STUDY MATERIAL FOR B.SC MICROBIOLOGY ENVIRONMENTAL ...

STUDY MATERIAL FOR B.SC MICROBIOLOGY ENVIRONMENTAL & AGRICULTURAL MICROBIOLOGY

SEMESTER - V, ACADEMIC YEAR 2020 - 21

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UNIT CONTENT PAGE Nr

I AERO MICROBIOLOGY 02

II SOLID WASTE MANAGEMENT 15

III DISTRIBUTION OF MICROORGANISMS IN NATURE 30

IV MICROBIAL ASSOCIATIONS 42

V MICROORGANISMS IN THE DECOMPOSITION OF ORGANIC MATTER

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UNIT - I AEROMICROBIOLOGY

In the 1930s, F.C. Meier coined the term aerobiology to describe a project that involved the study of life in the air. Since then, aerobiology has been defined by many as the study of the aerosolization, aerial transmission and deposition of biological materials. Others have defined it more specifically as the study of diseases that may be transmitted via the respiratory route. Despite the variations in definition, this evolving area is increasingly important in many aspects of diverse fields including public health, environmental science, industrial and agricultural engineering, biological warfare and space exploration. Aeromicrobiology involves various aspects of intramural (indoor) and extramural (outdoor) aerobiology, as they relate to the airborne transmission of environmentally relevant microorganisms, including viruses, bacteria, fungi, yeasts and protozoans. AEROSOLS

Particles suspended in air are called aerosols. These pose a threat to human health mainly through respiratory intake and deposition in nasal and bronchial airways. In addition, soil or dust particles can act as a “raft” for biological entities known as bioaerosols. Smaller aerosols travel further into the respiratory system and generally cause more health problems than larger particles. For this reason, the United States Environmental Protection Agency (USEPA) has divided airborne particulates into two size categories: PM10, which refers to particles with diameters less than or equal to 10 μm (10,000 nm), and PM2.5, which are particles less than or equal to 2.5 μm (2500 nm) in diameter. For this classification, the diameter of aerosols is defined as the aerodynamic diameter Atmospheric particulate concentration is expressed in micrograms of particles per cubic meter of air (μg/m3). Symptoms of particulate matter inhalation include: decreased pulmonary function; chronic coughs; bronchitis; and asthmatic attacks. Airborne particles can travel great distances. Smaller particles tend to travel greater distances than large particles. Stokes’ law is used to describe the fall of particles through a dispersion medium, such as air or water:

V = [D2 x (ρp - ρ1) x g]/18ρ where: V = velocity of fall (cm/s-1) g = acceleration of gravity (980 cm/s-2) D = diameter of particle (cm) Ρp = density of particle (density of quartz particles is 2.65 g/cm-3) ρ1 = density of dispersion medium (air has a density of about 0.001213 g/cm-3; water has a density of about 1 g/cm-3) ρ = viscosity of the dispersion medium (about 1.83x10-4 poise or g cm-1s-1 for air; 1.002x10-2 poise for water)

Using Stokes’ law, we can calculate the rate of fall of particles in air. Small particles are thus a greater concern than larger particles for several reasons. Small particles stay suspended longer and so they travel further and stay suspended longer. This results in an increased risk of exposure. Small particles also tend to move further into the respiratory system, exacerbating their effects on health. Stokes’ law explains why we can expect viruses to persist as a bioaerosollonger than bacteria, which are much larger. NATURE OF BIOAEROSOLS



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Biological contaminants include whole entities such as bacterial and viral human pathogens. They also include airborne toxins, which can be parts or components of whole cells. In either case, biological airborne contaminants are known as bioaerosols, which can be ingested or inhaled by humans. Bioaerosols vary considerably in size, and composition depends on a variety of factors including the type of microorganism or toxin, the types of particles they are associated with such as mist or dust, and the gases in which the bioaerosol is suspended. Bioaerosols in general range from 0.02 to 100 μm in diameter and are classified on the basis of their size. The smaller particles (,0.1 μm in diameter) are considered to be in the nuclei mode, those ranging from 0.1 to 2 μm are in the accumulation mode and larger particles are considered to be in the coarse mode, particles in nuclei or accumulation mode are considered to be fine particles and those in coarse mode are considered coarse particles.

The composition of bioaerosols can be liquid or solid, or a mixture of the two, and should be thought of as microorganisms associated with airborne particles, or as airborne particles containing microorganisms. This is because it is rare to have microorganisms (or toxins) that are not associated with other airborne particles such as dust or water. This information is derived from particle size analysis experiments, which indicate that the average diameter of airborne bacterial particles is greater than 5 μm. By comparison, the average size of a soil-borne bacterium, 0.3 to 1 μm, is less than one-fifth this size. Similar particle size analysis experiments show the same to be true for aerosolized microorganisms other than bacteria, including viruses.

Airborne droplet nuclei

Airborne droplet nuclei develop when the fluid of pathogenic droplets (1-5 µm in size;) evaporates. They are so small and light they may remain suspended in the air for several hours. Thus, they may also infect persons entering a room which has been left by a patient long ago. Also, airborne droplet nuclei can be widely dispersed by air currents. Tuberculosis, chickenpox, measles and possibly also influenza may be transmitted this way.



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➢ The influenza A virus is spread by droplet-nuclei from sneezing and coughing patients. ➢ Diphtheria is contagious, being spread by droplet nuclei and/or skin contact. ➢ Mycobacterium tuberculosis bacilli enter the respiratory route upon inhalation of droplet

nuclei expelled by infected hosts. The infection process begins when these droplet nuclei (particle size, <5 μm) containing tubercle bacilli reach the alveoli of the lungs.

➢ A productive TB infection originates following the inhalation of a single, aerosolized droplet nucleus containing 1–3 bacterial cells.

Droplet nuclei floating on the air may be carried by the movement of air. Entertainment

of air into neighboring airspaces may occur during the most innocuous daily activities; for example, as a result of people walking, or the opening of a door between a room and the adjacent corridor or space. Even a patient simply sitting in or beside the bed will create air temperature differences from their body heat. A higher air temperature directly above the patient's head (or body, if lying down) will create convective air currents that may entrain potentially infectious air from neighboring spaces into the higher temperature column rising air above the patient. Patients lying in bed, breathing or sleeping, may produce exhaled airflows that can reach the airspace of a patient in the neighboring bed, and even further in the presence of certain types of ventilation systems. In the same way, other mechanical devices, including fans, televisions and medical equipment may also disturb nearby airflows and disseminate air from nearby patients to the rest of the ward.

AIR POLLUTION

Microbes are involved in at least four areas of air pollution. First, microbes are the source of substantial quantities of gaseous pollutants; second, they serve as sinks for a variety of pollutants; third, they are, in themselves, pollutants, in that airborne microorganisms are involved in the aerial transmission of diseases and in allergic reactions; and fourth, after decomposition of air pollutants on and in terrestrial and aqueous environments, the microbiota has the potential of either transforming the contaminants into more or less toxic forms.



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Pollution caused by some microbes

S.No Pollutant Microbial source

1 CO Soil fungal metabolism Fresh water algae Diatoms Bacterial decomposition processes

2 H2S, NOx (Nitrogen oxides)

Bacterial decomposition processes

3 NH3 Bacterial deamination processes

4 N2O Anaerobic bacterial denitrification

As a primary natural biotic source of pollutants, microorganisms contribute large

amounts of both inorganic and organic gases and volatiles. These microbial emissions are either the end products or intermediates of metabolism. The major source of N2O is soil; an estimated 5.92 X 108 tons/year of N2O are emitted from soil as a result of anaerobic bacterial denitrification. Although the majority of soil denitrifying bacteria evolves N2O or molecular nitrogen by reduction of nitrates (NO3). For example, the bacterium, Nitrosomonaseuropaea, oxidizes NH4

+ to N2O, and the fungi, Aspergillusflavusand Penicilliumatrovenetum, emit N2O during nitrite (NO2) reduction. A variety of heterotrophic bacteria, such as Bacillus subtilis, Enterobacteraerogenes, and Escherichia coli, hitherto not implicated in denitrification, reduce NO3

-, with the subsequent release of N2O.

A large percentage of atmospheric sulfur is emitted in the form of hydrogen sulfide (H2S), produced by anaerobic microbial reduction of S04

-2. The main S04-2 reducers are bacteria of the

genus Desulfovibrio. Microbial S04-2 reductions occurs in waters, swamps, soil, and intertidal

flats, where microbial activity depletes the environment of available oxygen. In aerobic environments, H2S evolution occurs by bacterial decomposition of sulfur containing amino acids e.g., cysteine degradation by E. aerogenes, E. coli, Salmonella sp., andProteus sp. Microbes as Air Pollutants

Airborne microbes — bacteria, viruses, spores of lichens and fungi, small algae, and protozoan cysts — are, in themselves, pollutants. Aerial transmission of human diseases, such as pulmonary tuberculosis, pulmonary anthrax, staphylococcal and streptococcal respiratory infections, pneumococcal pneumonia, influenza, poliomyelitis, measles, smallpox, the common cold, aspergillosis, nocardiosis, histoplasmosis, sporotrichosis, cryptococcosis, blastomycosis, coccidiomycosis, and farmer's lurig disease; of cattle diseases, such as infectious bovine rhinotracheitis, pulmonary pasteurellosis, foot and mouth disease, rinderpest, and bovine contagious pleuropneumonia; and of poultry diseases, such as Marek's disease, avian infectious bronchitis, and Newcastle disease, is a potential hazard to the biosphere.

Airborne microbes also function as aeroallergens. Spores and hyphal fragments of species of Candida, Rhizopus, Mucor, Pénicillium, Aspergillus, Fusarium, Helminthosporium, Alternaría, andCladosporium are sources of airborne allergens. Fungal aeroallergens associated with asthma include Epicoccum, Cladosporium, andAlternaría. Some sensitive individuals are allergic to inhalation of Leptosphaeria-type ascospores and spores of the mirror yeasts and toadstools. Similarly, many plant pathogens are disseminated by air currents. For instance, conidia of Monilinialaxa, the causative agent of brown-rot blossom-blight of almond, spores of Sphaerothecapannosa, the causative agent of powdery mildew of plum, and spores of Pseudoperonosporahumuli, causative agent of downy mildew of hops, are disseminated by air



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currents. Intracontinental contamination, e.g., spread of the rust fungus (Pucciniapolyspora) through Africa, and intercontinental contamination, e.g., spread of coffee rust (Hemileiavastatrix) from Ethiopia to Ceylon and Brazil, of crops, through atmospheric transport of spores, has been suggested.

Hence, since airborne microorganisms can be pathogenic or allergenic, an understanding of their dispersal mechanisms into the atmosphere is of prime importance. Microorganisms, such as viruses, bacteria, algae, protozoans, and yeast, lack inherent dispersal mechanisms. Consequently, such organisms must rely upon some external, mechanical force for liberation to the atmosphere. Animal viruses become disseminated into the atmosphere during talking, sneezing, or coughing of infected individuals, or after shaking bed blankets used by infected individuals. Plant viruses and bacteriophages may enter the airborne state on water droplets or rafts of debris. Wind, animal activity, and mechanical disturbance of dust, e.g., during sweeping or land cultivation processes, emit bacteria laden dust particles into the atmosphère. Bacteria laden water droplets, produced by rain splash, ocean breakers, sea spray, air bubbles breaking at air-water interfaces, and trickling filter sewage treatment plants, also contribute to bacterial aerosolization. Algae, protozoa, and yeasts depend upon similar mechanisms for liberation into the atmosphere. Filamentous fungi are best adapted for aerial transmission.

Fungal launching mechanisms are of two types: violent discharge and passive liberation. In passive liberation processes, the fungus is adapted for spore liberation through use of an external force, such as raindrops or wind. For example, conidial fungi, such as Cladosporium, Pénicillium, Trichothecium, and Aspergillus, form aerial conidiophores containing dry spores that are easily released by atmospheric turbulence. Other fungi, which produce spores within a slime, depend upon raindrops, and the subsequent splatter, for spore liberation. Still other fungi, notably the bird's nest fungi, have developed specialized structures, termed splash cups, for the trapping of the energy engendered in falling raindrops. Cyathusstriatus, for instance, has a funnel-shaped basidiocarp with a mouth large enough to receive raindrops. The violent impaction of the raindrop in the cup and the upward thrust of the water result in the liberation of basidiospore units, termed peridioles. Large raindrops falling upon fructifications, such as those of the earth-stars (Geastntm) and puff-balls (Lycoperdon), liberate spores by a bellows action. Microbiological sources: FUNGI

Fungi include yeasts, molds, and mushrooms. Molds are ubiquitous organisms; their most common source is the outdoor environment. Building occupants are exposed to airborne molds outdoors and indoors. Indoor air quality complaints increase when mold growth proliferates in an indoor environment. Mold can cause discoloration and degradation of building materials, odor problems, and allergic reactions in building occupants. Some building occupants may be hypersensitive to certain species of fungi. The key to minimizing indoor exposure to airborne molds is to prevent the amplification and dissemination of the organisms.

Some patients in hospitals are highly susceptible to developing infections from airborne microorganisms. One such infection is aspergillosis, which is caused by several species of the fungus Aspergillus. Aspergillus is a ubiquitous organism in the indoor and outdoor environments. Aspergillosis is a fungal infection of the tissue in the lungs and respiratory tract. Patients with suppressed immune systems (e.g., bone marrow transplant and organ transplant recipients) are at increased risk of developing aspergillosis. Aspergillosis can be fatal in immune compromised patients. It may be associated with dust exposure from renovation and construction activities



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within hospitals; consequently, engineering and administrative controls should be in place to prevent patient dust exposure. AIR QUALITY ANALYSIS

Air pollutants are atmospheric substances—both naturally occurring and anthropogenic—which may potentially have a negative impact on the environment and organism health. With the evolution of new chemicals and industrial processes has come the introduction or elevation of pollutants in the atmosphere, as well as environmental research and regulations, increasing the demand for air quality monitoring.

Air quality monitoring is challenging to enact as it requires the effective integration of multiple environmental data sources, which often originate from different environmental networks and institutions. These challenges require specialized observation equipment and tools to establish air pollutant concentrations, including sensor networks, geographic information system (GIS) models, and the Sensor Observation Service (SOS), a web service for querying real-time sensor data. Air dispersion models that combine topographic, emissions, and meteorological data to predict air pollutant concentrations are often helpful in interpreting air monitoring data. Additionally, consideration of anemometer data in the area between sources and the monitor often provides insights on the source of the air contaminants recorded by an air pollution monitor.

Air quality monitors are operated by citizens, regulatory agencies, and researchers to investigate air quality and the effects of air pollution. Interpretation of ambient air monitoring data often involves a consideration of the spatial and temporal representativeness of the data gathered, and the health effects associated with exposure to the monitored levels. If the interpretation reveals concentrations of multiple chemical compounds, a unique "chemical fingerprint" of a particular air pollution source may emerge from analysis of the data. Air sampling

Passive or "diffusive" air sampling depends on meteorological conditions such as wind to diffuse air pollutants to a sorbent medium. Passive samplers have the advantage of typically being small, quiet, and easy to deploy, and they are particularly useful in air quality studies that determine key areas for future continuous monitoring.

