Methods of Studying Soil Micro-organisms

  Fertile soil contains a wide variety of microorganisms, so that no single method can be used for cultivating and enumerating all soil microorganisms.

• Factors influencing the size and variation in microbial population are :

1. Differences in composition of soil.
2. Differences in physical characteristics of soil.
3. Differences in the agricultural practices.

• Variation in any one of these conditions causes variability in soil microflora.

Methods of studying soil microflora

There are various methods  available for studying soil microflora such as

1. Direct Microscopic Method
2. Agar Plate Technique
3. Enrichment Culture Technique
4. Buried Slide Method.

1]. Direct Microscopic Method :

• Soil suspension is prepared in aqueous solution of agar for good fixation.
• 0.1 ml of soil suspension is transferred to the ruled area on a microscopic slide and spread out uniformly.
• It is stained with 1% rose bengal dissolved in 5% aqueous solution of phenol.
• Bacteria appear deep pink or rose in colour. Minerals and inorganic consistituents do not stain. Some of the dead organic matter appears light pink but most of it stains either yellow or not at all. The slide is examined under a calibrated oil Immersion objective and the number of organisms per field counted.
• An average of atleast 25 field count is taken for the calculation of total number of bacteria per gram of soil.

Advantage :

• It is a rapid technique many sample can be examined in a short period.

Disadvantages :

• Microscopic observation is tedious.
• Bad smear preparation gives wrong results.
• Distinction between live cells and soil particles, as well as live cells and dead cells is difficult.
• Accurate determination by this method needs considerable experience.

2]. Agar Plate Technique :

• This method is used for isolation and enumeration of bacteria, yeast and molds present in soil.
• Suitable modifications are made to meet the cultural requirements of organisms to be enumerated.
• A weighed sample of soil is mixed with a known volume of sterile water in a screw-cap bottle or tube.
• The sample is shaken vigorously to separate organisms from the colloidal material surrounding soil particles.
• The coarse particles are allowed to settle down and a series of dilution are prepared from the suspension.
• Aliquote from each dilution is plated onto suitable agar medium.
• The plates are incubated at 25°C for 2-14 days and the results are expressed as CFU/gm of soil.

Advantages :

An exact picture of total viable cells can be obtained.

Disadvantages :

• In a specific set of culture conditions only a part of the total microbial population can grow. Study of all types of microorganism cannot be done using a single medium and conditions for growth.
• Obligate anaerobes cannot grow under aerobic condition.
• Autotrophic bacteria cannot grow in organic media.
• Non-symbiotic N₂ fixers grow to a limited extent.
• Cellulose decomposers fail to grow on the commonly used media.
• Sulfate reducing organisms cannot grow in absence of sulfate in the medium.
• There is a great variety of molds found in soil. Number which appears on agar plate represents a small fraction of molds.

3]. Enrichment Culture :

• This method is useful for studying and obtaining a particular type of organism from a soil sample.
• Usually, a liquid or agar medium containing soil extract is used to enrich the rhizopods and ciliates type of protozoans.
• According to one technique, a short gram-negative, non-spore forming, bacterium such as Aerobacter sp. can be added to a minimal agar medium to cultivate protozoan, as it is easily digested by the protozoan fauna.

4). Buried Slide Method :

• The Buried Slide technique was introduced in the 1930s by Rossi and Cholodny for the direct microscopic study of soil microflora.
• This technique has also been described by Parkison et al. (1971).
• Clean, sterile glass-slides are inserted in situ by making slits in a soil by using a sterile knife. The soil may be dug out and used by filling it in a container in the laboratory.
• The soil is pressed around the slide for firm binding with glass-slide surface. It is then allowed to remain in place for 1-3 weeks.
• It is then removed and soil in cleaned from one surface. Gram staining or staining with phenolic aniline dyes is carried out after heat-fixation.
• Microscopic observation reveals a biofilm that represents soil microflora (colonies and single cells).
• It is possible that the microbial population may not be a representative of the soil.

Use of Winogradsky Column in Studying Microbial Diversity in Soil :

  The Russian microbiologist Sergei Winogradsky (1856-1953), first developed a simple technique to demonstrate microbial communities and their interactions.

The Winogradsky Column

• A Winogradsky column consists of a core soil sample placed in a glass cylinder alongwith cellulosic waste, and overlaid with saline water or freshwater, which facilitates isolation of various sulfur metabolizing bacteria.
• The height of the column allows the development of aerobic zone at the surface and microaerophilic and anaerobic zones below the surface.
• The column is exposed to light so that various photosynthetic populations develop.
• The column consists of mud, CaSO₄, plant tissues (as source of carbohydrate) and water.
• It is exposed to daylight and incubated at room temperature.

The microbiological events can be summarised follows :

Initially, a variety of heterotrophic microorganisms present in soil oxidise various substrates depleting the oxygen supply and creating anaerobic conditions. Clostridium sp. can produce fermentation products under anaerobic conditions :

• Organic matter + O₂ ➞  organic acid + H₂ + CO₂ ↑ (cellulose,starch etc.)

 Organic acids act as electron donors for reduction of sulfates and sulfites to H₂S by anaerobic sulfur reducing bacteria, e.g. Desulfotomaculum :

• Organic acids + SO₄²⁻ ➞ H₂S + CO₂

Photosynthetic microorganisms such as purple and green sulfur bacteria Chromatium and Chlorobium use H₂S, as the electron donor to reduce CO₂ :

• CO₂ + H₂S ➞ (CH₂O)x + S.
• The purple sulfur bacteria are more tolerant to sulfide than green sulfur bacteria.

The aerobic sulfur metabolizing bacteria Thiobacillus develop in the upper portion of the column and oxidise reduced sulfur compounds like sulfides, elemental sulfur and sulphite to produce sulfate :

• Reduced sulfur compound SO₄²⁻ + accumulation of 'S°' (FeS, H₂S, S°, SO₃-)

The nonsulfur purple bacteria includes :
Rhodospirullum, Rhodopseudomonas, Rhodomicrobium, etc.

  They are facultative phototrophs. They grow aerobically in the dark and anaerobically in the light. They can utilise sulfide at low levels, They use H₂ gas as electron donor in photosynthesis:

• CO₂ + H₂S ➞ (CH₂O)x + S°(in presence of light)
• CO₂ + 2H₂ ➞ (CH₂O)x + H₂O.

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Soil Microflora : Diversity, Function & Examples

  The soil flora is composed of a large number of microorganisms, root system of higher plants and animals such as rodents, insects and warm.

• A spoonful of fertile soil is said to contain more microorganisms than there are people in this world.
• Most soil organisms are found in the surface layer. Their number decreases with depth.
• A well aerated soil contains more organisms than one lacking abundance of oxygen. The number and diversity of organisms found in soil depends on the following factors.

1. Nature of soil
2. Depth
3. Season of the year
4. State of cultivation
5. Amount of organic matter
6. Degree of aeration
7. Temperature
8. pH of soil
9. Moisture

• The existence of roots and their extensiveness also influence the soil flora. The climatic conditions also have effects on various organisms in soil.

