Biotechnology: Beyond Borders 15

M. V. Deshpande and J. Ruiz-Herrera (Eds.), Published by CSIR-National Chemical Laboratory, Pune, India, 2013

3. Biological nitrogen fixation for sustainable

agriculture and global food security
Rai A. K.

Department of Botany, Banaras Hindu University, Varanasi 221005, Uttar

Pradesh, India. akrai.bhu@gmail.com

Abstract
Although during 1970s, Green Revolution knocked over the world food
crisis, once again experts alarm of a possible food crisis. From mid-2008,
there is an unexpected rise in food prices, the cause of which is still under
debate. The current world population of 7.1 billion is expected to reach 9.1
billion by 2050, and world food production will need to rise by 70 %, and
double in the developing world. Undoubtedly, the application of industrially
fixed nitrogen fertilizers has revolutionized the productivity, especially
wheat and rice worldwide. However, the large increase in the use of nitrogen
fertilizer for the production of crops has dramatically increased the emissions
of nitrous oxide, a powerful greenhouse gas. Hence, nitrogenous fertilizers
input must be reduced, if we are to reduce environmental degradation caused
by agricultural processes. The approach to improve soil fertility is to
stimulate biological nitrogen fixation. There exist indeed viable options to
achieve this goal if we consider the ways in which nature has evolved
successful system.

Green Revolution in the 1970s reversed the scenario of food crisis; while
population doubled food supply tripled. However gradually we began to
realize that this blessing has come at a substantial economic and
environmental cost. Once again, experts are visualizing a possible food
crisis. The rate of increase in grain yields per acre is on decline; possibly
most of the benefits of irrigation, seed, land, machinery, fertilizer and plant
breeding have already been realized. From mid-2008, there is an unexpected
rapid rise in food prices, the cause of which is still under debate.
It is paradoxical that far from decreasing, the number of hungry
people in the world is currently increasing. Another aspect is to match the
rapidly changing demand of affluent population for quality food with
increased nutrient content. According to Food and Agricultural Organization
(FAO) report, the number of hungry people in the world crossed the one
billion mark in 2009, of which 642 million live in Asia and the Pacific.
 Biotechnology: Beyond Borders 16

According to Indian Government documentation In a recent survey, it was

deduced that 22% of the Indian population is undernourished, whereas 40%
of children below the age of 3 years are underweight, majority of children
aged between 6 to 35 months are anaemic and 33% of the women aged
between 15-49 years have a BMI (body mass index, a measure of body fat
based on height and weight that applies to adult men and women) below
normal. The growth rate and the immunity level of the Indian population
have been declining considerably throughout these years. The presented
figure is despite the fact that country grows enough food for its people. The
reason may be argued for lack of warehouse, storage, transportation and
above all, lack of will power. Recently on 26 August 2013, Parliament of
India passed a National Food Security Bill, 2013, which aim to provide for
food and nutritional security in human life cycle approach, by ensuring
access to adequate quantity of quality food at affordable prices to people to
live a life with dignity and for matter connected therewith or incidental
thereto." This is to be accomplished through Public Distribution System, and
entitlements are Priority households are entitled to 5 kg of food grains per
person, and Antyodaya households (the 5% population that sleeps without
two square meals a day) to 35 kg per household per month at Rs 3/2/1 per kg
for rice/wheat/millets. These may be revised after three years.....
The current world population of 7.1 billion is expected to reach 9.1
billion by 2050, and world food production will need to rise by 70 %, and
double in the developing world. The problem is further aggravated with the
production of biofuel on good quality agricultural land, which will require 35
million hectares of land by 2030. In addition, the projected increase in food
production will have to overcome ever-escalating energy cost, depletion of
underground water, loss of cultivable land to urbanization and salinization,
increased drought, flooding, global greenhouse gas emissions and climate
change. Undoubtedly, the application of industrially fixed nitrogen fertilizers
has revolutionized the productivity, especially of grains worldwide.
However, a large increase in the use of nitrogen fertilizer has dramatically
increased the greenhouse effect, resulting in depletion of stratospheric ozone,
formation of smog, contamination of drinking water, acidified rain, and
eutrophication of water bodies and stressful ecosystems. The current
concentration, of nitrous oxide about 310 ppbv is about 10 % higher than the
value before this century, while the current rate of increase is about 0.8 ppbv
(0.3 %) per year. The values indicate the flow of fixed nitrogen among soils,
waterways, oceans, and the atmosphere (Schlesinger, 1997; Socolow, 1999).
Hence, nitrogenous fertilizers input must be reduced, if we are to reduce
environmental degradation caused by agricultural processes. The approach to
 Biotechnology: Beyond Borders 17

