Microorganism in Human Welfare

CHAPTER: 11

Microorganism in Human Welfare

Ø    Fermentation

Ø    Antibiotic

Ø    Biofertilizers

Ø    Biopesticides

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Microorganisms in Human Welfare:

Application of Microorganism:

                    Microorganisms play an important role in sustaining life on this planet and in our daily life through the following activities:

1.      Transformation of matter:

                    Microorganisms degrade dead organic matter and return to the atmosphere in inorganic form. They complete the cycle of matter and are responsible for transformation of C, N and S and other important elements which are essential for life.

2. Biological nitrogen fixation:

        They fix nitrogen from atmosphere and make it available to the plants in usable form. Important microorganisms under this category include, Rhizobium, Azotobacter, Azospirillum etc.

3. Mycorrhiza:

                    Association of roots of many plants with fungi forms a composite structure called mycorrhiza. Fungus helps in absorption of mineral salts from soil and plant in turn provides carbohydrates for the growth of fungus.

4. Silage:

                    This method is used to preserve feed with its characteristic flavor, taste and nutritive value. Leaves of green plants are compacted in size and some molasses is added. Lactic acid bacteria develop and produce lactic acid which helps to conserve the cattle feed.

5. Cellulose degradation in Rumen:

                    Ruminants feed on straw and grass which contains about 50 % cellulose. There is symbiotic association of microorganisms with rumen for degradation of cellulose and about 10¹⁰ – 10 ¹¹ cells/ml of different bacteria are usually present in the rumen. Most important of these include Ruminococcus and Clostridium.

6. Biogas:

                    Animal waste products and cellulose containing waste is fermented by microorganisms (Methanogens). Animal excreta is preserved in rotting sediment and methane gas so formed is used as a fuel.

7. Composting:

                    Decomposition of organic matter by microorganisms to convert it into nutrient rich manure is known as composting. Bacillus, Aspergillus and Thermoactinomyces are important in this process.

8. Industrial uses:

                    Different microorganisms are used for the production of wide range of products at industrial scale. These include alcoholic beverages, antibiotics, enzymes, pharmaceuticals etc.

Fermentation:

•Fermentation is a metabolic process that converts sugar to acids, gases and/or alcohol.

•It occurs in yeast and bacteria, but also in oxygen-starved muscle cells, as in the case of lactic acid fermentation.

•Fermentation is also used more broadly to refer to the bulk growth of microorganisms on a growth medium.

•Fermentation consists of inoculating the medium  of suspended or dissolved raw material with sufficient quantum of inoculum of the desired organisms, and allowing it to multiply and ferment for a period of time.

Types  Of Fermentation   Processes:

                     Industrial fermentation processes may be divided into two main types, with various combinations and modifications. These are batch fermentations and continuous fermentations.

1.      Batch fermentations:

•A tank of fermenter is filled with the prepared mash of raw materials to be fermented.

•The temperature and pH for microbial fermentation is properly adjusted, and occasionally nutritive supplements are added to the prepared mash.

•The mash is steam-sterilized in a pure culture process.

•The inoculum of a pure culture is added to the fermenter, from a separate pure culture vessel.

•Fermentation proceeds, and after the proper time the contents of the fermenter, are taken out for further processing.

•The fermenter is cleaned and the process is repeated. Thus each fermentation is a discontinuous process divided into batches.

2. Continuous fermentation:

•In continuous fermentation, the substrate is added to the fermneter continuously at a fixed rate.

•This maintains the organisms in the logari­thmic growth phase. The fermentation products are taken out conti­nuously.

•The design and arrangements for continuous fermentation are somewhat complex.

Application of fermentation technology:

1.     Alcoholic Fermentation:

•Ethyl alcohol can be produced by fermentation of any carbohy­drate containing a fermentable sugar, or a polysaccharide that can be hydrolysed to a fermentable sugar.

•The equation that describes the net result of alcoholic fermentation by yeast is :

                    C₆H₁₂ O₆  => 2C₂H₅OH + 2 CO₂

•It indicates that a sugar is the substrate and that the process is anaerobic.

