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DINITROGEN

BIOLOGICAL. DINITROGEN. FIXATION. Mengapa menjadi komponen dari Pertanian Berkelanjutan ?. THE NITROGEN CYCLE. ALIHRUPA NITROGEN DALAM TANAH. Mana saja yang merupakan proses MIKROBIOLOGIS ?. Suhu 1200 o C. 3 H 2. Tekanan 500 atm. N 2. N 2. NH 3. NH 3. NITROGENASE.

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DINITROGEN

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  1. BIOLOGICAL DINITROGEN FIXATION Mengapa menjadi komponen dari Pertanian Berkelanjutan ?

  2. THE NITROGEN CYCLE

  3. ALIHRUPA NITROGEN DALAM TANAH Mana saja yang merupakan proses MIKROBIOLOGIS ?

  4. Suhu 1200oC 3 H2 Tekanan 500 atm N2 N2 NH3 NH3 NITROGENASE Suhu & tekanan normal PROSES HABER-BOSCHVS PENAMBATAN N2 HAYATI   1.3 TON MINYAK UNTUK MEMPRODUKSI 1 TON NITROGEN DAMPAK : Melepaskan GRK (CO2 & NOX) serta pencemaran NO3- MIKROBA DIAZOTROF SIMBIOTIK & NON-SIMBIOTIK

  5. HUJAN ASAM & GRK NH4+ lepas lambat 5 ATP > 0.5 mg kg-1 tanah NH4+ N2O NH3 GDH Pathway NO < 0.5 mg kg-1 tanah Asam amino GS-GOGAT Pathway NO2- NH3 Nitrifikasi Nitrat Reduktase Mo 20 ATP NO3- AKUMULASI Pada tanah2 kahat Mo Methemoglobinemia (blue-baby syndrome)pada Konsumen Leaching, Menurunkan kejenuhan basa & memasamkan tanah, Pencemaran air, Eutrofikasi, Nitrosamin, Denitrifikasi NO3- Terjerap mineral lempung tipe 2 : 1 (Vermikulit, Illit, Mica butir halus & Smektit) 5 ADP 20 ADP

  6. TYPES OFBIOLOGICALNITROGEN FIXATION FREE-LIVING (ASYMBIOTIC): • Cyanobacteria • Azotobacter ASSOCIATIVE: • Rhizosphere–Azospirillum • Lichens–cyanobacteria • Leaf nodules SYMBIOTIC: • Legume-rhizobia • Actinorhizal-Frankia

  7. SYMBIOTIC NITROGEN FIXATION

  8. ESTIMATED AVERAGE RATES OF BIOLOGICAL N2 FIXATION

  9. QUESTION 1: WHAT ARE RHIZOBIA? Rhizobia are one of the groups of microorganisms living in soil. Rhizobia are bacteria which are single cells about one thousandth of a millimetre long. Rhizobia belong to a specific group of bacteria that form a close association with legume plants. This association is called a SYMBIOSIS. The symbiosis results in visible, ball-like structures being formed on roots - these structures are called NODULES. The nodules are formed by the plant IN RESPONSE TO the presence of the bacteria. Rhizobia belong to a family of bacteria called RHIZOBIACEAE. There are a number of groups (genera and species) of bacteria in this family.

  10. The bacteria take nitrogen from the air (which plants cannot use) and convert it into a form of nitrogen called ammonium nitrogen, which plants can use. The process is called NITROGEN FIXATION and these bacteria are often called "NITROGEN FIXERS“ = DIAZOTROPH. Rhizobia can be EXTREMELY BENEFICIAL to plants in areas WHERE THE SOILS ARE LOW IN NITROGEN, if they form associations with legumes in these soils. Rhizobia are found in soils of many natural ecosystems. They may also be present in agricultural areas where they are associated with both crop legumes (like soybean) and pasture legumes (like clover). Usually, the rhizobia in agricultural areas have been introduced when the legumes were sown.

