BIOLOGICAL NITROGEN FIXATION
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• Beberapa tumbuhan dapat membentuk senyawa yang mengandung N dari udara • Melalui bakteri yang memfiksasi N inilah N atmosfir memasuki biosfer (lingkungan makhluk hayati) • Bakteri pemfiksasi N mereduksi N atmosfir menjadi amonia (NH3) NH4+ • Terdapat 2 bakteri yang memfiksasi N yaitu: Bakteri yang hidup bebas dan Bakteri yang bersimbiosis Rhizobium sp. • Bakteri mampu memanfaatkan berbagai sumber nitrogen untuk sintesis protein • Nitrogen merupakan elemen dari molekul biologis, spt: asam amino, nucleotides, protein dan DNA KRT-2011
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The Nitrogen Cycle •
Mineralization: Organic nitrogen (mostly amino acids) → NH4+ (All organisms)
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Nitrification: NH4+ → NO2NO2- → NO32-
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(Nitrosomonas) (Nitrobacter)
Denitrification: NO3- → N2O N2O → N2 (Several species, including certain Pseudomonas and Bacillus) KRT-2011
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The Nitrogen Cycle •
Assimilatory Nitrate Reduction
NO32- → Organic Nitrogen (Many microbial species and plants)
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N2 fixation
N2 → NH4+ Free-living nitrogen fixers eg Azotobacter and Azospirillum Symbiotic nitrogen fixers eg Rhizobium and Bradyrhizobium Cyanobacteria attached to the cordgrass plant Spartina in salt marshes (=rawa?) KRT-2011
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Most plants depend on bacteria to supply nitrogen
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The Nitrogen Cycle
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General view of nitrogen metabolism
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Pathways Involved in Nitrogen Utilization 1. Protein Digestion – by proteinase & peptidase 2. Oxidative Deamination
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3. Reductive Deamination
4. Decarboxylation
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5. Transamination Reactions
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6. Nitrification 7. Denitrification
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Industrial Fixation • Di bawah tekanan tinggi, pada suhu 600oC, dan dengan penggunaan catalyst, nitrogen dan hydrogen atmosfir (biasanya turunan dari natural gas atau petroleum) dapat bergabung untuk membentuk ammonia (NH3). • Ammonia dapat digunakan secara langsung sebagai pupuk, tetapi umumnya diproses lebih lanjut menjadi urea dan ammonium nitrate (NH4NO3). KRT-2011
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Biological Fixation Kemampuan untuk memfiksasi nitrogen hanya ditemukan dalam bacteria tertentu. • Beberapa hidup dalam symbiotic relationship dengan tanaman dari famili legume (e.g., kedelai, alfalfa). • Beberapa ada yang bersimbiosis dengan tanaman selain legumes (e.g., alders). • Beberapa bakteri fiksasi-nitrogen hidup bebas dalam tanah • Nitrogen-fixing cyanobacteria penting untuk mempertahankan kesuburan dari lingkungan semiaquatic seperti lahan padi KRT-2011
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Biological Fixation cont. • Biological nitrogen fixation membutuhkan serangkaian enzymes dan penghasil ATP yang sangat besar. • Meskipun produk stabil pertama dari proses adalah ammonia, ini cepat diinkorporasi ke dalam protein dan senyawa nitrogen organic lain • Para ilmuwan memperkirakan bahwa biological fixation secara global menambah kira-kira 140 million metric tons nitrogen kedalam ecosystems setiap tahun.