•Selected strains of Saccharomyces cerevisiae are commonly employed for fermentation.

• It is imperative that the strain must have a high tolerance for alcohol, must grow vigorously and produce a large quantity of alcohol.

Distilled Alcoholic Beverages:

•Whisky, Gin, Rum, Brandy etc are alcoholic products of fermentation using different raw materials.

•The type of beverage produced is determined by the nature of the plant material.

•The alcohol is distilled and the distillate is aged in oak barrels for at least three years.

Malt  Beverages:

•Beer and ale are the principal malt beverages produced by the fermentation process with microorganisms.

Wine:

•This is the fermentation product of fruit juices, primarily grapes.

Production Of Vinegar:

•Vinegar may be defined as the condiment made from sugary or starchy material by alcoholic and subsequent acetic acid fermentaions.

•Vinegar is the product resulting from the conversion of ethyl alcohol to acetic acid by a group of widely distributed bacteria of the genus Acetobacter.

•The microorganisms that produce acetic acid from ethyl alcohol are species of Acctobacter—A. orkannt, A. orleansis, A.schutzenbachi, A.Aceti and others.

•The biochemical reaction by which they form acetic acid from ethanol is as follows :-_:

                    2CH₃ CH₂OH + 0₂      =>        2CH₃CHO + 2H₂O

                    2CH₃ CHO + O₂         =>        2CH₃COOH

Organic Acids:

                    Fermentation process with some bacteria is utilized for producing several organic acids.

1.      Lactic acids: Lactobacillus bulgaricus, or L. delbrueckii

2.      Citric acids: Aspergillus niger, A. wentii

3.      Fumaric acids: Rhizopus nigricans

Antibiotics:

•Large scales of antibiotics are also produced by fermentation process.

•The fermentation medium generally consists of glucose as carbon source, together with soyabean meal and mineral salts.

•The fermentation process is carried under submerged aerobic conditions.

•The separation of the antibiotics from the broth is accomplished through different procedures.

 Microorganisms & its Antibiotics:

1.      Streptomyces griseus:  Streptomycin

2.      S. aureofaciens:  Tetracycline

3.      S. venezuele:  Chloramphenicol

4.      S. nouresii:    Nystatin

5.      S. erythreus: Erythromycin

6.      S. fradiae:  Neomycin

Biofertilizers:

•A biofertilizer is a substance which contains living microorganisms which, when applied to seed, plant surfaces, or soil, colonizes the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients to the host plant.

•Bio-fertilizers add nutrients through the natural processes of nitrogen fixation, solubilizing phosphorus, and stimulating plant growth through the synthesis of growth-promoting substances.

•The microorganisms in bio-fertilizers restore the soil's natural nutrient cycle and build soil organic matter.

Important of Biofertilizers:

•help to get high yield of crops by making the soil rich with nutrients.

•Biofertilizers have replaced the chemical fertilizers as chemical fertilizers are not beneficial for the plants.

•Plant growth can be increased if biofertilizers are used, because they contain natural components which do not harm the plants.

•Destroy harmful components of the soil which cause disease in the plants.

•Are not costly

•Environment friendly.

Types of Biofertilizers:

1. Rhizobium - 

•This belongs to bacterial group and the classical example is symbiotic nitrogen fixation.

•The bacteria infect the legume root and form root nodules within which they reduce molecular nitrogen to ammonia which is reality utilized by the plant to produce valuable proteins, vitamins and other nitrogen containing compounds.

•The site of  symbiosis is within the root nodules.

2.  Azotobacter -

•It is the important and well known free living nitrogen fixing aerobic bacterium.

•It is used as a Bio-Fertilizer for all non leguminous plants especially rice, cotton, vegetables etc. 

•Azotobacter cells are not present on the rhizosplane but are abundant in the rhizosphere region.

•The lack of organic matter in the soil is a limiting factor for the proliferation of Azotobacter in the soil.

