Effect of various environmental factors on microbial growth- Effect of Temperature on the growth of microorganisms

The growth of microorganisms also is greatly affected by the chemical and physical nature of their surroundings. Microorganisms being mostly unicellular and poikilothermic (coldblooded), any change in the environment is easily reflected on them.  An understanding of environmental influences aids in the control of microbial growth and the study of the ecological distribution of microorganisms. 

Procaryotes are present anywhere life can exist. Many habitats in which procaryotes thrive would kill most other organisms. Some microorganisms can adapt to extreme and inhospitable environments. Procaryotes such as Bacillus infernus are even able to live over 1.5 miles below the Earth’s surface, without oxygen and at temperatures above 60°C. Microorganisms that grow in such harsh conditions are often called extremophiles.

Effect of Temperature on the growth of microorganisms 

Environmental temperature has a great effect on microorganisms since their temperature varies with that of the external environment.

Temperature has a major effect on microbial growth due to the temperature sensitivity of enzyme-catalysed reactions. Each enzyme has an optimum temperature at which it functions optimally. At temperatures below the optimum, it ceases to be catalytic. As the temperature rises from this low temperature, the rate of catalysis increases, till the optimal temperature is reached. The velocity of the reaction will roughly double for every 10°C rise in temperature. As the rate of each reaction increases, metabolism is more active, and the microorganism grows faster. However, beyond a certain point, further increases actually slow growth, and sufficiently high temperatures are lethal.

Microbial growth thus has distinct cardinal temperatures—minimum, optimum, and maximum growth temperatures. 

Based on their temperature ranges for growth, microorganisms  can be placed in one of five classes.

1. Psychrophiles grow well at 0°C and have an optimum growth temperature of 15°C or lower; the maximum is around 20°C. They are readily isolated from Arctic and Antarctic habitats; because 90% of the ocean is 5°C or colder, it is an enormous habitat for psychrophiles. Chlamydomonas nivalis is a psychrophilic algae seen in snowfield or glacier turning it pink with its bright red spores. Pseudomonas, Vibrio, Alcaligenes, Bacillus, Arthrobacter, Moritella, Photobacterium, and Shewanella are common psychrophiles  among bacteria. A psychrophilic archaeon, Methanogenium, has been isolated from Ace Lake in Antarctica.

Psychrophilic microorganisms have adapted to their environment in several ways. Their enzymes, transport systems, and protein synthetic mechanisms function well at low temperatures. The cell membranes of psychrophilic microorganisms have high levels of unsaturated fatty acids and remain semifluid when cold. Indeed, many psychrophiles begin to leak cellular constituents at temperatures higher than 20°C because of cell membrane disruption.

2. Many species can grow at 0 to 7°C even though they have optima between 20 and 30°C, and maxima at about 35°C. These are called psychrotrophs or facultative psychrophiles. Psychrotrophic bacteria and fungi are major factors in the spoilage of refrigerated foods.

3. Mesophiles are microorganisms with growth optima around 20 to 45°C; they often have a temperature minimum of 15 to 20°C. Their maximum is about 45°C or lower. Most microorganisms probably fall within this category. Almost all human pathogens are mesophiles, because their environment is a fairly constant 37°C.

4. Some microorganisms are thermophiles; they can grow at temperatures of 55°C or higher. Their growth minimum is usually around 45°C and they often have optima between 55 and 65°C. The vast majority are procaryotes although a few photosynthetic protists and fungi are thermophilic. These organisms flourish in many habitats including composts, self-heating hay stacks, hot water lines, and hot springs.

5. Hyperthermophiles are Thermophiles which can grow at 90°C or above and with maxima above 100°C. They usually do not grow well below 55°C.  Their growth optima can be between 80°C and about 113°C are called hyperthermophiles. Pyrococcus abyssi and Pyrodictium occultum are examples of marine hyperthermophiles found in hot areas of the seafloor.

High temperatures damage microorganisms by denaturing enzymes, transport carriers, and other proteins. Temperature also has a significant effect on microbial membranes. At high temperatures, the lipid bilayer melts and disintegrates. Thus, when organisms are above their optimum temperature, both function and cell structure are affected. At very low temperatures, membranes solidify.  If temperatures are  low, function is affected but not necessarily cell chemical composition and structure.

Thermophiles differ from mesophiles in many ways.

