholders, including water utility operators,
regulatory agencies, and the public.
Response and Management: Implementing appropriate management actions, such as water
treatment optimization, public health advisories, or regulatory enforcement, based on analysis
results and risk assessments.
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FOOD PRESERVATION METHODS
Food preservation methods are techniques used to prolong the shelf life of food products by
inhibiting microbial growth, delaying spoilage, and preventing deterioration. These methods aim
to maintain the quality, safety, and nutritional value of food over an extended period. Here are
some common food preservation methods:
1. Heat Processing
Canning: Heat treatment of food in sealed containers (e.g., cans, jars) to destroy
microorganisms, enzymes, and toxins.
Pasteurization: Mild heat treatment of liquid foods (e.g., milk, juice) to kill pathogens and
extend shelf life without significantly affecting flavor or nutritional value.
Blanching: Brief exposure of fruits and vegetables to steam or boiling water to inactivate
enzymes, preserve color, and facilitate freezing or canning.
2. Refrigeration
Cold Storage: Storing perishable foods at low temperatures (usually between 0-4°C) to slow
down microbial growth, enzymatic reactions, and spoilage.
Refrigerated Transport: Transporting food products under refrigerated conditions to maintain
freshness and prevent microbial proliferation during transit.
3. Freezing
Freezing: Lowering the temperature of food products below freezing point (-18°C or below) to
halt microbial growth, enzymatic activity, and chemical reactions.
Flash Freezing: Rapid freezing of food products using cryogenic gases or freezing tunnels to
minimize ice crystal formation and maintain texture and quality.
4. Drying
Dehydration: Removing moisture from food products through air drying, sun drying, or
mechanical methods to inhibit microbial growth and prevent spoilage.
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Freeze Drying: Freeze-drying food products under vacuum conditions to remove moisture while
preserving flavor, texture, and nutritional content.
5. Fermentation
Fermentation: Controlled microbial fermentation of food substrates by beneficial
microorganisms (e.g., bacteria, yeast) to produce lactic acid, alcohol, or other metabolites that
inhibit spoilage and enhance flavor.
Examples: Fermented dairy products (yogurt, cheese), fermented vegetables (sauerkraut,
kimchi), fermented beverages (beer, wine), and fermented condiments (soy sauce, miso).
6. Pickling
Pickling: Immersing food products in an acidic solution (e.g., vinegar, brine) to create an
inhospitable environment for spoilage microorganisms and extend shelf life.
Examples: Pickled cucumbers (pickles), pickled peppers, pickled fruits, and pickled eggs.
7. Curing and Smoking
Curing: Treating food products with salt, sugar, and/or nitrites to inhibit microbial growth,
dehydrate the product, and develop flavor and texture.
Smoking: Exposing food products to smoke from burning wood or other sources to impart
flavor, preserve the product, and inhibit microbial growth.
Examples: Cured meats (ham, bacon), smoked fish, and smoked cheeses.
8. Vacuum Packaging
Vacuum Sealing: Packaging food products in airtight containers or pouches and removing air to
create a vacuum, preventing oxidation, microbial contamination, and moisture loss.
Benefits: Extended shelf life, preservation of flavor, texture, and nutritional quality.
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9. Chemical Preservation
Use of Preservatives: Adding chemical preservatives such as salt, sugar, vinegar, and synthetic
additives (e.g., benzoates, sulfites) to inhibit microbial growth, prevent spoilage, and enhance
shelf life.
Examples: Salted fish, sugared fruits, vinegar-preserved vegetables, and processed foods
containing preservatives.
INDUSTRIAL MICROBIOLOGY
Industrial microorganisms are microorganisms (bacteria, fungi, yeast) that are utilized in various
industrial processes for their biochemical capabilities. These microorganisms are often
genetically modified or selected for specific traits to optimize their performance in industrial
settings. Here are some common types of industrial microorganisms and their applications:
1. Bacteria
Escherichia coli (E. coli):
Applications: Used in biotechnology for recombinant protein production, vaccine
manufacturing, and biochemical synthesis.
Bacillus species:
Applications: Produce enzymes such as amylases, proteases, and lipases for use in detergent
manufacturing, food processing, and textile industry.
Lactic Acid Bacteria (LAB):
Applications: Fermentation of dairy products (yogurt, cheese), meat products, and vegetable
fermentations (sauerkraut, pickles).
2. Fungi
Aspergillus species:
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Applications: Produce enzymes (e.g., amylases, cellulases, proteases) and organic acids (e.g.,
citric acid) for food, beverage, and pharmaceutical industries.
