Editor’s Note: A couple of months ago, I sent this link to Dr. Mae-Wan Ho to see if she had thoughts on the news from the University of Nottingham that they had found a way to manipulate plants so they can take nitrogen directly out of the atmosphere — bypassing the ‘need’ for chemical fertilisers. It has been hailed as a "ground breaking" solution to solving environmental problems and hunger. I wasn’t convinced…. Dr. Ho followed up with the piece below. The take-home message, before even considering many other unknown consequences for this new technology, is that the process means the symbiotic relationship between plant roots and micro-organisms would be compromised. This relationship is critical — not only for nitrogen fixation, but also for the uptake of other key nutrients and trace nutrients. In short, if we go down this road we are ignoring a perfectly successful and functional process in our obsessive desire to tinker, with potentially disastrous consequences.
The contribution to sustainable food production in substituting for chemical fertilizer is trivial compared with organic agroecological managements that enhance natural biological nitrogen fixation in soils, and at the same time, increase the availability of numerous other essential nutrients as well as water retention capacity.
by Dr Mae-Wan Ho
“World changing technology” announced with scant details
The University of Nottingham’s press release  “World changing technology enables crops to take nitrogen from the air”, announces the “ground breaking” discovery of a specific strain of nitrogen-fixing bacteria in sugar-cane that could get inside the cells of all major crop plants, with “enormous” implications for agriculture, as it enables plants to make use of nitrogen in the atmosphere directly, rather than expensive and environmentally damaging chemical nitrogen fertilizers. And it does not involve genetic modification.
Nitrogen is the most important element for organisms next to carbon, hydrogen and oxygen which make carbohydrates; for it enables carbohydrates to be turned into amino acids, the building blocks of proteins, and organic bases, the building blocks of nucleotides and nucleic acids and other essential molecules such as chlorophyll for photosynthesis. Although there is abundant N2 gas in the atmosphere (nearly 79 %) neither the plants nor the animals can use it. The nitrogen gas must first be fixed in combination with hydrogen in the form of ammonium ions (NH4+) or in combination with oxygen in the form of nitrate ions (NO3-).
The story was widely and favourably reported in the popular media, particularly as nitrogen fixation has been a major selling point and focus of genetic modification since the beginning , even though serious scientists knew it was not going to be an easy task .
The press release  was remarkably short on details, and it stated explicitly that no additional resources were available. My curiosity aroused, I began to search the web, especially after failing to get replies to my requests for further information.
Nitrogen-fixation energetically costly and genetically complex
The enzyme system responsible, nitrogenase (nif, nitrogen fixing), present in a wide variety of soil bacteria but not in plants or animals, only works under anaerobic conditions, as it is irreversibly inactivated by oxygen. It is energetically costly, requiring 16 ATP molecules to reduce one molecule of N2 into ammonia with the release of one molecule of hydrogen gas (see equation 1) .
N2 + 8H+ + 8e- + 16ATP ⇒ 2NH3 + H2 + 16 ADP + 16 Pi (1)
Genetic analysis of the best known system in the soil bacterium Klebsiella pneumoniae identified 8 operons (units of controlled gene expression in bacteria) and a total of 20 genes within a region of about 24kb of DNA; three encoding the nitrogenase enzyme subunits, while the others are involved in assembly and incorporation of Fe (iron) and Mo (molybdenum) ions into the nitrogenase subunits, or encode proteins involved in electron transport and regulation of expression of other genes .
The high energetic cost of the process, the nitrogenase enzyme requirement for anaerobic environment, plus the complexity and number of the genes involved, and the basic differences in the organization, transcription, and translation of genes in bacteria and plants, including distinct promoter sequences, operon structure in bacteria not recognized by plants, as well as plant genes involved in symbiosis, all go to make genetic modification of plants for nitrogen fixation little more than a pipe-dream to this very day . So, any advance in achieving the same goal without genetic modification seems eminently sensible, if not a welcomed development. But is it really?