  11. LEGUME-RHIZOBIUM SYMBIOSIS • The subfamilies of legumes (Caesalpinioideae, Mimosoideae, Papilionoideae), 700 genera, and 19,700 species of legumes, • Only about 15% of the species have been evaluated for nodulation, • Rhizobium : • Gram -, rod • Most studied symbiotic N2-fixing bacteria • Now subdivided into several genera • Many genes known that are involved in nodulation (nod, nol, noe genes)

  12. PENGELOMPOKAN BRADYRHIZOBIUM RHIZOBIACEAE BRADYRHIZOBIUM (Genus II) B.japonicum - Soybeans Bradyrhizobium spp. RHIZOBIUM (Genus I) leguminosorum Biovar : phaseoli - Bean, trifolli - Clover, viceae - Pea, meliloti - Alfalfa, loti - Lupines, Lotus, galegae - Galega, Rhizobium spp. (host plant) SINORHIZOBIUM (Genus III) S. fredii - Soybeans S. xinjiangensis AZORHIZOBIUM (Genus IV) A. caulinodans - Sesbania

  13. TAXONOMY OF RHIZOBIA

  14. QUESTION 2: WHAT IS THE NODULATION PROCESS? The nodulation process is a series of events in which rhizobia interact with the roots of legume plants to form a specialised structure called a ROOT NODULE. The process involves complicated signals between the bacteria and the roots. In the first stages, the bacteria multiply near the root and then adhere to it. Next, the small hairs on the root's surface curl around the bacteria and they enter the root. Alternatively, the bacteria may enter directly through points on the root surface. The method of entry of the bacteria into the root depends on the type of plant.

  15. Once inside the root, the bacteria multiply within thin threads. Signals stimulate cell multiplication of both the plant's cells and the bacteria and this repeated division results in a mass of root cells containing many bacterial cells. Some of these bacteria then change into a form that is able to convert gaseous nitrogen into ammonium nitrogen (that is, they can "fix" nitrogen). These bacteria are then called BACTEROIDS. The shape the nodules form are controlled by the plant and nodules can vary considerably - both in size and shape. Most plants need very specific kinds of rhizobia to form nodules. For example, the rhizobia that form nodules on peas cannot form nodules on clover.

  16. QUESTION 3: WHAT DOES SPECIFICITY MEAN? Specificity is the DEGREE TO WHICH A BACTERIA SPECIES CAN FIX ATMOSPHERIC NITROGEN IN ASSOCIATION WITH MORE THAN ONE HOST SPECIES. For example, the bacteria Bradyrhizobiumjaponicum can only form symbiotic associations with soy bean plants and is said to have HIGH SPECIFICITY. Whereas another bacteria in the genus Bradyrhizobium can form symbiotic associations with both lupins and serradella and so has lower specificity.

  17. QUESTION 4: WHAT IS NITROGENASE? NITROGENASE is the bacterial enzyme that allows atmospheric nitrogen to be converted to ammonium. Enzymes are special kinds of proteins that allow reactions to occur at faster speeds than normal or under less extreme conditions. Without nitrogenase, atmospheric nitrogen can only be converted to ammonium with HIGH PRESSURE and TEMPERATURES. Nitrogenase requires a low oxygen environment to function.

  18. NITROGENASE Fd(ox) FeMo Cofactor Fd(red) N2 + 8H+ 8e- 2NH3 + H2 nMgATP nMgADP + nPi 4C2H2 + 8H+ 4C2H4 Dinitrogenase reductase Dinitrogenase N2 + 8H+ + 8e- + 16 MgATP  2NH3 + H2 + 16MgADP

  19. GENETICS OF NITROGENASE

  20. QUESTION 5: WHAT IS LEGHAEMOGLOBIN? LEGHAEMOGLOBIN is an organic molecule made up of two protein molecules that control the flow of oxygen to the bacteria that fix atmospheric nitrogen. Leghaemoglobin gives the inside of nodules their REDISH PINK COLOURING.

  21. QUESTION 8: HOW ARE RHIZOBIA IDENTIFIED? Rhizobia are identified according to HOW FAST THEY GROW AND MULTIPLY ON ARTIFICIAL FOOD SOURCES. They are very difficult to identify just from their shape or size alone, because all of them are very small and all are shaped like short rods with rounded ends. SO YOU CANNOT TELL THE DIFFERENT TYPES APART JUST BY LOOKING AT THEM, EVEN WITH A VERY POWERFUL MICROSCOPE. Rhizobia are usually grouped into "FAST" and "SLOW" growers, based on HOW QUICKLY THEY GROW ON ARTIFICIAL FOOD SOURCES. Recently, new methods of characterisation of rhizobia have been developed using DNA from known rhizobia.