Penicillium species:
Applications: Used in the production of antibiotics such as penicillin and other β-lactam
antibiotics.
Saccharomyces cerevisiae (Baker's yeast):
Applications: Fermentation of alcoholic beverages (beer, wine) and leavening agent in bread-
making.
3. Yeast
Saccharomyces cerevisiae:
Applications: Used in the production of biofuels (ethanol), baking (bread, pastries), and
fermentation of alcoholic beverages.
Pichia pastoris:
Applications: Expression of recombinant proteins for pharmaceuticals, enzymes, and industrial
biotechnology.
4. Algae
Chlorella and Spirulina:
Applications: Production of biofuels, food supplements, pigments, and pharmaceuticals.
Haematococcus pluvialis:
Applications: Production of astaxanthin, a natural antioxidant used in food, cosmetics, and
aquaculture.
5. Actinomycetes
Streptomyce
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s species:
Applications: Production of antibiotics (e.g., streptomycin, tetracycline, erythromycin) and
enzymes (e.g., cellulases, amylases) used in pharmaceuticals and industrial processes.
6. Archaea
Methanogenic Archaea:
Applications: Anaerobic digestion of organic waste for biogas production (methane),
wastewater treatment, and renewable energy generation.
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7. Genetically Engineered Microorganisms (GEMs)
Applications: GEMs are engineered for specific industrial applications, such as bioremediation,
biofuel production, biopharmaceuticals, and bioplastics manufacturing.
8. Microbial Consortia
Applications: Synergistic interactions between multiple microorganisms are harnessed for
various industrial processes, including wastewater treatment, bioremediation, and bioconversion
of biomass.
FOOD PRODUCTION PROCESSES
Food production processes involve various stages and techniques used to transform raw
agricultural commodities into edible products ready for consumption. These processes
encompass activities such as harvesting, processes, packaging and distribution, and they vary
depending on the food product and its intended use.
YOGHURT PRODUCTION
Yoghurt is a fermented milk product that contains the characteristic bacterial cultures
Lactobacillus bulgaricus and Streptococcus thermophilus. All yoghurt must contain at least
8.25% solids not fat. Full fat yoghurt must contain not less than 3.25% milk fat, low-fat yogurt
not more than 2% milk fat, and nonfat yoghurt less than 0.5% milk.
The two styles of yoghurt commonly found in the grocery store are set type yoghurt and Swiss
style yoghurt. Set type yogurt is when the yogurt is packaged with the fruit on the bottom of the
cup and the yoghurt on top. Swiss style yoghurt is when the fruit is blended into the yoghurt
prior to packaging.
Ingredients
The main ingredient in yoghurt is milk. The type of milk used depends on the type of yoghurt –
whole milk for full fat yoghurt, low-fat milk for low-fat yoghurt, and skim milk for nonfat
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yoghurt. Other dairy ingredients are allowed in yogurt to adjust the composition, such as cream
to adjust the fat content, and nonfat dry milk to adjust the solids content. The solids content of
yoghurt is often adjusted above the 8.25% minimum to provide a better body and texture to the
finished yoghurt.
Stabilizers may also be used in yogurt to improve the body and texture by increasing firmness,
preventing separation of the whey (syneresis), and helping to keep the fruit uniformly mixed in
the yoghurt. Stabilizers used in yoghurt are alginates (carageenan), gelatins, gums (locust bean,
guar), pectins, and starch.
Sweeteners, flavors and fruit preparations are used in yoghurt to provide variety to the consumer.
Bacterial Cultures
The main (starter) cultures in yoghurt are Lactobacillus bulgaricus and Streptococcus
thermophilus. The function of the starter cultures is to ferment lactose (milk sugar) to produce
lactic acid. The increase in lactic acid decreases pH and causes the milk to clot, or form the soft
gel that is characteristic of yogurt. The fermentation of lactose also produces the flavor
compounds that are characteristic of yogurt. Lactobacillus bulgaricus and Streptococcus
thermophilus are the only 2 cultures required by law to be present in yogurt.
Other bacterial cultures, such as Lactobacillus acidophilus, Lactobacillus subsp. casei, and
Bifido-bacteria may be added to yogurt as probiotic cultures. Probiotic cultures benefit human
health by improving lactose digestion, gastrointestinal function, and stimulating the immune
system.
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General Manufacturing Procedure
The following flow chart and discussion provide a general outline of the steps required for
making yogurt.