• They have more heat-stable enzymes and protein synthesis systems, which function well at high temperatures.
• Heat-stable proteins have highly organized, hydrophobic interiors; more hydrogen bonds and other noncovalent bonds strengthen the structure. 
• Larger quantities of amino acids such as proline also make the polypeptide chain less flexible.
• The proteins are stabilized and aided in folding by special chaperone proteins.
• In thermophilic bacteria, DNA is stabilized by special histone like proteins.
• Their membrane lipids are more saturated, more branched, and of higher molecular weight. This increases the melting points of membrane lipids thus making them temperature stable.
• Archaeal thermophiles have membrane lipids with ether linkages, which protect the lipids from hydrolysis at high temperatures. Sometimes archaeal lipids actually span the membrane to form a rigid, stable monolayer.

 

The cardinal temperatures for a particular species are not rigidly fixed but often depend to some extent on other environmental factors such as pH and the available nutrients. For example, Crithidia fasciculate, a flagellated protist living in the gut of mosquitoes, will grow in a simple medium at 22 to 27°C. However, to grow at 33 to 34°C extra metals, amino acids, vitamins, and lipids are required.

The cardinal temperatures vary greatly between microorganisms. The temperature optimum is always closer to the maximum than to the minimum. The growth temperature range for a particular microorganism usually spans about 30 degrees. Some species (e.g., Neisseria gonorrhoeae) have a small range (stenothermal); others, like Enterococcus faecalis, will grow over a wide range of temperatures(eurythermal).

The major microbial groups differ from one another regarding their maximum growth temperatures. The upper limit for protists (protozoa/algae) is around 50°C. Some fungi can grow at temperatures as high as 55 to 60°C. Procaryotes can grow at much higher temperatures than eucaryotes.

Optima usually range from 0°C to 75°C, whereas microbial growth occurs at temperatures extending from less than ^-20°C to over 120°C. Some archaea can even grow at 121°C (250°F), the temperature normally used in autoclaves.

Methods of transmission of infections.

                      

1. Contact Transmission

Contact transmission include direct contact or indirect contact.

Direct contact

Person-to-person transmission is a form of direct contact transmission. Here the agent is transmitted by physical contact between two individuals through actions such as touching, kissing, sexual intercourse, or droplet sprays. Contact transmission may also be site-specific; for example, some diseases can be transmitted by sexual contact but not by other forms of contact. eg. syphilis, gonorrhea

Indirect contact

 Indirect contact transmission involves inanimate objects called fomites -clothes, beddings, towels etc., that become contaminated by pathogens from an infected individual or reservoir . For example, an individual with the common cold may sneeze, causing droplets to land on a fomite such as a tablecloth or carpet, or the individual may wipe her nose and then transfer mucus to a fomite such as a doorknob or towel.

2. Inhalation (Air-borne transmission)

Respiratory infections such as influenza, tuberculosis may be spread by inhalation of droplets (airborne droplet nuclei) containing microorganisms generated during coughing, sneezing and talking. These microorganisms land on another person, entering through  conjunctivae, nasal mucosa or mouth. 

 3. Inoculation

Vector-borne transmission and infections due to animal bites like rabies are transmitted by inoculation . Vectors  that are capable of transmitting diseases are flies, mites, fleas, ticks, rats, and dogs. The most common vector for disease is the mosquito. Mosquitoes are vectors for malaria, West Nile virus, dengue fever, and yellow fever. Tetanus spores implanted in deep wounds following soil contamination is another example.

 4. Ingestion

Intsetinal infections are generally by fecal-oral route transmission where microorganisms enter the body through ingestion of contaminated food (food-borne) and water (water-borne). Unclean hands can also lead to such infections. In the intestine, these microorganisms multiply and are shed from the body in feces. In the absence of proper hygienic and sanitation practices, the microorganisms in the feces may contaminate the water supply through inadequate sewage treatment and water filtration. Diseases caused by fecal-oral transmission include diarrhea, typhoid, cholera, polio and hepatitis.

5. Congenital transmission (Perinatal Mother-to-Child Transmission (PMTCT; Vertical Transmission)

Pathogens which cross the placental barrier, infect the fetus in utero. This is also called vertical transmission. Cytomegalovirus is the most common,  Rubella virus, HIV, Syphilis, Gonorrhea  is also transmitted vertically. If it causes malformations in the fetus, such diseases are termed teratogenic infections. Rubella, cytomegalovirus, varicella, herpes simplex, toxoplasma, syphilis, etc. are teratogenic infections.

6. Iatrogenic transmission

Infections caused by medical intervention or iatrogenic infections are mostly  by inoculation. There are many organisms that can be transmitted to health care workers in a clinical setting, then it is occupational threat.