3. Azospirillum:

•It belongs to bacteria and is known to fix the considerable quantity of nitrogen in the range of 20- 40 kg N/ha in the rhizosphere in non- non-leguminous plants such as cereals, millets, Oilseeds, cotton etc.

4. Cyanobacteria:

•Both free-living as well as symbiotic cyanobacteria (blue green algae) have been harnessed in rice cultivation.

• A composite culture of BGA having heterocystous Nostoc, Anabaena, Aulosira etc. is given as primary inoculum in trays, polythene lined pots and later mass multiplied in the field for application as soil based flakes to the rice growing field at the rate of 10 kg/ha.

5.     Azolla:

•Azolla is a free-floating water fern that floats in water and fixes atmospheric nitrogen in association with nitrogen fixing blue green alga Anabaena azollae. Azolla fronds consist of sporophyte with a floating rhizome and small overlapping bi-lobed leaves and roots.  

•Azolla is used as biofertilizer for wetland rice and it is known to contribute 40-60 kg N/ha per rice crop.

6.     Phosphate solubilizing microorganisms(PSM):

•Several soil bacteria and fungi, notably species of Pseudomonas, Bacillus, Penicillium, Aspergillus etc. secrete organic acids and lower the pH in their vicinity to bring about dissolution of bound phosphates in soil.

•Increased yields of wheat and potato were demonstrated due to inoculation of peat based cultures of Bacillus polymyxa and Pseudomonas striata.

•Currently, phosphate solubilizers are manufactured by agricultural universities and some private enterprises and sold to farmers through governmental agencies.

7. AM fungi-

•An arbuscular mycorrhiza (AM Fungi) is a type of mycorrhiza in which the fungus penetrates the cortical cells of the roots of a vascular plant.

•Transfer of nutrients mainly phosphorus and also zinc and sulphur from the soil to the cells of the root cortex is mediated by intracellular obligate fungal endosymbionts of the genera Glomus, Gigaspora, Acaulospora, Sclerocysts and Endogone which possess vesicles for storage of nutrients and arbuscles for funneling these nutrients into the root system.

8. Silicate solubilizing bacteria (SSB)- 

•Microorganisms are capable of degrading silicates and aluminum silicates.

•During the metabolism of microbes several organic acids are produced and these have a dual role in silicate weathering.

9. Plant Growth Promoting Rhizobacteria (PGPR)-

•The group of bacteria that colonize roots or rhizosphere soil and beneficial to crops are referred to as plant growth promoting rhizobacteria (PGPR).

Mass Inoculum Production method of some Biofertilizer:

1.      Rhizobium:

•Rhizobium forms white, translucent, glistening, elevated and comparatively small colonies on this medium.

•Moreover, Rhizobium colonies do not take up the colour of congo red dye added in the medium.

•Those colonies which readily take up the congo red stain are not rhizobia but presumably Agrobacterium, a soil bacterium closely related to Rhizobium.

Culture Media: Yeast Extract Mannitol Broth

Mass Inoculum Production of Rhizobium:

•Prepare appropriate media(Yeast extract Mannitol Broth) to the bacterial inoculant in 250 ml, 500 ml, 3 litre and 5 litre conical flasks and sterilize.

• 250 ml flask is inoculated with  bacterial  strain under aseptic condition

•Keep the flask in room temp. in rotary shaker (200 rpm) for 5- 7 days.

•Observe for growth of the culture which serves as the starter culture.

•Using the starter culture  inoculate the larger flasks .

•The above media is prepared in large quantities in fermentor, sterilized well, cooled and kept it ready.

•The media in the fermentor is inoculated with culture grown in 5 litre flask.

•The cells are grown in fermentor by providing aeration and given continuous stirring.

•The broth is checked for the population of inoculated organism and contamination if any at the growth period.

•The cells from the fermentor are harvested and mixed with carrier materials and finally packaging is done and marketed.

•The use of ideal carrier material is necessary in the production of good quality biofertilizer.

•Peat soil, lignite, vermiculite, charcoal, press mud, farmyard manure and soil mixture can be used as carrier materials.

