In pre-Roman times legume seeds were used and have been found in various sites. They are nitrogen-fixing plant/bacteria associations. The Romans developed the idea of crop rotation where legumes were grown every four years in a rotation with non-nitrogen-fixing crops. One year the fields were left unused or "fallow". This system replaced nitrogen in the soil and led to higher yields. This is still used as a major farm management practice in many areas of the world.
In so-called "developed" countries, the addition of nitrogen-based fertilizers has largely replaced the rotation system. There is increasing concern over the price and environmental damage (e.g. nitrate contamination of groundwater) resulting from nitrogen fertilizer use.
The advent of the l9th century experimental scientist led to the study of nitrogen in plants and soils. In 1813 Humphrey Davy observed that legumes "seemed to prepare the ground for wheat" and speculated that the nitrogen came from the atmosphere. Between 1820 and 1880 there was a very active research environment and a series of bitter arguments between some very prominent scientists. Early efforts indicated that soil, not atmospheric nitrogen, was the source of the nitrogen for plant growth.
In 1838, Boussingault did an elegant series of experiments and showed that legumes had higher nitrogen levels than cereals and, based on some crop rotation studies over 5 years, concluded that the atmosphere was the source of this nitrogen (it could have been particulate matter, nitrogen gas or ammonia - he did not specify which). In 1848, Liebig, an eminent chemist. argued, with no new evidence, that it was ammonia and this was accepted because of his reputation. At the same time he said that N was not an important part of fertilizers and that it was the "minerals" that accounted for the effects of fertilizers.
He was proved wrong both before (by Boussingault) and afterwards by workers at the Rothamstead Experimental Station in England. In an experiment that continues to this day, they proved the source of nitrogen in fertilizer to be atmospheric. These views were accepted but a series of controversies then followed when it was shown that "roasting" the soil removed the ability of the plants to get the nitrogen. At the same time Liebig's mineral theory was discredited.
There followed a period when emphasis was placed on a soil source for the additional nitrogen. In 1880 Hellriegel and Willfarth showed unequivocally that: a). Legumes could use atmospheric nitrogen b). Fixation of this nitrogen into the plant depended on bacteria in root nodules. A key series of experiments in nitrogen-poor soil showed this conclusively. Beijerinck in 1888 isolated the bacteria responsible - Rhizobium spp. Free-living nitrogen-fixing bacteria were discovered in soil and this led to an active research program in many countries.
The techniques available were inadequate to the task and scientists left the field to do easier experiments. Qualification for an organism to be "nitrogen-fixing" seems clear and straightforward. It should grow in the presence of dinitrogen gas (N2) without the presence of any fixed, available nitrogen source (ammonia, nitrate, organic nitrogen, etc.). This is an easy definition in theory, but was difficult to apply in practical terms because; a). many organisms can scavenge very low levels of ammonia from the air b). the ability to fix nitrogen is scattered among many genera and species of bacteria c). ignorance of conditions for nitrogen fixation to occur d). technical difficulties of assessing amount of nitrogen fixed.
The Kjeldahl nitrogen assay was the only means available to early investigators. It is not very sensitive nor is it very accurate. New techniques have since been developed. In 1948 the only widely accepted nitrogen fixing system were the Rhizobium-legume plant symbiosis, Azotobacter spp. and Clostridium spp. together with the "blue-green algae" (actually Cyanobacteria). Many different genera of bacteria are now recognized to contain N2-fixing species. No other types of organisms fix atmospheric dinitrogen. Some lichens fix dinitrogen but only because they contain certain blue-green bacteria in a symbiotic relationship. These blue-green bacteria (previously called "blue-green algae") can fix dinitrogen.
There are aerobic, anaerobic and facultative dinitrogen-fixing bacteria. There are also bacteria that fix atmospheric dinitrogen only in association with plant roots (e.g. Rhizobium and Frankia). In terms of physiological groups, nitrogen-fixing bacteria are present in almost every type of group and, therefore, in most environments.
(from : Biology 446 , Univ. of Waterloo Biology 446)
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