General Yogurt Processing Steps
Adjust Milk Composition & Blend Ingredients
Pasteurize Milk
Homogenize
Cool Milk
Inoculate with Starter Cultures
Hold
Cool
Add Flavors & Fruit
Package
1. Adjust Milk Composition & Blend Ingredients
Milk composition may be adjusted to achieve the desired fat and solids content. Often dry milk is
added to increase the amount of whey protein to provide a desirable texture. Ingredients such as
stabilizers are added at this time.
2. Pasteurize Milk
The milk mixture is pasteurized at 185°F (85°C) for 30 minutes or at 203°F (95°C) for 10
minutes. A high heat treatment is used to denature the whey (serum) proteins. This allows the
proteins to form a more stable gel, which prevents separation of the water during storage. The
high heat treatment also further reduces the number of spoilage organisms in the milk to provide
a better environment for the starter cultures to grow. Yoghurt is pasteurized before the starter
cultures are added to ensure that the cultures remain active in the yoghurt after fermentation to
act as probiotics; if the yoghurt is pasteurized after fermentation the cultures will be inactivated.
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3. Homogenize
The blend is homogenized (2000 to 2500 psi) to mix all ingredients thoroughly and improve
yoghurt consistency.
4. Cool Milk
The milk is cooled to 108°F (42°C) to bring the yogurt to the ideal growth temperature for the
starter culture.
5. Inoculate with Starter Cultures
The starter cultures are mixed into the cooled milk.
6. Hold
The milk is held at 108°F (42°C) until a pH 4.5 is reached. This allows the fermentation to
progress to form a soft gel and the characteristic flavor of yogurt. This process can take several
hours.
7. Cool
The yoghurt is cooled to 7°C to stop the fermentation process.
8. Add Fruit & Flavors
Fruit and flavors are added at different steps depending on the type of yogurt. For set style yogurt
the fruit is added in the bottom of the cup and then the inoculated yogurt is poured on top and the
yoghurt is fermented in the cup.
9. Package
The yoghurt is pumped from the fermentation vat and packaged as desired.
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BEER PRODUCTION PROCESS/ BEER MAKING/BREWING
Brewing / beer production process
Brewing is the process of production of malt beverages. Beers, ale and lagers are the main malt
beverages produced by a method called brewing. Brewing is a complex fermentation process. It
differs from other industrial fermentation because flavor, aroma, clarity, color, foam production,
foam stability and percentage of alcohol are the factors associated with finished product.
Steps involved in beer production are:
1. Malting:
Beer is produced from barley grains.
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Barley grains are first cleaned and then soaked in water for about 2 days. Then excess
water is drained away and the barley are incubated for 4-5 days to allow germination
The germination steps allow the formation of highly active α-amylase, β-amylase and
proteases enzymes as well as various flavor and color components
oMalt adjuncts:
Barley contains considerable amount of protein. So, if only barley are used
for beer production, the final beer will be dark and unstable. Therefore,
protein present in malt should be diluted by adding additional starch or
sugary materials.
Such sugary or starchy materials are called malt adjuncts and includes
dextrose sugar syrup.
2. Kilning:
The germinated seed are then killed by slow heating at 80° This process is called kilning.
The kilning temperature must not harm amylase enzyme. Furthermore, if kilning
temperature is higher, darker will be the beer produced.
3. Mailing:
The dried barley grains are then crushed between rollers to produced coarse powder
called grist
4. Mashing:
Grist is mixed with warm water and the resulting materials is maintained at 65°C for
about 1 hour.
In doing so, starch is hydrolyzed by amylase enzyme to produce single sugar, maltose,
dextrose etc. similarly, protein is hydrolyzed by proteolytic enzymes into small fragments
and amino acids.
The degree of enzymatic hydrolysis strongly depends on pH and temperature. β-amylase
has optimum activity at temperature 57-65°C whereas α-amylase has optimum activity at
temperature 70-75°
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The liquid obtained by mashing is called wort. The husks and other grains residue as well
as precipitated proteins are removed filtration.
5. Boiling of wort:
The filtrate is then boiled with stirring for 2-3 hours and hop flowers are added at various
interval during boiling.
Reasons for boiling of wort:
oFor extraction of hop flavor from hop flower
oBoiling coagulates remaining protein and partially hydrolyze protein and help in
removal of protein
oBoiling inactivates enzymes that were active during mashing, otherwise causes
caramelization of sugar
oBoiling also sterilize and concentrate the wort
6. Hops:
Hops are dried female flower of hop plant Humulus lupulus. Approximately one quarter
pound of hop flower is added per barrel of beer and up to 2 pound per barrel of ale.