•The neutralized peat soil/lignite are found to be better carrier materials for biofertilizer production

Ø  Azospirillum, Azotobacter and phosphobacteria Can also prepared in the same method as Rhizobium is prepared.

Ø  The culture media are:

1.      Azospirillum:          Dobereiner's malic acid broth with NH4Cl (1g per liter)

2.      Azotobacter:           Waksman medium No.77 (N-free Mannitol Agar Medium)

3.      Phosphobacteria:     Pikovskaya’s Broth

1.      Mass production of Mycorrhizal Biofertilizer:

Steps:

•A trench (1m x 1m x 0.3m) is formed and lined with black polythene sheet to be used as a plant growth tub.

•Mixed 50 kg of vermiculite and 5 kg of sterilized soil and packed in the trench up to a height of 20 cm

•Spread 1 kg of AM inoculum (mother culture) 2-5 cm below the surface of vermiculite

•Maize seeds surface sterilized with 5% sodium hypochlorite for 2 minutes are sown

•Applied 2 g urea, 2 g super phosphate and 1 g muriate of potash for each trench at the time of sowing seeds.  Further 10 g of urea is applied twice on 30 and 45 days after sowing for each trench

•Quality test on AM colonization in root samples is carried out on 30th and 45th day

•Stock plants are grown for 60 days (8 weeks).  The inoculum is obtained by cutting all the roots of stock plants.  The inoculum produced consists of a mixture of vermiculite, spores, pieces of hyphae and infected root pieces.

3. Mass production and field application of cyanobacteria:

I. Multiplication in trays

•Big metallic trays (6’x 3’x 6”lbh) can be used for small scale production

•Take 10 kg of paddy field soil, dry powder well and spread

•Fill water to a height of 3”

•Add 250 g of dried algal flakes (soil based) as inoculum

•Add 150 g of super phosphate and 30 g of lime and mix well with the soil

•Sprinkle 25 g carbofuran to control the insects

•Maintain water level in trays

•After 10 to 15 days, the blooms of BGA will start floating on the water sources

•At this stage stop watering and drain. Let the soil to dry completely

•Collect the dry soil based inoculum as flakes

•Store in a dry place.  By this method 5 to 7 kg of soil based inoculum can be obtained.

II. Multiplication under field condition

•Select an area of 40 m2 (20m x 2m) near a water source which is directly exposed to sunlight.

•Make a bund all around the plot to a height of 15 cm and give it a coating with mud to prevent loss of water due to percolation.

•Plot is well prepared and levelled uniformly and water is allowed to a depth of 5-7.5 cm and left to settle for 12 hrs.

•Apply 2 kg of super phosphate and 200 g lime to each plot uniformly over the area.

•The soil based composite starter culture of BGA containing 8-10 species @ 5 kg / plot is powdered well and broadcasted.

•Carbofuran @ 200 g is also applied to control soil insects occurring in BGA.

•Water is let in at periodic intervals so that the height of water level is always maintained at 5 cm.

•After 15 days of inoculation, the plots are allowed to dry up in the sun and the algal flakes are collected and stored.

4.Mass multiplication of Azolla under field conditions:

Procedure:

•Select a wetland field and prepare thoroughly and level uniformly.

•Mark the field into one cent plots (20 x 2m) by providing suitable bunds and irrigation channels.

•Maintain water level to a height of 10 cm.

•Mix 10 kg of cattle dung in 20 litres of water and sprinkle in the field.

•Apply 100 g super phosphate as basal dose.

•Inoculate fresh Azolla biomass @ 8 kg to each pot.

•Apply super phosphate @ 100 g as top dressing fertilizer on 4th and 8th day after Azolla inoculation.

•Apply carbofuran (furadan) granules @ 100 g/plot on 7th day after Azolla inoculation.

•Maintain the water level at 10 cm height throughout the growth period of two or three weeks.

•Observations

•Note the Azolla mat floating on the plot. Harvest the Azolla, drain the water and record the biomass.

Biopesticides:

•Biopesticides are certain types of pesticides derived from such natural materials as animals, plants, bacteria, and certain minerals.

