Utilizing Soil Microbes Objectives of Agriculture To meet the food requirements of the population To generate export earnings To provide raw materials for industry To eradicate poverty and unsanitary conditions of the countryside Conventional Agricultural Problems Over-specialization, monocropping, and excessive intensification Excessive dependence on external inputs Large-scale deforestation Salinization, erosion, compaction, and fertility loss of soils Unsustainable intensive factory farming systems of animal production Heavy rural-urban migration Effects of the Multidimensional Crisis State subsidies were reduced Purchasing capacity was reduced (up to 40 percent) Fuel price was increased (some even doubled) Fertilizers were reduced to 25 percent Pesticides were reduced to 40 percent Animal feed concentrates were reduced to 30 percent **All agriculture seriously affected Strategy to Survive Shift to a low external input form of agriculture, while at the same time boosting production Organic Farming Alternatives to high input agriculture Many traditional practices were remembered ―Agroecological‖—farms where agroecological concepts are applied and which promote sustainable production systems Practices of Organic Farming Soil Conservation, and Organic and Biofertilization Ecological Management of Pests, Diseases & weeds Crop rotation and polyculture Integrated farming systems Roles of Microbes in Organic Farming Microbes provide nitrogen required for the plants through biological nitrogen fixation. Phospobacteria provides phosphorus required for the plant growth by converting unavailable phosphorus to available form. Phosphorus uptake enhanced using Mycorrhiza fungi. Microbes provide plant growth promoting substances Microbes play an important role in controlling diseases of plants as Bio protectors. Making Microbes Available Biofertilizers , Biological Control Agents. Biofertilizers Inputs containing efficient strains of specific microorganisms which are capable of mobilizing nutritive elements required for the plants by fixing atmospheric nitrogen, solubilizing and enhancing up take of soil phosphorus and stimulating plant growth through synthesis of growth promoting substances. Totally harmless, pollution free and low-cost renewable agricultural inputs. Components of Biofertilizers Decomposers Reliable to break down organic waste and dead organisms. Releases key ions such as nitrates, phosphates and sulfates for use by other organisms. Composting relies on bacterial action. Many types of bacteria participate in the composting process, thriving at different temperatures and on different materials Mesophiles Thermophiles (hot) Components of Biofertilizers Nitrogen Fixing Bacteria Contained in the root nodules of legumes. Contained in the root nodules of alders. Free living. convert nitrogen from the atmosphere into ammonium (NH4) or nitrate (NO3) ions. Components of Biofertilizers Phosphorous Solubilizing Bacteria Solubilizingunavailable organic & inorganic forms of phosphorus (80%). Organic P slowly mineralized by the action of phosphatases. Inorganic P solubilized by the action of organic and inorganic acids. Components of Biofertilizers Nutrients Up Take Enhancing Fungus (Mycorrhizae) Maximizing plants absorption area (Mycorrhizae can penetrate smaller crevices than root hairs) Improve soil texture Components of Biofertilizers Nutrients Up Take Enhancing Fungus (Mycorrhizae) Increase water uptake in plant. Increase mineral uptake (especially P, Cu & Zn). Sequester heavy metals (Os, Pb). Limits uptake of Al, As, Ti, Ba & Cd). Mycorrhizae Ectomycorrhizae Form a sheath around the root The mass of the hyphae =mass of roots Hyphae grow between the spaces in the cortical root cell No cellular penetration by the hyphae Reproductive spores are formed in the soil only Members of the basidomycetes or the ascomycetes Infect Pinaceae, Fabaceae, and temperate forests Mycorrhizae Endomycorrhizae Hyphae form arbuscles (Bulb structure) where they penetrate plant cell Were called VAM (vesicular arbuscular mycorrhizae) Direct cell to cell exchange of nutrients Hyphae grow into intercellular spaces of cortical cells, but penetrate cell walls Spores can form inside or outside of plant cell Belong mainly to Glomalae family Infect grasses, most herbaceous dicots, many perennial shrubs and 6 genera of gymnosperms Biological Control Pest suppression with biological agents operating in a background of integrated control that does not depend on host resistance, sterilization of the target pathogen, or modification of pest behavior Biological Control Mechanisms Competition Antibiosis Parasitism Induced resistance Disease agent transfer Microbial Pesticide Microbial agent intended for preventing, destroying, repelling, or mitigating any pest, or intended for use as a plant regulator, defoliant, or dessicant, that: (1) Is a eucaryotic microorganism including, but not limited to, protozoa, algae, and fungi; (2) Is a procaryotic microorganism, including, but not limited