Introduction:
Nitrogen is an essential plant nutrient. Unfortunately, it is usually not present in soil at concentrations sufficient for agricultural production of commercial crops. It therefore must be provided to the crops in the form of fertilizer. Of the commercially important crops, cereals such as corn, wheat, rice and barley require particularly large amounts of fertilizer. For example, Azotobacter is used for the non legume crops; Rhizobium is needed for the legume crops. Similarly blue green algae are needed to grow rice while Acetobacter is used to grow sugarcane. It means almost all the crops need different types of biofertilizers depending on their needs.

The use of nitrogen-fixing bacteria called biofertilization crops or inoculation and is used with some success worldwide, but the main problem is the low efficiency of inoculants, so that their use in the field has not been extensive. The production of a biofertilizer industry represents a much lower cost to produce a fertilizer. For example, one hectare of beans is about 500 pesos needed to fertilize and to inoculate the same surface with bacteria, without reducing the performance, about 10 pesos. Then, an affordable alternative and more environmentally appropriate for achieving the objective in the field is the improvement of atmospheric nitrogen fixation occurs in the cultivation of plants

Azospirillum

Naturally occurring Azospirillum bacteria are free- living, aerobic, gram-negative, motile and nitrogen fixing. They generally prefer organic acids as carbon sources, e.g. malic or lactic acid, and fix nitrogen in the absence of a combined nitrogen source, under micro- aerophilic conditions (low oxygen tension). They also have relatively short shelf lives with low pectinolytic activity (the ability to break down pectin present in the cell walls of cereal plants so as to render the cell walls more permeable to minerals, hormones) and generally are not resistant to commonly used, commercial fungicides, herbicides and other pesticides or to antibiotics released by different soil microorganisms.

To overcome certain disadvantages associated with naturally-occurring Azospirillum strains, the novel strains having relatively improved survivability, or resistance to commonly used pesticides or increased pectinolytic activity or a combination of these traits. Such strains are able to enhance crop yields or reduce nitrogen fertilizer requirements, or both
Since pectinolytic activity can be correlated with usefulness as biofertilizer, novel strains of Azospirillum having pectinolytic activity relative to the pectinolytic activity of naturally-occurring strains in the range from about 1.5:1 to 20:1 have been created. Presently, the preferred strains are of the species brasilense. Optionally, they are available as biologically pure, stable cultures.

The fertilizer composition may comprise at least 5% by total bacterial content of the Azospirillum brasilense; at least 30% by total bacterial content of the Azospirillum brasilense; or at least 50% by total bacterial content of the Azospirillum brasilense. The fertilizer composition may have the bacterial consortium comprise Azospirillum brasilense in combination with at least two other bacteria selected from the group consisting of Ochrobacterium tritici sp.; Ensifer adhaerens; Sinorhizobium sp; Enterobacter sp.; Zooglea sp.; Brevibacillus sp.; Bacillus cereus; and Agrobacterium tumefaciens; or at least two bacteria selected from the group consisting of Ochrobacterium tritici sp.; Ensifer adhaerens; Sinorhizobium sp; Enterobacter sp.; Zooglea sp.; Brevibacillus sp.; and Bacillus cereus.

Azotobactor

Azotobacter naturally fixes atmospheric nitrogen in the rhizosphere. There are different strains of Azotobacter each has varied chemical, biological and other characters. However, some strains have higher nitrogen fixing ability than others. Azotobacter uses carbon for its metabolism from simple or compound substances of carbonaceous in nature. Besides carbon, Azotobacter also requires calcium for nitrogen fixation. Similarly, a medium used for growth of Azotobacter is required to have presence of organic nitrogen, micro-nutrients and salt in order to enhance the nitrogen fixing ability of Azotobacter.

The bacterial fertilizer (biofertilizer) comprised of a suspension which contains cells of new strains of the micro-organisms Azotobacter vinelandii CECT 4534 and Azospirillum brasilense. Said bacterial fertilizer is a suspension of micro-organisms in a water medium. The micro-organisms are obtained by a process which comprises: a) plate culture of the original strain; b) treatment of the former colonies of the prior culture with another culture means; and c) culture of the isolated colonies of the prior step.

The advantages of the use of strains Azospirillum Azotobacter over the known strains are:
1. The strains collected from Azospirillum Azotobacter and reproduce well at a greater range of pH and better than the known strains, supporting very alkaline media (pH = 10), thus avoiding problems of sterilization and contamination.
2. These strains are able to fix more N2 more easily and eliminate the ion NH4+ to the culture medium, while polysaccharides excreting more concentrated than known strains. These polysaccharides serve Azotobacter cells and Azospirillum as an energy reserve for further development on the ground while allowing them to adhere more easily to the roots of plants.
3. These variants Azospirillum Azotobacter and also have a greater capacity to assimilate the compounds formed by the exudates from the roots of plants, such as p-hydroxybenzoic acid, which is hardly used by other races.
4. The rate of reproduction and assimilation of the substrates is also higher in Azotobacter variants and strains Azospirillum known that, since the maximum dry weight attained by them are up to three times higher than the 52 hours of culture dipped to about 28-30 ° C.
5. The performance and effectiveness of these variants Azotobacter is far superior to known strains and the initial strain DSM 382, also synthesized with high gelling polysaccharides (alginates)

Rhizobium:

Producing a biofertilizer for plants with improved efficiency in nitrogen fixation has been the target of several research groups for several years because it represents economic benefits, agricultural and environmental important. Improving the process of nitrogen fixation could be obtained through the use of organisms such as Rhizobium bacteria genetically modified. However, despite the various strategies that have been conducted, researchers have reported limited success in its efforts to improve nitrogen symbiotic fixation.

The expression of construction in the Rhizobium strains tested were characterized by increased levels of expression of the enzyme complex nitrogenase in Rhizobium bacteria to provide a high transcription promoter region coupled to the full operon nifHDK (SEQ ID NO: 2, 3 and 4). It is important to mention that there is no report or strain, except for those covered by this application; they are able to increase the binding capacity of Rhizobium.

The potential use as biofertilizer Rhizobium agencies to contain the gene construct with which nitrogenase is overexpressed (pr. nifHDK c) is supported in field trials. Although the tests were performed on the bean crop, this is just one example of how to carry out the implementation of the invention. Additionally it can be applied in other legumes such as peanuts, soybeans, alfalfa, clover, lentils, beans, peas, etc.. and even in other crops such as sugar beets, wheat, maize and sorghum. In the construction of the strains always considered their possible use in the Mexican countryside and therefore meet all the requirements for approval of release of genetically modified organisms which marks the National Biosafety Committee, the competent authority in the matter.

Therefore, the main feature of Rhizobium strains containing this gene construct in association with a legume such as beans are reaching increased levels of seed production and nutrient content of the same and that in fixing more nitrogen, it is incorporated protein and amino acids in the seed in larger quantities.

Conclusion:

Use of the microbial strains as biofertilizer in the field is completely safe based on decades of research and application as bio-fertilizers and does not harm the environment as they do nitrogen fertilizers are usually applied. The preparation of bacteria for use as biofertilizer and application at planting is simple and very inexpensive, so the replacement of chemical fertilizer nitrogen represents a significant economic benefit for farmers. Additionally, the seed produced has better nutritional characteristics for the general consumer.

About Author / Additional Info:
A Research Engineer from Dextrasys Technologies.