Advantages of hop addition in beer are;
oProvide beer with its pungent and aromatic character
oProvide tannin which helps in coagulation of remaining protein
oContains α-resin and β-resin which gives bitter flavor as well as preservative
action against gram Positive bacteria
oContains pectin which is responsible for foam characteristic of beer
7. Fermentation:
Beer production utilize strain of Saccharomyces carlsbergens and S. varum which are
bottom yeast and S. cerevisiae which is a top yeast.
Yeast cells for inoculation are usually recover from previous fermentation tank by
treatment with phosphoric acid, tartaric acid or ammonium persulphate to reduce the pH
and removed considerable bacterial contamination.
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Fermentation is usually carried out at 3-4 °C but it may range from 3- 14° Fermentation
usually completes in 14 days.
During fermentation yeast converts sugar mainly into ethanol and CO2 plus some amount
of glycerol and acetic acid.
For fermentation open tank fermenter can be used however closed fermenter tank is
preferred, so that CO2 liberated during fermentation can be collected for later
carbonation step.
CO2 evolution is maximum by fifth day of fermentation, there is no evolution of CO2 by
7-9 days because yeast cells become inactive and flocculate.
Most beer contains 3.5-5% alcohol.
8. Finishing, Ageing, Maturation and Carbonation:
The young and green beer is stored in vat at 0°C for several weeks to several months.
During this period, precipitation of protein, yeast, resin and other undesirable substances
take place and beer become clear.
Ester and other compounds are also produced during ageing which gives taste and aroma.
After ageing, the beer is carbonated by carbon dioxide of 0.45-0.52%.
The beer is then cooled, clarified, filtered and packed in bottles, barrels and cans.
!BIOGAS PRODUCTION
Biogas production is a sustainable process that involves the conversion of organic materials,
primarily biomass, into a renewable energy source known as biogas. Biogas primarily consists of
methane (CH4) and carbon dioxide (CO2), along with small amounts of other gases such as
hydrogen sulfide (H2S) and traces of nitrogen (N2), ammonia (NH3), and water vapor. Here's an
overview of the biogas production process:
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1. Feedstock Collection and Preparation:
Organic Waste: Various organic materials can serve as feedstock for biogas production,
including agricultural residues (crop residues, manure), food waste, sewage sludge, animal
waste, and energy crops (corn silage, grass).
Feedstock Characteristics: Feedstock is selected based on its availability, composition,
moisture content, and biodegradability.
2. Anaerobic Digestion:
Fermentation Process: The feedstock is introduced into an anaerobic digester, a sealed
container or reactor where anaerobic microorganisms break down organic matter in the absence
of oxygen.
Microbial Activity: Anaerobic bacteria, predominantly methanogenic archaea, decompose
complex organic molecules into simpler compounds through biochemical reactions.
Biogas Formation: Methane-producing bacteria (methanogens) metabolize organic compounds
(e.g., carbohydrates, fats, proteins) to produce methane (CH4) and carbon dioxide (CO2) as
metabolic by-products.
3. Biogas Collection and Storage:
Gas Collection: Biogas produced during anaerobic digestion is collected from the digester using
a gas-tight cover or dome.
Gas Composition: The composition of biogas varies depending on the feedstock composition
and digester conditions, typically comprising 50-70% methane, 30-50% carbon dioxide, and
trace gases.
Gas Storage: Biogas can be stored in gas holders, tanks, or pipelines for subsequent use or
conversion into energy.
4. Biogas Utilization:
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Electricity Generation: Biogas can be combusted in a gas engine or turbine to generate
electricity, which can be used on-site or fed into the electrical grid.
Heat Production: Biogas can be burned in boilers, furnaces, or combined heat and power (CHP)
systems to produce heat for space heating, water heating, or industrial processes.
Vehicle Fuel: Biogas can be upgraded to biomethane through purification processes (e.g.,
removal of CO2 and impurities) and used as a renewable vehicle fuel (bio-CNG or bio-LNG).
5. Digestate Management:
Residual Material: After anaerobic digestion, the solid and liquid residues, known as digestate,
remain and can be used as organic fertilizer or soil conditioner.
Nutrient Recycling: Digestate contains valuable nutrients (e.g., nitrogen, phosphorus,
potassium) that can be recycled back to agricultural land, closing the nutrient cycle and
improving soil fertility.