to, eubacteria and archaebacteria; or (3) Is a parasitically replicating microscopic element, including but not limited to, viruses. Microbial Pesticides May control many different kinds of pests, although each separate active ingredient is relatively specific for its target pest[s]. Finding Biological Control Organisms Suppressive soils Old world Plant pathogens Strange and unusual places Bacillus thuringiensis An insecticidal bacterium for control of many important plant pests - mainly caterpillars of the Lepidoptera (butterflies and moths) but also mosquito larvae, and simuliid blackflies that vector river blindness in Africa. Bacteria produce a crystalline protein that is toxic to a number of insects when ingested Paecilomyces lilacinus A thermophile fungus Parasitize nematodes: attacks eggs, juveniles and females Other Known Bacteria for Biological Control Agents Agrobacterium radiobacter Bacillus subtilis Burkholderia cepacia Pseudomonas fluorescens Pseudomonas syringae Pseudomonas chloroaphis Streptomyces griseoviridis Ampelomyces quisqualis Other Known Fungus for Biological Control Agents Paecilomyces fumosoroseusTrichoderma harzianum Candida oleophila Trichoderma polysporum Coniothyrium minitans Trichoderma viride Beauveria bassiana Fusarium oxysporum Beauveria brogniartii Gliocladium virens Vertricillium dahliae Gliocladium catenulatum Verticillum lecani Phlebia gigantea Metarhizium anisopliae Pythium oligandrum Phlebiopsis gigantea Known Viruses for Biological Control Agents For Insect Control Granulovirus (Baculoviridae) for controlling Adoxophyes orana and Cydia pomonella Nucleopolyhedrovirus (Baculoviridae) for controlling Helicoverpa armigera and Spodoptera exigua Viruscides (Inoculation) Potyvirus (Potyviridae) for controlling Zucchini Yellow Mosaic Virus Other Microbial Agents Pseudomonas sp. & Bacillus megaterium PGPBs or PSBs Work through 1 of 2 mechanisms Phytohormones secretion improve root growth Bioremediation Agents Decomposers also: Eat toxic compounds like oil, battery acid, detergents, pesticides (DDT), some plastics, and even radioactive waste products that can accumulated in dumps, alongside roads Degrade sewage in treatment plants and septic tanks, helping to recycle the 11 billion pounds of dung that people on earth produce every day, and turning it into fertilizer that is then returned to the biosphere Bioremediation Natural – using what bacteria exist in the soil to break down toxic materials ―Nature's Way to a Cleaner Environment‖ Enhanced – using ―super bugs‖ or merely enhancing the environment Steps to Utilize Soil Microbes Isolation Identification Plant (for biofertilizers) or Target Pest (for Biological Control Agents) Test Pot Culture Test: Sand test, Sterilized Soil test, and Unsterilized Soil test Field Trial Inocula Development Monocultures • Processes requiring monocultures • Sources of monocultures • Preserving pure cultures Advantages and disadvantages of pure cultures • Advantages: easy to obtain (isolate or purchase); better control of products; can be patented • Disadvantages: subject to contamination and genetic change Processes Requiring Mono Cultures PURE CULTURE FERMENTATIONS industrial ethanol alcoholic beverages fermented foods pharmaceuticals acetone-butanol acetic acid single cell protein industrial enzymes biotech products (insulin, growth hormone) Biological Control Agents Biofertilizers Culture Collections Supply of Industrial Microorganisms Abbrev. Name Location ATCC American Type Culture Collection Rockville, MD, U.S CBS Centraalbureau voor Baarn, The Netherlands Schimmenlculturen CDDA Canadian Department of Agriculture Ottawa, Canada CMI Commonwealth Mycological Institute Kew, United Kingdom FAT Faculty of Agriculture, Tokyo University Tokyo, Japan IAM Institute of Applied Microbiology Tokyo, Japan University of Tokyo NCIB National Collection of Industrial Bacteria Aberdeen, Scotland NCTC National Collection of Type Cultures London, United Kingdom NRRL Northern Regional Research Laboratory Peoria, IL, United States PCC Pasteur Culture Collection Paris, France Inocula Development Mixed cultures • Processes requiring mixed cultures • Defined versus enrichment cultures • Sources of mixed cultures • Preserving mixed cultures Advantages and disadvantages of mixed cultures • Advantages: obtained by enrichment or purchased; can't be patented; contamination not as much of problem • Disadvantages: control of culture and products is less definite; Processes Requiring Mixed Cultures MIXED CULTURE FERMENTATIONS breads: sour dough, soda cracker wines vegetables: pickles, sauerkraut dairy products: yogurt, sour cream composting anaerobic digestion soil and groundwater remediation bioleaching microbial enhanced oil recovery microbial metals recovery waste treatment INOCULUM PRODUCTION DEFINITION OF INOCULUM Living organisms or an amount of material containing living organisms (such as bacteria or other microorganisms) that is added to initiate or accelerate a biological process CRITERIA