Functions of Soil Microflora : 

One of the most important functions of soil organisms is to decompose various kinds of organic matter of plant and animal origin. This produces four distinct effects upon soil processes and upon plant growth.

1. They provide inorganic nutrients to plants especially Nitrogen and Phosphorus.
2. They affect the physical conditions of soil especially the moisture holding and buffering capacities.
3. They provide certain specific elements that may be limiting factors for plant growth.
4. They favour development of organisms that secrete antagonistic substances that control the growth of certain plant pathogenic species.

Diversity of Soil Microflora :

  Soil is an excellent culture medium for the growth of many kind of micro-organisms. The microscopic life of soil includes :
  (1) Bacteria
  (2) Fungi
  (3) Algae
  (4) Protozoa
  (5) Viruses.

(1) Bacteria :

• Bacterial population of soil exceeds the population of all other groups of organisms.
• Direct microscopic count of soil bacteria is as high as several billions per gram.
• Viable plate count of soil shows only a fraction of this number (millions/gm). Forest soil contains about 4.8 x 10⁹ cells/cm³. 
• There is a great variety of nutritional and physiological types of bacteria in soil, and no single laboratory environment can support growth of every viable cell type.
• The soil bacteria may be autotrophs, heterotrophs, mesophiles, thermophiles, psychrophiles, aerobes, anaerobes, cellulose digesters, sulfur oxidizers, N2 fixers, protein digesters, etc.
• The most abundant bacteria found in soil belong to the actinomycetes group. They produce geosmin a compound that gives a characteristic earthy odour to soil. 
• They can degrade many complex organic substances present in soil and increase soil fertility. They produce antibiotics, which maintain equillibrium of soil microflora. Nocardia, Streptomyces and Micromonospora are the predominant genera of  actinomycetes found in soil. 
• The other bacteria present in abundance include Arthrobacter, Pseudomonas, Clostridium, Bacillus, Micrococcus and Flavobacter. The less common are Chromobacterium, Sarcina and Mycobacterium.

(2) Fungi :

• Many species of fungi inhabit the soil. 
• They are usually found on the surface of soil. 
• They exist both in spore and mycelial stage.
• Both unicellular and multicellular fungi are present in soil. 
• The unicellular fungi is known as yeast. 
• The multicellular fungi is known as mold. 
• The main function of fungi is to decompose plant dead organic material Including cellulose, lignin and pectin. Fungi improve the physical structure and water holding capacity of soil. 
• Some fungal are predatory, they eat amoeba and nematodes, thereby maintain the microbial equilibrium. There are certain plant pathogenic fungi responsible for causing diseases in plants. 
• The fungi form oxygen-impermeable structures called sclerotia and hyphal cords. Filamentous fungi which move nutrients and water over long distances are found within these structures.

(3) Algae :

• Algal population in soil is smaller than that of fungi or bacteria. The major types found in soil include: Green algae, Diatoms and Blue-green algae (Cyanobacteria).
• They are photosynthetic and some cyanobacteria can also fix atmospheric Nitrogen. They are found only on the surface of soil.

(4) Protozoa :

• Most soil protozoa are flagellates or amoeba. Their number per gram of soil ranges from a few hundred to several hundred thousand in moist soils rich in organic matter. 
• They eat bacteria and thereby maintain equilibrium of bacterial population.

(5) Viruses :

• Viruses are obligate intracellular parasites. Bacterial, plant and animal viruses are often found in soil. 
• Some viruses present in soil cause plant diseases, e.g., tobacco mosaic virus (TMV). 

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Municipal Wastewater Treatment Methods : Primary, Secondary, Advanced and Final treatment

 Municipal wastewater treatment is basically applicable to those wastewater which has been collected from various residential localities through the sewage system of a city and town, and is treated before being dispersed in open field.
  Municipal wastewater treatment plants carryout a series of treatment processes which may be summarized as follows:

i. Primary treatment :

  As the name indicates, it is a primary treatment or physical treatment. This treatment is carried out to remove coarse solids and settleable solids.

ii. Secondary treatment :

  It is known as biological treatment. It comes after primary treatment. The aim of secondary treatment is to oxidize organic constituents of the wastewater by means of microorganisms and reduction of BOD also takes place.

A Typical Sewage Treatment Plant

iii. Advanced treatment :

This treatment is carried out for the removal of additional objectionable substances to further reduce BOD. The treatment Includes removal of nutrients such as phosphorus and nitrogen that are responsible for algal blooms.

iv. Final treatment :

   It is also known as Chemical treatment. This is basically to disinfect and dispose of liquid effluents.
  Solid processing involves the treatment of sludge or biosolids for stabilization of its organic matter and water content. It can then be used as organic manure for agriculture.

(A) Primary Treatment and Secondary Treatment : Principles and Role of Microorganisms

i. Primary Treatment :

  This is also known as physical treatment. It consists of methods to remove solid materials and these methods are basically mechanical.

• Wastewater as it arrives at a wastewater disposal plant is first treated to remove coarse solid materials by a variety of mechanical techniques which include-
(a) Screening
(b) Grinding
(c) Floatation
(d) Sedimentation

(a) Screening :

• In this method, the wastewater collected from various residential areas is passed through various sizes of screens such as bar rock, fine screens and microscreens.

• The solid objects which are larger than the screens will be retained back.

(b) Grinding :

• Here, the solid mass is ground or broken down into smaller particles, which will settle down in grit chambers.

(c) Floatation :

• Certain substances which are light in weight and floats on the surface such as finely divided suspended solids, particles with densities close to that of water, oil and gases are removed by dissolved or induced air floatation.

(d) Sedimentation :

• Sedimentation units such as tanks, basins or mechanical device provide the means for concentrating and collecting the particulate material referred to as sludge.

• Following sedimentation, the sludge and the liquid effluent are processed separately.

• Secondary treatment does not taken place before removal of coarse and settleable particles. If they are not removed, then they cause interference in later stages of secondary treatment.

ii. Secondary Treatment :  

  Secondary treatment processes accomplish oxidation of the organic material in the liquid waste by microbial activity.

• During this process microorganisms transform organic compounds into simple compounds.

• For the complete breakdown of organic compounds, oxygen supply is necessary. If there is no supply of oxygen, then the process is anaerobic and the degradation is incomplete. For the efficient treatment, sufficient oxygen supply is required.

• Many different methods are designed for secondary treatment in such a way that  maximum amount of sewage is exposed to air because wastewater can hold a limited amount of oxygen.

• Oxygen is not provided by artificial way because it is costly.

• The ultimate aim of secondary treatment is to reduce BOD in the wastewater. About 80 to 90% of the BOD and pathogens are removed.

There are various methods for secondary treatment.

(a) Septic Tank :

  A septic tank is a sewage - settling tank designed to retain the solids of the sewage entering the tank long enough to permit adequate decomposition of the sludge.