6. Process Optimization and Monitoring:
Optimization: Monitoring and controlling factors such as temperature, pH, retention time, and
feedstock composition to optimize biogas production and digester performance.
Monitoring: Regular monitoring of biogas production rates, gas composition, digester
parameters, and environmental factors to ensure efficient operation and troubleshooting of
issues.
BIODEGRADATION
Biodegradation is the breakdown of organic matter or substances into smaller and simpler
substances via a biologically catalyzed reduction in the presence of living microorganisms.
Biodegradation is a general term used for any change or breakdown in a substrate that is
biologically mediated.
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Biodegradation
Biodegradable matter can be metabolically degraded by microbes in the presence of oxygen
(aerobically) or in the absence of oxygen (anaerobically). Almost any matter living or non-living,
is subjected to biodegradation. The only key element is the ‘duration’ of that matter breakdown.
European Union has set a standard biodegradability for materials that 90% of the original
substance to be degraded to water, mineral, and carbon dioxide by biological reactions within six
months.
The process is brought about by the metabolic and enzymatic actions of microorganisms like
bacteria, yeast, and fungi. Microorganisms follow any two modes for biodegradation based on
the type of matter and environment. They are Minerilization and Cometabolism. Mineralization
is a complete degradation of organic pollutants where organisms use matter as the sole source of
carbon to produce energy. Whereas in Cometabolism, it is observed that degradation is done by
the addition of a growth substrate as a primary source of carbon and energy to initiate the
breakdown of matter. Some naturally occurring microbes show excellent catabolic activity to
transform and degrade a wide range of compounds like polychlorinated biphenyls, hydrocarbons
(eg. oils), radionuclides, metals, and more.!
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Biodegradation is referred to as one of the most favored and sustainable measures of removing
any complex organic matter and is often regarded in terms of ecology, the natural environment,
and waste management.
Biodegradable Pollutants
Since the time of the industrial revolution and with an increased standard of living, numerous
highly toxic organic compounds such as fuels, polycyclic aromatic hydrocarbons (PAHs), dyes,
pesticides, and some synthetic chemicals like radionuclides have been synthesized and released
for use in the environment for a long period of time for direct or indirect application. These toxic
and complex compounds are difficult for the native flora to readily degrade upon release into the
environment. Some of the organic pollutants are mentioned below.
Polycyclic Aromatic Hydrocarbons (PAHs)
Hydrocarbons are organic molecules composed of hydrogen and carbon as the primary
functional units of a compound. They are either aliphatic in nature (linear and branched
compounds) or aromatic (compounds with a benzene ring). Aliphatic compounds include
alkanes, alkene, and alkynes, while aromatic compounds include a benzene ring, such as
phenols, toluene, etc.
PAHs have been categorized as an important organic pollutant in the ‘Hydrophobic Organic
Contaminants (HOCs)’ class that’s readily found in sediments, soils, and air.!
They are present naturally in gasoline, crude oil, and coal.
They can be formed by burning coal and wood and also while cooking meat in high heat.
PAHs can be easily bound to and form minute particles that can get accumulated in
organisms.
In the United States, a man-made PAH called the ‘Naphthalene’ are used to make
mothballs and other chemicals.
Cigarette smoking also releases many PAHs into the environment.
They can get accumulated in the fish and other aquatic organisms, which can be
transferred to humans when consuming seafood.
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In the NHANES (National Health and Nutrition Examination Survey) of 2003-2004,
CDC Scientists measured the urine sample of 2,504 individuals aged from six years old
to above and found ten different PAH metabolites.
Higher exposure to Naphthalene and other PAHs present in the air can irritate eyes and
nasal passages and can also cause liver problems.
Polychlorinated Biphenyls (PCBs)
PCBs are categorized as man-made chemicals with an oily texture, no taste or smell, and are
yellow in color. Found in air, water, sediments, and soil across the world.
They are chemically very stable mixtures, highly resistant to high temperatures, non-
flammable, and with electrical insulation properties.!
Due to these features, they are widely used in hydraulic and electrical equipment, as
plasticizers in rubber products, and as pigments in dyes.
Once released in the environment, they are easily spread across long distances via air and
soil, making them persistent in the atmosphere.
Humans are generally exposed to PCBs by consuming contaminated meat, fish, dairy
products, fruits, and vegetables.