Air pollution can also be assessed by biomonitoring with organisms that bioaccumulate air pollutants, such as lichens, mosses, fungi, and other biomass. One of the benefits of this type of sampling is how quantitative information can be obtained via measurements of accumulated compounds, representative of the environment from which they came. However, careful considerations must be made in choosing the particular organism, how it's dispersed, and relevance to the pollutant.

Other sampling methods include the use of a denuder, needle trap devices, and micro extraction techniques. Air Sampling Devices The various methods of air sampling include:

1. Impingement which is trapping of airborne particles in a liquid matrix. 2. Impaction which is forced deposition of airborne particles on a solid surface. 3. Centrifugation which is mechanically forced deposition of airborne particles using



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inertial forces of gravity. 4. Filtration which is trapping of airborne particles by size exclusion. 5. Deposition which is collection of airborne particles using only naturally occurring

deposition process. The two most commonly used devices for microbial air sampling are all glass AGI-30

impinger and Anderson 6-stage impaction sampler. Apart from these, the Reuter air sampler (based on centrifugation) is also commonly used for small-scale air sampling. 1.Impingement:

An impinger operates by drawing air through an inlet that is similar in shape to the

human nasal passage. The air is transmitted through a liquid medium where the air particles become associated with the fluid and are subsequently trapped. The AGI-30 impinger (All glass impinger) is a liquid-filled cylinder which collects particles by their impingement into a fluid. The capillary tip of the inlet tube inside the cylinder is located 30mm from the impinge bottom, thus the nomenclature. AGI-30 is easy to use, inexpensive, portable, and reliable, easily sterilized and has high biological sampling efficiency in comparison to many other sampling devices but to sheer force used to collect the air. The usual volume of collection medium is 20ml and the typical sampling duration is approximately 20 minutes which prevents evaporation during the sampling of warm climates or freezing of the liquid medium when sampling at lower temperatures. The liquid and suspended micro organisms can be concentrated or diluted by using this method of impingement.

A sample medium is 0.85% sodium chloride which is an osmotically balanced, sampling medium used to prevent osmotic shock of recovered organisms. Another medium in use is peptone (1%) which is used as a medium for stressed organisms. Finally, enrichment medium can be used to sample selectively for certain types of organisms. Drawback:

No particle size discrimination which prevents accurate characterization of the sizes of the airborne particles that are collected. 2.Impaction:



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The Anderson six-stage impaction sampler provides accurate particle size discrimination

in contrast to the impinger. This device was developed by Anderson in 1958 and the general operating principle is that air is sucked through the sampling port and strikes agar plates. Impaction procedure depends upon the internal properties of the particle (size, density), on the physical parameters of the impactors (inlet nozzle dimensions) and the airflow pathway.

The principle that underlies this sampling device is simple and ingenious. Air impinging onto the top agar plate is travelling at relatively low speed, and is deflected around the agar plate. Only the larger (heavier) airborne particles will have sufficient momentum (defined as mass x velocity) to break free from this air current and impact onto the top agar surface. But then the same volume of air is sucked through a series of small holes, so its velocity is increased and this enables smaller particles to impact onto the second agar plate and so on, down the series of plates with increasingly smaller holes, so that the momentum of the airborne particles is increased at each stage.

The result is a size is a size (mass) separation of the airborne particles, which is remarkably similar to that which occurs in the human respiratory tract. Large spores such as those that impact onto the topmost agar plate. Smaller spores (3-7 micrometers) will impact on the middle agar plates, and even very small spores (e.g the spores of actinomycetes, 1-2 micrometers diameter) will impact on the lowest agar plates.

When the apparatus is running the incoming air impinges onto the topmost agar plate, where airborne particles can impact on the agar surface. Then the air is drawn round this first agar plate, and through the first set of perforations, so that particles can impact on the agar plate, and so on down the stack.

Larger particles are collected on the first layer, and each successive stage collects smaller and smaller particles by increasing the flow velocity and consequently the impaction potential.



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The particle size distribution of the air particles can be directly related to the particle size distribution that occurs naturally in the lungs of animals. The lower stages correspond to the alveoli and the upper stages to the upper respiratory tract. After this apparatus has run for some time the agar plates are removed and incubated, to identify the organisms that grow on them. 3.Centrifugation:

Centrifugal samplers use circular flow patterns to increase the gravitational pull within the sampling device in order to deposit particles. The most common is the Reuter’s air sampler which is based on the centrifugal force. Air is sucked in through the propeller blades and as it traverses through the body of the sampler, it gets deposited onto the thin agar media which lines the inner wall of the sampler. After a known period of time the media is taken out and incubated for further studies.

There is another device based on this principle, the cyclone which is a tangential inlet and return flow sampling device. These samplers are able to sample a wide range of air volumes (1-400 L/min), depending on the size of the unit. The unit operates by applying suction to the outlet tube, which causes air to enter the upper chamber of the unit at angle. The flow of air falls into a characteristic tangential flow pattern which effectively circulates air around and down along the inner surface of the conical glass housing. As a result of the increased centrifugal forces imposed on particles in the air stream, the particles are sedimented out. Analysis is performed by rinsing the sample with an appropriate liquid medium, collection of the medium and subsequent assay by standard methodologies.

4.Filtration:

Filter sampling requires a vaccum source and involves passage of air through a filter, where the particles are trapped. After collection, the filter is washed to remove the organisms before analysis. Usually membrane filters are used for the purpose with varied pore sizes.



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5.Deposition:

Deposition sampling can be accomplished merely by opening an agar plate and exposing it to the wind, which results in direct impaction, gravity settling and other depositional forces. There are problems associated with this method of sampling. They have low overall sampling efficiency because it relies on natural deposition. It has no defined sampling rates or particle sizing, and poses an intrinsic difficulty in testing for multiple microorganisms with varied growth conditions.

Chapter – 3 AIR BORNE PATHOGENS Airborne diseases are caused by pathogenic microbes small enough to be discharged

from an infected person via coughing, sneezing, laughing and close personal contact or aerosolization of the microbe. The discharged microbes remain suspended in the air on dust particles, respiratory and water droplets. Illness is caused when the microbe is inhaled or contacts mucus membranes or when secretions remaining on a surface are touched.



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Bacteria: 1.Streptococcus pyogenes:

➢ These organisms cause Strep throat. ➢ This pathogen is resistant to phagocytosis. ➢ This organism can be detected in the throat of many people who are asymptomatic

carriers. ➢ This is characterized by local inflammation and fever with tonsillitis. ➢ Another complication is Otitis media. ➢ Penicillin is the drug of choice. ➢ This strain produces an erythrogenic (reddening) toxin the infection is called Scarlet

Fever. ➢ It is a communicable disease spread mainly by inhalation of infective droplets from an

infected person.

2.Corynebacterium diphtheria: ➢ It is a Gram positive, non-spore-forming, rod. ➢ This organism causes Diphtheria. ➢ Disease begins with a sore throat and fever followed by general malaise and

swelling of neck. ➢ The bacterium is well-suited to airborne transmissions and is very resistant to

drying.

3.Streptococcus pneumoniae: ➢ It is a Gram positive, capsulated ovoid bacterium in pairs. ➢ This organism causes pneumococcal pneumonia. ➢ It involves bronchi and alveoli with high fever, breathing difficulty and chest pain. ➢ This organism causes Otitis media

4.Haemophilusinfluenzae:

➢ It is Gram negative coccobacilli. ➢ This has similar symptoms as common cold. ➢ This organism causes Otitis media.

5.Moraxella catarrhalis:

➢ This organism causes Otitis media. ➢ This is one of the uncomfortable complications of common cold.

6.Staphylococcus aureus: ➢ This organism causes Otitis media. ➢ This is one of the uncomfortable complications of common cold.

7.Bordetella pertussis:

➢ It is a small obligately aerobic capsulated Gram negative coccobacillus. ➢ This organism causes Pertussis (whooping cough). ➢ Disease is transmitted by inhaling pathogens expelled by the coughing of the

infected person. ➢ It is primarily a childhood disease and can be quite severe. Initial stages resemble

a common cold.



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8.Mycobacterium tuberculosis: ➢ It is a slender acid-fast rod and obligate aerobes which is a slow grower. ➢ This organism causes Tuberculosis. ➢ Tuberculosis is the most commonly acquired by inhaling the tubercle bacillus.

9.Mycoplasma pneumoniae:

➢ They are fastidious organisms which lack cell wall and show very small colonies on solid media.

➢ This organism causes Mycoplasmal pneumonia. ➢ Transmitted by airborne droplets. ➢ This infects the upper respiratory tract initially with low fever, cough and

headache followed by the lower respiratory tract infection.

10.Legionella pneumophila: ➢ It is an aerobic Gram negative rod. ➢ This organism causes Legionellosis. ➢ Characterised by high fever of 105o F, cough and general symptoms of

pneumonia.

11.Coxiellaburnettii : ➢ It is a Rickettsial member. ➢ This organism causes Q fever. ➢ Symptoms are undulating fever (1-2 weeks) with chills, chest pain and severe

headache. ➢ This organism is resistant enough to survive airborne transmission.

Fungi: 1.Histoplasmacapsulatum :

➢ It is a dimorphic fungus. ➢ This causes Histoplasmosis. ➢ It resembles TB superficially. ➢ Symptoms are sub-clinical. ➢ Disease is acquired from airborne conidia produced under conditions of

appropriate moisture and pH. 2.Coccidioidesimmitis:

➢ It is a dimorphic fungus. ➢ This causes Coccidioidomycosis. ➢ In soil, it forms filaments which reproduce by formation of arthrospores which are

carried by wind to transmit the infection. 3.Pneumocystis carinii:

➢ It has characters of both a fungus and a protozoan but it is strongly related to certain yeasts.

➢ This causes Pneumocystis pneumonia. ➢ This organism is found in the alveoli of the human lungs.

4.Blastomycesdermatitidis:

➢ It is a dimorphic fungus. ➢ This causes Blastomycosis. ➢ Infection begins in the lungs and spreads rapidly.



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5.Aspergillusfumigatus: ➢ This causes Aspergillosis. ➢ Common among gardeners and farmers due to the spores which are widespread

in decaying vegetation. Virus: 1. Rhinovirus:

➢ Rhinoviruses thrive at a temperature slightly below that of normal body temperature.

➢ This causes Common cold. ➢ A single rhinovirus particle deposited on the nasal mucosa is sufficient to cause a

cold. ➢ Symptoms are sneezing, excessive nasal secretions and congestion.

2. Coronavirus:

➢ This causes Common cold. ➢ Symptoms are sneezing, excessive nasal secretions and congestion. ➢ SARS - respiratory disease caused by a coronavirus, last reported in 2004

3. Respiratory syncytial virus (RSV):

➢ Most common cause of viral respiratory disease in infants. ➢ Symptoms are coughing and wheezing lasting for more than a week. ➢ All children become infected by the age of two.

4. Influenza (viral flu):

➢ Characterised by chills, fever, headache and muscular ache. ➢ Caused by influenza virus.

5. Viral pneumonia:

➢ This occurs as a complication of influenza, measles or even chicken pox.



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UNIT - II SOLID WASTE MANAGEMENT

Sources and types of Solid waste

As long as human have been living in settled communities, solid waste or garbage has been an issue and modern societies generate for more solid waste than early humans ever did. Daily life in industrialized nations can generate several pounds of solid waste per consumer, not only directly in the home, but indirectly in factories that manufacture goods purchased by consumers. Solid waste can be classified into different type depending on their source,

➢ Municipal solid waste /House hold waste. ➢ Industrial waste /Hazardous waste. ➢ Biomedical waste /Hospital waste /Infections waste. ➢ E-waste.

Municipal solid waste consists of household waste construction and demolition debris,

sanitation residual and waste from street. This garbage is generated mainly from residential and commercial complexes with rising urbanization and change in lifestyle and food habits, the amount of municipal solid waste has been increasing rapidly and its composition changing. Over the last few years, the consumer market has grown rapidly leading to products being packed in cans, aluminium foils, plastics and other non-biodegradable item that cause in calculable harm to the environment. These domestic solid wastes are usually thrown in municipal garbage collecting cans or on road side open waste lands. They are dumped over a large area of land which becomes the breeding ground for files and rats usually they are not burnt to reduce the volume because burning would cause air pollution which is still more dangerous.

Industrial and hospital waste is considered hazardous as they may contain toxic substance. Certain types of household wastes are also hazardous. Hazardous waste could be highly toxic to humans, animals and plants, corrosive highly inflammable or explosive and react when exposed to certain things e.g.; gases. Ash from thermal power plants is mainly composed of oxide of iron, silica and aluminium and a low concentration of toxic heavy metals. Household wastes that can be categorized as hazardous waste include old batteries, shoe polish, paint, tins, old medicines and medicine bottles. Hospital waste contaminated by chemical used in hospitals are considerable hazardous. These chemicals include formaldehyde and phenols and mercury, which used as disinfectants or equipment blood pressure. Most hospitals do not have proper disposal facilities for these hazardous wastes.

In the industrial sector, the major generator of hazardous waste is the metals, chemical, paper, pesticide, dye, refining and rubbers goods industries. Direct exposure to chemicals in hazardous waste such as mercury and cyanide can be fatal. Most of the toxic industrial wastes are dumped on waste hands for slow and gradual decomposition; some of the effluents have heavy metals which pollute the monsoon season. One such most toxic heavy metal is cadmium which is present in traces in some fertilizer. Toxic chemicals destroy vegetation and produce many deformities in animals and human beings.

Hospital waste is generated during the diagnosis, treatment or immunization of human being or animals in research activities in these fields or in the production or testing of biological. It may include waste like sharps, solid waste, disposables, anatomical waste , cultures, discarded



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medicines, chemical waste etc., these are in the form of disposable syringes, swabs bandages, body fluids human excreta etc. this waste is highly infections and can be a serious threat to human health if not managed in a scientific and discriminate manner. The use of incinerators is crucial for disposal of hospital waste.

Chemical fertilizers increase soils fertility and gives better crop yield in lesser time. Shortly the land becomes saline acidic or alkaline and loses fertility. Pesticides and biocides used in crop field is not eco-friendly, they enter into crop and then into primary and secondary consumers even human beings are affected due to bio magnification. Excrete of man, animals, digested sewage sludge used as manure pollute the soil. Several germs present in such waste contaminate soil, vegetables and waste bodies causing severe health hazards.