Septic Tank

• Thus, the unit accomplishes two processes.

(1) Sedimentation, and
(2) Biological degradation of the sludge.

• As sewage enters this type of tank, sedimentation occurs from the upper portion, permitting a liquid with fewer suspended solids to be discharged from the tank.

• The sedimented solids are subjected to degradation by anaerobic bacteria, hence the end products are still unstable i.e. high in BOD and odorous.

• When microbial growth in completed, it forms a stable settleable structure called flocs.

• The effluent from the septic tank is distributed under the soil surface through a disposal field as shown in the figure.

• Septic tank is the most satisfactory method for disposing of sewage from small installations, especially individual dwellings and isolated rural buildings where public sewen are not available.

• They cannot however, be relied upon to eliminate pathogenic microorganisms carried in the sewage.

• Consequently, it is imperative that the drainage from the tank be prevented from contaminating the drinking water supply.

(b) Imhoff Tank :

The Imhoff tank was developed to correct the two main defects of the septic tank.

Imhoff Tank

 
- It prevents the solids once removed from the sewage from being mixed with it again, but still provides for the decomposition of these solids in the same unit.
- It provides an effluent amenable to further treatment.

• The Imhoff tank may be either circular or rectangular and is divided into three compartments: 

(1) The upper section or sedimentation compartment.
(2) The lower section known as the digestion compartment.
(3) The gas vents and scum section.

• It is desirable to be able to reverse the direction of flow to prevent excessive deposition of solids at one end of the sediment compartment.

• Periodically reversing the flow will result in an even accumulation of sludge across the bottom of the tank.

• In operation, all of the wastewater flows through the upper compartment.

• Solids settle to the bottom of this sloped compartment slide down and pass through an opening or slot to the digestion compartment.

• One of the bottom slopes extends at least 6 inches beyond the slot. This forms a trap to prevent gas or digesting sludge particles in the lower section from entering the waste stream in the upper section.

• The gas and any rising sludge particles are diverted to gas vents and scum section.

(c) Trickling Filter :

  The trickling filter consists of a bed of crushed stone, gravel, slage or synthetic material with drains at the bottom of the tank.

Trickling Filter 

• Trickling filters have a depth of 5 to 10 feet. The circular round of the trickling filter is filled with various sizes of stones.

• In the centre of trickling filter, there is an arm running on entire bed.

• The liquid sewage is sprayed over the surface of the bed either by a rotating arm or through nozzles.

• The spraying saturates the liquid with oxygen. The treatment in trickling filter is an aerobic process.

• Wastewater is kept on the bed of stones, stones are of different shapes so the air pockets between the arrangement of stones varies.

• The sewage trickles from these air pockets. Now the rotating arm is rotated and begins a new entire cycle. The time is noted for the sewage to pass through the bed.

• Sewage contains bacteria, that adheres on the stones. The sewage which falls on stones is rich in nutrition, organic compounds and air is sufficient in this system.

• Now the air pockets will totally be aerobic and microorganisms will grow, consume the oxygen as well as nutrients. Then microorganisms multiply slowly. This results in formation of film of microorganisms called zoogloeal film.

• Due to this layer the rate of filtration decreases. This layer is made up of millions of microorganisms, so Initially the rate of filtration is fast, but with time filtration rate decreases.

• The sewage falling from rotating arms is rich in organic compounds and nutrition, which gives nutrient for growth of microorganisms. The water collected from the bottom has about 80% reduction of BOD. This is a very good process for the treatment of wastewater.

• The slimy layer formed is composed of a mixed microbial community including bacteria such as Beggiatoa alba, Sphaerotilus natans, Achromobacter sp., Pseudomonas sp., Flavobacterium sp., and Zoogiea ramitera, etc., Fungi, protozoa, nematodes and rotiflers are also present .

(d) Activated Sludge Process :

  The activated sludge process is an aerobic suspension type of liquid waste treatment system, also known as aeration tank digestion.

Activated Sludge Process

• After primary treatment, the sewage containing dissolved organic compounds is introduced into an aeration tank and mixed with a slurry rich in bacteria which is called an activated sludge.

• Air injection and or mechanical stiring provides the aeration.

• The heterogenous nature of the organic substrates in the sewage allows the development of diverse heterotrophic bacterial populations Including gram-negative rods, predominantly Escherichia, Enterobacter, Pseudomonas, Achromobacter, Flavobacterium and Zooglea sp., other bacteria includes Micrococcus, Arthrobacter, Sphaerotilus and other large filamentous bacteria, and low numbers of filamentous fungi, yeasts and protozoa, mainly ciliates.

• The protozoa are important predators of the bacteria alongwith rotifers.

• The bacteria in the activated sludge tank occur in free suspension and as aggregates or flocs.

• The flocs are composed of microbial biomass held together by bacterial slimes produced by Zooglea ramigera and similar organisms.

• The floc is too large to be ingested by the ciliates and rotifers.

• In the raw sewage, suspended bacteria predominate, but during holding time in the aeration tank, their number decreases and at the same time those bacteria associated with flocs greatly increase in number.

• As a result, significant portion of the dissolved organic substrate is mineralized and another portion is converted to microbial biomass.

• In the advanced stage of aeration, most of the microbial biomass becomes associated with flocs that can be removed from suspension by settling in a settling tank.

• Sometimes because of lower O2 levels or due to too old or young microbial population proliferation of filamentous bacteria like Sphaerotilus, Beggiatoa, Thiothrix and other filamentous fungi causes bulking of sewage, which results in formation of flocs that do not settle this affects the effluent quality.

• A portion of the settled sewage sludge is recycled for use as the inoculum for the incoming raw sewage.

• The organisms present in the flocs reduces the time for treatment everytime it is recycled.

• During the treatment, BOD level is reduced by 85-90%.

(e) Oxidation Ponds :

  Oxidation ponds are also called Lagoons or stabilization ponds.

Oxidation pond 

• This pond is natural or artificial. It is basically shallow pond and having a large surface area and depth of 2 to 4 feet.

• In an oxidation pond, wastes are added at single point, either in the middle of the pond or at the edge of the pond and effluent is removed at the single point at the edge of the pond.

• An oxidation pond is an aerobic system for the simple secondary treatment of the waste water in small industrial units or villages. However, the pond is having both aerobic as well as anaerobic zones.

• Within an oxidation pond, heterotrophic bacteria degrade organic matter in the sewage, which results in production of cellular material, CO2 and minerals.

• The production of these substances supports the growth of algae in the oxidation ponds.

• The production of this oxygen by photosynthetic activity of algae replenishes the oxygen used by the heterotrophic bacteria.

• The performance of oxidation pond is strongly influenced by seasonal temperature.

• Oxidation ponds also tend to get filled due to the settling of bacteria and algal cells formed during the decomposition of sewage.

• Overall, oxidation ponds, are low-cost operations but they tend to be inefficient and requires large holding capacity and long retention time.

