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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Fiksasi Biologis N 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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Fiksasi Biologis N.. • 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 •
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Free living aerobic bacteria – Azotobacter – Beijerinckia – Klebsiella – Cyanobacteria (lichens)
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• Free living anaerobic bacteria – Clostridium – Desulfovibrio – Purple sulphur bacteria – Purple non-sulphur bacteria – Green sulphur bacteria
Free living associative bacteria – Azospirillum
Symbionts – Rhizobium (legumes) – Frankia (alden trees)
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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)
N2 + 8H+
Fd(red) 8e-
2NH3 + H2
nMgATP
nMgADP + nPi
4C2H2 + 8H+
Dinitrogenase reductase
4C2H2
Dinitrogenase
N2 + 8H+ + 8e- + 16 MgATP 2NH3 + H2 + 16MgADP KRT-2011
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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 μg g1 soil, 300 μM)
Oxygen • • • • • •
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
Actinorhizal Plant Hosts Family
Genera
Betulaceae
Alnus
Casuarinaceae
Allocasuarina, Casuarina, Ceuthostoma, Gymnostoma
Myricaceae
Comptonia, Myrica
Elaeagnaceae
Elaeagnus, Hippophaë, Shepherdia
Rhamnaceae
Ceanothus, Colletia, Discaria, Kentrothamnus, Retanilla, Talguenea, Trevoa Cercocarpus, Chamaebatia, Cowania, Dryas, Purshia Coriaria KRT-2011 31 Datisca
Rosaceae Coriariaceae Datiscaceae
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) KRT-2011
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Taxonomy of Rhizobia Genus
Species
Host plant
Rhizobium
leguminosarum bv. trifolii “ bv. viciae “ bv. phaseoli tropici etli
Trifolium (clovers) Pisum (peas), Vicia (field beans), Lens (lentils), Lathyrus Phaseolus (bean) Phaseolus (bean), Leucaena Phaseolus (bean)
Sinorhizobium
meliloti fredii saheli teranga
Melilotus (sweetclover), Medicago (alfalfa), Trigonella Glycine (soybean) Sesbania Sesbania, Acacia
Bradyrhizobium
japonicum elkanii liaoningense
Glycine (soybean) Glycine (soybean) Glycine (soybean)
Azorhizobium
caulinodans
Sesbania (stem nodule)
‘Meso rhizobium’
loti huakuii ciceri tianshanense mediterraneum
Lotus (trefoil) Astragalus (milkvetch) Cicer (chickpea)
[Rhizobium]
galegae
Galega (goat’s rue), Leucaena
Photorhizobium
spp.
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Cicer (chickpea) Aeschynomene (stem nodule)33
Rhizobium Root Nodules
The picture above shows a clover root nodule. Available from [Internet] KRT-2011
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Rhizobium Root Nodules
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A few legumes (such as Sesbania rostrata) have stem nodules as well as root nodules. Stem nodules (arrows) are capable of photosynthesis as well as nitrogen fixation. KRT-2011 36
Formation of a Root Nodule
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Nodulation in Legumes
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Infection Process • Menempel • Rambut akar menggulung • Degradasi dinding sel setempat • Infection thread • Differensiasi Cortical cell • Rhizobia melepas ke dalam cytoplasm • Bacteroid differentiation (symbiosome formation) • Induksi dari nodulins KRT-2011
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Role of Root Exudates General • Amino sugars, sugars
Specific • Flavones (luteolin), isoflavones (genistein), flavanones, chalcones • Inducers/repressors of nod genes • Berbeda tergantung species tanaman • Responsiveness varies by rhizobia species KRT-2011
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nod Gene Expression Common nod genes
Nod factor–LCO (lipo-chitin oligosaccharide) KRT-2011
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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 KRT-2011
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Synthesis of Cellular Components
Reaksi dikatalisis oleh enzim nitrogenase
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