• Using unsterile syringes/equipment
• Transfusion of infected blood, use of contaminated syringes, needles etc.
• Exchange transfusion, dialysis, organ transplant surgery- increased possibilities of cases of iatrogenic Hepatitis B and C
• Laboratory personnel handling infectious material (anthrax, plague, tuberculosis)

 

Sources of infections

Sources of infections

Mainly four- Humans, Animals, Insects, Environment (soil, food and water, fomites)

1.     Human reservoir

The most important source or reservoir of infection for humans is the man himself. He may be a case or carrier

a. Case (Patient)

A case is defined as “a person having the particular disease, health disorder or condition under investigation”.

b. Carriers

A carrier is defined as a person (or animal) that harbours a specific pathogen and serves as a potential source of infection for others.

“Typhoid Mary” is a classic example of a carrier

Carriers may be classified as:

(a) Healthy: Healthy carriers carrying the pathogens without suffering from the disease.

(b) Convalescent: A person who has recovered from the disease but continue to harbour the pathogens in the body and shed them (during the period of convalescence).

(c) Contact Carrier- Carrier who acquires the pathogen from a patient

(d) Paradoxical Carrier- Carrier who acquires a pathogen from another carrier

Depending on the duration of carriage, carriers can be

(e) Temporary: Temporary carriers are those who shed the infectious agent for short periods of time (less than six months)

(f) Chronic: A chronic carrier is one who excretes the infectious agent for indefinite periods- many years to life-long. Chronic carrier state occurs in a number of diseases, e.g., typhoid fever, hepatitis B, dysentery, malaria, gonorrhoea, etc.

Chronic carriers are far more important sources of infection than cases.

2. Animals

Animals are a good source of infection, they cause zoonoses (animal acquired infections in man). Some common examples:

Zoonoses

Bacterial- Plague, from rats

Viral- Rabies, from dogs

Protozoal – Toxoplasmosis, from cats

Helminthic – Tapeworm from cattle/pig

Fungal – Zoophilic dermatophytes from cats & dogs

Some animals act as reservoirs of human infections- they maintain the parasite/microorganism in nature but do not show any symptoms themselves. They are called reservoir hosts.

3. Insects

Insects are a good Sources of infection. Some germs rely on insect carriers — such as mosquitoes, fleas, lice or ticks — to move from host to host. These carriers are known as vectors. Mosquitoes can carry the malaria parasite or West Nile virus, and deer ticks may carry the bacterium that causes Lyme disease. Blood-sucking insects cause many arthropod-borne diseases.

Transmission may be

Mechanical- pathogen transmitted mechanically by the vector. Eg; Transmision of dysentery or typhoid bacilli by the domestic fly

Biological- Pathogen multiplies in the body of the vector, a part of their lifecycle completed in the body of insect vector eg. Aedes aegypti mosquito in dengue, Anopheles mosquito in malaria.

Extrinsic incubation period is the time period between the entry of the pathogen into a vector and till the vector become infective.

4. Food and water

Another source of infections are contaminated food and water. Cholera, Hepatitis A can be caused by drinking contaminated water. Salmonella contaminates water and food and symptoms occur in one to three days after consuming. Staphylococci can cause food poisoning by producing toxins.

5. Soil

Soil is a good Source of infection. Clostridium tetani is a common soil bacterium and the causative agent of tetanus. Fungi such as Histoplasma and parasites such as round worm, hookworm survive in soil and cause human infection.

6. Fomites- Inanimate objects such as unsterile instruments, contaminated objects, accidental injuries with materials or equipments used in patient care/diagnosis which can cause infections.

Effect of oxygen on the growth of microorganisms

 Based on the effect of oxygen on growth, microorganisms occupy different regions when grown  in a culture tube, as demonstrated in the figure.

The different relationships with O₂ appear due to 

•  the inactivation of proteins 
•  the effect of toxic O₂ derivatives.

Enzymes can be inactivated when sensitive groups like sulfhydryls are oxidized. An example is the nitrogen-fixation enzyme nitrogenase, which is very oxygen sensitive.

Oxygen accepts electrons and is readily reduced because its two outer orbital electrons are unpaired. Flavoproteins, several other cell constituents, and radiation promote oxygen reduction. The result is usually some combination of the reduction products superoxide radical, hydrogen peroxide, and hydroxyl radical.

O₂ + e– → O₂ ^– (superoxide radical)

O₂ ^– + e^– + 2H⁺ → H₂O₂ (hydrogen peroxide)

H₂O₂ + e^– + H⁺ → H₂O + OH^– (hydroxyl radical)

These reactive oxygen species (ROS) are extremely toxic because they are powerful oxidizing agents and rapidly destroy cellular constituents. 

Neutrophils and macrophages use these toxic oxygen products to destroy invading pathogens.

A microorganism must be able to protect itself against such oxygen products or it will be killed. Many microorganisms possess enzymes that afford protection against toxic O₂ products.

