Atosphere:79/100 (as N2), Plants:5-2/100(mostly organic), soil: 0.5-5/1000(mostlyorganic)
Loss and gain should be balanced.
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.
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)
|Type||Metals of dinitrogen reductase||H2/NH3 mol ratio||Activity (NH3 formed nmol/min/mg enzyme)||Organisms||Gene code|
|3(nitrogenase-3)||Fe||10:1||<50||Azotobacter, Rhodobacter, Rhodopseudomonas(photosynthetic)||anf|
Among three kingdoms:Eucarya, Procarya, Archea
Nitrogen fixing organisms or nif genes are not found in Eucarya.
List of well known free-living bacteria is shown in Table 1.
|Clostridium pasteurianum||Firmibacteria(Low GC GRAM positive)||Ana||Isolated first(1893)、cell 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|
(Low GC GRAM positive)
|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)
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
|Move to lower O2 pressure||Azospirillum|
|High Oxygen uptake by respiration||Azotobacter, Derxia|
|Hemoglobin combines efficiently with O2||In nodules of rhizobia-legume 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.
Bacteria (rhizobia)-leguminous plants
Actinomycetes (Frankia) -tree
Cyanobacteria (Nostoc) -plants
|Large group||Genera||Plant group||Tissue|| Inside or outside
| Legumes and
|Actinomycetes||Frankia|| Betulaceae and
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)
(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?)
Soybean root photo by K. Minamizawa of Tohoku University
Photos above are from Dr. Minamizawa, Tohoku University, Japan
|Rhizobium||Grow fast on culture media, and genes of nitrogen fixation and nodulation reside on Sym-plamid （except for R.loti）||Agrobacterium（Crown-gall inducing bacteria）|
|Bradyrhizobium||Grow slowly on culture media, and genes of nitrogen fixation and nodulation reside on chromosome||Rhodopseudomonas palustris(photosynthetic bacteria）|
|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|
|Genus||Species||hosts（Genus or species）||New genus name|
|R. meliloti||Medicago (Alfalfa) , Melilotus, Trigonella spp.||Sinorhizobium|
|R.fredii||Glycine max, (Soybean) 、 Glycine soja||Sinorhizobium|
|R. leguminosarum bv. viciae||Vicia fava (Faba bean) 、 Pisium sativa (pea) 、Lathyrus 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.セラトロ|
|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|
|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|
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/FONT>
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
|R.l.eguminosarum . bv. trifoli||D||5,7,3',4'-Tetra hydroxy,|
|R. leguminosarum bv..viciae||D||5,7,3',4'-Tetra hydroxy,|
|R. meliloti||D1||5,7,3',4'-Tetra hydroxy,|
5,7,4'-Trihydroxy -3'- methoxy
|R.sp NGR234||D||5,7,3',4'-Tetra hydroxy,|
|R.leguminosarum bv. viciae-RBL5560||2,3||C18△2 4 6 11,|
|R.meliloti AK41||1,2,3||C16△2 9,|
C16△2 4 9
C16△2 9, C16△2 4 9,
|B. japonicum USDA110||3||C18△9||H||2-O-Me-Fucosyl||H||H|
|B. japonicum USDA135||3||C18△9, C16||H, CH3CO||2-O-Me-Fucosyl||H||H|
|B. elkani USDA61||3||C18△9||H, CH3CO||Fucosyl-,|
|H, CH3||H, NH2CO|
|R.fredii USDA234||1,2,3||C18△11||H||Fucosyl, ||H||H|
|R. etli||5||C18△1, C18||H||CH3||NH2CO|
|R.loti NZP2037||5||C18△11, C18||NH2CO||4-O-Ac-Fucosyl||CH3||NH2CO|
|A. caulinodans ORS57||1,2,3||C18△11, C18||H, NH2CO||H, |
Rhijn van and Vanderleyden , Microbial Rev. 59,124-142
(1995)とHeidstra & Bisseling, New
From R.etli:Cardenaz et al. Plant Mol. Biol. 29, 453-464(1995)
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).
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
Crop rotation and leguminous plants
and Greenmanure crops
Application of organic matter (low in nitrogen content) stimulated non-symbiotic nitrogen fixation
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)
|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||Difference in 15N content is large||Change of 15N in time and space in soil||Moderate|
Wetland rice fields are fertile partly due to nitrogen fixation. See Table 10
|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|
|Introduced||kg N/ha per crop of rice|
|Aquatic legume |
(Sesbania, Croteralia, Aeschynomene, Indigofera etc.)
Azolla-an aquatic fern (pteridophyte) in symbiosis with cyanobacteria.
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