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Takrif Biofertilizers Preparations containing LIVE or LATENT CELLS of EFFICIENT strains of N2 N2 N2 N2 FIXING, PHOSPHATE SOLUBILIZING or CELLULOLITIC microorganisms used for applications to SEED, SOIL or COMPOSTING AREAS, with the OBJECTIVE of INCREASING THE NUMBERS of such microorganisms and ACCELERATE certain microbial PROCESSES to augment the extent of the AVAILABILITY of nutrients in a form which can be easily assimilated by plant (Subba Rao,NS “Biofertilizers in Agriculture” Oxford & IBH Publishing Company) TAKRIF (DEFINITION):

Pupu k Haya ti vs Pupu k Orga nik

Nitrogen Cycle The 2 major processes of N 2 transformation are: Nitrification:  NH 4 + → NO 3 - :  NH 4 + →NO 2 -  NO 2 - → NO 3 - By bacteria e.g. Nitrobacter, Bacillus, Paracoccus, Pseudomonas. Denitrification: – NO 3 - → N 2 By bacteria e.g. Azotobacter, Clostridium and Rhizobium. 5

Nitrogen Fixation N 2 is the most stable form of nitrogen and high energy is required to break the N-N triple bond. Therefore only microorganisms can fix nitrogen N 2 + 8H + +8e - →2NH 3 + H 2 N 2 gas is the greatest reserve of nitrogen. The productivity of many environs is limited by the short supply of nitrogenous compounds. Nitrogen fixation is important to agriculture and legumes such as soybean can fix atmospheric nitrogen. 6

Dentrification Denitrification is the reduction of nitrates to N 2 or NO 2. This process is detrimental because it removes nitrogen the environment. This is of particular importance to agriculture where nitrate fertilizers are used. If anoxic condition develop e.g. water logged soil. The nitrate is remove from the soil by dentrification. What do you unerstand by the term “anoxic conditions”? 7

Ammonification The decomposition of organic nitrogen compounds such as amino acids and nucleotides is called ammonification. In the soil much of this NH 3 is converted to amino acids by plants. Some NH 3 is lost by evaporation especially in dense animal populations. Globally this constitutes 15% of N 2 released to the atmosphere. 8

Nitrification Nitrification is the oxidation of NH 3 to NO 3 - by nitrifying bacteria. The nitrates produced is readily assimilated by plants. Nitrate is soluble and is quickly leached from the soil. NH 4 + is +vely charged and will adhere to –vely charged soil (clay) particles. NH 4 + is extensively used in nitrogenous fertilizers. Denitrification consumes N 2 while nitrification produces it. 9

NH 3 3 H 2 +

NH 4 + NO 3 -

Methemoglobinemia (blue-baby syndrome) pada Konsumen NO 3 - Mo Nitrat Reduktase 20 ADP 5 ADP Pada tanah 2 kahat Mo NO 2 - Terjerap mineral lempung tipe 2 : 1 (Vermikulit, Illit, Mica butir halus & Smektit) > 0.5 mg kg -1 tanah < 0.5 mg kg -1 tanah NH 4 + NO 3 -

Nitrogen Fixation by Legumes The association of nitrogen fixing bacteria with legumes is one of the most important bacteria plant interaction. Nitrogen fixing legumes include, soybean, bean, pea, clover and alfalfa are plants with beans in pods. Nitrogen fixing bacteria in plants include:  Rhizobium  Bradyrhizobium  Mesorhizobium  Azorhizobium 17

Nitrogen Fixation The symbiotic relationship between plant and nitrogen fixing bacteria results in the formation of a root nodule. In the nodule N 2 is converted by the enzyme nitrogenase to ammonia. The ammonia is used in the synthesis of amino acids and other cellular components. Under normal conditions neither Rhizobium nor the plant can fix nitrogen. 18

