Mass Production Technologies Of Biopesticides PDF

MASS PRODUCTION TECHNOLOGIES OF BIOPESTICIDES  Scope for Production of Biopesticides Though there are about 140 bio-pesticide production units existing in the country as on today, they are able to meet the demand of only less than 1% of cropped area. There exists a wide gap,...

MASS PRODUCTION TECHNOLOGIES OF BIOPESTICIDES  Scope for Production of Biopesticides Though there are about 140 bio-pesticide production units existing in the country as on today, they are able to meet the demand of only less than 1% of cropped area. There exists a wide gap, which can only be bridged by setting up of more and more units for production of Bio-pesticides. There is a scope to enhance production and use of biological control agents in the days to come as the demand is on the increase every year. This requires large scale investment and private participation  To achieve optimum results, bio-pesticide facilities are to be set up in areas which have appropriate climatic conditions. Because temperature control is less costly in locations where there are no extreme conditions. Besides the climatic conditions, the proximity of the location to the market is also important. However, care must be taken that the production facilities are set up at least a quarter of a mile away from farming areas, so as to prevent the contamination of production facilities by insecticides from the farming areas. Also, as air pollution can damage bio-pesticides, the production should be located away from industrial and urban areas.  Methods in production of biopesticides 1. Solid state fermentation Solid-State Fermentation (SSF) SSF utilizes solid substrates like bran, bagasse, and paper pulp. The main advantage of using these substrates is that nutrient-rich waste materials can be easily recycled as substrates. In this fermentation technique, the substrates are utilized very slowly and steadily, so the same substrate can be used for long fermentation periods. Hence, this technique supports controlled release of nutrients. SSF is best suited for fermentation techniques involving fungi and microorganisms that require less moisture content.  However, it cannot be used in fermentation processes involving organisms that require high aw (water activity), such as bacteria. The most used substrates are starchy and lignocellulosic materials. Some of the common substrates used in solid state fermentation are wheat bran, rice and rice straw, hay, fruit and vegetable waste, paper pulp, bagasse, coconut coir, and synthetic media. Spores produced using SSF were more stable and resistant to stress than those produced using liquid culture or on agar. Most of the microorganisms used in SSF are filamentous fungi due to their capability to grow on solid surfaces with low free water, but also some bacteria with biocontrol ability have been produced using SSF. 2. Liquid state fermentation (lf)/ submerged fermentation (smf) SmF utilizes free flowing liquid substrates, such as molasses and broths. The bioactive compounds are secreted into the fermentation broth. The substrates are utilized quite rapidly; hence need to be constantly replaced/supplemented with nutrients. This fermentation technique is best suited for microorganisms such as bacteria that require high moisture content. An additional advantage of this technique is that purification of products is easier. Some common substrates used in submerged fermentation are soluble sugars, molasses, liquid media, fruit and vegetable juices, and sewage/waste water.  SmF is primarily used in the extraction of secondary metabolites that need to be used in liquid form. SmF is usually implemented in case of bacterial enzyme production, due to the requirement of higher water potential. SSF is preferred when enzymes have to be extracted from fungi, which require lesser water potential. More than 75% of the industrial enzymes are produced using SmF, one of the major reasons being that SmF supports the utilization of genetically modified organisms to a greater extent than SSF. This is highly critical due to the fact that the metabolism exhibited by microorganisms is different in SSF and SmF, and the influx of nutrients and efflux of waste materials needs to be carried out based on these metabolic parameters. Any slight deviation from the specified parameters will result in an undesirable product.  Mass production of Trichoderma For mass multiplication of Trichoderma the following steps should be followed sequentially 1. Take about 200 gm of grains in autoclavable bags [71 (B) x 111 (H) and add equal amount of tap water. 2. After filling the bags, keep a 1.51 inches PVC pipe at the top of the cover and tied it with a rubber band. 3. Close PVC pipe mouth using cotton plug. 4. Boil the grains in a 10-20 liter pressure cooker with water inside it for a period of 40 minutes. 5. The grains are cooled at room temperature after sterilization. 6. Transfer the bags into inoculation chamber.  Inoculate with 1-2 bits of Trichoderma mother culture in each bag inside the chamber with all the help of inoculation loop/spatula. Shake the bags properly for mixing the fungal culture all over the grains. Keep the inoculated bags at the room temperature (25-30 oC) Observe the inoculated bags if there is mycelial growth, do not disturb the inoculated bag. If mycelial growth is not observed, shake the inoculated bag. Once Trichoderma sporulation (green color) takes place shake the bags every alternate day for about 5 to 7 days in order to spread and allow the Trichoderma growth and further sporulation.  Transfer the grains with fully grown Trichoderma mycelia & sporulation into cleaned plastic trays and cover it with blotter/newspaper. Keep these plastic trays for further sporulation and drying for about 3-4 days at room temperature. Mix the transferred Trichoderma colonized grains once in every day for up to 3-4 days with the help of spatula for enhancing sporulation and drying. The Trichoderma will be ready for use as soil application or the grounded fine powder for seed treatment and or foliar application. From 1 kg sorghum grains approximately 500 gm dried biomass of Trichoderma including grains can be produced, which could be utilized directly for soil application for one hectare after mixing in kg of well decomposed compost or Farm Yard Manure (FYM). The dry biomass powder along with 0.5% Carboxy Methyl Cellulose (CMC) can be utilized for seed treatment @ 10 g/kg seed. Mass production of Pseudomonas Preparation of mother culture using the king’s B medium Peptone : 20.0 g K2HPO4 : 1.5 g Mg SO4 : 1.5 g Glycerol : 10 ml Distilled water : 1000 ml Dispense the above broth into conical flasks and autoclave at 15 lb pressure for 15 minutes. After cooling inoculate with a loop of Pseudomonas and incubate for 2 days.  