They are absorbed and stored in the fatty tissue, which can cause cancer and act as an
endocrine disruptor.
PCBs were sold commercially by the name of Aroclor by the company named Monsanto
Inc. in the United States. The levels of PCBs have been declining in the food chain and
environment since its ban in 1977 due to its exposure which led to ill health effects.
Pesticides
The concept of a pesticide is a combination of poisonous compounds that target pathogens
and is safe for non-pathogens like humans and animals. They belong to a class of chemicals
that includes herbicides, fungicides, rodenticides, molluscicides, plant growth regulators,
insecticides, and nematicides, used to regulate the growth of weeds, killing of pests, and
prevention of diseases.!
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Pesticides use enhances the productivity of crops and the prevention of vector-borne
diseases.!
The United States is one of the world’s largest traders, producers, and consumers.
Pesticides cause a serious health hazard to non-target organisms because they are readily
fat solubility and bioaccumulation.
The introduction of pesticides leads to a decline in the population of zooplankton and
phytoplankton
They can be neurotoxic and carcinogenic and decrease fertility in fishes, invertebrates,
insects, mammals, and amphibians.
Regular use of their presence develops resistance to pests, reducing their toxicity.
The biodegradation of pesticides is mostly done by soil microorganisms using it as a food
source, whereas persistent xenobiotics become incorporated into the environment leading
to biomagnification.
Dyes
They have applications in a wide range of industries, such as cosmetics, textiles,
pharmaceuticals, rubber products, and more.
The most dye used are Azo dyes that contain diazotized amine coupled with phenol or
an amine group and single or more azo (–N=N–) groups.
Microorganisms have potential oxidoreductive enzymes and a dynamic metabolism
ability that allows them to use dyes with complex xenobiotic compounds as a substrate,
helping in the decolorization of polluted sites.
Radionuclides and Heavy metals
An atom has a highly unstable nucleus with the ability to impart excess energy during the
radiation process in which they undergo radioactive decay to produce subatomic alpha or beta
particles or emission of gamma rays. Microbial degradation of radionuclides results in the
formation of less toxic and more stable compounds.
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Heavy metals, such as arsenic, cadmium, chromium, etc, cannot be destroyed like organic
compounds but can be converted into stable forms. The mechanism applied by microbes
includes:
Bioleaching – mobilization of heavy metal through methylation reactions or excretion of
organic acids
Biosorption – metal absorption in the cell surface.
Enzyme-catalyzed redox reactions.
And Intracellular accumulation of heavy metals.
Extraction of heavy metals can be achieved by recovering the precipitate of metals from
microbial samples.
Microorganism in Biodegradation and their Impact
In nature, biodegradation is a natural process that is carried out to recycle wastes and break down
complex and toxic organic compounds into a much simpler form that can be reused as a food
source by other organisms. Microorganisms, such as bacteria, fungi, and yeast, are the most
common species that help in the biodegradation process.
Bacterial Strain
Bacteria constitute a key organism in the biodegradation process, which can be easily isolated
from any place.!
Bacterial strains from genus Klebsiella, Enterobacter, Bacillus, Staphylococcus,
Acinetobacter, and more belong to the hydrocarbon degradation category. Brevibacillus
and Pseudomonas species, anaerobically reduced nitrate-containing compounds, have
been isolated from the petroleum-contaminated soil.
The genera of gram-negative strains like Pseudomonas, Aeromonas, etc, carry out
biodegradation of aromatic hydrocarbons.
A mixed population of bacterial strains is better suited to degrade pollutants as they can
easily share genetic information related to degradative enzymes and pathways.
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PCB degradation is carried out aerobically and anaerobically by redox and
dehalogenation reactions. PCB degrading activity is shown by both Gram-positive (eg.
Rhodococcus, Bacillus, Microbacterium, and more) and Gram-negative strains (genera
Pseudomonas, Sphingomonas, Ralstonia, etc.).
PCB degradation incorporates these four enzymes biphenyl dehydrogenase, dihydro
diol dehydrogenase, 2,3-dihydroxy biphenyl dehydrogenase, and hydrolase.
Pesticides such as DDT (Dichlorodiphenyltrichloroethane) are degraded by the strains
Staphylococcus and Stenotrophomonas bacteria, and ‘Chlorpyrifos’ pesticide degrading
bacteria is Providencia stuartii.
Shewanella decolorations are identified as a single bacterium species that can efficiently
remove azo dyes.!
Heavy metals are transformed via a dissimilatory metal reduction process where bacterial
species use metals as a terminal electron acceptors during respiratory reactions.
