6. Nitrification 7. Denitrification

KRT-2011 1

6. Nitrification

7. Denitrification

KRT-2011 2

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)

KRT-2011 3

Some nitrogen fixing organisms

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 Disulfoto- Rhodospirillum locust, etc Comptorinia

Bacillus maculum Rhodopseudo- In association Casurina

polymyxa monas with a bacterium in assocation

Mycobacterium Rhodo- of the genus with

flavum microbium Rhizobium or actinomycetes

Azospirillium Rhodobacter Bradyrhizobium of the genus

lipoferum Heliobacterium Frankia

Citrobacter freundii Some

Methylotrophs

Free living Symbiotic

Aerobes Anaerobes

KRT-2011 4

Estimated Average Rates of Biological N

Fixation

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

Free-living microorganisms Cyanobacteria

Azotobacter

Clostridium pasteurianum

0.325

0.1-0.5 Grass-Bacteria associative symbioses

Azospirillum 5-25

Cyanobacterial associations Gunnera

Azolla Lichens

10-20 40-80300

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

KRT-2011 5

Rank of Biological Nitrogen Fixation

N

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

KRT-2011 8

Types of Biological Nitrogen Fixation

Free-living (asymbiotic)

• Cyanobacteria

• Azotobacter

Associative

• Rhizosphere–Azospirillum

• Lichens–cyanobacteria

• Leaf nodules

Symbiotic

• Legume-rhizobia

• Actinorhizal-Frankia

Examples of N₂ fixing plants

Mesquite, Acacia, Palo Verde

Symbionts are Rhizobium & Bradyrhizobium Lupines - Bradyrhizobium

Root nodules

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Free-living N

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 g^-1 soil, 300 μM)

Oxygen

• Menghindar (anaerobes)

• Microaerophilly

• Respiratory protection

• Specialized cells (heterocysts, vesicles)

• Spatial/temporal separation

• Conformational (=penyesuaian diri?) protection

KRT-2011

Heterocyst

KRT-2011 14

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 17

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.

KRT-2011 18

Actinorhizal Plant Hosts

Family Genera

Betulaceae Alnus Casuarinace

ae

Allocasuarina, Casuarina, Ceuthostoma, Gymnostoma

Myricaceae Comptonia, Myrica Elaeagnacea

e Elaeagnus, Hippophaë, Shepherdia Rhamnacea

e 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

COnly about 15% of the species have been evaluated for nodulation

CRhizobium

CGram -, rod

CMost studied symbiotic N₂-fixing bacteria CNow subdivided into several genera

CMany genes known that are involved in nodulation (nod, nol, noe genes)

KRT-2011 20

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)

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]

KRT-2011 22

Rhizobium Root Nodules

KRT-2011 23

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.