The latest solid waste that has appeared in last twenty years commonly known as e-waste is no less harmful. Frequently more efficient and user friendly electronic items appear in the market thus discarding the old generation equipment, Irreparable computers and electronic goods which simply become garbage or solid waste. Over half the e-waste generation in developed countries are exported to developing countries where they ultimately increase the e-garbage proportions, where metals like copper, iron, silicon, nickel and gold are recovered during recycling process. Recycling of e-waste in developing countries often involves manual participation, thus exposing workers to toxic substances present in e-wastes. E-waste are buried in landfills or incinerated. COMPOSTING: Composting is a method of combined disposal of refuse and night soil or sludge. It is a process of nature whereby organic matter breaks down under bacterial action resulting in the formation of relatively stable humus like material, called the compost which has considerable manurial value for the soil. The principal by-products are carbon dioxide, water and heat. The heat produced during composting (60°C or higher), over a period of several days destroy eggs and larvae of flies, weed seeds and pathogenic agents. The end product, compost, contains few or no disease producing organisms, and is a good soil builder containing small amounts of the major plant nutrients such as nitrates and phosphates. The following methods of composting are now used: (1) Bangalore Method (Anaerobic method) (2) Mechanical composting (Aerobic method). Bangalore Method (Hot Fermentation Process):

It is recommended as a satisfactory method of disposal of town wastes and night soil. Trenches are dug 90 cm (3ft) deep, 1.5 to 2.5m broad and 4.5 -5.1m long, depending upon the amount of refuse and night soil to be disposed of. Depths greater than 9 cm are not recommended because of slow decomposition. The pits should be located not less than 800m from city limits. The composting procedure is as follows; First a layer of refuse about 15 cm thick is spread at the bottom of the trench. Over this, night soil is added corresponding to a thickness of 5 cm. Then alternate layers of refuse and night soil are added in the proportion of 15 cm and 5 cm respective, till the heap rises to 30 cm above the ground level. The top layer of refuse should be a t least 25cm thickness. Then the heap is cover with excavated earth. If properly laid, a man is legs will not sink when walking over the compost mass. Within 7 days as a result of bacterial action considerable heat (OVER 60°C) is generated in the compost mass. This intense heat which persists over 2 or 3 weeks, serves to decompose the refuse and night soil and to destroy all pathogenic and parasitic organisms. At the end of 4-6



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months, decomposition is complete and the resulting manure is a well decomposed, odourless, innocuous material value ready for application to the land. Mechanical Composting (Indore Process): Another method of composting known as mechanical composting is becoming popular. In this compost is literally manufactured on a large scale by processing raw materials and turning out a finished product. The refuse is first cleared of salvable materials such as rages, bones, metal, glass and items which are likely to interfere with the grinding operation. It is then pulverised in pulverising equipment in order to reduce the size of particle to less than 2 inches. The pulverised refuse is then mixed with sewage, sludge or night soil in a rotating machine and incubated. The factors which are controlled in the operation are a certain carbon nitrogen ratio, temperature, moisture, pH and aeration. The entire process of composting is composting is complete in 4-6 weeks. This method of composting is in vogue in some of the developed countries. Composting is a microbial process that converts putrefiable organic waste materials into stable, sanitary, humus like product that is reduced in bulk and can be used for soil improvement. Composting is accomplished in static piles, aerated piles, or continuous feed reactors. The static pile process is simple but relatively slow, typically requiring many months for stabilisation. Odour and insect problems can be controlled by covering the piles with a layer of soil, finished compost, or wood chips. Unless turned several times, the finished compost is rather uneven in quality. The aerated piles process achieves substantially faster composting rates through improved aeration. The aeration is maintained by suction of air through perforated pipes buried inside the compost pile. This design achieves at least partial oxygenation of the pile, but temperature control is inadequate. The aerated pile process goes to completion in about 3 weeks.

Composting can be accomplished more rapidly in a bioreactor. It requires about 20,000 cubic feet of air per ton or organic matter per day for efficient composting. This process forms a uniform and stable product, but it also requires a high initial investment. Composting in the reactor is accomplished in 2-4 days.

The composting process is initiated by mesophilic heterotrophs. As the temperature rises, these are replaced by thermophilic forms. Thermophilic bacteria prominent in the composting process are Bacillus stearothermophilus, Thermomonospora, Thermoactinomyces and Clostridium thermocellum. Important fungi in the thermophilic composting process are Geotrichumcandidum, Aspergillusfumigatus, Mucorpusillus, Chaetomium thermophile, ThermoascusauranticusandTorulathermophila. Vermicomposting

Vermicomposting is a method of preparing enriched compost with the use of earthworms. It is one of the easiest methods to recycle agricultural wastes and to produce quality compost. Earthworms consume biomass and excrete it in digested form called worm casts. Worm casts are popularly called as Black gold. The casts are rich in nutrients, growth promoting substances, beneficial soil micro flora and having properties of inhibiting pathogenic microbes.

Vermicompost is stable, fine granular organic manure, which enriches soil quality by



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improving its physicochemical and biological properties. It is highly useful in raising seedlings and for crop production. Vermicompost is becoming popular as a major component of organic farming system. Vermicomposting materials

Decomposable organic wastes such as animal excreta, kitchen waste, farm residues and forest litter are commonly used as composting materials. In general, animal dung mostly cow dung and dried chopped crop residues are the key raw materials. Mixture of leguminous and non-leguminous crop residues enriches the quality of vermicompost. There are different species of earthworm viz. Eiseniafoetida (Red earthworm), Eudriluseugeniae (night crawler), Perionyxexcavatusetc. Red earthworm is preferred because of its high multiplication rate and thereby converts the organic matter into vermicompost within 45-50 days. Since it is a surface feeder it converts organic materials into vermicompost from top.

Types of vermicomposting

The types of vermicomposting depend upon the amount of production and composting structures. Small-scale vermicomposting is done to meet the personal requirement and farmer can harvest 5-10 tons of vermicompost annually. While, large-scale vermicomposting is done at commercial scale by recycling large quantity of organic waste with the production of more than 50 – 100 tons annually Methods of vermicomposting

Vermicomposting is done by various methods, among them bed and pit methods are more common. Bed method:

Composting is done on the pucca / kachcha floor by making bed (6x2x2 feet size) of organic mixture. This method is easy to maintain and to practice Pit method:

Composting is done in the cemented pits of size 5x5x3 feet. The unit is covered with thatch grass or any other locally available materials. This method is not preferred due to poor aeration, water logging at bottom, and more cost of production



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Process of vermicomposting Following steps are followed for vermicompost preparation.

➢ Vermicomposting unit should be in a cool, moist and shady site ➢ Cow dung and chopped dried leafy materials are mixed in the proportion of 3: 1and are

kept for partial decomposition for 15 – 20 days. ➢ A layer of 15-20cm of chopped dried leaves/grasses should be kept as bedding material

at the bottom of the bed. ➢ Beds of partially decomposed material of size 6x2x2 feet should be made ➢ Each bed should contain 1.5-2.0q of raw material and the number of beds can be

increased as per raw material availability and requirement. ➢ Red earthworm (1500-2000) should be released on the upper layer of bed ➢ Water should be sprinkled with can immediately after the release of worms ➢ Beds should be kept moist by sprinkling of water (daily) and by covering with gunny

bags/polythene ➢ Bed should be turned once after 30 days for maintaining aeration and for proper

decomposition. ➢ Compost gets ready in 45-50 days. ➢ The finished product is 3/4th of the raw materials used.

Harvesting

When raw material is completely decomposed it appears black and granular. Watering should be stopped as compost gets ready. The compost shout be kept over a heap of partially decomposed cow dung so that earthworms could migrate to cow dung from compost. After two days compost can be separated and sieved for use. Preventive measures

➢ The floor of the unit should be compact to prevent earthworms’ migration into the soil. ➢ 15-20 days old cow dung should be used to avoid excess heat. ➢ The organic wastes should be free from plastics, chemicals, pesticides and metals etc. ➢ Aeration should be maintained for proper growth and multiplication of earthworms. ➢ Optimum moisture level (30-40 %) should be maintained ➢ 18-25°C temperature should be maintained for proper decomposition.

Advantages There are many advantages of vermicompost:

➢ It provides efficient conversion of organic wastes/crop/animal residues. ➢ It is a stable and enriched soil conditioner. ➢ It helps in reducing population of pathogenic microbes. ➢ It helps in reducing the toxicity of heavy metals. ➢ It is economically viable and environmentally safe nutrient supplement for organic food

production. ➢ It is an easily adoptable low cost technology.

SOLID WASTE DISPOSAL BY SANITARY LANDFILL

The term “sanitary landfill” is too often used to refer to a solid waste operation that is little better than an open dump. Actually, sanitary landfill means an installation where a satisfactory, nuisance-free solid waste disposal operation is being carried out in accordance with recognized standard procedures. The operation of a sanitary landfill requires skill and



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knowledge. It is a scientific method and should be treated as such. Engineering and planning is needed to operate a satisfactory sanitary landfill. Sanitary Landfill:

A method of disposing of solid waste on land without creating nuisances or hazards to public health or safety, by utilizing the principles of engineering to confine the solid waste to the smallest practical area, to reduce it to the smallest practical volume, and to cover it with a layer of earth at the conclusion of each day's operation or at more frequent intervals. SITE SELECTION

Selection of a sanitary landfill site depends upon evaluation of the site itself and upon community acceptance of the site for solid waste disposal purposes. Ordinarily, selection of a site starts with a search for conveniently located waste land or low-value land. Solid waste disposal sites in the state now include borrow pits, ravines, areas adjacent to water courses, and low-lying swampy land. Topographic Maps

Clear plans and procedures are essential to the efficient and successful operation of any sanitary landfill. Topographic maps will show to all those involved (planners, legislative bodies, health officials, supervisors, equipment operators, etc.) the existing situation and the sequence of operations planned. The design of a sanitary landfill should include one or more topographic maps at a scale of not over 200 feet to the inch with five-foot contour intervals. These maps should show: the proposed fill area; any borrow area; access roads; grades for proper drainage of each lift required and a typical cross-section of a lift; special drainage devices, if necessary; fencing; equipment shelter; existing and proposed utilities; employee facilities; and all other pertinent information to clearly indicate the orderly development, operation, and completion of the sanitary landfill. Cover material

Cover material is spread over the solid waste and compacted to form a tight seal or cover. This barrier prevents flies from laying eggs on the waste or rodents from invading the fill. It seals in odors, prevents infiltration of rainwater, and minimizes the blowing and scattering of material. It reduces the fire hazard, and helps to produce a dense, stable fill. An ideal soil for cover material is a combination of approximately 50 percent clay-silt and 50 percent sand. Such a sandy-loam mixture is porous, compacts well, and is not subject to cracking upon drying. Clay, when it becomes dry, will crack, giving rodents and insects an access to the covered solid waste. In addition, when clay becomes wet it is difficult to handle. Landfill Operation

Detailed engineering planning and control, both prior to and during operation, are necessary to insure efficient operation and maximum site utilization. Careful planning and control will pay dividends in economy, trouble-free operation, and full capacity utilization of the landfill site. There are basically two methods of operating a sanitary landfill; the area method and the trench method. The method selected will depend upon subsurface conditions, drainage, and topography of the land. Area Landfill

The solid waste is placed on the land; a bull-dozer or similar equipment spreads and compacts the waste; then the waste is covered with a layer of earth; and finally the earth cover



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is compacted. The area method is best suited for flat areas or gently sloping land, and is also used in quarries, ravines, pits, or where other suitable land depressions exist. Normally the earth cover material is hauled in or obtained from adjacent areas. Trench Landfill

A trench is cut in the ground and the solid waste placed in it. The waste is then spread in thin layers, compacted, and covered with earth excavated from the trench. The trench method of landfilling will consist of any one of the following three methods: Progressive Excavation:

The distinguishing feature of this method is its continuity. Cover material is excavated from the area directly in front of the working face of the landfill and is put over the previously compacted solid waste behind. The cover is excavated as required and the process goes on almost continuously. Cut and Cover:

A cut-and-cover landfill is one in which trench-type excavations are made on the site to hold the solid waste and get cover material. The trenches are usually parallel to each other in order to use the site efficiently. The trenches should be near as possible perpendicular to the prevailing winds to minimize the scattering of loose material. The width of the trench should be approximately twice the width of the tractor used in order to obtain maximum compaction of the material. Unlike the progressive excavation method, solid waste at a cut-and-cover fill is usually discharged at the top of the working face, although in some cases it may be desirable to discharge it at the bottom. Imported Cover:

The imported cover method of operating a landfill is not a single method but rather several, and is used when cover material is obtained from a source outside the site.

The trench method is best suited for flat land where the water table is not near the ground surface. Normally the material excavated can be used for cover with a minimum of hauling. A disadvantage is that more than one piece of equipment may be necessary.



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Access Roads Access roads to the landfill should be regularly maintained under all weather conditions

to permit a smooth flow of traffic. Spreading and Compacting

A successful sanitary landfill operation depends upon the adequate compaction of the waste. Solid waste should be placed at the top or bottom of the working face, spread in layers about two feet thick, and compacted. Further compaction is provided by repeated travel of landfill equipment over the layers and, if necessary, by the use of special compacting equipment. Additional compaction is also achieved by routing collection vehicles so that they travel repeatedly over the finished portion of the fill. Depth of Cells

The total depth of a landfill is governed by the characteristics of the site, the desired elevation of the completed site, and good engineering practice. Eight feet is generally recommended as a maximum single cell depth because deeper cells usually result in fills that have excessive settlement and surface cracking. Cover Daily Cover:

A compacted layer of at least six inches of suitable cover material should be placed on all exposed solid waste by the end of each working day. This is to prevent fly and rodent attraction, blowing of papers, production of odors, fire hazards, etc. Intermediate Cover:

For intermediate cover on lifts which will not have additional lifts placed on them within a year, a minimum of 12 inches of compacted soil is recommended. Final Cover:

A layer of suitable cover material compacted to a minimum thickness of two feet should be placed over the entire surface of each portion of the final lift not later than one week following the placement of waste within that portion. Where trees will be planted on the completed fill, a depth of three feet or more of compacted earth has been found necessary. Hazardous Materials

Although it is not common or recommended practice, hazardous materials, such as sewage sludge, radioactive wastes pathological wastes, explosive wastes, and chemicals, can be disposed of at sanitary landfills under special conditions. Present disposal methods for hazardous materials are: 1) mixing with soil, 2) evaporation, 3) infiltration, and 4) sanitary landfill. LIQUID WASTE MANAGEMENT

Urban areas (municipal corporations) which are sewered areas have to handle large proportions of wastes and are quite complex. Large scale sewage treatment involves three process namely (A) primary /preliminary/physical treatment (B) secondary/biological treatment (C) tertiary/ chemical treatment. A) PRIMARY/ PHYSICAL/ PRELIMINARY TREATMENT;

The primary treatment process consists of screening and sedimentation processes.



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Screening The purpose of screening is to remove larger floating solids and and larger organic solids

do which not aerate and decompose (become septic), and to skim grease and fatty oils. This process is accomplished by screeners which are of three types;

➢ Rock or coarse screens which are made of parallel bars 75mm apart. It involves more area and hence is more effective.

➢ Medium screens having bars or mesh with a width of 12.5-40mm. ➢ Fine screens made of fine wire no perforated metal with openings less than 13mm. they

get easily clogged. To prevent overloading with collected solids, the screen is slowly rotated so that solids

can be removed at regulars. Screenings are usually disposed by burial or incineration or they are passed through a macerator which shreds them to small sizes.