(B) Advanced Treatment, Final or Tertiary Treatment and Efficiency of Waste Treatment Procedures :

i. Advanced Treatment :

  The advanced treatment is needed when the organic matter in sewage which has passed through primary and secondary treatment is not yet completely degraded.

• Various kinds of advanced treatments are there which depends on the type of incoming sewage.

• Unit processes have been developed to remove nutrients, simple organic substances and complex synthetic organic compounds.

• Processes Include biological as well as physico-chemical methods.

• Examples are, Biological nitrification-denitrification, filtration, reverse osmosis, carbon adsorption, chemical addition and lon-exchange, etc.

• The major disadvantage of advanced treatment processes Is its high cost.

(a) Biodisc system or rotating biological contactor

This process is an example of a more advanced type of aerobic film-flow treatment system. In this process closed packed plastic discs, partially submerged in a trough containing the sewage are rotated.

• Microbial slime forms on the surface, which becomes thick and finally sloughs off when the Inner most microbes die and the film detaches from the plastic discs.

• It is useful In treatment of domestic as well as industrial wastewater.

(b) Modified or non-conventional activated process

This process is employed for removal of nitrogen and phosphorus, which are generally removed during tertiary treatment.

• A single sludge system comprising of a series of aerobic and anaerobic tanks is one method, In which methanol or settled sewage is used as the carbon source by denitrifiers.

• In multisludge system three separate systems are involved in carbonaceous oxidation, nitrification and denitrification.

• Bardenpho process removes nitrogen as well as phosphrus during nitrification-denitrification process. It consists of two aerobic and two anoxic tanks followed by a sludge settling tank.

ii. Final or Tertiary Treatment and Efficiency of Waste Treatment Procedures:

  After final treatment, the water is to be released in water bodies which is to be used by living creatures.

• Disinfection is commonly accomplished by chlorination. However, current research has proved the serious impact chlorinated waters have on the aquatic life of the receiving water.

• This has led to the development of several disinfection alternatives. The use of ozone and ultra-violet light is becoming more prevalent.

• Many facilities that continue to employ chlorine for disinfection now include dechlorination prior to discharge into a water body.

• Dissolved oxygen may also be added to the treated wastewater prior to final discharge. This process is termed post aeration that is accomplished by mechanical means or a cascading slow techniques, which minimizes the decrease in dissolved oxygen of the receiving water bodies.

• Activated carbon filters are normally used for the removal of P and N from secondary treated effluents.

• Phosphate is removed by precipitation as calcium, aluminium or Iron phosphate. Its presence in effluents causes eutrophication of receiving water bodies. It can be achieved by addition of lime.

• Ammonia (NH3) is removed by Break-point chlorination process, in which it is converted to form dichloramine and then to molecular nitrogen.

• Nitrogen can also be removed by denitrification. Here during aerobic treatment nitrate is produced by nitrifying bacteria. Nitrate is then reduced to nitrogen gas and nitrous oxide (N2O) by denitrifying bacteria that use it as an electron acceptor during degradation of organic matter under low oxygen condition.

• Anammox process is an anaerobic nitrogen removal process in which ammonium ion (used as the electron donor) is allowed to react with nitrite (the electron acceptor) produced due to partial nitrification. It can convert about 80% of the ammonium ion initially present to nitrogen gas.

• Tertiary treatment processes are expensive and hence used only where it is justified.

Removal of pathogens :

• Pathogens present in raw sewage are successfully removed mainly during the activated sludge process.
• It removes viruses, enteric bacterial pathogens (e.g. Salmonella), Glardia and Cryptosporidium.
• Chlorination, ozonization and U.V. rays are employed during tertiary treatment process for killing pathogens.

(C) Solid Waste Processing : Anaerobic Sludge Digestion and Composting

  A major cost at modern large scale wastewater treatment is associated with additional processing. of effluent after tertiary treatment.
Solid waste or sludge processing, removal of pathogens, composting, land-farming, etc. are involved in this activity.

i. Anaerobic Sludge Digestion :

  The biosolids or sludge is produced during the sewage treatment stages is rich in organic matter.

• The primary sludge contains about 3.8% solids, whereas secondary sludge contains 0.5 to 2.0% solids.

• The steps involved in treatment of sludge includes:
Thickening : reduction in its volume, by settling in a tank or by centrifugation.
  Digestion : this is for stabilization of the organic matter by microbial activities. It also causes destruction of pathogens due to higher temperatures reached during the process.

• It is carried out by both anaerobic or aerobic digestion.

• The sludge which accumulates during various treatment stages is collected and pumped into a separate tank designed for its digestion under controlled conditions.

• Initially, for a few hours, air is supplied for aerobic degradation. Aerobic microorganisms degrade the sludge reducing the solids content, and create anaerobic condition.

• Anaerobic digestion takes place for a period of 2-3 weeks in a large covered tank, it results in the formation of gases namely, methane (60-70%), CO2 (20-30%), small amounts of hydrogen and nitrogen. 

• Methane produced is used as a biofuel (biogas).

• This causes further stabilization of the sludge.

• Ripe sludge which contains an actively growing population of diverse microorganisms is used to seed fresh sludge collected in tanks. This helps is shortening the time Ens noj required for establishment of microbes and digestion.

• Conditions such as pH, temperature, nutrients, etc. which affect microbial metabolism and growth have to be controlled to achieve proper digestion.

• The process of adding chemicals such as lime, alum, ferric chloride, etc. for reduction of solids, precipitation of phosphorus, etc. is employed.

 • Finally, air drying, centrifugation or vacuum filtration are employed for dewatering the solids, to decrease its volume and reduce transportation cost.

• Land farming is a practice for disposal of biosolids produced during wastewater treatment onto agricultural land.

• Processes for reduction of pathogens are used to treat sludge before adding to soil.

• The blosolids may be liquid or solid in nature for soll application. It adds water and nutrients to soil that promotes soil fertility and plant growth.

• Various types of sanitary landfill, are employed for disposal of municipal solid waste (MSW) generated in major cities. Both organic and Inorganic solid wastes are deposited in a low-laying waste-land, and covered with a layer of soil over everyday's waste added.

• Finally, a developed landfill may be used for recreation or construction purpose.

ii. Composting

• Composting is a process for biodegradation or decomposing organic solid waste by aerobic, mesophilic and thermophilic microorganisms.

• Dewatered sludge or solid organic wastes after removal of inorganic fraction is ground, mixed with sludge or bulking agents such as wood chips, shredded newspaper, resins, etc. and composted.

• Microbial degradation activities converts the waste Into a stable humus like product.

• In Window method the solid waste is piled up in long rows, covered and allowed to decompose. The material is turned over at regular intervals.

• The Static pile or Aerated pile method waste is piled up on perforated pipes through which air is pumped. This speeds up the composting process.

• At some modern facilities, an enclosed reactor composting under controlled conditions is carried out in an industrial bioreactor or fermenter. The composting occurs in 2-4 days, however, it is expensive.