Acidithiobacillus ferrooxidans, an acidophilic iron bacteria, and sulfur-oxidizing bacteria
are found to have high concentrations of Arsenic, Cadmium, Zinc, Cobalt, and Copper
from contaminated soils.
Plant Growth Promoting Bacteria (PDPB and PGBR)
These bacteria can be generally found in the plants naturally, in roots, or near them. They are
non-pathogenic to plants and help in the growth and development of plants. Plants and plant
growth-promoting bacteria live in mutualistic relationships, wherein plants give shelter, and food
sources, and bacteria, in return, help in metabolizing toxic substances that can be used by plants.!
Pseudomonas species and Lysini bacillus show PAH hydrocarbon degrading activity
that’s released from plants.
Strains of the genus Luteibacter, Williamsia, and Rhodobacter have shown a promising
PCB degradation activity present in the contaminated soil.
Azospirillum lipoferum, isolated from the rhizoplane of crop plants, is observed to
degrade Malathion, an organophosphorus insecticide.
Fungal Species
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They are considered the principal microorganism important for the carbon decomposition in the
atmosphere. They can easily survive in the low pH and moisture areas. They are highly equipped
with special extracellular multienzyme complexes helping in the degradation of natural
polymeric compounds.!
With the aid of their hyphal structures, they are able to colonize and easily penetrate
substances, helping them to redistribute nutrients across the mycelium.!
Recycling of recalcitrants like lignin is implicated by the degradative mechanisms of
fungi.
Toluene-degrading fungi include deuteromycetes species belonging to the genera
Exophiala, Leptodontium, and Cladophialophora, and an ascomycete strain
Pseudeurotium zonatum, that utilize toluene for carbon and energy sources.
Filamentous fungi Cladosporium and Aspergillus show the degrading activity of aliphatic
hydrocarbon, whereas fungi that belong to Penicillinum, Fusarium, and Cunninghamella
degrade aromatic hydrocarbons.
Aspergillus niger has shown biodegrading activity towards PCBs.
Fungi detoxify metals via mechanisms like active uptake, transformation, and extra or
intracellular precipitation. Examples include Rhiloprzs arrhizus and Aspergillus niger.
Yeast
Yeast species have been heavily studied to degrade Poly Aromatic Hydrocarbons (PAHs) and
use them for energy-dependent uptake systems. Trichosporum cutaneum, a soil yeast, has been
seen to degrade phenols.
Alkanes of 10 to 20 carbons are degraded by species like Candida lipolytica,
Rhodotorula aurantiaca, Candida ernobii, and more.
Candida methanosorbosa BP-6 species have been reported to potentially break down azo
dye like aniline.
Saccharomyces cerevisiae, Candida biodinii, and several others can transform
plasticizers, insecticides, fungicides, and polychlorinated biphenyls (PCBs) into simpler
forms.
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Yeast strains such as Hansenula polymorpha, Cyberlindnera fabianii, Rhodotorula
pilimanae, etc, accumulate heavy metals like Cr, Co, Ni, Mg, and other heavy metals and
reduce metals via redox reactions.
Genetically-Modified Microorganisms (GMM)
Genetically-modified microorganisms have altered genetic material inspired by the
microorganisms’ gene transfer methods using genetic engineering like DNA Recombinant
Technology. GMMs have shown great potential with enhanced biodegradation capabilities for
various chemical contaminants.!
The main aspects to be kept in mind for GMM development in the biodegradation application
are:
1. Enzyme specificity and affinity modifications.
2. Regulation and construction of metabolic pathway.
3. Development, monitoring, and control of Bioprocesses.
4. Bioreporter sensory application for toxicity reduction, chemical sensing, and end-point
analysis.
Construction of upgraded genetic modules and new catabolic pathways for biodegradation in
microbes require separate plasmids for every toxic compound.! These plasmids are categorized
into four groups:
1. OCT plasmids – degradation of hexane, octane, and decane.
2. XYL plasmids – degrade toluenes and Xylene.
3. CAM plasmids – decomposition of Camphor compound.
4. NAH plasmids – degradation of Naphthalene.
Examples of genetically-engineered microorganisms (GEM) include:
GMM Pseudomonas putida with multi-plasmid capabilities is often referred to as
Superbug (Oil Eating Bug) that contains pKF 349 for salicylate toluene, pAC 25 (for 3-
cne chlorobenxoate degradation), in addition to XYL, NAH, OCT, and CAM plasmids.