Obligate aerobes and facultative anaerobes usually contain the enzymes superoxide dismutase (SOD) and catalase, which catalyze the destruction of superoxide radical and hydrogen peroxide, respectively. Peroxidase also can be used to destroy hydrogen peroxide.

2O₂ ^– + 2H+ O₂ → H₂O₂ (superoxide dismutase)

 2H₂O₂ → 2H₂O + O₂ (catalase)

H₂O₂ + NADH + H⁺ → 2H₂O + NAD (peroxidase)

Aerotolerant microorganisms may lack catalase but almost always have superoxide dismutase. The aerotolerant Lactobacillus plantarum uses manganous ions instead of superoxide dismutase to destroy the superoxide radical.

All strict anaerobes lack both enzymes or have them in very low concentrations and therefore cannot tolerate O₂. 

Although strict anaerobes are killed by O₂, they may be recovered from habitats that appear to be aerobic. In such cases they associate with facultative anaerobes that use up the available O₂ and thus make the growth of strict anaerobes possible. For example, the strict anaerobe Bacteroides gingivalis lives in the mouth where it grows in the anaerobic crevices around the teeth.

Different approaches must be used when growing aerobes and anaerobes since aerobes need O₂ and anaerobes are killed by O₂. When culturing aerobic microorganisms, either the culture vessel is shaken to aerate the medium or sterile air must be pumped through the culture vessel.

With anaerobes, all O₂ must be excluded using

(1) Special anaerobic media containing reducing agents such as thioglycollate or cysteine may be used. The reducing agents will eliminate any dissolved O₂ remaining within the medium so that anaerobes can grow beneath its surface.

(2) The medium is boiled during preparation to dissolve its components; boiling also drives off oxygen very effectively.

(3) Oxygen also may be eliminated from an anaerobic system by removing air with a vacuum pump and flushing out residual O₂ with nitrogen gas or CO₂. Many anaerobes require a small amount of CO₂ for best growth.

(4) One of the most popular ways of culturing small numbers of anaerobes is by use of a GasPak/Gas generator envelope. Water is added to chemicals in envelope to generate Hydrogen and carbon dioxide. Carbon dioxide promotes more rapid growth of microorganisms. The palladium catalyst catalyzes the formation of water from hydrogen and oxygen, thereby removing oxygen if at all it is present.

(5) Plastic bags or pouches can be used when only a few samples are to be incubated anaerobically. These have a catalyst and calcium carbonate to produce an anaerobic, carbon-dioxide rich atmosphere. A special solution is added to the pouch’s reagent compartment; petri dishes or other containers are placed. Anaerobic indicator strip Methylene blue becomes colorless in absence of O₂.

 Reference: Prescott's Microbiology

Media components- Carbon sources

 Carbon Sources

A carbon source is required for all biosynthesis leading to reproduction, product formation and cell maintenance.  In most fermentations it also serves as the energy source. 

Carbon requirements may be determined from the biomass yield coefficient (Y), an index of the efficiency of conversion of a substrate into cellular material.

Y_carbon(g/g) =   Biomass produced (g)

                      carbon substrate utilized (g)

 

For commercial fermentations, the determination of yield coefficients for all nutrients is usually essential.  As most carbon substrates also serve as energy sources, the organism’s efficiency of adenosine triphosphate (ATP) generation and its utilization are key factors.  Carbohydrates are traditional carbon and energy sources for microbial fermentations, although other sources may be used, such as alcohols, alkanes and organic acids.  Animal fats and plant oils may also be incorporated into some media, often as supplements to the main carbon source.

Molasses Pure glucose and sucrose are rarely used for industrial-scale fermentations, primarily due to cost.  Molasses, a by-product of cane and beet sugar production, is a cheaper and more usual source of sucrose.  Molasses are concentrated syrups or mother liquors recovered at any one of several steps in the sugar refining process with different names depending on the step from which it is recovered.  Blackstrap molasses from sugar cane is the cheapest and most used sugar source for industrial fermentation.  This is the residue remaining after most of the sucrose has been crystallized from the plant extract.  It is a dark-coloured viscous syrup containing 50-60% (w/v) carbohydrates, primarily sucrose, with 2% nitrogenous substances, along with some vitamins and minerals.  Overall composition varies depending upon the plant source, the location of the crop, the climatic conditions under which it was grown and the factory where it was processed.  The carbohydrate concentration may be reduced during storage by contaminating microorganisms. 