Patented and licensed technology based on a power point presentation and one paper
My search turned up a power point presentation , a paper published in 2006 , and a patent  claiming invention for “systemic non-nodular endosymbiotic nitrogen fixation in plants.” Most of the information was provided (unreferenced, and unpublished) in the power point presentation  from Azotic Technologies, which has licensed the technology to fix nitrogen for 200 crops from Nottingham University. The nitrogen fixing bacterium Gluconaetobacteria diazotrophicus was isolated from sugarcane in 1988 (by other researchers ); its natural host range includes high sugar crops such as sweet potato, pineapple, sweet sorghum and mango. It fixes nitrogen and produces indoleacetic acid (IAA), a plant growth hormone. A strain of the bacterium IMI501986  “under certain conditions (patented technology)” was shown to colonize wheat, rice, maize cotton, oilseed rape, white clover, tomato and grass “intracellularly”, and that was the first key discovery; as the bacterium in nature colonizes the intercellular spaces and dead tissues in the plant roots.
A second key discovery, it seems, is that no intracellular colonization took place when the usual 106 bacteria was used in inoculation; that only happened when very low numbers, around 5 bacteria or a single bacterium was applied to the root zone of each plantlet growing in an agar medium with some sucrose in it .
According to Azotics , penetration of the plant cell wall is facilitated by bacterial genes encoding the protein b-expansin (that presumably softens the plant cell wall ) and an endo-1, 4-glucanase (that breaks down cellulose ). The plant cell membrane then envelops the bacteria in a membrane bound vesicle inside the cell . As nitrogen fixation requires the absence of oxygen, the bacteria enclose themselves further in a mucus levan fructan produced by the bacterial levansucrase from sucrose  that keeps oxygen out, so nitrogen fixation can take place in a wide range of oxygen concentrations; and half of the nitrogen fixed is excreted for the plant cell to use.
Nitrogenase is demonstrated to be present and active inside the membrane-bound and mucus-insulated vesicles . And in maize colonized by the bacterium, up to 30 % of the total nitrogen in the plant was shown to be derived from atmospheric nitrogen by the N-fixation in young plants. Oilseed rape and grass inoculated with the bacterium grew for many months in the absence of synthetic nitrogen fertilizer, forming green shoots and leaves.
The results of pilot studies presented as graphs without further details in the power point showed an encouraging 30 to 40 % increase in maize cob dry weight of inoculated plants compared with non-inoculated controls; the same for Lolium perenne (perennial rye grass) grown in compost comparing seeds coated with the bacterial preparation to non-coated seeds.
The company is planning field trials in 2013 on grass, wheat, and oilseed rape in the UK and on corn in Canada, with the assurance that Gluconaetobacteria diazotrophicus is a Biosafety Level 1 (nonpathogenic) micro-organism, and is not known to be invasive in any territory.
However, the bacteria, once inside the plant root cell, can translocate throughout the plant and become systemic. Is the plant material safe to eat? Although Gluconaetobacteria diazotrophicus is not known to be a pathogen, and the levan fructan is not reported to be toxic, neither has been present in substantial amounts in food so far. Moreover, the intracellular colonization is bound to alter the plant’s gene expression and metabolic profile in ways that may impact on safety. No feeding trials have been carried out, and none has been planned. In addition, the effects of such intracellular nitrogen-fixing crop plants on the ecosystem, in particular, the soil ecosystem need to be thoroughly studied. There is a cost to the plant in providing sucrose and other organic substrates for the bacteria, which would mean less is extruded through the plant root zone to support the growth of soil microorganisms, many of which are involved in recycling a host of nutrients other than nitrogen and in disease control. I asked for further details from the company, but have received no reply.
Is intracellular colonization necessary?
The major selling point of the technology is intracellular colonization, achieved mainly by reducing the number of bacteria presented to the roots of plantlets down to 1-5. It is not clear, however, that intracellular colonization is crucial for benefiting the plant. After all, green manure is routinely created by rotating nitrogen fixing crops with other crops, or intercropped with non-nitrogen fixing crops, as many legumes can serve as fodder for cattle (see  Paradigm Shift Urgently Needed In Agriculture, SiS 60). The Brazilian sugarcane from which the bacterium was isolated appeared to derive 60 to 80 % of its N from the intercellular association ; while experimental inoculation of wheat plants (Triticum aestivum L.) with a nitrogen-fixing Klebsiella pneumoniae was able to relieve N deficiency symptoms and increase total N concentration in the plant, even though the bacterium remained in the intercellular space of the root cortex .