•For example, canola oil and baking soda have pesticidal applications and are considered biopesticides.

Types of Biopesticides:

Biopesticides fall into three major classes:

•Microbial pesticides

•Plant pesticides

•Biochemical pesticides

1.      Microbial pesticides:

•consist of a microorganism (e.g., a bacterium, fungus, virus, or protozoan) as the active ingredient.

•Microbial pesticides can control many different kinds of pests, although each separate active ingredient is relatively specific for its target pest[s]. For example, there are fungi that control certain weeds, and other fungi that kill specific insects.

•The most widely used microbial pesticides are subspecies and strains of Bacillus thuringiensis, or Bt.

•Each strain of this bacterium produces a different mix of proteins, and specifically kills one or a few related species of insect larvae.

• While some Bt's control moth larvae found on plants, other Bt's are specific for larvae of flies and mosquitoes.

•The target insect species are determined by whether the particular Bt produces a protein that can bind to a larval gut receptor, thereby causing the insect larvae to starve.

2. Plant pesticides:

•These are pesticidal substances that plants produce from genetic material that has been added to the plant.

•For example, scientists can take the gene for the Bt pesticidal protein and introduce the gene into the plant's own genetic material. Then the plant, instead of the Bt bacterium, manufactures the substance that destroys the pest.

3. Biochemical pesticides:

•These are naturally occurring substances that control pests by non-toxic mechanisms.

• Conventional pesticides, by contrast, are generally synthetic materials that directly kill or inactivate the pest.

•Biochemical pesticides include substances, such as insect sex pheromones that interfere with mating as well as various scented plant extracts that attract insect pests to traps.

Bacillus thuringiensis:

•It is a well-known insecticide example.

•The toxin from B. thuringiensis (Bt toxin) has been incorporated directly into plants through the use of genetic engineering.

•it has little effect on otherorganisms, and is more environmentally friendly than synthetic pesticides.

• However, at least one scientific study has suggested there may be a negative impact on the liver and kidneys of mammals with Bt toxin in their diet.

Ø  Other microbial control agents include products based on:

•entomopathogenic fungi

•(e.g.Beauveria bassiana, Lecanicillium spp., Metarhizium spp.),

•plant disease control agents: include Trichoderma spp. and Ampelomyces quisqualis (a hyper-parasite of grape powdery mildew); Bacillus subtilis is also used to control plant pathogens.

•entomopathogenic viruses (e.g.. Cydia pomonella granulovirus).

•weeds and rodents have also been controlled with microbial agents.

Ø  Various naturally occurring materials, including fungal and plant extracts, have been described as biopesticides. Products in this category include:

•Insect pheromones and other semiochemicals

•Fermentation products such as Spinosad (a macro-cyclic lactone)

•Chitosan: a plant in the presence of this product will naturally induce systemic resistance (ISR) to allow the plant to defend itself against disease, pathogens and pests.

•Biopesticides may include natural plant-derived products, which include alkaloids, terpenoids, phenolics and other secondary chemicals. Certain vegetable oils such as canola oil are known to have pesticidal properties. Products based on extracts of plants such as garlic are also cosidered as pest controller

•Naturally occurring minerals such as baking soda may also have pesticidal applications.

Ø  The most commonly used plants are neem (Azadirachta indica), pongamia (Pongamia)and mahua (madhuca indica).

Application of Biopestcides:

•Use against crop diseases

•Can be use under moderate to severe disease pressure

•Can be apply with mixing fungicide into grape production

•Used to control soil-borne fungal pathogens

•Used to control internal seed borne fungal pathogen.

Advantages of Biopesticides:

•Harmful residues not detected

•Can be cheaper than chemical pesticides when locally produced.

•Can be more effective than chemical pesticides in the long-term.

•Biodegradable

Disadvantages of Biopesticides:

•High specificity: require exact pests or pathogens

•Slow speed action

•Variable efficacy due to influence of biotic and abiotic factors

•Pests that undergo mutation is difficult to control