Made on 2000.3.30, Based from lecture in Cantho University, Vietnam BY IWAO WATANABE File name---BNF.html Revised on May 15 2000

Biological Nitrogen Fixation and its Use in Agriculture (outline)

Iwao WATANABE (JICA/Cantho Univ. expert Mar-Apr. 2000)

Countth visitor since May 15 2000


1) Nitrogen Cycle

Nitrogen content
Atosphere:79/100 (as N2), Plants:5-2/100(mostly organic), soil: 0.5-5/1000(mostlyorganic)

Loss and gain should be balanced.

Fig 1 Nitrogen Cycle


2) Industrial and biological nitrogen fixation

Some organisms(microorganisms) can assimilate atmospheric N2. And some plants assimilate N2 by the help of accompanying microorganisms capable of assimilating N2.Most well known plants of this capacity are leguminous plants. Leguminous plants have been widely used in agriculture.

Assimilation atmospheric N2: Biological Nitrogen Fixation (BNF)
In 1910-16, Habar-Bosch process to synthesize ammonia from N2 and H2 was established, and nitrogen fertilizer industry started : Industrial Nitrogen Fixation

Both processes have common characters
N2 + (6H) =2NH3
Biological: 30 deg. 1 atmosphere, enzyme catalysts-nitrogenase. Reducing agents are organic substances.
Industrial : 300-400 deg. 500 atm. chemical catalysts-Fe, Al oxides. Reducing agent is hydrogen.

3) Nitrogenase reaction (1)

Enzymes catalyzing BNF is nitrogenase (isolated since 1962)

3-1. Two components

I: Dinitrogen reductase (Fe-Mo protein): Combine N2 and reduce N2 to NH3
II: Dinitrogenase reductase (Fe protein):Accepting reducing power and reduce Dinitrogen reductase

Fig 2 Nitrogenase reaction model

3-2 Substrates specificity

Wide substrates containing double or triple bonds are reduced.

N2, N3,N3O, HCN, CH3NC, C2H2 (acetylene), H+
Acetylene reduction assay (ARA)
Acetylene+2H=ethylene(C2H4): easy to determine by gas chromatograph, and very sensitive.
Hydrogen evolution: Always evolved during N2 reduction,

N2 + 8HR=2NH3 + H2+ 8R (RH:reducing substances)

Genes: many genes (nif genes) are involved. In one microorganism (Klebsiella pneumoniae) most extensively studied, 20 genes are related to nitrogen fixation as shown in Fig. 3.

There are alternate nitrogenases in some organisms, expressed in the absence of Mo, or V (Table 2)

Fig 3 Nif genes map in Klebsiella pneumoneae and their roles


(Table 2)

Table 2 Alternate Nirogenase

Type Metals of dinitrogen reductase H2/NH3 mol ratio Activity (NH3 formed nmol/min/mg enzyme) Organisms Gene code
1(Mo-nitrogenase) Fe,Mo 2:1 1000 All nif
2(V-nitrogenase) Fe,V 5:1~2:1 1000~300 Clostridium,Azotobacter,Anabaena? vnf
3(nitrogenase-3) Fe 10:1 <50 Azotobacter, Rhodobacter, Rhodopseudomonas(photosynthetic) anf
Remark:Acitivities of pure enzyme. Alternate nitrogenases can reduce acetylene up to ethane.

4) Organisms that can fix nitrogen

Among three kingdoms:Eucarya, Procarya, Archea
Nitrogen fixing organisms or nif genes are not found in Eucarya.

4-1 Free-living and symbiotic nitrogen fixation

List of well known free-living bacteria is shown in Table 1.