There are other reasons why intracellular colonization is neither necessary nor desirable.
Biological nitrogen fixation predominates globally
Biological nitrogen fixation predominates by far globally, producing more than twice as much as non-biological fixation and 3-4 times as much as the industrial Haber Bosch process that makes chemical nitrogen fertilizers (Table 1) at great energetic costs as well as carbon emissions andenvironmental costs. A 50-year agricultural trial found that the application of synthetic nitrogen fertilizer resulted in all the carbon residues from the crop disappearing, plus an average loss of around 10 tonnes of soil carbon per hectare (see ). Nitrogen fertilizer is also responsible for the majority (70 % in some estimates) of greenhouse gas emissions associated with the production of crops both through the fossil energy used in its manufacture and N2O emissions from the soil subsequent to its application. Thus, there is no doubt that chemical nitrogen fertilizers should be phased out; just as there is no doubt that biological nitrogen fixation can already substitute for chemical nitrogen fertilizer 3 or 4 times over without the need for intracellular nitrogen fixation in crop plants. Global biological nitrogen fixation could be even more abundant if organic agroecological farming were to replace all intensive industrial agriculture reliant on soil-destroying chemical fertilizers and pesticides.
|Table 1 Estimates of nitrogen fixed on a global scale |
|Type of fixation||Mtonnes N2 per year|
|Forest & non-agricultural land||~50|
Numerous free-living as well as symbiotic bacteria inhabit healthy organically managed soils free from chemical fertilizers and other agrochemicals that can fix plenty of nitrogen, especially in the root zones, so as to be readily available to plants. There is really no need to put them inside the cells of crop plants, especially on the basis of such incomplete knowledge and scientific research.
Organically managed soil provides much more than nitrogen
It is important to recognize that organically managed soils do not just involve avoiding chemical fertilizers or planting crops that fix nitrogen, it entails a whole system of practices that returns organic matter to the soil on which soil health and crop productivity depends. Organic matter in the soil not only sequesters carbon, it is crucial for soil fertility, positively influencing soil structure, the diversity and activity of soil organisms that recycle nutrients, the availability of nutrients, and the soil’s water retention capacity .
Many microorganisms fix nitrogen, often in association with plants: cyanobacteria associated with lichens, the water fern Azolla (in rice fields), and cycads; Rhizobium species in symbiotic relationship with legumes, Frankia, the filamentous actinomycetes bacteria that form nodules with the roots of woody actinorhizal plants such as alder (Alnus species), and sea buckthorn (Hippophae rhamnoides).
Free living cyanobacteria also fix nitrogen, and are mainly responsible for the nitrogen fixed at sea . In the soil, many heterotrophic bacteria (those that depend on oxidising organic molecules released by other organisms or from decomposition) fix significant amounts of nitrogen without directly interacting with other organisms. These include species of Azotobacter, Bacillus, Clostridium, and Klebsiella . A study in Australia of an intensive wheat rotation farming system showed that free-living microorganisms contributed 20 kg/ha /year (30-50 %) of the cropping system’s nitrogen requirements  (the rest could be supplied by decomposing organic matter in the soil). The maintenance of wheat stubble and reduced tillage provided the high-carbon, low nitrogen environment that optimizes nitrogen fixation. Azospirillum species can form close associations with Paoceae grasses that include rice, wheat, corn, oats and barley, fixing substantial amounts of nitrogen within the plant root zone, where oxygen is low and host plants can provide organic substrates exuded from the roots to feed the bacteria .
Biological nitrogen fixation is carried out by numerous free-living, associative as well as symbiotic species of bacteria, providing 3 to 4 times the nitrogen for plants as the environmentally damaging chemical fertilizers produced by the energy intensive and high greenhouse gas emitting industrial process. Plants growing in healthy organic soils free from agrochemicals are not limited by nitrogen; furthermore, such soils provide all other nutrients including the capacity to retain water. Introducing intracellular nitrogen-fixation into plants to substitute for chemical fertilizers is trivial in terms of promoting plant growth, and with yet unknown effects on health and the environment.