improve soil fertility is to stimulate biological nitrogen fixation. The N2-

fixing organisms convert molecular nitrogen (N2) in the field, in the soil,
near the root system, where it is needed. Thus, biofertilizers not only help to
produce more food, but save money, time and energy on the mineral
fertilizers, and lead to a reduction in greenhouse gas emissions and runoff
from fertilizers that pollute aquatic habitats.
There exist indeed viable options to achieve this goal. The habitat of
rice fields are optimum condition for N2 fixing cyanobacteria and Azolla
(agronomically most important symbiont). Maximum benefit may be derived
by inoculating the fields with competent N2-fixing cyanobacteria and Azolla
tolerant to adverse conditions such as drought, radiation, temperature and
salinity (Dubey and Rai, 1995; Rai and Rai, 1999; Ladha and Reddy, 2003).
By treating seeds with special bacteria that allow a plant to produce its own
fertilizer on its roots and engineering crops (with nif genes) that fixes
nitrogen themselves to sustain their growth and yield. The transfer of
nitrogen fixation (nif) genes to cereals is one option to achieve this goal.
The nitrogenase complex responsible for the conversion of elemental
nitrogen (N2) to fixed forms of nitrogen is encoded by the nif genes (Roberts
et al., 1978). Apparently, 20 different nif genes encode the complex. Some of
nif genes are nifH that codes for the structural unit of dinitrogenase
reductase, and nifD and nifK for the structural units of dinitrogenase. nifB,
nifEN and nifH encode the synthesis of FeMo-co. Flavodoxin is a nifF
product, while pyruvate:flavodoxin oxidoreductase involved in electron
transport is a nif J gene product. nifM, nifS,and nifU are needed for the
synthesis of active Fe protein. A limited number of prokaryotes possess nif
genes and are blessed with the ability to fix dinitrogen, other creatures
exploit these organisms. Even in the prokaryotes, this character is not
universal. The nif genes are present in free-living and symbiotic bacteria and
cyanobacteria, and Frankia. The free-living diazotrophs are either aerobic
such as Beijerinckia, Spirillum, Derxia and cyanobacteria, facultative
(Klebsiella, Rhodopseudomonas) or anaerobic (Clostridium, Chromatium).
Azatobacter and Azospirillum are associated with roots of tropical grasses.
Cyanobacteria and symbiotic diazotrophs, Rhizobium and Bradyrhizobium
(forming nodules in leguminous plants) are agronomically much important.
Azorhizobium caulinodans forms N2-fixing nodules on the roots and
stems of the tropical legume Sesbania rostrata. In simpler terms, symbiosis
between rhizobia and legumes starts with the induction of bacterial nod
genes and synthesizing nodulation factor (Nod factor) responsible for the
developmental changes in the host plant. Cortical cells divide to form the
nodule primordia, bacteria penetrate via host-derived infection threads, and
 Biotechnology: Beyond Borders 18

Nitrogen fixers

Free- living Symbiotic

Legumes
Facultative
Rhizobium
K.pnuemoniae
Aerobic Azorhiz obium Non-legumes
Rhodopseudomonas
Cyanobacteria Bradyrhizobiu m
Beijerinckia Anaerobic
Spir illum Clos tridium Insects Tropical grasses:
Derxia Chroomatium gut of termites and Azotobacter
gland of deshayes Azospirillum
shipworms-
Trichonympha agilis,
Teredinibacter turnera e

Lichen: Moss: Cycads: Endophytic:

Diatoms: Peltigera, Sphagnum- Cycas- Nostoc, Su garcane,
Rhizosolenia Collema-Nos toc, Haplosiphon Azoarcus -
Anabaena
- Richelia Calothrix (superficial) Gluconacetobacter
(root cortex, E )
(in cephalodia,E) Fern: Angiosperm: diazotro phicus
Liverworts: Blaz ia- Azolla-Anabaena Gunnera - Nostoc Herba spirillum s pp
Nostoc (open leaf (clos ed leaf cavivty E) (leaf base glands,I)
cavity, E)