Table 1 Well-studied free-living nitrogen-fixing bacteria

Species Super family Aerobic-Anaerobic
Clostridium pasteurianum Firmibacteria(Low GC GRAM positive) Ana Isolated first(1893)Acell free-extract. nitrogenase was first made(1962)
Klebsiella pneumoniae Proteobacteria- gamma Ana Close to E.coli. Genetics was first studied
Klebsiella oxytoca Proteobacteria- gamma Ana Isolated from rice root in Japan
Bacillus polymyxa Firmibacteria
(Low GC GRAM positive)
Ae GRAM-positive bacteria
Azotobacter chroococcum Proteobacteria- gamma Ae Azotobacter was isolated next.(1901). Widely used for research
Az. vinelandii Proteobacteria- gamma Ae  
Azospirilium brazilense Proteobacteria- alfa Ae Isolated from rhizosphere of C4-plant. Widely studied as rhizosphere bacteria
Azospirillum lipoferum Proteobacteria- alfa Ae Widely studied as rhizosphere bacteri
Rhodopspirillum rubrum Proteobacteria- alfa Ana Non-S-photosynthetic bacteria. Active in H2 production
Rhodobacter capsulatus Proteobacteria- alfa Ana Non-S-photosynthetic bacteria. Active in H2 production
Azoarcus sp. Proteobacteria- beta Ae Isolated from salt-tolerant plant. Enter into root tissue
Acetobacter diazotrophicus Proteobacteria- alfa Ae Isolated from sugarcane stalks. Tolerant to 30% sucrose
Herbaspirillum seropedicae Proteobacteria- beta Ae Isolated inside plant tissue(endophyte)
Methanosarcina barkeri Archea Ana Methane forming Archea. Discovered in 1984
Anabaena sp. 7120 Cyanobacteria Ae Heterocyst-forming . Most well studied among cyanobacteria
Gloethece sp. Cyanobacteria Ae Uni-cellular cyanobacteria. Fix N2 at night.

Free living: Product of BNF :ammonia is assimilated by themselves.

< FONT COLOR="red">Symbiotic: Live in association with partner, and ammonia is given to the partner. Efficiency (how much energy is consumed per mol of NH3 formed) is higher in symbiotic nitrogen fixation.(10-50 g N/kg glucose)

4-2 Nitrogenase reaction (2)

Other characteristics of nitrogenase to understand nitrogen fixation
a) Energy requirement

ATP is required (at least 16 mole ATP per mol of N2 reduced) Aerobic (under the presence of oxygen) respiration is most efficient in ATP production.:Dilemma!

b) Oxygen damage:

Nitrogen fixing organisms developed various ways to protect nitrogense from oxygen. Strategies: See Table 3

Table 3 Mechanism escaping O2 damage

Avoid (Escape)    
  Move to lower O2 pressure Azospirillum
O2 scavenging    
  High Oxygen uptake by respiration Azotobacter, Derxia
  Hemoglobin combines efficiently with O2 In nodules of rhizobia-legume symbiosis,
Casuarina-Frankia symbiosis
  Some proteins protect nitrogenase under O2 exposure Azotobacter
Separation in space    
  Nitrogenase in heterocysts (Heterocysts do not have photosynthetic ability, do not evolve O2) Cyanobacteria
  in Vesicles in Frankia-nodules symbiosis Frankia
  In colony, differentiate into photosynthetic filamants and nitrogen fixing filaments marine Scenedesmium
Separation in time    
  Photosynthesis daytime, and nitrogen fixation at night. Uni-cellular Cyanobacteria

Generally, Nitrogen Fixation is active under low (<0.1% v/v) O2 pressure

c) Ammonia inhibition

When product-ammonia-is available, cells stop nitrogen fixation.

5) Symbiotic nitrogen fixation

There are 3 groups in symbiosis of nitrogen fixing microorganisms with higher plants

Bacteria (rhizobia)-leguminous plants

Actinomycetes (Frankia) -tree

Cyanobacteria (Nostoc) -plants

Table 4 Symbiotic Nitrogen Fixation

Microorganisms Host plants Location Isolated
Large group Genera Plant group Tissue Inside or outside
plant cell
Legumes and
Inside Yes
Actinomycetes Frankia Betulaceae and
8 family(trees)
Inside Yes
Cyanobacteria Nostoc Bryophytes
(Antheros etc.)
Leaf cavity Outside Yes
Leaf cavity Outside No
Nostoc Cycadophyta
(Cycas,Macrozamia etc.)
Collaroid root Outside Yes
Nostoc Angiosperm
Gland tissue Inside Yes

Legume-rhizobia symbiosis is probably most specialized (developed) , and most extensively studied.