• For optimal composting the C: N ratio must be 40:1, temperature 50 - 60°C, and moisture 50-60%.

• Mesophilic bacteria and fungi grow first followed by thermophilic organisms such as Thermomonospora spp., Clostridium spp., Aspergillus fumigatus, Geotrichum spp., Torula thermophila, etc.

• The high temperature kills most of the enteric pathogens present during composting of solid waste.

• The composted waste is screened for removal and recycling of wood chips, resins, etc. and humus like material is used as a soil conditioner as a commercial product, especially for organic farming. 

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Transcription process in Prokaryotes

  The transfer of genetic information from double standard template DNA molecule to a single standard RNA molecule by usual process of complementation base pairing catalysed by enzyme RNA polymerase.
  Transcription occurs unidirrectionally in which RNA (transcript) is synthesized from the 5' to 3' direction.

Introduction

• Crick in 1958 reported, replication is auto catalytic function of DNA. Transcription and translation are heterocatalytic function of DNA. 

• Central doma : This name was given by Crick to the 2 step process.
DNA ➞ RNA ➞ Protein
DNA to RNA by Transcription and RNA to DNA by Translation.

• Transcription Unit
  Transcription is a selective process. Each transcript segment of DNA is called transcription unit there are two types of transcription unit :
a. monocistronic transcription unit
In Eukaryotes, transcription unit typically carries the information of just one gene.
b. Polycistronic transcription unit  
In prokaryotes, a set of adjacent genes is transcribed as a unit termed Polycistronic transcription unit.

• Transcription occurs throughout the cell cycle depends on need.

• Transcription involves rewriting of genetic message in DNA and RNA molecule and results in the formation of mRNA (messenger RNA) complementary to the gene sequence of one of two strands of double stranded DNA helix.

• The lifespan of mRNA is very short, it present only during protein synthesis. 

• Transcription is catalyzed by RNA polymerase in prokaryotes and eukaryotes.

• RNA synthesis takes place in 5'➞3' direction. RNA polymerase can add nucleotides to the 3' OH end of the polynucleotide.
- Start point is the first base pair at which transcription starts. RNA polymerase moves along template and elongate until reaches terminator sequence.

• During transcription only 3' to 5' strand (one strand) of DNA is transcribed. This strand is called coding strand which is comple complementary to the template strand from which RNA is transcribed 5' to 3' strand.
The template strand or non coding strand is the 3' to 5' strand from which the RNA is transcribed.

• RNA synthesis can start without primer (De-novo). First nucleotide at 5' end of RNA transcript is purine triphosphate (GTP or ATP).

• Primary transcript :
   The immediate product of transcript is called the primary transcript, that is processed to yield either single type or more than one type of mRNAs depending on transcription unit is simple or complex.

Mechanism of Transcription in Prokaryotes

Requirements
• Template DNA
• Ribonucleotides (GTP,CTP, UTP, ATP)
• Mg+² ions (as cofactor).
• RNA polymerase (DNA dependent RNA polymerase).

RNA polymerase
• This is a DNA dependent RNA polymerase
• Discovered by Samuel B. Weiss and Jerard Hurwitz in 1960.
• In prokaryotes different types of RNA ( mRNA, rRNA, tRNA) are synthesized by single RNA polymerase.

  Bacterial RNA polymerase is a complex holoenzyme (4,80,000 M.W.). It is made up of core enzyme + Sigma(σ) subunit.

RNA Polymerase Subunits and Functions

Subunit	Function
2α	Assembly of core enzyme & Promoter recognition
β	Catalytic centre
β'	Catalytic centre
ω	Assembly of RNA polymerase β' unit to be stable
Sigma  Subunit	Promoter recognition & transcription initiation

Core enzyme : formed of 5 chain (α,α,β,β',ω). It binds non specifically to DNA template strands and catalyse process of chain elongation.
Sigma subunit(σ) is a loosely attached to the core enzyme. It recognises start signal on DNA and directs core enzyme to bind promoter region.

• RNA polymerase lack proofreading 3' to 5' exonuclease activity.
  In every 10⁴ to 10⁵ nucleotide added, a mistake may be made for one nucleotide it is not serious because of high turnover and wobble pairing during transcription.

Operon in prokaryotes

  Prokaryotic DNA is a polycistronic. The length of DNA transcribed into single functional RNA molecule is known as operon in prokaryotes, the segment of operon contains,
- a promoter region
- a short Codon or initiation sequence
- a colony segment
- a terminator sequence

Prokaryotic Pramoter
• Promoter region is a upstream regulatory DNA segment. The initiation codon of gene located before 5' end off coding DNA.
- Strong promoter supports high rate of transcription initiation.
• RNA polymerase binds at Pramoter site.
• Pramoter is highly variable but has 2 to 3 important functional sequence.

Promoter in Prokaryotes

1) -10 Sequence or Pribnow box :
- This is a Consensus sequence of 6 to 10 bases (TATAAT).
- Located upstream to initiation site
- It orients RNA polymerase on promoters
- This sequence helps the enzyme to locate the precise bases at which transcription should initiate. second.

2). -35 region
- A consensus sequence about 35 base above the initiation site (TTGACA)
- Sigma (σ) subunit of RNA polymerase get bound at this site.

3). Upstream promoter element
- AT rich recognition element in promoter
- present only in certain highly expressed genes
- located between -40 to -60 base sequences from initiation codon.

Transcription process in prokaryotes :

Transcription process in Prokaryotes can be divided in 3 step
1. Initiation
2. Elongation and
3. Termination

1). Initiation of transcription :
• Only RNA polymerase (holoenzyme) can initiate transcription.
• Sigma factor recognised consequence sequences (-35 & -10 region of promoter)
• Sigma factor changes the DNA binding properties of RNA polymerase. so that it's affinity for a consensus sequence in promoter is increases.
• The core enzyme does not differ between promoter and other DNA sequence.

Transcription bubble

Closed binary complex :

• RNA polymerase holoenzyme binds at the promoter.
• The enzyme first form a closed complex in which the two DNA strands remain fully base paired.
• RNA polymerase covers 75-80 base pair extending from -55 to +20 bp.

Open reading complex :
• In the next step closed complex is converted to open complex. In this DNA helix open by meeting of short DNA sequence bound by enzyme.
• The template DNA is expressed for initiation of an RNA chain.

• The next step is to add first two nucleotides than a phosphodiester bond forms between them. This generates tertiary complex that contain RNA, DNA and enzyme which is generate transcription bubble.

2). Elongation of transcription
• Sigma factor now dissociate and only core enzyme moves along.
• Elongation involves movement of transcription bubble. As the enzyme moves it unwinds the DNA helix to expose a new segment of template in single stranded DNA.
• Nucleotides are covalently added to 3' end of growing RNA chain forming RNA-DNA hybridize
• The length of RNA-DNA helix with in open complex is 8-9 base pair.
• Overall reaction rate is 30 to 50 nucleotide per second at 37°C.