Fig. 1. Free-living and associative molecular nitrogen fixers. E, extracellular; I,

intracellular
invade the plant cell cytoplasm, differentiate into bacteroids and fixes N2,
provide ammonium to the host in return for photosynthates (Broughton et al.,
2000; Suzuki et al., 2007). What is needed to understand is the signal
exchange between rhizobia and legumes, and the physiological mechanism
targeting the maturation and maintenance of the nodules. Suzuki et al (2007)
used transposon induced A. caulinodans mutants to inoculate the stems of S.
Rostrata, and found that roles of many genes are not identical in different
symbiotic systems, while some have no roles in the symbiosis. The
identification of typical symbiosis-related genes will pave the way in
understanding the maturation and maintenance mechanisms of nodules and
construct new symbiotic crop systems.
Since the diet of termites and Shipworm is rich in carbon but
deficient in nitrogen, N2-fixing bacteria occur in the gut of termites, and
considerable rate of nitrogen fixation has been recorded (Lechene et al.,
2007; Breznak, 2000). Likewise, a diazotrophic bacterium Teredinibacter
turnerae has been isolated in pure form from the cells (bacteriocytes) of
shipworm gills (Waterbury et al. 1983; Distel et al, 2002) and its presence in
 Biotechnology: Beyond Borders 19

the gill has been confirmed by in situ hybridization and quantitative

polymerase chain reaction analysis (Luyten et al,, 2006).
Cyanobacterial symbioses are found with almost all classes of
flowering and non-flowering plants and involve taxonomically diverse hosts.
Richelia intracellularis and Calothrix rhizosoleniae are intracellular
cyanobionts in several diatom genera, including Hemiaulus, Rhizosolenia
and Chaetoceros. The rates of N2-fixation were 171420 times higher under
symbiotic state compared to those of the cells living freely, revealing the role
of the hosts (diatoms) in the growth and metabolism of cyanobionts (Foster
et al., 2011). These intracellular cyanobionts must possess a high degree of
regulation and adaptation to maintain the mutualistic symbiosis. These
symbioses may serve a key model to study the establishment of nitrogen
fixation in eukaryotic hosts and regulation of nutrient exchange.
A detailed study of the cyanobiont-host interaction will make
possible to understand the complex molecular mechanisms underlying the
evolution of obligate endosymbionts. Lichen, Peltigera and Collema have
association with Nostoc and Calothrix (in cephalodia), liverwort Blazia with
Nostoc (open leaf cavity), moss Sphagnum with Haplosiphon (superficial),
the marine sponge Lamellodysidea chloroea with host-specific Oscillatoria
spongeliae, fern Azolla with Anabaena (closed leaf cavity), cycads with
Nostoc, Anabaena (root cortex) and the only angiosperm Gunnera with
Nostoc (in leaf base glands and is intracellular). The intracellular interaction
within Gunnera and cyanobiont Nostoc is unique in flowering plants and
may provide an insight to develop novel symbioses between crop plants and
cyanobacteria. Almost all the diazotroph and host plant associations undergo
some morphological alteration to accommodate the symbiont as well as
physiological adaptations in their metabolism for the exchange of
metabolites.
Thus, with the molecular techniques available, the goal should be
within reach, especially if we consider the ways in which nature has evolved
successful system. Endophytic diazotrophs such as Gluconacetobacter
diazotrophicus and Herbaspirillum spp. inhabit the stems and leaves of
sugarcane, and Azoarcus, the roots of Kallar grass (Leptochloa fusca). G.
diazotrophicus colonizes the roots of rice, wheat and other crop plants
(Ladha and Reddy, 2003). Azotobacter vinelandii mutant with deletion of the
nifL gene expressed nitrogenase constitutively, and excreted ammonium into
the surrounding medium making available to other plants (Bali et al., 1992).
The treatment of wheat roots with the synthetic auxin, 2,4-
dichlorophenoxyacetic acid (2,4-D) led to the formation of tumour-like
 Biotechnology: Beyond Borders 20

structures (para-nodules), which loosely arranged cells were readily

colonized by cyanobacteria Gantar and Elhai, 1999).
Protection of nitrogenase complex from oxidative damage is
necessary. Plastids may serve favourable sites for nif gene expression.
Nitrogenese can be protected from oxygen by functioning in dark; some
cyanobacteria perform photosynthesis at separate times. Mitochondria may
provide low-oxygen environment to allow nitrogenase to function.
Moreover, cyanobacteria dierentiate heterocysts, which protect nitrogenase
against O2 with no need for the low O2 tensions maintained within their
nodules by legume plants. However, Sesbania rostrata has stem nodules as
well as root nodules. Stem nodules are capable of photosynthesis as well as
nitrogen fixation. Though challenging, there exists a widespread optimism
that recently developed molecular technologies and holistic approaches can
allow the substantial impact of biological nitrogen fixation in agriculture.