What characters are common in symbiosis?

I : specificity II: In harmony (one partner never surpasses another)

5-1 How specificity is decided from bacteria, from plant?

(How plant allows invading by bacteria, and supports its activity? in contrast to plant-parasites relationship)

Communicate each other by signal molecules (molecular ID cards?)

Fig 4 Soybean nodules(left) and Fig 5 Stem nodules of Aeschynomene afraspera (right)

soybean stemnodule

Soybean root photo by K. Minamizawa of Tohoku University

Fig 6 Nodules section(left) and Fig 7 Bacteroid tissues(right)



Photos above are from Dr. Minamizawa, Tohoku University, Japan

6 Legume-rhizobia relationship

6-1 Specificity

Table 5 Major groups of rhizobia

Genus Characteristics Closest relatives
Rhizobium Grow fast on culture media, and genes of nitrogen fixation and nodulation reside on Sym-plamid iexcept for R.lotij AgrobacteriumiCrown-gall inducing bacteriaj
Bradyrhizobium Grow slowly on culture media, and genes of nitrogen fixation and nodulation reside on chromosome Rhodopseudomonas palustris(photosynthetic bacteriaj
Azorhizobium Grow slowly on culture media, and genes of nitrogen fixation and nodulation reside on chromosome. Capble of free-living nitrogen fixation. Isolated from the stem nodules of Sesbania rostrata Aquabacter spiritensis

Table 6@Rhizobia species and their major hosts

Genus Species hostsiGenus or speciesj New genus name
  R. meliloti Medicago (Alfalfa) , Melilotus, Trigonella spp. Sinorhizobium
  R.fredii Glycine max, (Soybean) A Glycine soja Sinorhizobium
  R. leguminosarum bv. viciae Vicia fava (Faba bean) A Pisium sativa (pea) ALathyrus spp.  
  R. leguminosarum bv. trifolii Trifolium spp. (clovers)  
  R. leguminosarum bv. phaseoli Phaseolus vulgaris (common bean)  
  R.tropici Phaseolus vulgaris, (common bean) Leucoena spp (Ipil Ipil(in Philippines)) . Macroptilium spp.Zg  
  R. etli Phaseolus vulgariscommon bean  
  R.galegae Galega officinalis, G.orientalis  
  R.loti Lotus spp. (Birdfoot trefoil) Mesorhizobium
  R.huakuii Astragalus sinicus (Chinese milk vetch or Renge) Mesorhizobium
  R.ciceri Cicer arietinum Mesorhizobium
  Rhizobium sp. strain NGR234 tropical legumes, Parasponiaetc.  
  B. japonicum Glycine max (soybean) , Glycine soja (wild soybean) etc  
  B.elkani Glycine max, Glycine soya, Macroptilium spp. (Seratro) ,  
  Bradyrhizobium sp. Vigna (cowpea) , Arachis (peanut) and many tropical legumes  
  Bradyrhizobium sp. strain Parasponia Parasponia spp. etc.  
  A.caulinodans Sesbania rostratastem nodules  
Often a word -"rhizobia" is used to include three groups

Certain bacteria form nodule to a limited number of host plants. (though the range of host plants varies with rhizobia-wide to narrow). Generally rhizobia from tropical leguminous plants have wide host range.

Three group of rhizobia genus.(rhizobia refers to three groups) Rhizobium (incld. Mesorhizobium, and Sinorhizobium), Bradyrhizobium, and Azorhizobium
(see Table 5, and 6)

6-1-1 Nodulation process

a) Invasion from root hair, or crack of epidermis layer, or epidermis cell

b) Formation infection thread(in most case)

c) Cell division in cortex layer

d) Bacteria enter into plant cells of nodule tissue, and spread within them

e) Nitrogen fixing genes are activated, and start nitrogen fixation in matured nodules.

