Nitrogen Fixation

N2-fixing Organisms and Rates of N2 Fixation

The ability to fix N2 is restricted to prokaryotic organisms, but within this group occurs in species that are taxonomically diverse. This is evident in the following partial listing:

  • Heterocystous (Anabaena, Nostoc) and non-heterocystous (Trichodesmium, Gloeocapsa ) cyanobacteria
  • Actinomycetes (Frankia), and
  • Heterotrophic (Azotobacter, Bacillus), autotrophic (Thiobacillus; ), aerobic (Pseudomonas, Methylobacter ), anaerobic (Clostridium ; Desulfovibrio ) and phototrophic (Chlorobium, Rhodospirillum ) bacteria.

N2-fixing species can be free-living, loosely associated with other organisms, or symbiotic and housed within specific structures made by the host. Free-living organisms depend on sources of energy in their environment (Azotobacter, soil, utilization of carbon compounds from soil: Rhodospirillum, water, light energy and photosynthesis). Associative and symbiotic organisms are supplied nutrients by their host. Young (1992) provides a full listing of N2-fixing species.  The diversity of organisms known to fix N2, and the extent to which the genes for N2 fixation are conserved among markedly different species, would suggest that N2 fixation is an ancient trait. However nif genes in some organisms are located on plasmids, and could have been horizontally transferred between species.

Rates of N2 fixation also vary, even for organisms that are free-living in nature. Thus in culture Azotobacter fixes up to 350 ug N ml-1day-1, and grows well without N addition to the medium, whereas N2 fixation in Clostridium may be only 13.6 ug N mL-1day-1 with added N needed for growth. It is surprizing, but most symbiotic nitrogen fixing rhizobia fix little or no nitrogen when grown in pure culture. Non-symbiotic N2 fixation in most soil environments is limited by energy availability, and so is usually less than 10 kg ha-1annum-1. Higher levels of nitrogen fixation are common in rice paddies (30-50 kg ha-1annum-1) and mud flats where cyanobacteria and photosynthetic bacteria are present.

Quite a range of organisms can contribute to N2 fixation in rice. Under irrigated conditions these include photo-synthetic organisms in the irrigation water, anaerobes in the flooded bulk soil, and aerobes in the rhizosphere where oxygen  is available through aerenchyma cells that connect to above ground parts of the plant. In studies at IRRI where rice has been grown for many years with no N fertilization, yields remain constant at about 4 tons ha-1.

Associative N2 fixation in sugar cane can reach 150 kg N ha-1annum-1, and provide up to one third of the N needed for plant growth. The organism involved, Gluconacetobacter diazo-trophicus, can tolerate high sucrose levels, a pH of around 3, and still fix at high levels of nitrate in soil. It survives poorly in soil, but persists in the host because of the practice of propagating sugar cane through the use of setts. N2 fixation in sugar cane is cultivar dependent, as shown in Table 2.

Table 2: Influence of plant cultivar on nitrogen fixation in sugar cane.

Cultivar
15N enrichment
N accumulated kg ha-1
CB47-89
0.191
61.4
CB45-3
0.166
84.3
NA56-79
0.198
57.8
IAC52-150
0.188 
59.6
SP70-1143
0.146 
77.5
SP79-2312
0.198
63.6
Chunee 
0.227 
33.0
Krakatau 
0.133 
102.8
from Urquiaga et al., 1992

In other C4 grasses, rates of N2 fixation are commonly much lower, though in sorghum, rates of up to 80 kg ha-1 have been reached in some studies. Organisms involved in such associative associations include Beijerinkia, Herbaspirillum, and Azospirillum. These organisms are commonly microaerophilic, and best recovered from tissue by growth in semi-soft media with malate as energy source.