Removal of grit (grit/Detritus Chamber)

The detritus chamber removes heavy inorganic matter like grit, sand, gravel, road scrapings and ashes. These particles are discrete particle that do not decay but create nuisance. They may injure pumps and make sludge digestion difficult. Grit particles are of large size and hence high density compared to organic matter. Thus, they are removed by differential settling. Grit particles (0.20mm) settle at a velocity of 1.2m/min whereas suspended solid (faeces) have considerably lower settling velocities. The grit settles 30 cm in 16 sec, if the velocity is 30 cm/sec and the detention period is 1 min. Length of the tank should be 18 m. Storage space is provided at the bottom of the tank such that the clearing period in average is 2 weeks. Removal of fatty oils (skimming tanks)

Sewage contains lot of grease and fatty oils (which forms a scum in sedimentation tanks and interferes with oxidation process in aeration tanks) which are removed. Skimming tanks or primary setting basins are about 1 m deep and the scum accumulations are removed manually or buried or burnt. This method is not of much use in India. Sedimentation (primary sedimentation)

Sewage from the primary settling basin is now admitted into a huge tank called the primary sedimentation tank.



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It is a very large rectangular tank. Nearly 50-70% of the solids settle down under influence of gravity. A reduction between 30-40% in the number of coliforms is obtained. The organic matter which settles down is called sludge and is removed by mechanically operated devices (scrapper) without disturbing the operation in the tank. Simultaneously a small amount of biological action also takes place in which microbes present in the sludge attack complex organic solid and break them down to simpler soluble substances and ammonia. A small amount of fat and grease rises to the surface to form a scum which is removed from time to time and disposed. Purpose of sedimentation is to remove suspended solids and thereby reduce the strength of the sewage. Sedimentation or clarification is the settling and removal of suspended impurities which occur when water stands still or flows slowly through a basin or tank. There is negligible turbulence and hence particle of high density tend to settle down (gravity) and form a sludge layer at the bottom of the tank whereas clarified water will be collected through the outlet. Hence sedimentation units play dual role;

➢ Removal of settleable solids. ➢ Concentration of removed solids into sludge.

There are types of sedimentation tanks;

➢ Vertical flow tanks. ➢ Horizontal flow tanks ➢ Circular flow tanks or radial flow tanks which receive sewage in the centre and

their flow is towards the sides. SECONDARY TREATMENT

The effluent from the primary sedimentation tank, still contain a proportion of organic matter in solution or colloidal stats and numerous living organisms. Its BOD is high hence it is subjected to aerobic oxidation by various methods. It is necessary to oxidize the organic matter present in the effluent before it is being discharged into the body of water (diluting). This oxidation is either carried out on land (naturally) or in bacterial beds (artificially) such as tricking filter or aerators. The preliminary treatment reduces the BOD while the secondary treatment satisfies the demand of BOD with the help of bacterial beds. Secondary treatment relies on microbial activity. Since these oxidation processes are carried out by microorganisms, they are called as biological oxidators. There are four different types of oxidation processes; Fixed film sewage treatment

➢ Biological filter or trickling filters ➢ Rotating biological contractor or biodisc system

Suspended cell sewage treatment

➢ Oxidation ponds ➢ Activated sludge process

Trickling Filter system

This is a fixed film sewage treatment method. It is a simple method but very expensive. It works on the principle of filtration. Sewage is distributed by a sprinkler revolving over a bed of porous material which is normally an artificially constructed bed consisting of broken stones, bricks or other suitable material. The aerobic life that flourishes on the surface of such material oxidises and nitrifies the organic matter present. Whenever sewage flows over a contact surface, aerobic bacteria lying dormant in the liquid become active and start breeding readily in favourable condition and form a film called the zooglial layer on the surface. Sewage percolates



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the porous bed and the effluent is collected at the bottom. Dense slimy bacterial grow and coat the porous material. Zoogloearamigera has a principal role in generating slime matrix through secretion of exopolysaccharide which accumulates a heterogeneous microbial community (bacteria, fungi, protozoa, nematodes and rotifers). This community absorbs and mineralises the dissolved organic nutrients in the sewage thus reducing the BOD of the effluent. Aeration is passively provided by the porous material. A food web is established based upon the microbial biform. Insects consume the excess biomass generated (only when the sprinkling is shut off) Sewage is re-circulated several times through the same filter to further clear the sewage. Drawbacks

Nutrient overload may lead to excess microbial slime reducing aeration and percolation rates and leads to removal of trickling filter bed. Trickling filter method cannot be used during cold winters when the temperature is very low since the growth rate of the organisms becomes very low.

Oxidation Lagoons

Oxidation ponds are also known as stabilisation ponds or lagoons. They are used for simple secondary treatment of sewage effluents. Within an oxidation pond, heterotrophic bacteria degrade organic matter in the sewage which results in production of cellular material and minerals which supports the growth of algae in the oxidation pond. Growth of algal populations allows further decomposition of the organic matter by producing oxygen. The production of this oxygen replenishes the oxygen used by the heterotrophic bacteria. Typically oxidation ponds need to be less than 10 feet deep in order to support the algal growth. In addition, the use of oxidation ponds is largely restricted to warmer climatic region because they are strongly influenced by seasonal temperature change. Oxidation ponds also tend to decomposition of the sewage. Overall, oxidation ponds tend to be inefficient and require large holding capacities and retention times. The degradation is relatively slow and the effluents containing the oxidised products need to be periodically removed from the ponds.

Microbes grow as suspended particles within the water column rather than as biofilms. As oxygenation is usually achieved by diffusion, and by the photosynthetic activity of algal these systems need to be shallow. Sewage is subjected to primary settling and is subsequently channelled through a series of oxidation ponds. Treated sewage is discharged into surface waters. Partially treated sewage is used for ground water. After settling of most of the algal and bacterial biomass, the water is transferred to large shallow infiltration a ponds. From these ponds, water flows through sand and soil layer and slowly returns underground. Clogging of



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infiltration pond (undegraded microbial polysaccharide and accumulation of iron sulphide) is a recurrent problem periodic rest periods for infiltration basins which allow degradation of the excess polysaccharides and re- aerates the sediment of oxidation pond with the oxidation of clogging iron sulphide.

Activated Sludge Process This is the most common option in secondary treatment and is also a method of suspended cell sewage treatment method. It starts with aeration that encourages the growth of microbes in the waste. The microbes feed on the organic material, which then allows solid to settle out. Bacteria –containing activated sludge is continually re-circulated back to the aeration basin to increase the rate of organic decomposition. After primary settling, sewage containing dissolved organic compound is introduced into an aeration tank. Aeration is provided by air injection or mechanical stirring by allowing compressed air continuously from the bottom of the aeration tanks.

Microbial activity is maintained at high levels by introduction of most of the settle

activated sludge (also called returned sludge which is rich in culture of aerobic bacteria) thus the name activated sludge process. During the process of aeration, organic matter of the sewage gets oxidised with the help of aerobic bacteria in tank which are suspended in the sewage water. The typhoid and cholera organism are definitely destroyed and the coliforms are greatly reduced. During the holding period in the tank, there is vigorous developing development of diverse heterotrophic bacterial population like Micrococcus, Achromobacter, Athrobacter, Flavobacterium, Zoogloea. Filamentous bacteria like Sphaerotilus, Mycobacteria are also common. Filamentaous fungi and yeast occur in low numbers. Protozoa are represented by ciliates. They are important predator of bacteria.

Bacteria occur individually in free suspension aggregates as floccules by Zoogloearamigera. Floc is too large to be ingested by protozoa hence considered as a defence mechanism. In raw sewage, bacteria predominate during the holding time in the aeration tank. The number of suspended bacteria decrease but those associated Withfloc greatly increase in number. During the holding period, a portion of dissolved organic substrate is mineralised. Another portion is converted to microbial biomass. In the advanced stage of aeration, most of the microbial biomass is associated with floc that can be removed from suspension by settling.



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Poor settling characteristic are associated with the bulking of the sewage sludge, a

problem caused by proliferation of filamentous bacteria like Sphaerotilus, Beggiatoa, Thiothrixand Bacillus, filamentous fungi such as Geotrichum, Cephalosporium, Cladosporiumand Penicillium. Bulking may be caused by a high C: N and C: P ratios and low dissolved oxygen concentration. A portion of the settled sewage is recycled for inoculation of incoming raw sewage. Excess sludge required incineration or addition of treatment by anaerobic digestion and composting or disposal as landfills. ASP tends to reduce the BOD to 5 -15% of raw sewage. Treatment drastically reduces the number of intestinal pathogens in sewage (through competition, adsorption, predation and settling). Numbers of E.coli and enteroviruses are 90 – 99% lowered in the effluent by the ASP than in the incoming raw sewage. Growth of sludge bacteria (floc) must be equal to the sludge wastage. The presence of attached ciliates is critical for the control of disposal bacteria and removal of portion of BOD. The types and number of protozoa associated with the floc can be used as an indicator of sludge condition and thus treatment performance can be monitored. Sludge is in poor condition when few ciliates and many flagellate are seen. Ciliates predominate in good sludge. ASP is efficient and flexible and is able to withstand in sewage flow rate and concentration and is widely used for the treatment of domestic waste and industrial effluent. It produces large volume of sludge.

SEPTIC TANK

It is a water-tight masonry tank into which household sewage is admitted for treatment. It is a satisfactory mean of disposing excreta and liquid waste from individual dwellings, small group of house and institutions which have adequate water supplies but do not have access to public sewerage system. Septic tanks are settling tanks and are based on the sedimentation principle. They are plain single storeyed tanks. It is simplest form of anaerobic treatment system. It is a kind of sewage settling tank. It is an enclosed concrete box into which waste flows from the house. The organic matter accumulates at the bottom of tank whereas the water rises to the outlet pipes which empty into the surrounding area. The tank is to be pumped out regularly as there is no absorption of the digested organic matter into the earth. Complex organic matters are anaerobically decomposed to simple organic molecules and fermentation gases. The tank accomplishes two processes sedimentation and biological degradation of the sludge which are achieved by anaerobic digestion and aerobic oxidation.



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Anaerobic digestion and Aerobic oxidation

The human excreta consists of 65 percentage mineral matter (which do not undergo any chemical changes in a septic tank) and 35 percentage organic matter of which only 20-40 percentage of organic matter are liquefied or gasified in the septic tank. The heavier matter (sludge) settles at the bottom and the lighter matter (grease and fats) forms a layer called scum on the top. Solids are attacked by the bacteria and are broken down into simpler compounds. This is the first stage of purification called anaerobic digestion. The sludge is much reduced in volume and is rendered stable and inoffensive. A portion of the solid is transferred into liquid and gases (mostly methane) which rises to the surface in the form of bubbles. The liquid which passes out of the outlet pipe from time to time is called effluent. It contains numerous bacteria, cysts, helminthic ova and organic matter in solution or fine suspension. The effluent is allowed to percolate into the subsoil. There are millions of aerobic bacteria in the upper layers of the soil which attack the organic matter present in the effluent. Thus the organic matter is oxidised into stable end products like nitrates, carbon-di-oxide and water. This stage of purification is the aerobic oxidation. Thus two stages are involved in the purification of sewage; First stage (anaerobic digestion) takes place in the septic tank and the second stage (aerobic oxidation) takes place outside the septic tank is the subsoil. Sedimented sludge accumulated at the bottom of septic tank is removed and may be dried, ground and spread on soil as a fertiliser. In typical septic tanks, about 80% of the incoming suspended solids will be removed and the BOD will be reduced. However, this method cannot be relied upon to eliminate pathogenic microorganism in sewage. The effluent will contain large number of bacteria, and discharge from aseptic tank may contain even E.coli /100ml. Hence it is important that the tank should not be in the proximity of water supplies. TERTIARY TREATMENT

All the preliminary and secondary treatment reduces the BOD levels of the sewage. The aim of the tertiary treatment is to remove non- biodegradable toxic organic pollutants such as chlorophenols, polychlorinated biphenyls and other synthetic pollutants. They are removed by activated carbon filters. Phosphate is removed by precipitation as calcium phosphate. Nitrogen is removed by volatilisation as ammonia. Ammoniacal nitrogen can also be removed by breakpoint chlorination by adding hypochlorous acid in 1:1 ratio. Removal of ammoniacal nitrogen lowers the BOD because nitrification would consume oxygen dissolved in the remaining water. Removal of heavy metals like mercury, lead, chromium and cadmium also occur during



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tertiary treatment. The absorbed metal ions are generally converted into either toxic products or residues that remain associated with the microbe biopolymer matrix and are either release during sludge treatment or are remobilised after sludge disposal. The general tendency of bacteria to concentrate heavy metals in their biomass is favourable to effluent quality, but it complicates the disposal of sludge (rectified to some extent by microbial mining. e.g. acid produced by thiobacilli would solubilise heavy metals and leach them from sludge). Heavy metals can be subsequently fed and reprocessed for use or permanently immobilised. Disinfection is the final step in tertiary treatment. This is to kill escaped bacteria or viruses. This is accomplished by chlorination (chlorine gas, hypochloride and sodium hypochlorite).

Cl2 + H2O HOCl + HCl (Hypochlorous acid)

CaO(Cl2) + 2H2O 2HOCl + Ca(OH)2 (Calcium hypochlorite)

This hypochlorous acid is the actual disinfectant. It is a strong oxidant which is designated as antibacterial in nature. It is desirable to remove nitrogen or other contaminants during the secondary treatment before chlorination. Disadvantages

More resistant types of organic molecule including some lipids and hydrocarbon are not oxidised completely but instead become partially chlorinated. Chlorinated hydrocarbons tend to be toxic and are difficult to mineralise. Alternative means of disinfections are more expensive, hence chlorination remains the principle mean of sewage disinfectant



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UNIT – III DISTRIBUTION OF MICROORGANISMS IN NATURE

MICROBIAL FLORA OF SOIL BIOTA Bacteria

Bacteria are single-cell organisms and the most numerous denizens of agriculture, with populations ranging from 100 million to 3 billion in a gram. They are capable of very rapid reproduction by binary fission (dividing into two) in favourable conditions. One bacterium is capable of producing 16 million more in just 24 hours. Most soil bacteria live close to plant roots and are often referred to as rhizobacteria. Bacteria live in soil water, including the film of moisture surrounding soil particles, and some are able to swim by means of flagella. The majority of the beneficial soil-dwelling bacteria need oxygen (and are thus termed aerobic bacteria), whilst those that do not require air are referred to as anaerobic, and tend to cause putrefaction of dead organic matter. Aerobic bacteria are most active in a soil that is moist (but not saturated, as this will deprive aerobic bacteria of the air that they require), and neutral soil pH, and where there is plenty of food (carbohydrates and micronutrients from organic matter) available. Hostile conditions will not completely kill bacteria; rather, the bacteria will stop growing and get into a dormant stage, and those individuals with pro-adaptive mutations may compete better in the new conditions. Some gram-positive bacteria produce spores in order to wait for more favourable circumstances and gram – negative bacteria get into a "nonculturable" stage. Bacteria are colonized by persistent viral agents (bacteriophages) that determine gene word order in bacterial host. Actinobacteria

Actinobacteria are critical in the decomposition of organic matter and in humus formation, and their presence is responsible for the sweet "earthy" aroma associated with a good healthy soil. They require plenty of air and a pH between 6.0 and 7.5, but are more tolerant of dry conditions than most other bacteria and fungi. Fungi

A gram of garden soil can contain around one million fungi, such as Yeasts and Mold. Fungi have no chlorophyll, and are not able to photosynthesize. They cannot use atmospheric carbon dioxide as a source of carbon, therefore they are chemo-heterotrophic meaning that, like animals, they require a chemical source of energy rather than being able to use light as an energy source, as well as organic substrates to get carbon for growth and development.

