Biological Nitrogen Fixation and its Use in Agriculture (outline)

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Outline

• 1) Nitrogen Cycle
• 2) Industrial and biological nitrogen fixation(BNF)
• 3) Nitrogenase reaction (1)
• 4) Organisms that can fix nitrogen
• 5) Symbiotic nitrogen fixation
• 6) Legume-rhizobia relationship
• 7) Associative nitrogen fixation
• 8) Agricultural use of BNF

1) Nitrogen Cycle

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

• N gain process: From N2 to microorganisms, and eventually to plant
• N oxidation: ammonia is oxidized to nitrate (nitrification)
• N loss process: Nitrate is ultimately lost as N2 (return to atmosphere)

Loss and gain should be balanced.

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)

3-1. Two components

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)

(Table 2)

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.

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!

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

• b-1) Stop synthesizing of nitrogenase, when cells are exposed to oxygen
• b-2) Making oxygen concentration around cells low
• b-3) Seperate oxygen evolution and nitrogen fixation in 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

Bacteria (rhizobia)-leguminous plants

Actinomycetes (Frankia) -tree

Cyanobacteria (Nostoc) -plants

Table 4 Symbiotic Nitrogen Fixation

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?)

Soybean root photo by K. Minamizawa of Tohoku University

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

6 Legume-rhizobia relationship

6-1 Specificity

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, 5,7,3'-Trihydroxy, 7,4'- Dihydroxy	5,7,4'-Trihydroxy
R. leguminosarum bv..viciae	D	5,7,3',4'-Tetra hydroxy, 5,7,3'-Trihydroxy, 7,3',4'-Trihydroxy	5,7,4'-Trihydroxy, 5,7,3',4'-Tetra hydroxy, 5,7,3'-Trihydroxy-4'-methoxy
R. meliloti	D1	5,7,3',4'-Tetra hydroxy, 7,3',4'-Trihydroxy, 5,7,4'-Trihydroxy -3'- methoxy			4,4'-Dihydroxy-2'-methoxychalcone
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, 7,4'-Dihydroxy, 5,7- Dihydroxy	5,7,4'-Trihydroxy,
Br. japonicum	D			5,7,4'-Trihydroxy 7,4'-Dihydroxy
R.sp NGR234	D	5,7,3',4'-Tetra hydroxy, 5,7,3'-Trihydroxy, 7,4'-Dihydroxy, 5,7-Dihydroxy	5,7,4'-Trihydroxy 5,7,3'-Trihydroxy-4'-methoxy	5,7,4'-Trihydroxy 7,4'-Dihydroxy	5,7,3'-Trihydroxy-flavonol, 5,7,4'-Trihydroxy-flavonol
Az. caulinodans	D		7,4'-Dihydroxy

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Bone structure of Nod factors

Table 8　Nod Factors of rhizobia

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rhizobia	n	Q	R1	R2	R3	R4, 5
R.leguminosarum bv. viciae-RBL5560	2,3	C18△2 4 6 11, C18△11	CH3CO	H	H	H
R.meliloti AK41	1,2,3	C16△2 9, C16△2 4 9	H, CH3CO	HSO3	H	H
R.meliloti 2011	2,3	C16△9, C16△2 9, C16△2 4 9, (Ω-1)-OH C18-26	H,CH3CO	HSO3	H	H
R.tropici CFN299	3	C18△11	H	HSO3	CH3	H
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-, 2-O-Me-Fucosyl	H, CH3	H, NH2CO
R.fredii USDA234	1,2,3	C18△11	H	Fucosyl, 2-O-Me-Fucosyl	H	H
R.sp. NGR234	3	C18△11,C16	H	2-O-Me-Fucosyl, 2-O-Me-4-O-SO3H-Fucosyl, 2-O-Me-3-O-CO-CH3-Fucosyl	H	H, NH2CO
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, D-Arabinosyl	CH3	H

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From:

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

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

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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.

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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

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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

10-90%?

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.	Lowest
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	High-low
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

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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.)	20-260

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8-4 Azolla and its use

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

WEB SITE is available
Click "ABC of Azoll"

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