Refinery blackstrap molasses is a similar product obtained from the recrystallization refining of crude sucrose.  High test or invert molasses is produced after whole cane juice is partially inverted/partially hydrolyzed to monosaccharides to prevent sugar crystallization. It contains approximately 70-75% sugar and is preferable to blackstrap molasses as it has lower levels of non-fermentable solids. Beet molasses are produced from beetroot, in a similar process as for sugarcane.  However, it may be limiting in biotin for yeast growth and a small amount of cane molasses may need to be added in these fermentations. Hydrol molasses, a by-product of maize starch processing primarily contains glucose (60%) and a relatively high salt concentration.

Malt Extract Aqueous extracts of malted barley can be concentrated to form syrups that are useful carbon sources for the cultivation of filamentous fungi, yeast and actinomycetes. The composition of malt extracts varies to some extent, but they usually contain approximately 90% carbohydrate, on a dry weight basis.  This contains 20% hexoses (glucose and small amounts of fructose), 55% disaccharides (mainly maltose and traces of sucrose), along with 10% maltotriose, a trisaccharide.  These products contain a range of branched and unbranched dextrins (15-20%), which may or may not be metabolized, depending upon the microorganism.  Malt extracts also contain some vitamins and approximately 5% nitrogenous substances, proteins, peptides and amino acids.

Sterilization of media containing malt extract must be carefully controlled to prevent overheating which produce Maillard reaction products. These are brown condensation products due to the reaction of amino groups of amines, amino acids and proteins with the carboxyl groups of reducing sugars, ketones and aldehydes. This occurs when reducing sugars and amino acids are heated at low pH. Such reactions cause loss of fermentable materials, colour change and some reaction products may inhibit microbial growth.

Starch and Dextrins These polysaccharides are not as readily utilized as monosaccharides and disaccharides. It can be metabolized by amylase-producing microorganisms, particularly filamentous fungi which hydrolyze the substrate to a mixture of glucose, maltose or maltotriose. The product composition is similar to that of malt extracts.  Maize starch is most widely used, but may also be obtained from other cereal or root crops. Starch is usually converted into sugar syrup, containing mostly glucose before use in fermentations.  It is first gelatinized and then hydrolyzed by dilute acids or amylolytic enzymes, often microbial glucoamylases.

Sulphite Waste Liquor Sulphite waste liquor is the product of the paper pulping industry. It is obtained after wood for paper manufacture is digested to cellulose pulp.  It can be used as a dilute fermentation medium for ethanol production by S. cerevisiae and the growth of Torula utilis for feed.   Waste liquors from coniferous trees contain 2-3% (w/v) sugar, which is a mixture of hexoses (80%) and pentoses (20%).  Hexoses include glucose, mannose and galactose, whereas the pentose sugars are mainly xylose and arabinose.   The liquors derived from deciduous trees contain mainly pentoses.  Sulphite Waste Liquor requires processing before use as it contains sulphur dioxide or calcium hydroxide or calcium carbonate which need to be stripped or removed by precipitation with lime. Supplementation with sources of nitrogen and phosphorous is also required.

Cellulose Cellulose is mainly present as lignocellulose in plant cell walls, which has cellulose, hemicellulose and lignin.  Lignocellulose is available from agricultural, forestry, industrial and domestic wastes.  Very few microorganisms can utilize it directly, as it is difficult to hydrolyze.  The cellulose component is encrusted with lignin and provides little surface area for enzyme attack.  It is mainly used in solid-substrate fermentations to produce various mushrooms. Preliminary processing by hydrolysis may be required in case of cellulose to expose fermentable sugars.  It has potential as a valuable renewable source of fermentable sugars particularly in the bioconversion to ethanol for fuel use.  

Whey Whey is an aqueous by-product of the dairy industry.  The annual worldwide production is over 80 million tonnes, containing over 1 million tonnes of lactose and 0.2 million tonnes of milk protein.  It is expensive to store and transport.  Milk proteins can be removed from whey to be used as food supplements. Whey is then evaporated and lactose concentrates are obtained which can be used for later fermentation. Lactose is generally less useful as a fermentation foodstock than sucrose, as it is metabolized by fewer organisms.  S. cerevisiae, for example, does not ferment lactose.  It was used extensively in penicillin fermentations and is still employed for producing ethanol, single cell protein, lactic acid, xanthan gum, vitamin B12 and gibberellic acid.

Alkanes and Alcohols n-Alkanes of chain length C10-20 are readily metabolized by certain microorganisms.  Mixtures, rather than a specific compound, are usually most suitable for microbial fermentations.  However, their industrial use is dependent upon the prevailing price of petroleum.  Methane is utilized as a carbon source by a few microorganisms, but its conversion product methanol is often preferred for industrial fermentations as it presents fewer technical problems.  High purity methanol is readily obtained and it is completely miscible with water.  Methanol has a high percent carbon content and is relatively cheap, although only a limited number of organisms will metabolize it.  Also unlike many other carbon sources, only low concentrations, 0.1-1% (v/v), are tolerated by microorganisms, higher levels being toxic.  During fermentations on methanol, the oxygen demand and heat of the fermentations are high, but this is even more problematic when growing on alkanes.  Several companies used methanol in microbial protein production in the 1970s and early 1980s, but these processes are currently uneconomic.