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Some nitrogen fixing organisms • Free living aerobic bacteria – Azotobacter – Beijerinckia – Klebsiella – Cyanobacteria (lichens)
• Free living associative bacteria – Azospirillum
• Free living anaerobic bacteria • Symbionts – Clostridium – Rhizobium (legumes) – Desulfovibrio – Frankia (alden trees) – Purple sulphur bacteria – Purple non-sulphur bacteria – Green sulphur bacteria
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Some nitrogen fixing organisms Free living
Symbiotic Aerobes Anaerobes Leguminous Non Heterotrophs Phototrophs Heterotrophs Phototrophs plants leguminous plants Azotobacter spp. Various Clostridium spp Chromatium soybeans, Alnus, Myrica Klebsiella Cyanobacteria Desulfovibrio Chloribium clover, Ceanthus Beijerinckia DisulfotoRhodospirillum locust, etc Comptorinia Bacillus maculum Rhodopseudo- In association Casurina polymyxa monas with a bacterium in assocation Mycobacterium Rhodoof the genus with flavum microbium Rhizobium or actinomycetes Azospirillium Rhodobacter Bradyrhizobium of the genus lipoferum Heliobacterium Frankia Citrobacter freundii Some Methylotrophs KRT-2011 16
Estimated Average Rates of Biological N2 Fixation
Organism or system
Free-living microorganisms Cyanobacteria Azotobacter Clostridium pasteurianum
N2 fixed (kg ha-1 y-1) 25 0.3 0.1-0.5
Grass-Bacteria associative symbioses Azospirillum
5-25
Cyanobacterial associations Gunnera Azolla Lichens
10-20 300 40-80
Leguminous plant symbioses with rhizobia Grain legumes (Glycine, Vigna, Lespedeza, Phaseolus) Pasture legumes (Trifolium, Medicago, Lupinus)
50-100 100-600
Actinorhizal plant symbioses with Frankia Alnus Hippophaë Ceanothus Coriaria KRT-2011 Casuarina
40-300 1-150 1-50 50-150 50
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Rank of Biological Nitrogen Fixation N2 fixing system Rhizobium-legume
Nitrogen Fixation (kg N/ha/year) 50 - 600
Cyanobacteria- moss
10 - 300
Rhizosphere associations Free- living
5 - 25 0.1 - 25 KRT-2011
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Nitrogen Fixation • Semua bacteria fiksasi nitrogen menggunakan conserved enzyme complex yang disebut Nitrogenase • Nitrogenase tersusun dari dua subunits: an iron-sulfur protein dan a molybdenum-iron-sulfur protein • Aerobic organisms face special challenges to nitrogen fixation karena nitrogenase inactivasi bila oxygen bereaksi dengan iron component of the proteins KRT-2011
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Nitrogenase FeMo Cofactor
Fd(ox)
Fd(red)
N2 + 8H+
8e-
2NH3 + H2
nMgATP nMgADP + nPi
4C2H2 + 8H+
Dinitrogenase reductase
4C2H2
Dinitrogenase
N2 + 8H+ + 8e- + 16 MgATP
2NH3 + H2 + 16MgADP
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Nitrogenase
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Types of Biological Nitrogen Fixation Free-living (asymbiotic) • Cyanobacteria • Azotobacter
Associative • Rhizosphere–Azospirillum • Lichens–cyanobacteria • Leaf nodules
Symbiotic • Legume-rhizobia • Actinorhizal-Frankia KRT-2011
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Free-living N2 Fixation Energy
• 20-120 g C used to fix 1 g N
Combined Nitrogen
• nif genes tightly regulated • Terhambat pada NH4+ and NO3- rendah (1 g g1 soil, 300 M)
Oxygen
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Menghindar (anaerobes) Microaerophilly Respiratory protection Specialized cells (heterocysts, vesicles) Spatial/temporal separation Conformational (=penyesuaian diri?) protection KRT-2011
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Heterocyst
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Associative N2 Fixation • • • • • •
Phyllosphere or rhizosphere (tropical grasses) Azosprillum, Acetobacter 1 to 10% of rhizosphere population Beberapa berada dalam akar Same energy and oxygen limitations as free-living Acetobacter diazotrophicus hidup dalam internal tissue dari tebu, tumbuh dalam 30% sucrose, dapat mencapai populasi 106 sampai 107 cells g-1 tissue, dan memfiksasi 100 to 150 kg N ha-1 y-1
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Phototrophic N2-fixing Associations • Lichens–cyanobacteria and fungi • Mosses and liverworts–some have associated cyanobacteria • Azolla-Anabaena (Nostoc)–cyanobacteria dalam batang water fern Gunnera-Nostoc–cyanobacteria dalam batang nodule dari dicot Cycas-Nostoc–cyanobacteria dalam akar gymnosperm
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Azolla pinnata (left) 1cm. Anabaena from crushed leaves Of Azolla.
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Simbiosis Anabaena-Azolla
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Frankia and Actinorhizal Plants • Actinomycetes (Gram +, filamentous); septate hyphae; spores in sporangia; thick-walled vesicles
Frankia vesicles showing thick walls that confer protection from oxygen. Bars are 100 nm. KRT-2011
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Alder and the other woody hosts of Frankia are typical pioneer species that invade nutrient-poor soils. These plants benefit from the nitrogenfixing association, while supplying the bacterial symbiont with photosynthetic products. KRT-2011 30