Mass production For mass multiplication of Pseudomonas the following steps should be followed sequentially as noted below: Prepare Kings B Medium and transfer into conical flask or fermentor depending on the requirement. Sterilize the medium at 15 lb pressure for 15 minutes. The media should be cooled at room temperature after sterilization. Inoculate the conical flask/ fermentor with Pseudomonas mother culture @ 5% Keep the inoculated vessels at the room temperature (30- 35oC)  Once bacterial growth starts, shake the flasks at every 4-6 hours for about 3 to 4 days in order to spread and allow the bacterial growth. After 7-8 days Pseudomonas will be ready to use. Transfer the liquid media with bacterial growth into cleaned plastic trays and add fine talc material @ 1:3 (bacterial media: talc). Mix the material properly and allow them to dry at room temperature. The mixed formulation will be ready for use as soil application or for seed treatment and or foliar application Mass production of Beauveria bassiana and Metarhizium anisopliae Cut the carrot into small pieces (40 g), wash in potable water Transfer the carrot pieces in to conical flask (250 ml) and add 150 ml of water. Plug the conical flasks with cotton and autoclave for 15 min at 15 psi. Allow the flasks to cool and take to laminar flow chamber for inoculation. Transfer the loopful of B. bassiana or M. anisopliae aseptically. Incubate the flasks at room temperature. Harvest the spores in 12-15 days. Mass production of Bacillus thuringensis The fermentation of the different isolates of B.t., regardless of subspecies, have some general characteristics in common. They all use sugar (usually glucose, molasses, or starch), producing acid during the fermentation. In general, they have similar requirements for proteins or protein hydrolysates, can use NH4+ salts, and respond similarly to minerals. However, the individual isolates are unique entities, and a particular medium that may support good growth or toxin production by one isolate may be less satisfactory for another. Different isolates of B.t. may produce toxins with different spectra of activities. The "log-phase" of any bacterial fermentation is that period during which the organism is vigorously growing and rapidly dividing. This first phase lasts 16-18 hours. Sporulation is complete 20-24 hours after inoculation, although the cells have not yet lysed. Lysis is complete by 35-40 hours.  Mass production of nucleopolyhedrosis virus (NPV) Mass production of Nuclear Polyhedrosis Virus (NPV) on commercial scale is restricted to in vivo procedures in host larvae which are obtained by Field collection from cotton, pigeon pea and chickpea – H. armigera Mass culturing in the laboratory in semi synthetic diet – H. armigera Some small scale producers use field – collected larvae for mass production of NPV in spite of the following constraints. Collection of a large number of larvae in optimum stage (late IV / early V instars) is time-consuming and can be expensive in terms of labour and transportation costs. Wild populations of insects may carry disease causing organisms like microsporidians, cytoplasmic polyhedrosis virus, stunt virus and fungal pathogens which will affect both virus production and quality. Transportation of a large number of larvae with cannibalistic behaviour will be a difficult task. Parasitized larvae collected from the field will die prematurely yielding little virus.  Production procedure The NPV of H. armigera is propagated in early fifth instar larvae. The dose of the inoculum used is 5 x 10 5 polyhedral occlusion bodies (POB) in 10 ml suspension. The virus is applied on to the semisynthetic diet (lacking formaldehyde) dispensed previously in 5 ml glass vials. A blunt end polished glass rod (6 mm) is used to distribute the suspension containing the virus uniformly over the diet surface. Early fifth instar stage of larvae are released singly into the glass vials after inoculation and plugged with cotton and incubated at a constant temperature of 25 oC in a laboratory incubator. When the larvae exhausted the feed, fresh untreated diet is provided. The larvae are observed for the development of virosis and the cadavers collected carefully from individual bottles starting from fifth day. Approximately, 200 cadavers are collected per sterile cheese cup (300 ml) and the contents are frozen immediately. Depending upon need, cadavers are removed from the refrigerator and thawed very rapidly by agitation in water.  Processing of NPV The method of processing of NPV requires greater care to avoid losses during processing. The cadavers are brought to normal room temperature by repeatedly thawing the container with cadaver under running tap water. The cadavers are homogenized in sterile ice cold distilled water at the ratio 1: 2.5 (w/v) in a blender or precooled all glass pestle and mortar. The homogenate is filtered through double layered muslin and repeatedly washed with distilled water. The ratio of water to be used for this purpose is 1: 7.5-12.5 (w/v) for the original weight of the cadaver processed. The left over mat on the muslin is discarded and the filtrate can be semi-purified by differential centrifugation. The filtrate is centrifuged for 30-60 sec. at 500 rpm to remove debris. The supernatant is next centrifuged for 20 min at 5,000 rpm. Then the pellet containing the polyhedral occlusion bodies (POB) is suspended in sterile distilled water and washed three times by centrifuging the pellet in distilled water at low rpm followed by centrifugation at high rpm. The pellet finally collected is suspended in distilled water and made up to a known volume, which is necessary to calculate the strength of the POB in the purified suspension.