 Ethanol is less toxic than methanol and is used as a sole or co-substrate by many organisms, but it is too expensive for general use as a carbon source.  However, its biotransformation to acetic acid by acetic acid bacteria remains a major fermentation process.

Fats and Oils

Hard animal fats that are mostly composed of glycerides of palmitic and stearic acids are rarely used in fermentations.  However, plant oils (primarily from cotton seed, linseed, maize, olive, palm, rape seed and soya) and occasionally fish oil, may be used as the primary or supplementary carbon source, especially in antibiotic production.  Plant oils are mostly composed of oleic and linoleic acids, but linseed and soya oil also have a substantial amount of linolenic acid.  The oils contain more energy per unit weight than carbohydrates.  In addition, the carbohydrates occupy a greater volume because they are usually prepared as aqueous solutions of concentrations no greater than 50% (w/v).  Consequently, oils can be particularly useful in fed-batch operations as less spare capacity is needed to accommodate further additions of the carbon source.

References

1. Industrial Microbiology: An Introduction. Michael J. Waites, Neil L. Morgan, John S

1. Principles of Fermentation Technology- Peter Stanbury, Allan Whitaker, Stephen Hall

 

Biopesticides - Fungal and Viral

Fungal Biopesticides

Entomopathogenic fungi parasitise insects, and can kill or seriously disable them. They are considered natural, environmentally-safe control agents, so used worldwide for biological control of insects and other arthropod pests. Fungal biopesticides are so diverse in nature and their means of affecting the target pest can be diverse. 

The most common modes of action are through competitive exclusion, mycoparasitism, and production of metabolites. Some fungi can exhibit all of these modes of action. 

The most common commercial fungal biopesticides are Trichoderma spp., Metarhizium anisopliae, Verticillium lecanii and Beauveria bassiana. Each are frequently used in the nursery, ornamental, vegetable, field crop, and forestry industries to control a variety of pests.

Generally, entomopathogenic fungi have a common mode of action.  When the spores of the fungus come into contact with the body of an insect host, they germinate and form an infection bulb or appressorium. From it hyphae emerge and penetrate the cuticle. The fungus then develops inside the body, forming mycelium which invades throughout the insect body thus draining the insect of its nutrients. Eventually insect is killed, after a few days. This lethal effect is aided by the production of specific insecticidal toxins-destruxins, bassianolides etc. Spores are liberated from the dead cadaver of insect and the cycle continued.

Metarhizium anisopliae formerly known as Entomophthora anisopliae, is an entomopathogenic fungus that grows naturally in soils throughout the world. It is commonly called green muscardine fungi. Mechnikov named it after the insect species it was originally isolated from, the beetle Anisoplia austriaca. The disease caused by the fungus is sometimes called green muscardine disease because of the green colour of its spores. Initially, fungal hyphae appear white, but, as conidia form and mature they often take on a characteristic olive green color.  When the spores of the fungus come into contact with the body of an insect host, they germinate and the hyphae that emerge penetrate the cuticle. The fungus then develops inside the body, draining the insect of nutrients, eventually killing it after a few days. This lethal effect is aided by the production of insecticidal cyclic peptides (destruxins). 

Metarhizium species are also known to produce compounds that are toxic to arthropods. Other insects that come in contact with infected insects also become infected with the fungus. M. Anisopliae and its related species are used to control a number of pests such as termites, thrips, etc. M. Anisopliae does not appear to infect humans or other animals and is considered safe as an insecticide. The microscopic spores are typically sprayed on affected areas. 

Beauveria bassiana, named after the Italian entomologist Agostino Bassi, who discovered it in 1815 as the cause of white muscardine disease in insects is a fungus that acts as a parasite on many insect species. B. Bassiana can attack a broad range of insects. When the microscopic spores of the fungus come into contact with the body of an insect host, they germinate, penetrate the cuticle, and grow inside the insect’s body, feeding on internal tissues and releasing insect toxins like beauvericin, beauverolides, bassianolide etc. As the insect dies, it changes color to pink or brown (due to oosporein pigment) and eventually the entire body cavity is filled with fungal mass killing the insect within a matter of days. 