Direction of Transcription

• During its nucleotides edition
- Beta and gamma phosphates are removed from incoming nucleotide.
- OH group is removed from 3' carbon of nucleotide present at the end of the chain.
- During the elongation when RNA polymerase transcribes DNA unwinding and rewinding occurs.
- As RNA polymerase moves ahead generate positive supercoiling ahead (Gyrase removes the positive supercoils and develop negative supercoiling).
- As RNA polymerase moves ahead generate negative supercoiling behind ( Topoisomerase I  removes negative supercoiling).

3). Termination of transcription
• Termination of prokaryotic gene transcription is signalled by controlling elements called terminators.
• At this point, the enzymes stop adding nucleotides to the growing RNA and release the completed product and dissociates from the DNA template.

Terminators are 2 type
1) Intrinsic Terminator
2) rho(ρ) dependent Terminator

1) Intrinsic Terminator
• Intrinsic terminator include palindromic sequences that form hairpin structure by forming a complementary base pairing.

The inverted repeats forming stem and loop structure and terminal U-sequence at the 3 terminal or mRNA molecule

- Hairpins vary in length from 7 to 20 basepairs.
- The stem loop structure includes a G-C rich region and is followed by a U-rich region.
   Typical distance between hairpin and U-rich region is 7-9 bases.

2) Rho(ρ) dependent Terminator
• Rho(ρ) dependent Terminator sequence require activity of a protein called Rho(ρ).
- Rho is an ATP dependent RNA stimulated helicase that disrupt the new ones and RNA DNA complex.
- It binds to RNA at rut site and translocate along RNA in 5' to 3' direction until riches RNA DNA hybrid in RNA polymerase where is release RNA from DNA
- Rut site is rich in C residues and  poor in G residues.

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Biosurfactants - Types, Producers, Advantages, Applications & Role in Bioremediation

Biosurfactants are surface active molecules or chemical compounds synthesized by micro-organisms. These are amphiphilic compounds having both hydrophobic and hydrophilic domains. Biosurfactants are usually either anionic or neutral.

• The hydrophilic moiety can be a carbohydrate amino acid, or a phosphate group or some other compounds.
• The hydrophobic moiety is mostly long carbon chain fatty acid.
• Amphiphilic compounds are produced on living surfaces, mostly on microbial cell surfaces, or excreted extracellularly.

Emulsification of Biosurfactants molecules

• The amphiphilic molecules confer the ability to accumulate between fluid phases, thus reducing surface and interfacial tensions at the surface and interface respectively. It Reduce surface and interfacial tensions in both aqueous solutions and hydrocarbon mixtures.
• This property of biosurfactants make them potential candidates for enhancing oil recovery.

Producers of Biosurfactants

  A lot of microorganisms produce several classes of biosurfactants such as glycolipids, lipopeptides, phospholipids, neutral lipids or fatty acid and polymeric biosurfactants.

• Several microorganisms are known to produce biosurfactants that can vary in structure and chemical composition. These variations depend on the producing microorganism and raw materials used.
• Biosurfactants are Produced during the growth of microorganisms on water soluble and water insoluble substrates.
• Micro organisms utilise a variety of organic compounds as the source of carbon and energy.
• When carbon source is an insoluble substrate like a hydrocarbon, microorganisms facilitate their diffusion in to the cell by producing a variety of biosurfactants.
• Some bacteria and yeasts excrete ionic surfactants which emulsify the CnHn substrates in the growth medium. Eg : rhamnolipids produced by different pseudomonas sp.
• Some other microorganisms are capable of changing the structure of their cell wall, which they achieve by synthesizing liposaccharides or non-ionic surfactants in their cell wall. Eg; Candida lipolytica and Candida tropicalis

Other effective biosurfactants are:

1. Mycolates and Corynomycolates produced by Rhodococcus sp.,Corynebacteria sp., Mycobacteria sp., Nocardia sp.
2. Ornithinlipids produced by Pseudomonas rubescene, Gluconobacter cerinus and Thiobacillus ferroxidans

Types of Biosurfactants

Biosurfactants are Categorised mainly by their chemical composition and microbial origin :
a) Low-molecular mass molecules

1. Glycolipids
2. Lipoproteins
3. Phospholipids

b) High-molecular mass molecule 

1. Polyanionic heteropolysaccharide.

a] Low-molecular mass molecules

This molecule has Efficiently low surface and interfacial tension.

1).Glycolipids

• These are the most common biosurfactants.
• Glycolipids are conjugates of carbohydrates and fatty acids.
• Linkage is by means of either ether or an ester group.
• Eg. Rhamnolipids, Trechalolipids etc.

2).Lipopeptides and lipoproteins

• Lipopeptides and Lipoproteins Consists of a lipid attached to a polypeptide chain.

Two of them are
I). Surfactin

• Surfactin have superior surface activity and belongs to a group of cyclic lipoheptapeptides containing B-hydroxyl fatty acids and amino acid residues
• Produced by Bacillus sp. 

II). Lichenysin

• Bacillus licheniformis produces several biosurfactants which act synergistically and exhibit excellent temperature and pH and salt stability.
• Capable of lowering surface tension of water.

3). Fatty acid, phospholipids and neutral lipids

• Several bacteria and yeast produce large quantities of fatty acids and phospholipid surfactants during growth on n-alkanes.
• The amphiphilic balance id directly related to the length of the hydrocarbon chain in their structure.

b]. High-molecular mass molecule

These are the more effective emulsion stabilizing agents 1).Polymeric biosurfactants

• These are high molecular weight compounds
• Best studied polymeric biosurfactants are emulson, liposon, alasun, lipomannan and other polysaccharide-protein complexes.

Emulson

• It is a complex extracellular acylated polysaccharide synthesized by the gram negative bacterium Acinetobacter calcoacetius
• An effective emulsifying agent for hydrocarbons in water.

Particulate Biosurfactants

These are of two types:
1). Extracellular vesicles uptake -

• These partition hydrocarbons to form microemulsions which play an important role in hydrocarbon uptake by microbial cells.

2). Whole microbial cell uptake -

• Whole bacterial cell act as surfactant.

Advantages of Biosurfactants

1. Biodegradability

• Biosurfactants are biodegradable in nature.
• Biosurfactants are able to broken down into more simple compounds by microbes through natural processes.
• Biosurfactants are environment friendly way and suited for bioremediation.

2. Low toxicity

• Biosurfactants are don't caue serious damage/harm of the biotic ecosystem since their toxicity level is low.
• They are non-toxic and on-mutagenic.

3. Biocompatibility and Digestibility

• Biosurfactants are well tolerated by living organisms.
• These when interact with living organisms do not change bioactivity of the organisms.

4. Availability of raw materials

• Biosurfactants can be produced from cheap raw materials like rapeseed oil, potato process effluent, oil refinery waste, cassava flour waste water, curd whey and distiller waste, sunflower oil etc. Which are available in large quantities.
• The carbon source may come from hydrocarbons, carbohydrates and /or lipids, which may be used separately or in combination with each other.