6-1-2 Bacteria genes related to nitrogen fixation, and nodulation

nif, nod, fix genes: nod genes: nodulation, nif genes: common with free-living nitrogen fixation, fix genes; Unique to symbiotic nitrogen fixation

In most of Rhizobium, these genes sets are located a large plasmid (satellite DNA-often transferable to other bacteria cells)-Sym-plamid.

6-1-3 Sequence of molecular communication

a) Plant excrete certain flavonoid compounds(differ between plants)

b) Rhizobia recognize certain flavonoids(gene nod D product is a sensor)(See Table 7)

c) If NOD-D protein (product of nodD genes)recognizes right flavonoids, switch of other nod genes on, and products of nod genes coded proteins are formed--Nod factors (oligochitin compounds)

d) Plant, in return, recognize right Nod factors.(See Table 8.)
Early processes of nodulation is triggered by Nod factors.

e) In addition to Nod factors, extracellular polysaccharides of bacteria may function in recognition of bacteria at later process of nodulation (bacteria spreading inside plant cells)

Chemical structures of various flavonoids compounds and Nod factors, recognized and Recognized by corresponding bacteria are shown in Tables 7, and figures.

Positions of residues are shown in molecular structures Note:Bradyrhizobium japonicum, and Rhizobium fredii ,forming nodules to soybean respond to similar flavonoids, and synthesized similar Nod factors, despite difference in taxonomical position of two groups of soybean bacteria

Bone structure of flavonoids

Table 7 Activation of NodD protein by flavonoid compounds

From (Rhijn van P, and Janderleyden J (1995)

Bacteria NodD Flavones Flavanones Isoflavones Others
R.l.eguminosarum . bv. trifoli D 5,7,3',4'-Tetra hydroxy,
7,4'- Dihydroxy
R. leguminosarum bv..viciae D 5,7,3',4'-Tetra hydroxy,
5,7,3',4'-Tetra hydroxy,
R. meliloti D1 5,7,3',4'-Tetra hydroxy,
5,7,4'-Trihydroxy -3'- methoxy
R. meliloti D2       4,4'-Dihydroxy-2'-methoxychalcone
R.leguminosarum bv.phaseoli D2 5,7,3'-Trihydroxy 5,7,4'-Trihydroxy 5,7,4'-Trihydroxy  
R.tropici D1 5,7,3' Trihydroxy,
5,7- Dihydroxy
Br. japonicum D     5,7,4'-Trihydroxy
R.sp NGR234 D 5,7,3',4'-Tetra hydroxy,
Az. caulinodans D   7,4'-Dihydroxy    

Bone structure of Nod factors

Table 8@Nod Factors of rhizobia

rhizobia n Q R1 R2 R3 R4, 5
R.leguminosarum bv. viciae-RBL5560 2,3 C182 4 6 11,
R.meliloti AK41 1,2,3 C162 9,
C162 4 9
R.meliloti 2011 2,3 C169,
C162 9, C162 4 9,
(-1)-OH C18-26
R.tropici CFN299 3 C1811 H HSO3 CH3 H
B. japonicum USDA110 3 C189 H 2-O-Me-Fucosyl H H
B. japonicum USDA135 3 C189, C16 H, CH3CO 2-O-Me-Fucosyl H H
B. elkani USDA61 3 C189 H, CH3CO Fucosyl-,
R.fredii USDA234 1,2,3 C1811 H Fucosyl, 2-O-Me-Fucosyl H H
R.sp. NGR234 3 C1811,C16 H 2-O-Me-Fucosyl,
R. etli 5 C181, C18 HCH3 NH2CO
R.loti NZP2037 5 C1811, C18 NH2CO 4-O-Ac-Fucosyl CH3 NH2CO
A. caulinodans ORS57 1,2,3 C1811, C18 H, NH2CO H,


