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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

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The Nitrogen Cycle• Mineralization:

Organic nitrogen (mostly amino acids) NH4+

(All organisms)

• Nitrification:NH4

+ NO2- (Nitrosomonas)

NO2- NO3

2- (Nitrobacter)

• Denitrification:NO3

- N2O

N2O N2

(Several species, including certain Pseudomonas and Bacillus)

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The Nitrogen Cycle• Assimilatory Nitrate Reduction

NO32- Organic Nitrogen

(Many microbial species and plants)

• N2 fixationN2 NH4

+

Free-living nitrogen fixers eg Azotobacter and Azospirillum

Symbiotic nitrogen fizers eg Rhizobium and Bradyrhizobium Cyanobacteria attached to the cordgrass plant Spartina in salt marshes (=rawa?)

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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).

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Biological FixationKemampuan 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 semi-aquatic seperti lahan padi

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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 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

Leguminous Non Heterotrophs Phototrophs Heterotrophs Phototrophs plants leguminous plantsAzotobacter spp. Various Clostridium spp Chromatium soybeans, Alnus, MyricaKlebsiella Cyanobacteria Desulfovibrio Chloribium clover, CeanthusBeijerinck ia Disulfoto- Rhodospirillum locust, etc ComptoriniaBacillus maculum Rhodopseudo- In association Casurina polymyxa monas with a bacterium in assocationMycobacterium Rhodo- of the genus with flavum microbium Rhizobium or actinomycetesAzospirillium Rhodobacter Bradyrhizobium of the genus lipoferum Heliobacterium Frank iaCitrobacter freundiiSome Methylotrophs

Free living SymbioticAerobes Anaerobes

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Estimated Average Rates of Biological N2 Fixation

Organism or system N2 fixed (kg ha-1 y-1)

Free-living microorganismsCyanobacteria Azotobacter Clostridium pasteurianum

250.3

0.1-0.5

Grass-Bacteria associative symbiosesAzospirillum 5-25

Cyanobacterial associationsGunnera AzollaLichens

10-20300

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

Casuarina

40-300

1-150

1-50

50-150

50

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Rank of Biological Nitrogen Fixation

N2 fixing system Nitrogen Fixation (kg N/ha/year)

Rhizobium-legume

50 - 600

Cyanobacteria- moss

10 - 300

Rhizosphere associations

5 - 25

Free- living 0.1 - 25

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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

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Nitrogenase

FeMo Cofactor

N2 + 8H+

2NH3 + H2

8e-

4C2H2 + 8H+ 4C2H2

DinitrogenaseDinitrogenase

reductase

Fd(red)

Fd(ox)

nMgATP

nMgADP + nPi

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

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Free-living N2 Fixation

Energy• 20-120 g C used to fix 1 g NCombined Nitrogen• nif genes tightly regulated• Terhambat pada NH4

+ and NO3- rendah (1 μg g-

1 soil, 300 μM)Oxygen• Menghindar (anaerobes)• Microaerophilly• Respiratory protection• Specialized cells (heterocysts, vesicles)• Spatial/temporal separation• Conformational (=penyesuaian diri?) protection

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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.

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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 nitrogen-fixing association, while supplying the bacterial symbiont with photosynthetic products.

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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

Rosaceae Cercocarpus, Chamaebatia, Cowania, Dryas, Purshia

Coriariaceae Coriaria

Datiscaceae Datisca

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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)

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Taxonomy of Rhizobia

Genus Species Host plantRhizobium leguminosarum bv. trifolii

“ bv. viciae “ bv. phaseolitropicietli

Trifolium (clovers)Pisum (peas), Vicia (field beans), Lens (lentils), LathyrusPhaseolus (bean)Phaseolus (bean), LeucaenaPhaseolus (bean)

Sinorhizobium melilotifrediisaheliteranga

Melilotus (sweetclover), Medicago (alfalfa), TrigonellaGlycine (soybean)Sesbania Sesbania, Acacia

Bradyrhizobium japonicumelkaniiliaoningense

Glycine (soybean)Glycine (soybean)Glycine (soybean)

Azorhizobium caulinodans Sesbania (stem nodule)

‘Meso rhizobium’ lotihuakuiiciceritianshanensemediterraneum

Lotus (trefoil)Astragalus (milkvetch)Cicer (chickpea)

Cicer (chickpea)

[Rhizobium] galegae Galega (goat’s rue), Leucaena

Photorhizobium spp. Aeschynomene (stem nodule)

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Rhizobium Root Nodules

The picture above shows a clover root nodule. Available from [Internet]

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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.

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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

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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

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nod Gene Expression

Commonnod genes

Nod factor–LCO(lipo-chitin

oligosaccharide)

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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

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Synthesis of Cellular Components

Reaksi dikatalisis oleh enzim nitrogenase