5. Acceptable production economics

• Depending on the application, biosurfactants can also be produced from industrial wastes and byproducts.
• This would be of particular interest for bulk production of biosurfactants which is economically acceptable.

6. Use in environmental control

• Biosurfactants are efficiently used in handling industrial emulsions, control of oil spills, biodegradation and detoxification of industrial effluents and in bioremediation of contaminated soil.

7. Specificity

• Being complex organic molecules with specific functional groups, are often specific in their action.
• This property used in detoxification of specific pollutants, de-emulsification of industrial emulsions, specific cosmetic pharmaceutical and food applications.

Disadvantages of Biosurfactants

• It is not produce at Large scale and as well as the products is very costly.
• Large quantities are particularly needed in petroleum and environmental applications, which, due to the bulk use may be expensive.
• Lack of obtaining pure substances which is of particular importance in pharmaceutical, food, and cosmetic applications.

Applications of Biosurfactants

1. Anti-adhesive agent

• Biosurfactants act as an anti adhesive agents.
• eg. surfactants from Streptococcus thermophilus, Pseudomonas fluorescens.

2. In food formulations

• In controlling agglomeration of fat globules, stabilization of aerated systems, improving texture and shelf life of starch containing products and improve consistency and texture of fat based products.
• Also used in bakery and ice-cream formulations where they act by controlling consistency, retarding staling and solubilizing flavor oils. Eg. rhamnolipids surfactants

3. Therapeutic and biomedical applications

• Antimicrobial activity - Eg: Pseudomonas aeruginosa Bacillus subtilis, Bacillus licheniformis, Candida antartica
• Anticancer activity - Eg: mannosylerythrintol, rhamnolipid, Sophorose lipid
• Anti-HIV and sperm -immobilising activity - Eg: sporolipid produced by Candid bombicola

4. Oil storage tank cleaning

• Biosurfactants used for reducing the viscosity of heavy oils, thereby facilitating recovers, transportation and pipelining.
• Eg: glycolipid

5. Biosurfactant used in mining

• Used for the dispersion of inorganic minerals in mining and manufacturing processes.
• Eg: biodispersan produced by Acinetobacter caleoaceticus.

6. Biosurfactants for agricultural use

• Maintaining soil health and protecting crops from various diseases.
• Low toxicity and biodegradability of biosurfactants have made these compounds superior to synthetic ones.
• They are used as biocontrol agents too.
• Needed for the hydrophilization of heavy soils to obtain good wettability and also to achieve equal distribution of fertilizers and pesticides in soils.
• Also used for formulating the poorly soluble organophosphorus pesticides.
• Biosurfactants used for the biodegradation of chlorinated pesticide and β endosulfan by 30%-40%.
• Eg: Bacillus subtilis
• Also mobilized the residual endosulfan isomers towards biodegradation.

Biosurfactants and Bioremediation

  Bioremediation aims at providing cost effective, contaminant specific treatments to reduce or removal of the concentration of individual or mixed environmental contaminants.

• The process of bioremediation can be carried out by utilizing plants microbes and or microbial products.
• Biosurfactants are used for the purpose of bioremediation.
• Biosurfactants in comparison to chemical surfactants have lower possible toxicity and shorter persistence in the environment. Eg: pseudomonas aeruginosa degrade polycyclic aromatic compounds.

Microbial Interaction with Pollutants

There is this three step mechanism for the uptake of pollutants by microbes

1. Uptake of pollutants dissolved in water
2. Direct uptake of the pollutant from the liquid-liquid interface.
3. Uptake of pseudosolubilized compounds.

Role of Biosurfactants in Bioremediation (How Biosurfactants are works?)

  Two mechanisms through which microbial surfactants can enhance bioremediation are:

• The increase of the substrate availability for microorganisms.
• Interaction with the cell surface to increase the hydrophobicity.
• Biosurfactants have the potential to promote the growth of bacteria on hydrocarbons.
• They do this by increasing the surface area between oil and water by emulsification, and by increasing pseudosolubility of hydrocarbons.
• Organic contaminants in soil have the tendency to be strongly adsorbed to soil particles. This can lead to extended remediation time.
• This time can be overcome through the application of biosurfactants. This relates to the process of mobilization
• The process of mobilization occurs at concentration below the CMC.
• At this concentration,  biosurfactants reduce the surface and interfacial tension, thus increase the contact of biosurfactant with soil, oil systems.
• Solubilization occurs at concentration above CMC.

• At above CMC concentration the hydrophobic tails of microbial surfactants connect together inside the micelle, while the hydrophilic heads are directed towards the aqueous phase.
• They are also able to increase the hydrophobicity of degrading micro organisms, thereby facilitating cells to access hydrophobic substrates more easily. It will allow microorganisms to directly contact oil drops and solid hydrocarbons.
• Another aspect is the enhanced bioremediation of heavy metal contaminated sites.
• Heavy metals are nonbiodegradable and they can only be transferred from one chemical form to another, which will be less toxic or biodegradable.
• Biosurfactants form complexes with heavy metals. These complexes can be removed by washing process.
• Biosurfactants are efficient in the biodegradation of aliphatic and aromatic hydrocarbons.

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B-Cell Receptors

 B-cells are originate and mature in the bone marrow. VDJ recombination occurs during the early development of B-cells. The light and heavy chains are selected by gene rearrangement and they together form the B-cell receptors.
  B-cell receptors are expressed on the cell membranes of the B cells.

B Cell Receptors (BCRs)
B-cell receptors are also known as membrane-bound immunoglobulins. This is because their structure is almost identical to the secreted antibodies, one difference is that B-cell receptors have an extra protein sequence at the C terminal of each heavy chain. This sequence anchors B cell receptors to the B cell membrane.
  It consists of a transmembrane domain and a cytoplasmic domain. 'That these extra domains are absent in the secreted antibodies'.

  Each mature naive B-cell Express membrane bound immunoglobulin M (Ig M) and membrane-bound immunoglobulin D (Ig D) as its B-cell receptors. Both of these receptors are of same antigenic specificity. This means both recognise the same epitope on an antigen.
• The function of these B-cell receptors is to recognise and bind the specific antigen.

For B-cell activation the nucleus of the b-cell should get the signal that specific antigen has been recognised and bound by the B-cell receptors.
• The cytoplasmic tails of B-cell receptors are very short.
• They cannot convey antigen recognition signal to the nucleus by themselves.

Signal to the nucleus is conveyed by two accessory proteins, which are associated with these B-cell receptors. These accessory proteins are Ig α (alpha) and Ig β (beta).
• Together they form a heterodimer held together by a disulfide bond.
• Ig α & Ig β chains are glycoproteins and they are transmembrane molecules.
  Their extracellular portions consist of single Ig like domain. this heterodimer conveys the intracellular signals, indicating that antigen has been found.
• Ig α and Ig β molecules are expressed only in B-cells. the designation IgE in these accessory proteins represents that these proteins are associated with the immunoglobulins.