Beauveria bassiana can be used as a biological insecticide to control termites, whiteflies, aphids, grasshoppers, flies, beetles, caterpillars etc. The fungus rarely infects humans or other animals, so it is generally considered safe as an insecticide. 

Verticillium lecanii

Verticillium lecanii is an entomopathogenic fungus. The mycelium of this fungus produces a toxin called bassianolide and other insecticidal toxins such as dipicolinic acid, which infect aphids, whiteflies, rust fungi, scale insects and lead to the death of the host. The spores of this fungus when come in contact with the cuticle (skin) of target insects, germinate and grow directly through the cuticle to the inner body of their host. The fungus proliferates throughout the insect’s body, draining the insect of nutrients, and eventually killing it in around 48-72 hours.

Paecilomyces species

Paecilomyces fumosoroseus, is considered a very promising biological pesticide due to its extensive host range which includes insects in over 25 different families, including moth, Russian wheat aphid, silver leaf whitefly (Bemisia argentifolii) and wide range of mites.  Paecilomyces lilacinus, is another naturally occurring fungus found in many kinds of soils throughout the world. As a pesticide active ingredient, Paecilomyces lilacinus is applied to soil to control nematodes that attack plant roots. It acts against plant root nematodes by infecting eggs, juveniles, and adult females.

Non-entemopathogenic fungi such as Trichoderma spp. (Trichoderma viride, T. harzianum) are some of the most common fungi in nature. These microbial biofungicides can out-compete pathogenic fungi for food and space, and can stimulate plant host defenses and affect root growth. Many beneficial Trichoderma have the ability to readily colonize plant roots, without harming the plant. This close relationship with the plant that make these species excellent biocontrol agents.  They are highly active on root & stem rots caused by Schlerotinia, Rhizoctonia, wilts caused by Fusarium and blights/leaf spots caused by Alternaria, Downy Mildews & powdery mildews, Trichoderma viride has been highly effective where conventional fungicides are not able to control the root diseases. 

Viral Biopesticides 

Microbial biopesticides known as baculoviruses are a family of naturally-occurring viruses known to infect and destroy a number of important plant pests only insects and some related arthropods. They are particularly effective against the lepidopterous pests of cotton, rice and vegetables. They are so specific in their action that they infect and kill only one or a few species of Lepidopteran larvae (caterpillars), making them good candidates for management of crop pests with minimal off-target effects. 

Baculoviruses used as microbial biopesticides consist of DNA surrounded by a protein coat (nucleocapsid), which is embedded in a protein “microcapsule” or occlusion body (OB) that provides some protection from degradation in the environment. Depending on the virus, OBs may contain a single nucleocapsid (granulovirus, or GV) or multiple nucleocapsids (nucleo/cytoplasmic polyhedrovirus, or PV).

Upon ingestion by a susceptible caterpillar, OBs are dissolved within the alkaline midgut, releasing nucleocapsids that infect the cells lining the midgut. The viral DNA replicates in the nuclei of the host cells and then spreads throughout the body of the larvae, essentially turning it into a “virus factory.” The infected insect stops feeding within a few days, dies and disintegrates, releasing billions of new OBs which can be ingested and cause new infection of neighbouring larvae.

 

The granulovirus of the codling moth Cydia pomonella, or CpGV, is a good example of a commercially successful viral insecticide.

Baculoviruses are of two types

1) Nuclear/Cytoplasmic Polyhedrosis                                           2) Granulosis Virus 

1. Nuclear Polyhedrosis Virus (NPV)

∙ Develop in host cell nuclei

∙ Rod shaped, double stranded; virions occluded as groups in inclusion bodies

. Highly host specific-∙ Enters through injection into insect gut through mouth &cuticle

 

∙ Symptoms in larvae are: - Discoloration (brown and yellow) -Stress -Decomposition (liquification) -Lethargy -Infected larvae hang invertedly from twigs - host will become visibly swollen with fluid containing the virus and will eventually die turning black with decay 

 

2. Cytoplasmic Polyhedrosis Virus

∙ Develop only in cytoplasm of host midgut epithelial cells

∙ Virions occluded singly in polyhedral inclusion bodies

∙ Infection confined to midgut and does not spread to other tissues

∙ Infection not always lethal but shows larval growth reduction

∙ Continuously shed infective polyhedra in faeces 

3. Granulosis Virus (GV)

∙ Develop either in the nucleus/cytoplasm/ tracheal matrix / epithelial cells of host 

∙ Virions are occluded singly in small inclusion bodies called capsules; Rod shaped virion, ds DNA

∙ They enter through ingestion, similar to NPV

∙ Fat body is the major organ invaded

∙ Diseased larvae – less active, flaccid, fragile, wilted prone to rupture in later stages,  death in 6-20 days 