Rhijn van and Vanderleyden , Microbial Rev. 59,124-142 (1995)Heidstra & Bisseling, New Phytol133:25(1996)

From R.etli:Cardenaz et al. Plant Mol. Biol. 29, 453-464(1995)

Fig 8 ID cards molecules exchange in Rhizobium-legume root interaction


First, host plants excrete flavonoids, and bacteria NOD-protein recognize proper flavonids, and initiate synthesis of Nod factor by a series of nod genes products.
Nodfactors, in return, initiate early processes of nodulation. In addition to Nodfactors, cell surface polysaccharides may be involved in the later prosesses of nodule symbiosis.

6-1-4 Events in mature nodules.

Nitrogen fixation proceeds only in mature nodules. Hemoglobin(leg-hemoglobin--red protein) acts as O2 storage protein and keeping free O2 concentration very low in nodule tissues

Plant transport mainly sugars to nodules, and bacteria assimilate only organic acids. Ammonia (product of nitrogen fixation) is assimilated by plant, and transported to leaves as asparagine (pea) or as ureido compounds(soybean).

Fig 9 Functions of nitrogen fixing mature nodule


7 Associative nitrogen fixation

Nitrogen fixing bacteria are present around root, or inside plant tissues, and fix nitrogen, and contribute to plant nitrogen nutrition. This process is (may be) not negligible in tropical grasses (C4-Gramineae), and wetland rice.

In sugarcane, 20-50% of nitrogen in plant are originated by associative nitrogen fixation.

Bacteria are not so specific, no specialized organ like nodule is formed. Probably direct transfer of ammonia formed by nitrogen fixing bacteria does not occur

8) Agricultural use of BNF

8-1 History:

Used as

Crop rotation and leguminous plants

and Greenmanure crops

Application of organic matter (low in nitrogen content) stimulated non-symbiotic nitrogen fixation

8-2 Quantity fixed by various system

World-wide estimation :BNF:15-18 million ton, Industrial :8 million ton. The 2/3 of BNF are symbiotic nitrogen fixation

a) Estimated by nitrogen balance method

N gain = Input - output(Crop removal + Soil N loss)

In long-term fertilizer experiments revealed N gains amounting to 20-40 kgN/ha.per year Phosphate application encouraged N gains

b) Contribution of nitrogen fixation in symbiotic systems


In soybean seeds in Japan average :50% ( For methods of estimating the quantity of N2-fixation See Table 9)

8-3 Nitrogen fixation in wetland rice fields

Table 9 Comparison of methods of estimating nitrogen fixation

Difference in 15N content is large
Methods Advantages Disadvantages Sensitivity
1. Total N balance Simplest Low sensitivity
ncluding other inputs.
2. 15N2 incorporation Most direct Expensive, only for short period High-moderate
3. Acetylene reduction Simple, highly sensitive Indirect, semi-quantitative High
4. 15N dilution Throuout growing season Only N Fixation in plant
Varies with reference plants
4a. Natural abundance Simple, no disturbance to system only slight difference in 15N content Low
4b. Substrate addition Change of 15N in time and space in soil Moderate

Wetland rice fields are fertile partly due to nitrogen fixation. See Table 10

Table 10 Range of estimates of N2 fixed by various agents in wetland rice fields.

(Modified from Roger and Ladha 1992)
Indigenous kg N/ha per crop of rice
N2-fixation associated with rice rhizosphere 1-7
N2-fixation associated with straw 2-4
Total heterotrophic N2-fixation 1-31
Cyanobacteria(surface) 0-80
Introduced kg N/ha per crop of rice
Azolla 10-50(fields)
Aquatic legume
(Sesbania, Croteralia, Aeschynomene, Indigofera etc.)

8-4 Azolla and its use

Azolla-an aquatic fern (pteridophyte) in symbiosis with cyanobacteria.

WEB SITE is available
Click "ABC of Azoll"