Many fungi are parasitic, often causing disease to their living host plant, although some have beneficial relationships with living plants, as illustrated below. In terms of soil and humus creation, the most important fungi tend to be saprotrophic; that is, they live on dead or decaying organic matter, thus breaking it down and converting it to forms that are available to the higher plants. A succession of fungi species will colonise the dead matter, beginning with those that use sugars and starches, which are succeeded by those that are able to break down cellulose and lignins.

Fungi spread underground by sending long thin threads known as mycelium throughout the soil; these threads can be observed throughout many soils and compost heaps. From the mycelia the fungi is able to throw up its fruiting bodies, the visible part above the soil (e.g., mushrooms, toadstools, and puffballs), which may contain millions of spores. When the fruiting body bursts, these spores are dispersed through the air to settle in fresh

https://en.wikipedia.org/wiki/Flagellum

https://en.wikipedia.org/wiki/Aerobic_organism

https://en.wikipedia.org/wiki/Putrefaction

https://en.wikipedia.org/wiki/Soil

https://en.wikipedia.org/wiki/Soil_pH

https://en.wikipedia.org/wiki/Micronutrient

https://en.wikipedia.org/wiki/Mutation

https://en.wikipedia.org/wiki/Bacteriophage



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environments, and are able to lie dormant for up to years until the right conditions for their activation arise or the right food is made available. Mycorrhizae

Those fungi that are able to live symbiotically with living plants, creating a relationship that is beneficial to both, are known as Mycorrhiza. Plant root hairs are invaded by the mycelia of the mycorrhiza, which lives partly in the soil and partly in the root, and may either cover the length of the root hair as a sheath or be concentrated around its tip. The mycorrhiza obtains the carbohydrates that it requires from the root, in return providing the plant with nutrients including nitrogen and moisture. Later the plant roots will also absorb the mycelium into its own tissues. Algae

Algae are present in most of the soils where moisture and sunlight are available. Their number in soil usually ranges from 100 to 10,000 per gram of soil. They are photoautotrophic, aerobic organisms and obtain CO2 from atmosphere and energy from sunlight and synthesize their own food. They are unicellular, filamentous or colonial. Soil algae are divided in to four main classes or phyla as follows:

1. Cyanophyta (Blue-green algae) 2. Chlorophyta (Grass-green algae) 3. Xanthophyta (Yellow-green algae) 4. Bacillariophyta (diatoms or golden-brown algae)

Out of these four classes / phyla, blue-green algae and grass-green algae are more

abundant in soil. The green-grass algae and diatoms are dominant in the soils of temperate region while blue-green algae predominate in tropical soils. Green-algae prefer acid soils while blue green algae are commonly found in neutral and alkaline soils. The most common genera of green algae found in soil are: Chlorella, Chlamydomonas, Chlorococcum, Protosiphon etc. and that of diatoms are Navicula, Pinnularia. Synedra, Frangilaria.

Blue green algae are unicellular, photoautotrophic prokaryotes containing Phycocyanin pigment in addition to chlorophyll. They do not posses flagella and do not reproduce sexually. They are common in neutral to alkaline soils. The dominant genera of BGA in soil are: Chrococcus, Phormidium, Anabaena, Aphanocapra, Oscillatoria etc. Some BGA posses specialized cells known as "Heterocyst" which is the sites of nitrogen fixation. BGA fixes nitrogen (non-symbiotically) in puddle paddy/water logged paddy fields (20-30 kg/ha/season). There are certain BGA which possess the character of symbiotic nitrogen fixation in association with other organisms like fungi, mosses, liverworts and aquatic ferns Azolla, eg Anabaena-Azolla association fix nitrogen symbiotically in rice fields. Functions / role of algae or BGA:

1. Plays important role in the maintenance of soil fertility especially in tropical soils. 2. Add organic matter to soil when die and thus increase the amount of organic carbon in

soil. 3. Most of soil algae (especially BGA) act as cementing agent in binding soil particles and

thereby reduce/prevent soil erosion. 4. Mucilage secreted by the BGA is hygroscopic in nature and thus helps in increasing water

retention capacity of soil for longer time/period.



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5. Soil algae through the process of photosynthesis liberate large quantity of oxygen in the soil environment and thus facilitate the aeration in submerged soils or oxygenate the soil environment.

6. They help in checking the loss of nitrates through leaching and drainage especially in un-croppedsoils.

7. They help in weathering of rocks and building up of soil structure. Nematodes

Nematodes are microscopic, wormlike organisms that live in water films and water-filled pore spaces in the soil. Typically, they are most abundant in the upper soil layers where organic matter, plant roots, and other resources are most abundant.

Soil is an excellent habitat for nematodes, and 100 cc of soil may contain several thousand of them. Because of their importance to agriculture, much more is known about plant-parasitic nematodes than about the other kinds of nematodes which are present in soil. Most kinds of soil nematodes do not parasitize plants, but are beneficial in the decomposition of organic matter. These nematodes are often referred to as free-living nematodes. Juvenile or other stages of animal and insect parasites may also be found in soil. Although some plant parasites may live within plant roots, most nematodes inhabit the thin film of moisture around soil particles. The rhizosphere soil around small plant roots and root hairs is a particularly rich habitat for many kinds of nematodes. Nematode Feeding Habits

Nematodes can be classified into functional groups based on their feeding habits, which can often be deduced from the structure of their mouthparts. In agricultural soils, the most common groups of nematodes are the bacterial-feeders, fungal-feeders, plant parasites, predators, and omnivores. Predatory nematodes feed on protozoa and other soil nematodes. Omnivores feed on different foods depending on environmental conditions and food availability; for example, omnivorous nematodes can be predators, but in the absence of their primary food source, they can feed on fungi or bacteria.

Nematodes can be classified into different feeding groups based on the structure of their mouthparts. (a) bacterial feeder, (b) fungal feeder, (c) plant feeder, (d) predator, (e) omnivore.



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PHYSICAL PROPERTIES OF SOIL Horizonation Soil “horizons” are discrete layers that make up a soil profile. They are typically parallel with the ground surface. In some soils, they show evidence of the actions of the soil forming processes. O horizons are dominated by organic material. Some are saturated with water for long periods or were once saturated but are now artificially drained; others have never been saturated. A horizons are mineral layers that formed at the surface or below an O horizon, that exhibit obliteration of all or much of the original rock structure, and that show one or both of the following:

➢ An accumulation of humified organic matter intimately mixed with the mineral fraction and not dominated by properties characteristic of E or B horizons

➢ Modification as a result of the actions of cultivation, pasturing, or similar kinds of disturbance

E horizons are mineral layers that exhibit the loss of silicate clay, iron, aluminum, humus, or some combination of these, leaving a concentration of sand and silt particles. These horizons exhibit obliteration of all or much of the original rock structure. B horizons are mineral layers that typically form below an A, E, or O horizon and are dominated by obliteration of all or much of the original rock structure and show one or more of the following:

➢ illuvial concentration of silicate clay, iron, aluminum, humus, carbonate, gypsum, or silica, alone or in combination

➢ evidence of removal of carbonates ➢ residual concentration of sesquioxides ➢ coatings of sesquioxides that make the horizon conspicuously lower in value, higher in

chroma, or redder in hue than overlying horizons without apparent illuviation of iron ➢ alteration that forms silicate clay or liberates oxides or both and that forms granular,

blocky, or prismatic structure if volume changes accompany changes in moisture content; or brittleness



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C horizons are mineral layers which are not bedrock and are little affected by pedogenic processes and lack properties of O, A, E or B horizons. The material of C layers may be either like or unlike that from which the overlying soil horizons presumably formed. The C horizon may have been modified even if there is no evidence of pedogenesis. R horizons are layers of hard bedrock.

Transitional horizons are dominated by properties of one master horizon, but have subordinate properties of another. AB and B/C are examples of transitional horizon designations. Soil Color

In well aerated soils, oxidized or ferric (Fe+3) iron compounds are responsible for the brown, yellow, and red colors you see in the soil.

When iron is reduced to the ferrous (Fe+2) form, it becomes mobile, and can be removed from certain areas of the soil. When the iron is removed, a gray color remains, or the reduced iron color persists in shades of green or blue.

Upon aeration, reduced iron can be reoxidized and redeposited, sometimes in the same horizon, resulting in a variegated or mottled color pattern. These soil color patterns resulting from saturation, called “redoximorphic features”, can indicate the duration of the anaerobic state, ranging from brown with a few mottles, to complete gray or “gleization” of the soil.

Soils that are dominantly gray with brown or yellow mottles immediately below the surface horizon are usually hydric.

Soil color is typically described using some form of color reference chart, such as the Munsell Color Chart. Using the Munsell system, color is described in reference to the color’s “hue”, “value”, and “chroma”. Hue describes where in the color spectrum the soil color exists, which for soils includes the colors yellow, red, blue, green, and gray. Value describes the lightness of the color. Chroma indicates the strength of the color. In a Munsell notation, the color is written in the order hue-value-chroma. The color “5YR 4/3” is an example of a Munsell notation, where 5YR is the hue, 4 is the value, and 3 is the chroma. Soil Texture

Soil texture refers to the proportion of the soil “separates” that make up the mineral component of soil. These separates are called sand, silt, and clay. These soil separates have the following size ranges:

➢ Sand = <2 to 0.05 mm ➢ Silt = 0.05 to 0.002 mm ➢ Clay = <0.002 mm

Sand and silt are the “inactive” part of the soil matrix, because they do not contribute to a

soil’s ability to retain soil water or nutrients. These separates are commonly comprised of quartz or some other inactive mineral.

Because of its small size and sheet-like structure, clay has a large amount of surface area per unit mass, and its surface charge attracts ions and water. Because of this, clay is the “active” portion of the soil matrix.



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For all mineral soils, the proportion of sand, silt, and clay always adds up to 100 percent. These percentages are grouped into soil texture “classes”, which have been organized into a “textural triangle”.

Soil texture can affect the amount of pore space within a soil. Sand-sized soil particles fit together in a way that creates large pores; however, overall there is a relatively small amount of total pore space. Clay-sized soil particles fit together in a way that creates small pores; however, overall there are more pores present. Therefore, a soil made of clay-sized particles will have more total pore space than a will a soil made of sand-sized particles. Consequently, clayey soils will generally have lower bulk densities than sandy soils.

Collectively, the soil separates of sand, silt, and clay are called the “fine-earth fraction”, and represent inorganic soil particles less than 2mm in diameter. Inorganic soil particles 2mm and larger are called “rock fragments”.

When the organic matter content of a soil exceeds 20 to 35% (on a dry weight basis) it is considered organic soil material, and the soil is called an organic soil. As this material is mostly devoid of mineral soil material, they cannot be described in terms of soil texture. However, the following “in lieu of” texture terms can be used to describe organic soils:

➢ “peat”; organic material in which the plant parts are still recognizable ➢ “muck”; highly decomposed organic material in which no plant parts are recognizable ➢ “mucky peat”; decomposition is intermediate between muck and peat

Soil Structure

The soil separates can become aggregated together into discrete structural units called “peds”. These peds are organized into a repeating pattern that is referred to as soil structure. Between the peds are cracks called “pores” through which soil air and water are conducted. Soil structure is most commonly described in terms of the shape of the individual peds that occur within a soil horizon.

Types of Soil Structure

Graphic Example

Description of Structure Shape

Granular – roughly spherical, like grape nuts. Usually 1-10 mm in diameter. Most common in A horizons, where plant roots, microorganisms, and sticky products of organic matter decomposition bind soil grains into granular aggregates

Platy – flat peds that lie horizontally in the soil. Platy structure can be found in A, B and C horizons. It commonly occurs in an A horizon as the result of compaction.

Blocky – roughly cube-shaped, with more or less flat surfaces. If edges and corners remain sharp, we call it angular blocky. If they are rounded, we call it subangular blocky. Sizes commonly range from 5-50 mm across. Blocky structures are typical of B horizons, especially those with a high clay content. They form by repeated expansion and contraction of clay minerals.



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Prismatic – larger, vertically elongated blocks, often with five sides. Sizes are commonly 10-100mm across. Prismatic structures commonly occur in fragipans.

Columnar – the units are similar to prisms and are bounded by flat or slightly rounded vertical faces. The tops of columns, in contrast to those of prisms, are very distinct and normally rounded.

"Structureless" Soil Types

Graphic Example

Description of Structure Shape

Massive – compact, coherent soil not separated into peds of any kind. Massive structures in clayey soils usually have very small pores, slow permeability, and poor aeration.

Single grain – in some very sandy soils, every grain acts independently, and there is no binding agent to hold the grains together into peds. Permeability is rapid, but fertility and water holding capacity are low.

Soil Consistence

Soil consistence refers to the ease with which an individual ped can be crushed by the fingers. Soil consistence, and its description, depends on soil moisture content. Terms commonly used to describe consistence are: Moist soil:

➢ loose – non-coherent when dry or moist; does not hold together in a mass ➢ friable – when moist, crushed easily under gentle pressure between thumb and

forefinger and can be pressed together into a lump ➢ firm – when moist crushed under moderate pressure between thumb and forefinger, but

resistance is distinctly noticeable. Wet soil:

➢ plastic – when wet, readily deformed by moderate pressure but can be pressed into a lump; will form a “wire” when rolled between thumb and forefinger

➢ sticky – when wet, adheres to other material and tends to stretch somewhat and pull apart rather than to pull free from other material

Dry Soil:

➢ soft – when dry, breaks into powder or individual grains under very slight pressure ➢ hard – when dry, moderately resistant to pressure; can be broken with difficulty between

thumb and forefinger Bulk Density



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Bulk density is the proportion of the weight of a soil relative to its volume. It is expressed as a unit of weight per volume, and is commonly measured in units of grams per cubic centimeters (g/cc).

Bulk density is an indicator of the amount of pore space available within individual soil horizons, as it is inversely proportional to pore space: Pore space = 1 – bulk density/particle density CHEMICAL PROPERTIES OF SOIL Cation Exchange Capacity (CEC)

Some plant nutrients and metals exist as positively charged ions, or “cations”, in the soil environment. Among the more common cations found in soils are hydrogen (H+), aluminum (Al+3), calcium (Ca+2), magnesium (Mg+2), and potassium (K+). Most heavy metals also exist as cations in the soil environment. Clay and organic matter particles are predominantly negatively charged (anions), and have the ability to hold cations from being “leached” or washed away. The adsorbed cations are subject to replacement by other cations in a rapid, reversible process called “cation exchange”.