Root Nodules 19

Nitrogen Fixation Rhizobium can only fix N 2 under microaerophilific (reduced O 2 ) conditions. This is because O 2 is needed by Rhizobium but O 2 also inhibits nitrogenase. 20

Leghemoglobin In the nodule O 2 level is reduced by leghemoglobin. Leghemoglobin is synthesized only after interaction of the plant and Rhizobium. 90% of legumes will fix nitrogen. However each nitrogen fixing bacteria will only associate with certain legumes. 21

Leghemoglobin 22

Steps in Nodule Formation 1.Recognition of the correct partner by both plant and bacteria. 2.Attachment of the bacteria to the plant root. 3.Invasion of the root hair by bacteria through the formation of an infection thread. 4.Growth to the main root via the infection thread. 5.Formation of bacteroids (deformed bacteria cells) and development of nitrogen fixing state. 6.Continued division of plant and bacteria cell and formation of mature root nodule. 23

Steps in Formation of Root Nodule 24

Steps in Formation of Root Nodule 25

Nitrogen Fixation 26

Bioremediation Microorganisms are the Earth’s greatest chemists. They can be used to: ▲ Extract valuable metals from low grade ore (microbial leaching). ▲ Clean up the environment (bioremediation). Bioremediation is the use of microorganisms to clean up pollution created by human activity e.g. petroleum and pesticides. 27

Bioremediation of Petroleum Petroleum is a rich hydrocarbon (HC) source and many organisms including bacteria, mold yeast, cyanobacteria and blue green algae is capable of aerobically oxidize it. This type of microbial activity is important in the cleanup of oil and other pollutants. In a large oil spill the volatile HC fractions will evaporate. Hydrocarbon oxidizing microorganisms develop quickly on the oil film and attach to the aliphatic and aromatic components. The will oxidize these HC in the oil to CO 2. 28

Hydrocarbon Oxidizing bacteria 29

Bioremediation of Xenobiotics It has been shown that HC oxidizing bacteria can increase in # to shortly after an oil spill. Xenobiotics are chemically synthesized compounds that are not naturally occurring. They include pesticides, polychlorinated biphenyls (PCB, used in electric generation) dyes and many chlorinated solvents. Many xenobiotics are structurally related to naturally occurring compounds and as thus can be degraded by microorganisms. 30

Bioremediation of Pesticides Other Xenobiotics are different and therefore degradation in nature is very slow. The most common xenobiotics are pesticides. Over 1000 pesticides are market for chemical pest control e.g. herbicide, insecticide and fungicide. Pesticides include variety of chemicals types including chlorinated compounds, aromatic rings and nitrogen and phosphorus containing compounds. Some of these compounds are suitable carbon source and electron donors for some microorganisms. 31

Bioremediation of Pesticides If the pesticide can be degraded by microorganisms then it will prevent toxic build up in the soil and water table. Chlorinated pesticides are recalcitrant and can persist for more than 10 years 32

Persistence of Herbicides and Insecticides in Soil 33

Bioremediation and Plastics Another major environmental concern is the disposal of solid waste particularly plastic. The plastic industry produce 40 billion kg of plastic each year, 40% of which end up in landfills. Plastics are xenobiotics polymers of various types including: ╬ Polyethylene ╬ Polypropylene ╬ Polystyrene 34

Bioremediation and Plastics 35

Many of these polymers are recalcitrant and remain in the landfill for decades. One solution is the use of biodegradable polymers such as photobiodegradable plastics, starch-linked and microbially synthesized plastics. Photobiodegradable plastics are attacked by UV light (from sunlight) generating polymers which are amenable to microbial attack. 36 Bioremediation and Plastics

Biodegradable Plastics Starch-based plastics incorporate starch as a for a biodegradable polymer. Starch digesting bacteria in the soil attack the starch releasing polymer fragments which are degraded by other microorganisms. Microbially synthesized plastic e.g. poly-β- hydroxyalkonate (PHA) is synthesized by microbial cells and can be degraded by microorganisms. 37