References
Bali A, Blanco G, Hill S and Kennedy C. Excretion of ammonium by a nifL mutant
of Azotobacter vinelandii fixing nitrogen. Appl Environ Microbiol 1992; 58:1711-
1718.
Breznak JA, 2000. In Termites: Evolution, Sociality, Symbioses, Ecology, T. Abe, D.
E. Bignell, M. M. Higashi, Eds., Kluwer Academic, Dordrecht, Netherlands, pp.
209232.
Broughton WJ, Jabbouri S, and Perret X. Keys to symbiotic harmony. J Bacteriol
2000; 182:56415652.
Distel DL, Morrill W, MacLaren-Toussaint N, Franks D, and Waterbury J.
Teredinibacter turnerae gen. nov., sp. nov., a dinitrogen-fixing, cellulolytic,
endosymbiotic gamma-proteobacterium isolated from the gills of wood-boring
molluscs (Bivalvia: Teredinidae). Int J Syst Evol Microbiol 2002; 52:2261-2269.
Dubey AK, Rai AK. Application of algal biofertilizers (Aulosira fertilissima Tenuis
and Anabaena doliolum Bhardawaja) for sustained paddy cultivation in Northern
India. Israel J Plant Sci 1995; 43:41-51.
Foster RA, Kuypers MMM, Vagner T, Paerl RW, Musat N and Zehr J P. Nitrogen
fixation and transfer in open ocean diatomcyanobacterial symbioses. The ISME J
2011; 5:14841493.
Gage DJ. Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia
during nodulation of temperate legumes. Microbiol Mol Biol Rev 2004; 68:280
300.
 Biotechnology: Beyond Borders 21

Gantar M and Elhai J. Colonization of wheat para-nodules by the N2-fixing

cyanobacterium Nostoc sp. Strain 2S9B. New Phytol 1999; 141: 373-379.
Ladha JK and Reddy PM. Nitrogen fixation in rice systems: state of knowledge and
future prospects. Plant and Soil 2003; 252:151167.
Lechene CP, Luyten Y, McMahon and. Distel GL. Quantitative imaging of nitrogen
fixation by individual bacteria within animal cells. Science 2007; 317:1563 -1566.
Luyten YA, Thompson JR, Morrill W, Polz M F, Distel DL. Extensive variation in
intracellular symbiont community composition among members of a single
population of the wood-boring bivalve Lyrodus pedicellatus (Bivalvia: Teredinidae)
Appl Environ Microbiol 2006; 72:412-417.
Rai V and Rai AK. Growth behaviour of Azolla pinnata at various salinity levels and
induction of high salt tolerance. Plant Soil 1999; 206:79-84.
Roberts GP, Macneil T, MacNeil D and Brill WJ. Regulation and characterization
of protein products coded by the nif (nitrogen fixation) genes of Klebsiella
pneumonia. J Bacteriol 1978, 136:267-279.
Schlesinger WH. Biogeochemistry: An Analysis of Global Change (Academic, New
York), 1997; 2nd Ed.
Socolow RH. Nitrogen management and the future of food: Lessons from the
management of energy and carbon. Proc Natl Acad Sci USA 1999; 96:60016008.
Suzuki S, Aono T, Lee K-B, Suzuki T, Liu C-T, Miwa H, Wakao S, Iki T, and
Oyaizu H. Rhizobial factors required for stem nodule maturation and maintenance in
Sesbania rostrata-Azorhizobium caulinodans ORS571 symbiosis. Appl Environ
Microbiol 2007; 73:6650- 6659.
Waterbury JB, Calloway CB, Turner RD. A cellulolytic nitrogen-fixing bacterium
cultured from the gland of deshayes in shipworms (bivalvia: teredinidae). Science
1983; 22:14011403.