Cations leaving the exchange sites enter the soil solution, where they can be taken up by

plants, react with other soil constituents, or be carried away with drainage water.

The “cation exchange capacity”, or “CEC”, of a soil is a measurement of the magnitude of the negative charge per unit weight of soil, or the amount of cations a particular sample of soil can hold in an exchangeable form. The greater the clay and organic matter content, the greater the CEC should be, although different types of clay minerals and organic matter can vary in CEC.

Cation exchange is an important mechanism in soils for retaining and supplying plant nutrients, and for adsorbing contaminants. It plays an important role in wastewater treatment in soils. Sandy soils with a low CEC are generally unsuited for septic systems since they have little adsorptive ability and there is potential for groundwater. Soil Reaction (pH

By definition, “pH” is a measure of the active hydrogen ion (H+) concentration. It is an indication of the acidity or alkalinity of a soil, and also known as “soil reaction”.

The pH scale ranges from 0 to 14, with values below 7.0 acidic, and values above 7.0 alkaline. A pH value of 7 is considered neutral, where H+ and OH- are equal, both at a concentration of 10-7 moles/liter. A pH of 4.0 is ten times more acidic than a pH of 5.0.

The most important effect of pH in the soil is on ion solubility, which in turn affects microbial and plant growth. A pH range of 6.0 to 6.8 is ideal for most crops because it coincides with optimum solubility of the most important plant nutrients. Some minor elements (e.g., iron) and most heavy metals are more soluble at lower pH. This makes pH management important in controlling movement of heavy metals (and potential groundwater contamination) in soil.

In acid soils, hydrogen and aluminum are the dominant exchangeable cations. The latter



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is soluble under acid conditions, and its reactivity with water (hydrolysis) produces hydrogen ions. Calcium and magnesium are basic cations; as their amounts increase, the relative amount of acidic cations will decrease.

Factors that affect soil pH include parent material, vegetation, and climate. Some rocks and sediments produce soils that are more acidic than others: quartz-rich sandstone is acidic; limestone is alkaline. Some types of vegetation, particularly conifers, produce organic acids, which can contribute to lower soil pH values. In humid areas such as the eastern US, soils tend to become more acidic over time because rainfall washes away basic cations and replaces them with hydrogen. Addition of certain fertilizers to soil can also produce hydrogen ions. Liming the soil adds calcium, which replaces exchangeable and solution H+ and raises soil pH. Lime requirement, or the amount of liming material needed to raise the soil pH to a certain level, increases with CEC. To decrease the soil pH, sulfur can be added, which produces sulfuric acid. BIOLEACHING

Leaching process was first observed in pumps and pipelines installed in mine pits containing acid water. This process was later on employed for recovering metals from ores containing low quantity of the metal. Presently certain metals from sulfide ores and other ores are extracted by employing only leaching method. Extraction of metals from low-grade ores by employing microorganism is called as bioleaching. Large quantities of low-grade ores are produced during the separation of higher-grade ores and are generally discarded in waste heaps. Metals from such ores cannot economically be processed with chemical methods. There are large quantities of such low-grade ores especially copper ores, which can be processed profitably by bio-leaching.

Copper and Uranium are presently produced commercially by employing bioleaching process. However, problems may creep in when the large scale bioleaching process of a waste dump is improperly managed because seepage of leach fluids containing low pH and metals into natural water supplies and ground water causing metal pollution. Mechanism of Bioleaching: The process of bioleaching is accomplished by two ways:

i. Direct bioleaching ii. Indirect bioleaching

i.Direct Bioleaching:

Thiobacillusferrooxidans is oftenly used in microbial leaching. It is an autotrophic, aerobic, gram (-) negative rod shaped bacterium. It synthesizes its carbon substances by CO2 fixation. It derives the required energy for CO2 fixation either from the oxidation of Fe2+ to Fe3+or from the oxidation of elemental sulphur or reduced sulphur compounds to sulfates.

Thiobacillusthiooxidans oxidizes insoluble sulphur to sulphuric acid, which takes place in the periplasmic space. It is possible to dissolve iron through direct bacterial leaching as shown in the above reactions. ii.Indirect Bioleaching

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This leaching process takes place without direct involvement of microorganisms but they indirectly support the leaching by producing agents responsible for oxidation of minerals. It can be explained by the process of oxidation of pyrite. Pyrite is a common rock mineral that is found in association with many ores. The pyrite is initially oxidized to elemental sulphur equation 4, which is subjected to further oxidation by Thiobacillusferrooxidans due to which sulphuric acid is formed which is shown in equation 2.

ThiobacillusthiooxidansandThiobacillusferrooxidans are generally seen associated with leaching dumps. In pilot plant reactors of 50 liter capacity, leaching can be performed continuously in a cascade series with recycling of cells and leachates.

In the laboratory better yields of bioleaching products can be obtained under optimal

conditions, like control of temperature, O2 and CO2 adjustments, maintenance of pH between 2 and 3, and eh around – 300mv with very finely ground ores in a tower (percolator). However, conditions and yield cannot be achieved in a commercial scale because it is expensive.

Thiobacillusthiooxidans and Thiobacillusferrooxidans are generally used in bioleaching methods. However, a number of other microorganisms such as Thiobacillusconcretivorus, Pseudomonas florescens, P.putida, Achromobacter, Bacillus licheniformis, B. cereus, B luteus, B. polymyxa, B.megaterium and several thermophilic bacteria like Thiobacillusthermophilica, Thermothrixthioparus, ThiobacillusTH1 and Sulfolobusacidocaldarius. Because of more rapid growth rate the thermophilic bacteria may significantly accelerate the bioleaching process. Processes of Bioleaching: Commercial Processes: Methods described below are generally employed in large scale bioleaching processes: Slope Leaching:

In this method finely powdered ore, approximately 10,000 tons are made into large piles along the slopes of a mountain, and water containing Thiobacillus is continuously sprinkled. Metals are extracted from the water that collects at the bottom of the mountain. The water is recycled again after metal extraction and regeneration of the bacteria in an oxidation pool (Fig. 12.1a).

Heap Leaching:

The ore is arranged in a big heap, which is treated with water as in slope leaching. The recovery of metals and other processes are conducted just like in slope leaching (Fig. 12.1b). In-Situ Leaching:

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This process is carried with an ore which remains in its original location in the earth. The permeability of ore is increased by sub-surface blasting. Several passages, as shown in Fig. 12.1c are drilled through the ore. A well like pit is also made out at the bottom of the ore. Now acidic water containing Thiobacillusis pumped through drilled passages of the ore. The acidic water percolates through the ore and collects in the pit at the bottom of the ore. The water is pumped out from the pit and the minerals are extracted. The water after extraction of minerals is reused after regeneration of bacteria. Copper Leaching:

Covellite, chalcocite and chalcopyrite are generally used as copper ores for bioleaching processes. Apart from containing copper, the ores also contain other elements like iron, zinc and sulphur. For example – Chalcopyrite contains 26% copper, 25.9% iron, 20.5% zinc and 33% sulphur. Mechanism of Copper Leaching: During the oxidation of Chalcopyrite the following reaction occurs: 2CuFeS2 + 8 ½ O2 + H2SO4 → 2 CuSO4 + Fe2 (SO4)3 + H2O Similarly covellite is oxidized to copper sulphate CuS + 2O2 → CuSO4

Generally heap leaching process is employed in copper leaching process but sometimes a combination of heap leaching and in situ leaching processes are used. The solution (Sulphate/Fe3 solution) is sprinkled over the heap which percolates through the ore and collects at the bottom pit. The solution collecting in the bottom pit will include copper metal, which is removed by precipitation. The remaining water with Fe3+ is used again in the leaching process after adjusting the pH to 2.0 with the help of H2SO4.

Bioleaching of copper has been used in the United States, Australia, Canada, Mexico, South Africa, Portugal, Spain and Japan. About 5% of the world copper production is obtained through bioleaching.

Uranium Leaching:

In the uranium leaching process, insoluble tetravalent uranium is oxidized with a hot H2SO4/Fe3+ solution to soluble hexavalent uranium sulfate.

UO2 + Fe2 (SO4)3 → UO2SO4 + 2FeSO




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Uranium bioleaching process is more significant economically. In situ microbial leaching is greater acceptance since it eliminates the expense of moving vast amounts of material. For instance thousand tons of uranium ore must be handled in other than bioleaching processes, to obtain one ton of uranium. This is an indirect leaching process since the microbial attack is not on uranium ore directly but on the Iron oxidant. Ferric sulphate and sulfuric acid can be produced by Thiobacillusferrooxidans from the pyrite within the uranium ore.

2FeS2 + H2O + 7.5 O2 → Fe2 (SO4)3 + H2SO4 For the initial production of the Fe3+ leach solution the pyrite reaction is used. For

carrying this reaction pilot plants with surface reactors are used which are similar to trickling filters used in sewage operations. For getting optimum uranium leaching the incoming air should passes a pH of 1.5-3.5, temperature of 35°C and CO2 0.2%. However, certain thermophilic strains require a temperature optimum of 45-50°C. The dissolved uranium is extracted from the leach liquor, in commercial processes with organic solvents like tributyl phosphate and the uranium is subsequently precipitated from the organic phase. The organic solvents which remain in the water system after extraction of uranium may be toxic and hence cause problems when the microbiological system is reused.

Microbial leaching of refractory precious metal ores to enhance recovery of gold and

silver is one of the most promising applications. Gold is obtained through bioleaching of arsenopyrite/pyrite ore and its cyanidation process. Silver is more readily solubilized than gold during microbial leaching of iron sulphide. Similarly silica is leached from ores like magnesite, bauxite, dolomite and basalt by Bacillus licheniformis. The silica is accumulated by B.licheniformis by process of adsorption which is readily separated. Ore leaching by microbes has potential for use in the extraction of other metals such as zinc, cobalt and nickel. New reactor systems are likely to be developed to increase the efficiency of bioleaching in terms of cost and kinetics. These innovations are expected to extend the scope of bleaching applications.



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UNIT - IV MICROBIAL ASSOCIATIONS

Interactions among soil Microorganisms

The microbial ecosystem is the sum of biotic and abiotic components of soil. it includes the total microbial flora together with the physical composition and physical characteristics of the soil. The microorganisms that inhabit the soil exhibit many different types of interactions or associations. Some interactions are indifferent or neutral; while some are positive and some are negative in nature. The associations existing between different soil microorganisms, whether of a symbiotic or antagonistic nature, influence the activity of micro-organisms in the soil. Neutral association

Neutral association or neutralism is the association between microorganisms, where two different species of microorganisms occupy the same environment without affecting each other. Such an association might be transitory; as condition change in the environment, like nutrients availability, there might be a change in the relationship. Positive associations There are three types of positive association exist between microorganisms, which are below: Mutualism

It is the symbiotic relationship in which each organism is benefited from the association. This is an obligatory relationship in which each organism is metabolically dependent on each other. Lichens are the association between specific ascomycetes (the fungus) and certain genera of either green algae or cyanobacteria. In lichen, the fungal partner is termed the mycobiont and the algal or cyanobacterial partner, the phycobiont. Because the phycobiont is a photoautotroph- dependent only on light, carbon dioxide, and certain mineral nutrients- the fungus can get its organic carbon directly from alga or cyanobacterium. The fungus often obtains nutrients from its partner by haustoria (projections of fungal hyphae) that penetrate the phycobiont cell wall. It also uses the O2produced during phycobiont photophosphorylation in carrying out respiration. In turn, the fungus protects the phycobiont from excess light intensities, provides water and minerals to it, and creates a firm substratum within which the phycobiont can grow protected from environmental stress. Phycobiont- Cyanobacteria ( Chlorophyta, xanthophyta) Mycobiont- Ascomycota, Basidiomycota

The Rhizobiumin the root nodules, where they differentiate morphologically into bacteroids, fix nitrogen from the atmospheric N2, into a plant-usable form, ammonium, using the enzyme nitrogenase. In return, the plant supplies the bacteria with carbohydrates, organic acids (principally as the dicarboxylic acids and succinate) as a carbon and energy source, proteins, and sufficient oxygen. Proto-cooperation/ Synergism

Protocooperation is a mutually beneficial relationship, similar to that which occurs in mutualism, but in protocooperation, this relationship is not obligatory. An example of protocooperation happens between soil bacteria or fungi, and the plants that occur growing in the soil. None of the species rely on the relationship for survival, but all of the bacteria, fungi, and higher plants take part in shaping soil composition and fertility. Soil bacteria and fungi interrelate with each other, forming nutrients essential to the plants survival. Plants utilize these



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microorganism synthesized nutrients through root nodules thereby decomposing organic substances. Soil bacteria and fungi help in improving the fertility of the soil and shaping of the soil. Plants get essential carbon dioxide and nutrients. Nutritional proto-cooperation between bacteria and fungi has been reported for various vitamins, amino and purines in the terrestrial ecosystem are very useful in agriculture. Commensalism

Commensalism is a relationship in which one symbiont,the commensal, benefits while other (sometimes called the host) is neither harmed nor helped.This happens commonly in soil with respect to degradation of complex molecules like cellulose and lignin. For example, many fungi can degrade cellulose to glucose, which is utilized by many bacteria. Many bacteria are unable to utilize cellulose, but they can utilize the fungal breakdown products of cellulose, e.g., glucose and organic acids.

Another example of commensalism is that of a change in the substrate produced by the combination of species and not by individual species. For example, lignin which is the major constituents of woody plants and is usually resistant to degradation by most of the microorganisms. But in forest soils, lignin is readily is degraded by a group of Basidiomycetous fungi and the degraded products are used by several other fungi and bacteria which cannot utilize lignin directly. Negative associations Antagonism

It is the relationship in which one species is inhibited or adversely affected by another species in the same environment. The relationship is known as antagonism. The species which adversely affects other is said to be antagonistic. Such organisms may be of great practical importance since they often produce antibiotics or the other inhibitory substances which affect the normal growth processes or survival of other organisms.

Antagonistic relations are most common in nature. One example of which is the antagonistic nature of both Staphylococcus aureusandPseudomonas aeruoginosatowards the fungus Aspergillusterreus. Competition

Soil is inhabited by different kinds of microorganisms, and therefore they exhibit competition among themselves for nutrients and space. In this kind of situation, the best adapted microorganism will predominate or eliminate the others which are dependent upon the same limited nutrient substance. The organisms with inherent ability to grow fast are better competitors.

Exogeneous nutrients are required for the germination of chlamydospores of Fusarium, Oospores of Aphanomycesand conidia of Verticilliumdahlaein soil. But other fungi and soil bacteria deplete these critical nutrients required for spore germination and thereby hinder the spore germination resulting into the decrease in population. Soil bacteria compete for space and suppress the growth of the fungal population. Parasitism

Parasitism is the relationship between two organisms, in which one organism lives in or on another organism. The parasite is dependent upon the host and feeds on the cells, tissues or



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fluids of the host organism. The parasite lives in intimate physical contact with the host and forms metabolic association with the host. All major groups of plants, animals, and microorganisms are susceptible to attack by microbial parasites.