Healthy, active state - minimize lag period Available in sufficient quantities Suitable morphological form Free of contamination Stable - retain its product forming properties CHOICE OF MICROORGANISM Nutritional characteristics - cheap medium Optimum environmental conditions Productivity - substrate conversion, product yield, rates Ease of handling and safety (suitability) SAFETY LAMINAR FLOW CABINETS used; to limit exposure of operators to aersols and other possible infections to protect the culture material from contamination ASEPSIS MUST BE MAINTAINED CORRECT STANDARD MUST BE APPLIED SAFETY STANDARD CLASS 1 - none or minimal hazard CLASS 2 - ordinary potential hazard CLASS 3 - Special hazard, require special containment CLASS 4 - Extremely dangerous, may cause epidemic disease CLASS 5 - Pathogens excluded by law STORAGE AND PRESERVATION Isolates/cultures should retain desirable characteristics over long periods of time METHODS Storage at reduced temperatures Slopes - refrigerator (4 oC), freezer (-20 oC), deep freezer (-80 oC) Fungal spores in water (5 oC) Liquid nitrogen (-150 to -196 oC) Storage in dehydrated form Soil + culture dried. Used for fungi Lyophilization\freeze drying. Freezing of culture followed by drying under vacuum which results in sublimination of cell water QUALITY CONTROL OF PRESERVED CULTURES Each batch must be routinely tested Whatever method is used in preservation of stock cultures it is important to assess the quality of the stocks Each batch of cultures should be routinely checked to ensure the propagated strains retain the correct growth charatertistics, morphology and product forming properties Stability and performance of a culture Influenced by Mode of substrate feeding Nutrients Temperature Osmotic pressure Oxygen Intracellular product accumulation FUNGAL INOCULA Spore suspension - used at early stages, small pellets in subsequent transfers Inoculum affected by morphology of fungus - can influence size of pellet or floc Spore Production Solidified media e.g. agar media + roll-bottle technique Solid media e.g. cereal grains, bran, malt, flaked maize etc. (amount of water, relative humidity of air, temp. are important) Submerged culture - influenced by media Problem with Mycorrhizae Mycelium better inoculant in many cases Difficult to grow in culture Formulation of Inocula The ecological competence (the ability of microbial cells/inocula to compete and survive in nature) of laboratory/bioreactor prepared inocula is important to commercial exploitation of biotechnological processes initiated by the addition of microbial cultures to natural habitats. It caused by inability to regulate the process environment stringently Formulation of Inocula Inocula systems will require, as a first step, an efficient formulation and delivery system, based on microenvironmental control, directed at minimizing the lag period and maximizing competitive advantage to the introduced microorganisms The use of polymer gels, for example alginate, to immobilize cells has allowed the development of spatially organized microenvironments with control on the degree of protection afforded, the rate of cell release and the positioning of cells with nutrients and/or selective agents or chemicals Types of Inoculant Comparison of Sterile and Nonsterile Peat-based Inoculants Comparisons of Alginate and Peat Inoculant Comparisons of Alginate and Peat Inoculant Other materials as potential carriers for bacterial inoculants Methods of inoculation with peat- based inoculants Methods of inoculation with peat-based inoculants Factors Influencing Bacterial Survival in Soils Grass Root Colonization by Four Bacterial Species (B. cepacia P2, Flavobacterium sp. strain F4, P. fluorescens, and Alcaligenes sp. Maximum Abundance Percentage of Actinobacterial Genera in the Roots of Wheat Grown from Seed Inoculated with Endophytes (Microbispora sp. [EN2], Streptomyces sp.[EN27], N. albus [EN46]) Maximum Abundance Percentage of Actinobacterial Genera in the Roots of Wheat Grown from Seed Inoculated with Bacterial Inoculum (Nutrilife) FIELD TRIAL Biofertilizers Rhizobium , Azetobactor , Azospirillium and Phosphate solubilising organisms A view of untreated (control) field of Pea Bio Control Agents Fungal antagonists (AGAINST ROOT-ROT FUNGI) A view of biofertilizer & biocontrol agent treated field of pea Biofertilizers and Biological Control Agent Remember that it is a living organism. Living organisms have preferences (Give a correct inoculant for a certain crop or pest). Organisms may die off in package (Make sure it hasn’t expired, fresher better). Storage affect lifespan (store in a cool, dark place until used). Use ASAP after inoculating. Example of Problem Reduced Nodulation Improper storage of bacteria (heated, dried). Fertilizer will kill bacteria if mix granular and fertilizer in seeding operation. Fertilizer desiccates bacteria. Herbicide residue (glyphosate that inhibits root hairs will reduce nodulation. Plant stress. Saline, inadequate soil fertility, low pH.