 

3

Media components

 The media components usually are,

■       Water

■       Carbon source

■       Nitrogen source

■       Oxygen

■       Sources of P, S, minor and trace elements

■       Vitamins – Biotin, riboflavin etc

■       Buffers

■       Antifoam

■       Precursor

■       Inducer

■       Inhibitor

Most fermentations, except those involving solid substrates, require large quantities of water in which the medium is formulated. General media requirements include a carbon source, which provides both energy and carbon units for biosynthesis, and sources of nitrogen, phosphorus and sulphur. Other minor and trace elements must also be supplied, and some microorganisms require added vitamins, such as biotin and riboflavin. Aerobic fermentations require oxygen, and even some anaerobic fermentations require initial aeration of media, e.g. beer fermentations. As the fermentation proceeds different metabolic products are formed which may change the pH of the media and it can interfere with the growth or product formation, So, to adjust the pH, media should contain buffers or the pH is controlled by addition of acid/ alkali. Antifoam agents are also required to control foam production. Some processes require compounds like precursor, inducer or inhibitor which are added certain stages of the fermentation.

The composition of a fermentation medium may be simple to complex, depending on the particular microorganism and its fermentation.  Autotrophic microorganisms require only the simplest of inorganic media (inorganic salts, water, nitrogen source, carbon source is fulfilled by CO₂ or by carbonates) and are capable of synthesizing all the complex organic compounds required to sustain life.  Fastidious microorganisms on the other hand lack the ability to synthesize many of their sustenance and growth requirements.  They require the presence of many simple to complex preformed nutrients in the medium and must have an organic carbon supply to provide for synthesis of cell substances and release of metabolic energy.  So they require complex media.

Simple and complex media are further subdivided into two categories: synthetic and crude. In a synthetic medium, all the components are specifically defined and known compounds.  Each component is relatively pure and the exact concentrations are known.  Synthetic medium has defined components and concentrations and are expensive due to the relatively pure ingredients used. The concentration of one or several can be varied in order to determine the effect on cell growth and product yield. Individual components may be added or deleted as well. This has advantages in certain types of studies. However, yields derived from these media are relatively low.  Crude media contain crude factors or ill-defined sources of nutrients and growth.  They usually allow much higher yields.

The media adopted also depend on the scale of the fermentation. For small-scale laboratory fermentations pure chemicals are often used in well-defined media. However, this is not always possible due to cost, as media components may account for up to 60-80% of process expenditure. Industrial-scale fermentations primarily use cost-effective complex crude substrates, where many carbon and nitrogen sources are almost indefinable.  Most are derived from natural plant and animal materials, often by-products of other industries, with varied and variable composition.  The effects of such batch-to-batch variations must be determined.  Small scale trials are usually performed with each new batch of substrate, to examine the impact on product yield and product recovery.

            The main factors that affect the final choice of individual raw materials are as follows:

1 Cost and availability: ideally, materials should be inexpensive and of consistent quality and year round availability.

2 Ease of handling in solid or liquid forms, along with associated transport and storage costs, e.g., requirements for temperature control.

3 Sterilization requirements and any potential denaturation problems.

4 Formulation, mixing, complexing and viscosity characteristics that may influence agitation, aeration and foaming during fermentation and downstream processing stages.

5 The concentration of target product to be attained, its rate of formation and yield per gram of substrate utilized.

6 The levels and range of impurities and the potential for generating further undesired products during the process.

7 Overall health and safety implications.

Composition of a typical fermentation media

The composition of fermentation media is dependent on a number of factors characteristic of the particular fermentation.  The major ingredients of a typical fermentation media are:

■       Water

■       Carbon source

■       Nitrogen source

■       Sources of P, S, minor and trace elements

■       Vitamins – Biotic, riboflavin etc

■       Buffers

■       Antifoam

■       Precursor

■       Inducer

■       Inhibitor 

Water

Water is a major component of all fermentation media except solid-substrate fermentation. It also provides trace mineral elements. Water is also important for ancillary equipment and cleaning. Supply of large quantities of clean water, of consistent composition, is therefore essential. The quality of water in terms of pH, dissolved salts and effluent contamination is important.  The mineral content is important in brewing (mashing step) and has influenced the location of breweries and types of beer produced.  Before use, suspended solids, colloids and microorganisms should be removed. If the water is ‘hard’, it should be treated to remove salts such as calcium carbonate. Iron and chlorine should also be removed. For some fermentations, especially, plant and animal cell culture, the water must be highly purified. Since, water is becoming increasingly expensive, its recycle/reusage wherever possible should be encouraged. This minimizes water costs and reduces the volume requiring waste-water treatment.

References