The bacterial parasite of Gram-negative bacteria Bdellovibriobacteriovoruswhich is widespread in soil and sewage attaches to a host cell at a special region and eventually causes the lysis of that cell. As a consequence, plaque like areas of lysis appears when these parasites are plated along with their host bacteria. Parasitism is widely spread in soil communities. Viruses which attack bacteria (bacteriophages), fungi, and algae are strict intracellular parasites since they cannot be cultivated as free-living forms. There are also many strains of fungi which are parasitic on algae and other fungi by penetration into the host. Fungi with antagonistic activity toward plant pathogens have an essential role in plant growth and health. Mycoparasites and presumptive mycoparasites have biocontrol potential; some are responsible for natural suppressiveness of soils to certain plant pathogens. Several species of Trichodermawere used successfully against certain pathogenic fungi. Trichodermasp. was used as commercial bio-fungicides to control a range of economically important soil-borne fungal plant pathogens. Soils contain a large number and great diversity of oospore parasites, which may have the potential to reduce populations of plant pathogenic Phycomycetes in soil. Predation

Predation is an association in which predator organism directly feed on and kills the pray organism. Predators may or may not kill their prey prior to feeding on them, but the act of predation often results in the death of its prey and the eventual absorption of the prey's tissue through consumption.

Many species of the soil-dwelling myxobacteria are predators of other microbes. Many myxobacteria, e.g., Myxococcusxanthus, exhibit several complex social traits, including fruiting body formation and spore formation cooperative swarming with two motility systems, and group predation on both bacteria and fungi. Myxobacteria use gliding motility to search the soil matrix for prey and produce a wide range of antibiotics and lytic compounds that kill and decompose prey cells and break down complex polymers, thereby releasing substrates for growth. The nematophagous fungi are the best predatory soil fungi. Species of Arthrobotrytisand Dactylellaare known as nematode-trapping fungi. RUMEN MICROBIOLOGY

Bacteria, protozoa, and fungi exist together in the cow’s rumen. Bacteria make up about half of the living organisms but do more than half of the rumen’s digestive work. Rumen bacteria are classified into fiber digesters, starch and sugar digesters, lactate using bacteria, and hydrogen-using bacteria. They cooperate togetherand cross feed. Bacteria

Bacteria make up about half of the living organisms inside of the rumen. However, they do more than half of the work in the rumen. The bacteria work together, some breakdown certain carbohydrates and proteins which are then used by others. Some require certain growth factors, such as B-vitamins, which are made by others. Some bacteria help to clean up the rumen of others’ end products, such as hydrogen ions, which could otherwise accumulate and become toxic to other organisms. This is called “cross-feeding”. Classification of Rumen Bacteria



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Fiber – digesting or cellulolytic bacteria The fiber-digesters are some of the “fussiest” bacteria in the rumen. They are very

sensitive to acid. When a cow has acidosis (pH<6.0), the rumen produces a lower proportion of acetate to propionate because the fiber-digesters who primarily make acetate are not working well. Also, high levels of rumen available fat (generally over 5% of the diet) reduce the growth of the fiber-digesters. The exact reason for fat’s negative effect on the fiber-digesters is not known. Some think that it reduces the microbe’s ability to move nutrients into and out of its body. Others think that the fat coats fiber particles making it difficult for the fiber-digesting microbes to get in to do their work. Bacterial species:

Ruminococcusflavefacians, Ruminococcusalbus, Bacteriodessuccinogenes, Butyrivibriofibrisolvens Growth Requirements: Cellulose, Hemicellulose, Pectin Many Also Require: Ammonia, Isoacids (Branched-Chain Amino Acids), Starch, B-vitamins - Fermentation Products: Acetate, Butyrate, Hydrogen (H2), Carbon Dioxide (CO2) Rumen pH Requirement: High pH (above 6.0) Fat Tolerance: Low Susceptibility to Ionophores (Bovatec and Rumensin): Some are susceptible Reproduction Speed: Slow Starch and Sugar-Digesting (or Amylolytic) Bacteria:

Starch and sugar-digesters make up a significant part of the rumen’s bacterial population. Generally, high-producing dairy cows are fed diets containing more than 30% starches and sugars, so these bacteria are greatly needed. Even if a cow is on an all-straw diet, the fiber-digesters still never account for more than 25% of the rumen bacterial population. Starch and sugar-digesters are still present, cross-feeding off of the fiber-digesters’ byproducts. Bacterial species:

Bacteriodesruminocola, Bacteriodesamylophilus, Selenomonasruminantium, Streptococcus bovis, Succinomonasamylolytica Growth Requirements: Sugar, Starch, Peptides, Amino Acids. Many Also Require: Ammonia, B-vitamins Fermentation Products:

Propionate, Butyrate, Acetate, Lactate, Hydrogen (H2), Carbon Dioxide (CO2) Rumen pH Requirement: Tolerate a lower (more acidic) pH (5.7) Fat Tolerance: Higher than fiber digesters Susceptibility to Ionophors (Bovatec and Rumensin): Most aren’t susceptible Reproduction Speed: Faster than fiber digesters

Streptococcus bovis is present only when large amounts of starch or sugars are fed and pH is low. It produces lactic acid; a stronger acid than many of the other VFA’s produced in the rumen. When conditions are favorable for Streptococcus bovis, it will grow explosively (doubling every 13 minutes). This type of growth produces rumen acidosis. Streptococcus bovis is controlled by ionophores. This is one of the major reasons for the favorable growth responses seen by the addition of ionophores to the diets of feedlot cattle. Some bacteria, such as Streptococcus bovis, produce a strong acid called lactic acid. Megasphaeraelsdenii uses lactic



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acid to grow. This helps to clean up the rumen a bit and raise rumen pH, aiding the growth of the acid-intolerant fiber-digesters. Hydrogen-Using (or Methane) Bacteria: Under normal rumen conditions, hydrogen (H2) does not accumulate in the rumen because it’s used by hydrogen-using bacteria, such as Methanobacteriumruminantium. Growth Requirements: Carbon dioxide and hydrogen Fermentation products: Methane The methane bacteria commonly produce methane in this way: 4H2 + CO2 ---------> CH4 + 2H2O Protozoa

As much as 50% of the microbial mass in the rumen can be made up of protozoa. However, their role, as compared to the rumen bacteria, is not as significant. The protozoa are actually predators to the bacteria in the rumen --- they eat the bacteria for dinner! Protozoa are about 40 times the size of rumen bacteria. The rumen protozoa produce fermentation end-products similar those made by the bacteria, particularly acetate, butyrate, and hydrogen. Rumen methane bacteria actually attach and live on the surface of rumen protozoa for immediate access to hydrogen. Rumen protozoa eat large amounts of starch at one time and can store it in their bodies. This may help to slow down the production of acids that lower rumen pH, benefiting the rumen. Rumen protozoa multiply very slowly in the rumen --- over 15-24 hours – as opposed to the bacteria that may take as little as 13 minutes to multiply. For this reason, the rumen protozoa hide out in the slower moving fiber mat of the rumen so that they aren’t washed out before they have a chance to multiply. Low roughage diets reduce the retention of fiber in the rumen and may decrease the number of protozoa in a cow’s rumen. Rumen Fungi

Fungi are known to exist in the rumen (up to 8% of the total mass) but they are poorly understood. They attach to feed particles and they reproduce very slowly. They may help out the fiber-digesting bacteria by doing some of the initial work of splitting fibrous material apart and making it more accessible for the bacteria. Higher numbers of fungi have been found in the rumens of cows fed very poorly digestible sub-tropical forages



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UNIT – V NITROGEN CYCLE

Nitrogen Cycle The nitrogen cycle is the biogeochemical cycle by which nitrogen is converted into

various chemical forms as it circulates amongthe atmosphere and terrestrial and marine ecosystems. The conversion of nitrogen can be carried out through both biological and physical processes. Important processes in the nitrogen cycle include fixation, ammonification, nitrification, and denitrification. The majority of Earth's atmosphere (78%) is nitrogen, making it the largest source of nitrogen. However, atmospheric nitrogen has limited availability for biological use, leading to a scarcity of usable nitrogen in many types of ecosystems. The nitrogen cycle is of particular interest to ecologists because nitrogen availability can affect the rate of key ecosystem processes, including primary production and decomposition. Human activities such as fossil fuel combustion, use of artificial nitrogen fertilizers, and release of nitrogen in wastewater have dramatically altered the global nitrogen cycle.

Nitrogen is present in the environment in a wide variety of chemical forms including organic nitrogen, Ammonium (NH+4), nitrite (NO−2), nitrate (NO−3), nitrous oxide (N2O), Nitric oxide (NO) or inorganic nitrogen gas (N2). Organic nitrogen may be in the form of a living organism, humus or in the intermediate products of organic matter decomposition. The processes of the nitrogen cycle transform nitrogen from one form to another. Many of those processes are carried out by microbes, either in their effort to harvest energy or to accumulate nitrogen in a form needed for their growth. For example, the nitrogenous wastes in animal urine are broken down by nitrifying bacteria in the soil to be used as new. The diagram besides shows how these processes fit together to form the nitrogen cycle.



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Nitrogen fixation

Atmospheric nitrogen must be processed, or "fixed", in a usable form to be taken up by plants. Between 5x1012 and 10x1012 g per year are fixed by lightning strikes, but most fixation is done by free-living or symbiotic bacteria known as diazotrophs. These bacteria have the nitrogenase enzyme that combines gaseous nitrogen with hydrogen to produce ammonia, which is converted by the bacteria into other organic compounds. Most biological nitrogen fixation occurs by the activity of Mo-nitrogenase, found in a wide variety of bacteria and some Archaea. Mo-nitrogenase is a complex two-component enzyme that has multiple metal-containing prosthetic groups. An example of the free-living bacteria is Azotobacter. Symbiotic nitrogen-fixing bacteria such as Rhizobium usually live in the root nodules of legumes (such as peas, alfalfa, and locust trees). Here they form a mutualistic relationship with the plant, producing ammonia in exchange for carbohydrates. Because of this relationship, legumes will often increase the nitrogen content of nitrogen-poor soils. A few non-legumes can also form such symbioses. Today, about 30% of the total fixed nitrogen is produced industrially using the Haber-Bosch process, which uses high temperatures and pressures to convert nitrogen gas and a hydrogen source (natural gas or petroleum) into ammonia. Assimilation

Plants take nitrogen from the soil by absorption through their roots as amino acids, nitrate ions, nitrite ions, or ammonium ions. Most nitrogen obtained by terrestrial animals can be traced back to the eating of plants at some stage of the food chain. Plants can absorb nitrate or ammonium from the soil via their root hairs. If nitrate is absorbed, it is first reduced to nitrite ions and then ammonium ions for incorporation into amino acids, nucleic acids, and chlorophyll. In plants that have a symbiotic relationship with rhizobia, some nitrogen is assimilated in the form of ammonium ions directly from the nodules. It is now known that there is a more complex cycling of amino acids between Rhizobia bacteroids and plants. The plant provides amino acids to the bacteroids so ammonia assimilation is not required and the bacteroids pass amino acids (with the newly fixed nitrogen) back to the plant, thus forming an interdependent relationship. While many animals, fungi, and other heterotrophic organisms obtain nitrogen by ingestion of amino acids, nucleotides, and other small organic molecules, other heterotrophs (including many bacteria) are able to utilize inorganic compounds, such as ammonium as sole N sources. Utilization of various N sources is carefully regulated in all organisms. Ammonification

When a plant or animal dies or an animal expels waste, the initial form of nitrogen is organic. Bacteria or fungi convert the organic nitrogen within the remains back into ammonium (NH+4), a process called ammonification or mineralization. Enzymes involved are:

➢ GS: GlnSynthetase (Cytosolic & Plastic) ➢ GOGAT: Glu 2-oxoglutarate aminotransferase (Ferredoxin & NADH-dependent) ➢ GDH: Glu Dehydrogenase: ➢ Minor Role in ammonium assimilation. ➢ Important in amino acid catabolism.

Nitrification



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The conversion of ammonium to nitrate is performed primarily by soil-living bacteria and other nitrifying bacteria. In the primary stage of nitrification, the oxidation of ammonium (NH+4) is performed by bacteria such as the Nitrosomonas species, which converts ammonia to nitrites (NO−2). Other bacterial species such as Nitrobacter are responsible for the oxidation of the nitrites (NO−2) into nitrates (NO−3). It is important for the ammonia (NH3) to be converted to nitrates or nitrites because ammonia gas is toxic to plants.

Due to their very high solubility and because soils are highly unable to retain anions, nitrates can enter groundwater. Elevated nitrate in groundwater is a concern for drinking water use because nitrate can interfere with blood-oxygen levels in infants and cause methemoglobinemia or blue-baby syndrome. Where groundwater recharges stream flow, nitrate-enriched groundwater can contribute to Eutrophication, a process that leads to high algal population and growth, especially blue-green algal populations. While not directly toxic to fish life, like ammonia, nitrate can have indirect effects on fish if it contributes to this eutrophication. Nitrogen has contributed to severe eutrophication problems in some water bodies. Since 2006, the application of nitrogen fertilizer has been increasingly controlled in Britain and the United States. This is occurring along the same lines as control of phosphorus fertilizer, restriction of which is normally considered essential to the recovery of eutrophiedwaterbodies. Denitrification

Denitrification is the reduction of nitrates back into nitrogen gas (N2), completing the nitrogen cycle. This process is performed by bacterial species such as Pseudomonas and Clostridium in anaerobic conditions. They use the nitrate as an electron acceptor in the place of oxygen during respiration. These facultatively anaerobic bacteria can also live in aerobic conditions. Denitrification happens in anaerobic conditions e.g. waterlogged soils. The denitrifying bacteria use nitrates in the soil to carry out respiration and consequently produce nitrogen gas, which is inert and unavailable to plants. Dissimilatory nitrate reduction to ammonium

Dissimilatory nitrate reduction to ammonium (DNRA), or nitrate/nitrite ammonification, is an anaerobic respiration process. Microbes which undertake DNRA oxidise organic matter and use nitrate as an electron acceptor, reducing it to nitrite, then ammonium (NO3

−→NO2−→NH4

+). Both denitrifying and nitrate ammonification bacteria will be competing for nitrate in the environment, although DNRA acts to conserve bioavailable nitrogen as soluble ammonium rather than producing dinitrogen gas. Anaerobic ammonia oxidation

In this biological process, nitrite and ammonia are converted directly into molecular nitrogen (N2) gas. This process makes up a major proportion of nitrogen conversion in the oceans. Other processes

Though nitrogen fixation is the primary source of plant-available nitrogen in most ecosystems, in areas with nitrogen-rich bedrock, the breakdown of this rock also serves as a nitrogen source. CARBON CYCLE

The carbon cycle is one of the major biogeochemical cycles describing the flow of essential elements from the environment to living organisms and back to the environment again.



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This process is required for the building of all organic compounds and involves the participation of many of the earth's key forces. The carbon cycle has affected the earth throughout its history; it has contributed to major climatic changes, and it has helped facilitate the evolution of life.