  B-cell receptor complex is made up of a membrane bound immunoglobulin and Ig α and Ig β heterodimer.
• Antigen recognition and binding is performed by the membrane bound immunoglobulin that is the B-cell receptor.
• Signal to the nucleus is conveyed by heterodimer Ig α and Ig β

Another important requirement of B-cell to generate the activation signal is that
- Multiple B-cell receptors must be brought close together on the surface of the B cell.
  Antigen generally has multiple copies of the same epitope on its surface. Each epitope can bind adjacent B-cell receptors on one B-cell. Once bound surface b-cell receptors begin to cluster. This process activates the accessory proteins like Ig α and Ig β and initiate the signaling process. 

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Antibiotics - Mode of Action

   The antibiotics are the chemotheraputic agents. The target of antibiotics are microorganisms more precisely bacteria. once treated with the correct antibiotics the bacterial infection is eliminated. In general antibiotics do not function against viruses or fungi instead they are specifically directed against bacteria.

  The first antibiotic was discovered by Sir Alexander Fleming in 1928. He observed a spot in his petri dish where bacteria(staphylococcus aureus) did not grow around fungi(Penicillium notatum). These fungi produced an antibacterial substance which killed the surrounding microbes. It is commonly known under the term penicillin. This discovery is often described as one of the greatest victories ever achieved over disease.

General Mode of Action of Antibiotics

  The antibiotics act in general by killing the pathogen or inhibiting their growth. This is achieved by following general modes of attack.

1. By Inhibition of cell wall synthesis.
2. By Inhibition of protein synthesis.
3. By Interfering with nucleic acid synthesis.
4. By interfering with cell membrane structure and function.
5. By inhibition specific enzyme system of the cell.

Inhibition of cell wall synthesis

  Many Antibiotics are able to act by inhibiting the synthesis of peptidoglycan of bacterial cell wall.
  Peptidoglycan is the principal chemical constituent of the bacterial cell walls. Inhibition of synthesis of peptidoglycan, therefore, Interferes with the formation of new cell wall.

  Thus, if the bacteria are allowed to grow in presence of the agents that Inhibit peptidoglycan synthesis, cells will continue to grow and divide, but without formation of new cell wall material. As a result, the cells will be ultimately converted Into spheroplasts and protoplasts.

  These, spheoplasts and protoplasts are highly fragile and get lysed. Thus, the cells, growing in presence of agents inhibiting cell wall synthesis are lysed.

  Since, peptidoglycan is found only in bacteria and not in other cukaryotic cells, these agents possess selective toxicity for bacteria and become sultable for the chemotherapeutic application.

  Further, these antibiotics are usually more effective on gram- positive bacteria. This is due to the higher content of peptidoglycan in cell walls of gram-positive bacteria as compared to gram-negative bacteria.
  The table shows different antibiotics inhibiting peptidoglycan synthesis at different stages of its biosynthesis.

Antibiotics Inhibiting cell wall biosynthesis

Inhibition of protein synthesis

  Proteins are important constituent of all living cells. They may be

• Structural proteins, being part of various cell structures.
• Functional proteins, mainly enzymes, responsible for various metabolic activities performed by cells.

Inhibition of protein synthesis, therefore, results into the inhibition of growth of microorganisms.

 Synthesis of proteins takes place on ribosome involving following major steps. 

1. Charging of tRNA with amino acids.
2. Formation of mRNA-ribosome complex.
3. Binding of aminoacyl tRNA to ribosome.
4. Formation of peptide bond involving a peptidal transfer reaction where a new amino acid is transferred from aminoacyl tRNA to a growing peptide chain.
5. Translocating step where deacylated tRNA is removed from ribosome and site is made vacant for new aminoacyl tRNA to bind on ribosome so that the cycle leading to growth of peptide chain continues.
6. Release of grown peptide chain from ribosome, when complete mRNA is translated. This signal for termination of protein synthesis is provided by nonsense codons of mRNA. This is followed by dissociation of ribosome and mRNA.

  This shows that any agent, which can bind with ribosome, will be able to inhibit protein synthesis. Most antibiotics, capable of inhibiting protein synthesis are able to bind either 30S or 50S submit of 70S ribosome of prokaryotes. Eukaryotes have 80S ribosome.

  These antibiotics are not able to bind with them and hence do not interfere with their functioning. Thus, the antibiotics affecting protein synthesis have selective toxicity for bacteria and therefore, they possess chemotherapeutic value.
  Table shows various antibiotics that inhibit protein synthesis at difference stages.

Examples of Antibiotics interfering protein synthesis

Inhibition of nucleic acid synthesis

  Like proteins, nucleic acids are also vital macromolecules of cells. Two classes of nucleic acids occur in cell.

1. RNA, which mainly participate in the protein biosynthesis.
2. DNA, which carries the entire genetic information for the characters to be expressed by the organisms. by acting as the hereditary material.

  Inhibition of the synthesis of nucleic acids is, therefore, able to inhibit the growth of microorganisms.

1) Inhibition of RNA synthesis

  Certain antibiotics are able to bind with the key enzyme involved in RNA synthesis; RNA polymerase. Bindings of antibiotics to this enzyme Interfere with the functioning of the enzyme and prevent RNA synthesis. e.g. rifamycin and rifampicin.

Certain other antibiotics bind with GC pair of the DNA and prevent unfolding of DNA. required for transcription (RNA synthesis). Thus, they Inhibit RNA synthesis. e.g. Mitomycin C, Actinomycin D.

2) Inhibition of DNA synthesis

  Certain antibiotics are able to inhibit replication of chromosomal DNA, by

• binding to the enzyme DNA polymerase, required for chromosome replication or
• binding to GC pair of DNA and prevent its unfolding. essential for DNA replication.

 Table summarizes agents that inhibit Nucleic acid synthesis.

Antibiotics targeting nucleic acid synthesis and functioning

Damage to cytoplasmic membrane

  Certain chemotherapeutic agents are able to inhibit or kill the microorganisms by damaging the functioning of cell membrane. Cell membrane is a selectively permeable membrane and it maintains concentration of the cytoplasm by control of entry and exit of molecules across the membrane.

  Binding of agents to cell membrane, therefore, interferes with cell's permeability barrier and causes leakage of cellular molecules, leading to cell's death. These antibiotics possess toxicity to tissues. Therefore they have limited application in chemotherapy.
e.g. Polymyxins, Gramicidin. Tyrocidines. Nystatin, Amphotericin B.

Inhibition of specific enzyme system

  Certain chemotherapeutic agents are able to bring about inhibition of a specific enzyme reaction, which may be essential for the operation of a specific biochemical pathway leading to the biosynthesis of a vital substance essential for the growth.

Inhibition of this pathway, therefore, deprives cell from availability of this vital substance essential for growth (which may be a vitamin, amino acid, coenzyme etc.). As a result, the growth of organisms is inhibited. 

  Table summarizes some of such chemotherapeutic agents.

Examples of Antibiotics agent targeting action of enzyme

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