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For the removal of Chromium in industrial wastewater, Alcaligenes eutrophus AE104
(pEBZ141) are used for degradation.
Rhodopseudomonas palustris, a recombinant photosynthetic bacterium, is used for
mercury removal from wastewater.
PCB (polychlorinated biphenyls) are degraded by using GEM Achromobacter sp.
LBS1C1 and A. denitrificans JB1.
The use of GMM strains for biodegradation and bioremediation purposes can cause competition
with wild-type species in a mixed culture. The controversy surrounding GMM release into the
environment must be overcome by proper field testing, biosafety, and the reduction in potential
damage to the ecosystem.
Stages of Biodegradation
The biodegradation process can be subdivided into three processes – Biodeterioration, Bio-
fragmentation, and Assimilation. Biodeterioration refers to the process of mechanical
weakening of complex structures. In Bio-fragmentation, microorganisms break down toxic and
complex compounds. And in Assimilation, old structures are transformed into new compounds.
Biodeterioration
The process involves the mechanical, physical, and chemical weakening of the compound
structure. The abiotic factors, such as light, temperature, and chemicals of the environment,
initiate these changes.!
Bio-fragmentation
After the weakening of the structure, the cleavage of polymeric bonds leads to the transformation
of oligomers and monomers. The fragmentation is achieved in aerobic as well as in anaerobic
conditions. Aerobic digestion of compounds leads to the formation of water, carbon dioxide,
and simple molecules that are utilized as a nutrient source. Anaerobic digestion reduces the
mass and volume of complex material, such as the production of natural gases. Anaerobic
reactions are used widely used in waste management facilities as a source of renewable energy.
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Assimilation
The newly formed molecules are taken up by the microorganisms via membrane carriers. The
molecules are then used as an energy source in the form of ATP (Adenosine Triphosphate) or as
cell structural elements.
Factors Affecting Biodegradation
Biodegradation of materials and its rate of degradation by microorganisms is influenced by
nutritional requirements and environmental factors like soil, water, etc. The efficiency and rate of
degradation depend on the compounds’ concentration, nature, bioavailability, and
physicochemical properties.!
Environmental Factors
Biodegradation occurs at the ground, so the soil type and organic matter potentially affect the
adsorption and absorption of pollutants on the soil surface.!
Absorption of the contaminant by the soil matrix reduces the bioavailability to
microorganisms, and the proportion of metabolic breakdown is reduced consequently.
Porosity variations are seen in the saturated and unsaturated zone of the aquifer matrix,
influencing the fluid movement and migration of pollutants in groundwater.
In fine-grained soil with water saturation, the transmission of gases such as CO2,
methane, and oxygen gets reduced; hence the biodegradation process gets slower.
Microbes can oxidize pollutants in soil with more redox potential, thus increasing
electron transport. This indicates aerobic conditions, hence low electron density.
In anaerobic conditions, high electron density can be observed in the soil, indicating the
reduction potential of the microorganisms.
Biological Factors
The biological factor is the metabolic potential which includes the inhibition of enzymatic
factors of an organism to degrade any contaminant.!
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Inhibition of enzymatic activity can occur due to competition for the availability of
limited carbon sources between microorganisms or predation by the bacteriophages and
protozoa.
The degradation rate also depends on the concentration of the contaminant present in the
surroundings and the number of microorganisms present with the ability to produce
proper enzymes to degrade the contaminant.
The affinity of the contaminant to the specific enzymes can contribute to the rapid
metabolism of pollutants by the organisms.
Biological enzyme-catalyzed reactions for biodegradation have an optimum pH of
approx. 6.5 – 8.5, temperature and moisture influence the rate of metabolism, amount of
soluble materials, and osmotic pressure in the terrestrial and aquatic systems.
Conclusion
The end goal is the same for both processes, which is to clean the environment and achieve
sustainable development goals as much as possible. Since the beginning of industrialization and
technological advancement, humans have been releasing toxic chemicals into the environment.
These toxic compounds have become pollutants and have proven to be hazardous to human
health and to the ecosystem in general, which has affected the balance of nature immensely. To
curb this situation, nature has been doing its job of removing wastes via biodegradation
employing microorganisms to metabolize these toxic chemicals. Microorganisms showed the
super abilities and specific digestive machinery to transform synthetic compounds into a stable
form. Microbes use the pollutants as their carbon source, which helps them gain energy and, in
turn, cleans up the ecosystem.
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