  22. EXAMPLES OF NITROGEN-FIXING BACTERIA (*DENOTES A PHOTOSYNTHETIC BACTERIUM)

  23. FREE-LIVING N2 FIXATION ENERGY • 20-120 g C used to fix 1 g N COMBINED NITROGEN • nif genes tightly regulated • Inhibited at low NH4+ and NO3- (1 μg g-1 soil, 300 μM) OXYGEN • Avoidance (anaerobes), • Microaerophilly, • Respiratory protection, • Specialized cells (heterocysts, vesicles), • Spatial/temporal separation, • Conformational protection,

  24. ASSOCIATIVE N2FIXATION • Phyllosphere or rhizosphere (tropical grasses), • Azosprillum, Acetobacter, • 1 to 10% of rhizosphere population, • Some establish within root, • Same energy and oxygen limitations as free-living, • Acetobacter diazotrophicus lives in internal tissue of sugar cane, grows in 30% sucrose, can reach populations of 106 to 107 cells g-1 tissue, and fix 100 to 150 kg N ha-1 y-1

  25. PHOTOTROPHIC N2-FIXING ASSOCIATIONS • Lichens–cyanobacteria and fungi • Mosses and liverworts–some have associated cyanobacteria • Azolla-Anabaena (Nostoc)–cyanobacteria in stem of water fern C Gunnera-Nostoc–cyanobacteria in stem nodule of dicot C Cycas-Nostoc–cyanobacteria in roots of gymnosperm

  26. ACTINORHIZAL PLANT HOSTS

  27. ROLE OF ROOT EXUDATES GENERAL • Amino sugars, sugars SPECIFIC • Flavones (luteolin), isoflavones (genistein), flavanones, chalcones • Inducers/repressors of nod genes • Vary by plant species • Responsiveness varies by rhizobia species

  28. nodGene Expression Common nod genes Nod factor–LCO (lipo-chitin oligosaccharide)

  29. INFECTION PROCESS • Attachment, • Root hair curling, • Localized cell wall degradation, • Infection thread, • Cortical cell differentiation, • Rhizobia released into cytoplasm, • Bacterioid differentiation (symbiosome formation), • Induction of nodulins.

  30. NODULE METABOLISM Oxygen metabolism • Variable diffusion barrier • Leghemoglobin Nitrogen metabolism • NH3 diffuses to cytosol • Assimilation by GOGAT • Conversion to organic-N for transport • Carbon metabolism • Sucrose converted to dicarboxylic acids • Functioning TCA in bacteroids • C stored in nodules as starch

  31. ROOT NODULE FORMATION

  32. legume Fixed nitrogen (ammonia) Fixed carbon (malate, sucrose) rhizobia

  33. Nodulation in Legumes

  34. PHYSIOLOGY OF A LEGUME NODULE

  35. ATMOSPHERE N2 N2 Atmosphere Soil Nitrate and nitrogenousorganiccompoundsexported inxylem toshoot system Nitrogen-fixingbacteria N2 Denitrifyingbacteria H+ (From soil) NH4+ NH3 (ammonia) Soil NO3– (nitrate) NH4+ (ammonium) Nitrifyingbacteria Ammonifyingbacteria Organicmaterial (humus) Root

  36. SUMBER PERBANYAKAN INOKULAN

  37. R. leguminosarum nodules Pink color is leghaemoglobin a protein that carries oxygen to the bacteroids

  38. Azorhizobiumcaulinodans on Sesbania (Secang)

  39. A few legumes (such as Sesbaniarostrata) have stem nodules as well as root nodules. Stem nodules (arrows) are capable of photosynthesis as well as nitrogen fixation.

  40. CURRENT APPROACHES TO IMPROVING BIOLOGICAL NITROGEN FIXATION Enhancing survival of nodule forming bacterium by improving competitiveness of inoculant strains, Extend host range of crops, which can benefit from biological nitrogen fixation, Engineer microbes with high nitrogen fixing capacity. WHAT EXPERIMENTS WOULD YOU PROPOSE IF YOU WERE TO FOLLOW EACH OF THESE APPROACHES?

  41. Root Nodules YEMA Medium

  42. Komposisi YEMA (per 1 liter) Sumber : Subba Rao (1995) Media diautoklaf pada 120o C selama 15 menit. Kalsium karbonat (CaCO3) sebanyak 3 g per liter dapat ditambahkan bila dikehendaki senyawa untuk menetralisir asam yang terbentuk.

  43. REAKSI-REAKSI YANG DAPAT DIKATALISIS OLEH NITROGENASE (Zuberer, 2005).

  44. Metoda untuk mengatasi faktor-faktor kesuburan tanah yang menghambat proses penambatan N2 (Dierlolf et al., 2001).

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