The carbon cycle is one of the earth's fastest recycling processes--each atom of carbon has been recycled numerous times. For this reason, the carbon cycle has no specific beginning or ending point on the diagram. The carbon cycle passes through three main stages: reservoirs, assimilation, and release.

Much of the earth's carbon is contained in the atmosphere which serves as a reservoir, and that is where we will begin our explanation. Atmospheric carbon consists mostly of carbon dioxide and has two major sinks: terrestrial ecosystems and marine ecosystems, both of which deal with photosynthesis as a part of assimilation and respiration as a part of release.

Terrestrial ecosystems draw carbon dioxide from the atmosphere and use it in

photosynthesis. The equation, C02 + H20 + light => C6H12O6 + O2 + energy, shows how carbon dioxide is broken down and used to produce glucose for the plants and oxygen as a byproduct. All plants act as a sink for carbon dioxide because it is a necessary gas for photosynthesis. Of the terrestrial ecosystems, forests have the highest rates of productivity, thus utilizing carbon at a higher rate compared to oceans.

Marine ecosystems are separated into two areas: coastal ecosystems and the open

ocean. Coastal ecosystems include estuaries, wetlands, and continental shelves. Open oceans are considered all areas beyond the shelves. Both have the capacity to store significant amounts of carbon in sediments and also are able to sequester carbon in photosynthesis or chemosynthesis through phytoplankton, seaweeds, and other marine algae. Most storage of carbon is in marine sediments and rocks, although some carbon is used by marine life in the formation of calcium carbonate.

Another carbon sink is the weathering of mountains and other rock formations formed by plate tectonics, mainly silicate weathering. Carbon dioxide is consumed from silicate weathering as seen in this equation:

CaSiO3 + 2CO2 + 2H2O => CaCO3 + SiO2 + CO2 + 2H2O

Here one mol of CO2 is sequestered for each mol of Ca from the silicate dissolution. The equation begins with two mols of CO2, then the calcium silicate is broken up and calcium carbonate is formed. This uses up one mol of carbon dioxide leaving only one mol at the end of the reaction. A major source of atmospheric CO2 is degassing from volcanic activity which acts as a release of carbon dioxide. Conversely, the process of subduction of crust provides a sink for CO2. Another important source of carbon in the atmosphere is in the decomposition of organicmaterial. Carbon dioxide is captured by plants throughout their lives and heterotrophic organisms in turn obtain a part of this carbon. The element is transferred from organism to organism when plants are eaten by herbivores which are in turn eaten by carnivores along the food chain. All these organisms go through respiration, excrete organic waste, and eventually die and decompose which releases carbon into the soil either as carbonates or fossil fuels. The carbon dioxide drawn into marine ecosystems is eventually released through oceanic respiration. Through time and pressure, the organic material buried in soil and sediments may



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eventually become fossil fuels such as coal and oil, which then become additional sources of carbon. When these are burned, they emit tremendous amounts of carbon back into the atmosphere. The burning of fossil fuels, however, is occurring at much higher rates than is their production.

Thus the atmospheric carbon that is sequestered through weathering and the terrestrial and marine ecosystem sinks is eventually recycled through the processes of decomposition, respiration, and tectonic forces, releasing them once more into the atmosphere.

The carbon cycle also has major effects on global climate:

The burning of fossil fuels at the presently alarming rate increases global warming. This

is due to the increasing amount of greenhouse gases, specifically CO2, which capture heat easily thus increasing global temperature. Although alarming now, this had a positive effect in periods such as those between the Permian and Triassic where there was heavy glaciation periods followed by a large release of carbon.

At present, there is a fear of the greenhouse effect increasing global warming to an

almost dangerous high. The excess carbon dioxide from the burning of fossil fuels is responsible for the gradual yet constant rise in temperature over the past few decades. Certain wave lengths coming from the sun, mainly infrared radiation, are trapped by greenhouse gases and kept in the earth's atmosphere. These gases block the re-radiation of infrared waves back out into space, causing the global temperature to increase.



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PHOSPHOROUS CYCLE

Phosphorus is an important element for all forms of life. As phosphate (PO4), it makes up an important part of the structural framework that holds DNA and RNA together. Phosphates are also a critical component of ATP—the cellular energy carrier—as they serve as an energy release’ for organisms to use in building proteins or contacting muscles. Like calcium, phosphorus is important to vertebrates; in the human body, 80% of phosphorous is found in teeth and bones.

The phosphorus cycle differs from the other major biogeochemical cycles in that it does not include a gas phase; although small amounts of phosphoric acid (H3PO4) may make their way into the atmosphere, contributing—in some cases—to acid rain. The water, carbon, nitrogen and sulfur cycles all include at least one phase in which the element is in its gaseous state. Very little phosphorus circulates in the atmosphere because at Earth’s normal



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temperatures and pressures, phosphorus and its various compounds are not gases. The largest reservoir of phosphorus is in sedimentary rock.

It is in these rocks where the phosphorus cycle begins. When it rains, phosphates are

removed from the rocks (via weathering) and are distributed throughout both soils and water. Plants take up the phosphate ions from the soil. The phosphates then moves from plants to animals when herbivores eat plants and carnivores eat plants or herbivores. The phosphates absorbed by animal tissue through consumption eventually returns to the soil through the excretion of urine and feces, as well as from the final decomposition of plants and animals after death.

The same process occurs within the aquatic ecosystem. Phosphorus is not highly soluble, binding tightly to molecules in soil; therefore it mostly reaches waters by traveling with runoff soil particles. Phosphates also enter waterways through fertilizer runoff, sewage seepage, natural mineral deposits, and wastes from other industrial processes. These phosphates tend to settle on ocean floors and lake bottoms. As sediments are stirred up, phosphates may reenter the phosphorus cycle, but they are more commonly made available to aquatic organisms by being exposed through erosion. Water plants take up the waterborne phosphate which then travels up through successive stages of the aquatic food chain.

While obviously beneficial for many biological processes, in surface waters an excessive concentration of phosphorus is considered a pollutant. Phosphate stimulates the growth of plankton and plants, favoring weedy species over others. Excess growth of these plants tend to consume large amounts of dissolved oxygen, potentially suffocating fish and other marine animals, while also blocking available sunlight to bottom dwelling species. This is known as eutrophication.

Humans can alter the phosphorus cycle in many ways, including in the cutting of tropical rain forests and through the use of agricultural fertilizers. Rainforest ecosystems are supported primarily through the recycling of nutrients, with little or no nutrient reserves in their soils. As the forest is cut and/or burned, nutrients originally stored in plants and rocks are quickly washed away by heavy rains, causing the land to become unproductive. Agricultural runoff provides



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much of the phosphate found in waterways. Crops often cannot absorb all of the fertilizer in the soils, causing excess fertilizer runoff and increasing phosphate levels in rivers and other bodies of water. At one time the use of laundry detergents contributed to significant concentrations of phosphates in rivers, lakes, and streams, but most detergents no longer include phosphorus as an ingredient. SULPHUR CYCLE

Sulfur (S), the tenth most abundant element in the universe, is a brittle, yellow, tasteless, and odorless non-metallic element. It comprises many vitamins, proteins, and hormones that play critical roles in both climate and in the health of various ecosystems. The majority of the Earth’s sulfur is stored underground in rocks and minerals, including as sulfate salts buried deep within ocean sediments.

Steps of the sulfur cycle are:

➢ Mineralization of organic sulfur into inorganic forms, such as hydrogen sulfide (H2S), elemental sulfur, as well as sulfide minerals.

➢ Oxidation of hydrogen sulfide, sulfide, and elemental sulfur (S) to sulfate (SO42−).

➢ Reduction of sulfate to sulfide. ➢ Incorporation of sulfide into organic compounds (including metal-containing derivatives).

These are often termed as follows:

➢ Assimilative sulfate reduction in which sulfate (SO42−) is reduced by plants, fungi and

various prokaryotes. The oxidation states of sulfur are +6 in sulfate and –2 in R–SH. ➢ Desulfurization in which organic molecules containing sulfur can be desulfurized,

producing hydrogen sulfide gas (H2S, oxidation state = –2). An analogous process for



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organic nitrogen compounds is deamination. ➢ Oxidationof hydrogen sulfide produces elemental sulfur (S8), oxidation state = 0. This

reaction occurs in the photosynthetic green and purple sulfur bacteria and some chemolithotrophs. Often the elemental sulfur is stored as polysulfides.

➢ Oxidation of elemental sulfur by sulfur oxidizers produces sulfate. ➢ Dissimilative sulfur reduction in which elemental sulfur can be reduced to hydrogen

sulfide ➢ Dissimilative sulfate reduction in which sulfate reducers generate hydrogen sulfide from

sulfate

The sulfur cycle contains both atmospheric and terrestrial processes. Within the terrestrial portion, the cycle begins with the weathering of rocks, releasing the stored sulfur. The sulfur then comes into contact with air where it is converted into sulfate (SO4). The sulfate is taken up by plants and microorganisms and is converted into organic forms; animals then consume these organic forms through foods they eat, thereby moving the sulfur through the food chain. As organisms die and decompose, some of the sulfur is again released as a sulfate and some enters the tissues of microorganisms. There are also a variety of natural sources that emit sulfur directly into the atmosphere, including volcanic eruptions, the breakdown of organic matter in swamps and tidal flats, and the evaporation of water.

Sulfur eventually settles back into the Earth or comes down within rainfall. A continuous loss of sulfur from terrestrial ecosystem runoff occurs through drainage into lakes and streams, and eventually oceans. Sulfur also enters the ocean through fallout from the Earth’s atmosphere. Within the ocean, some sulfur cycles through marine communities, moving through the food chain. A portion of this sulfur is emitted back into the atmosphere from sea spray. The remaining sulfur is lost to the ocean depths, combining with iron to form ferrous sulfide which is responsible for the black color of most marine sediments.

Since the Industrial Revolution, human activities have contributed to the amount of sulfur that enters the atmosphere, primarily through the burning of fossil fuels and the processing of metals. One-third of all sulfur that reaches the atmosphere—including 90% of sulfur dioxide—stems from human activities. Emissions from these activities, along with nitrogen emissions, react with other chemicals in the atmosphere to produce tiny particles of sulfate salts which fall as acid rain, causing a variety of damage to both the natural environment as well as to man-made environments, such as the chemical weathering of buildings. However, as particles and tiny airborne droplets, sulfur also acts as a regulator of global climate. Sulfur dioxide and sulfate aerosols absorb ultraviolet radiation, creating cloud cover that cools cities and may offset global warming caused by the greenhouse effect.

XENOBIOTIC DEGRADATION

Biodegradation of naturally occurring organic compounds follows their synthesis. In contrast, man-made compounds, also known as xenobiotics, are often refractory to degradation. The main reason is that they cannot be recognized by naturally present organisms and therefore do not enter common metabolic pathways. The physical and chemical characteristics of the compounds, as well as environmental factors, may influence their biodegradability. Some compounds may be transformed only in the presence of another compound which appears as a carbon and energy source. Very often compounds are degraded sequentially through the activity of a series of different organisms. The main degraders in nature are microorganisms, mostly bacteria and some fungi. These organisms, due to their rapid rates of multiplication and great



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metabolic potential, are able to adapt to new substrates. Selection of degradative potent microorganisms and their successive adaptation to a naturally persistent compound might be a powerful means for environmental detoxification. Although numerous laboratory experiments have given positive results, very few are applicable on a large scale. It is necessary to select microorganisms or microbial communities capable of controlled degradation of persistent organic chemicals without their transformation to other, more hazardous compounds. Better understanding of metabolic pathways for the biodegradation of specific organic compounds as well as more thorough knowledge of degrading microorganisms will make purposeful application of biodegradation possible. The broad categories of xenobiotic pollutants are:

➢ Recalcitrant hydrocarbons. ➢ Haloalkyl propellants and solvents. ➢ Recalcitrant nitroaromatic compounds. ➢ Polychlorinated biphenyls (PCBs) and dioxins. ➢ Synthetic polymers. ➢ Alkyl benzyl sulphonates. ➢ Petroleumhydrocarbons ➢ Acid mine drainage.

HALOALKYL PROPELLANTS

The haloalkanes are a group of chemical compounds derived from alkanes containing one or more halogens. They are a subset of the general class of halocarbons. They are used as flame retardants, fire extinguishants, refrigerants, propellants, solvents, and pharmaceuticals. Subsequent to the widespread use in commerce, many halocarbons have also been shown to be serious pollutants and toxins. For example, the chlorofluorocarbons have been shown to lead to ozone depletion. Methyl bromide is a controversial fumigant. Only haloalkanes which contain chlorine, bromine, and iodine are a threat to the ozone layer, but fluorinated volatile haloalkanes in theory may have activity as greenhouse gases. Methyl iodide, a naturally occurring substance, however, does not have ozone-depleting properties. Haloalkane or alkyl halides are the compounds which have the general formula "RX" where R is an alkyl or substituted alkyl group and X is a halogen (F, Cl, Br, I).

Chloroethane was produced synthetically in the 15th century. The systematic synthesis of such compounds developed in the 19th century in step with the development of organic chemistry and the understanding of the structure of alkanes. Methods were developed for the selective formation of C-halogen bonds. Especially versatile methods included the addition of halogens to alkenes, hydrohalogenation of alkenes, and the conversion of alcohols to alkyl halides. These methods are so reliable and so easily implemented that haloalkanes became cheaply available for use in industrial chemistry because the halide could be further replaced by other functional groups.

While most haloalkanes are human-produced, non-artificial-source haloalkanes do occur

on Earth, mostly through enzyme-mediated synthesis by bacteria, fungi, and especially sea macroalgae (seaweeds). More than 1600 halogenated organics have been identified, with bromoalkanes being the most common haloalkanes. Brominated organics in biology range from biologically produced methyl bromide to non-alkane aromatics and unsaturates (indoles, terpenes, acetogenins, and phenols). Halogenated alkanes in land plants are rarer, but do occur, as for example the fluoroacetate produced as a toxin by at least 40 species of known plants.



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Specific dehalogenase enzymes in bacteria which remove halogens from haloalkanes, are also known.

ALKYLBENZYL SULPHONATES

They are main components of anionic detergents. They are surface active with polar sulphate and nonpolar alkyl end in their molecule. Due to their characteristic of emulsification of fatty substances and thereby cleaning occurs while these molecules make a monolayer around lipophilic droplets or particles. Their molecules orient with their nonpolar end towards the lipophilic substance and the sulphonate end towards water. Nonlinear alkylbenzylsulphonates are recalcitrants and resistant to biodegradation and cause foaming in the rivers in plenty.

Though AlkylBenzylSulphonates are easier to manufacture and bear superior detergent properties but the methyl branching of alkyl chain in them interferes with biodegradation of ABS. It is specifically because tertiary carbon atom blocks the normal p oxidation sequence in the molecule. That is why detergent industry turned from ABS to LAS (linear alkylbenzylsulphonates), which are biodegradable.

STUDY MATERIAL FOR B.SC MICROBIOLOGY ENVIRONMENTAL ...

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