MX2010014426A - Novel nitrogen-fixing biofertilizing compositions. - Google Patents

Abstract

The present invention describes biofertilizer composition which comprises a photosynthetic microbial consortia (MC) with a high capacity to promote the fixation of atmospheric nitrogen in agricultural crops. The MC is stable to the repeated spread and dry, and does not present important variations in the constitution thereof. The MC is formed by no more than 20 different microorganisms, where about 40% of the total population are cianobacteria (Aphanizomenon aphanizomenoides, Leptolyngbya and Anabaena oscillarloides) and the rest of the population is formed by proteobacteria (alpha, beta, gamma and delta) and bacteriodetes (Flavobacterium).

Description

Nitrogen-fixing novel biofertilizing compositions Field of the invention.

The invention belongs to the field of biotechnology and refers to the application of studies of microbial ecology in environmental and agroindustrial aspects, specifically to obtain biofertilizing compositions from stable microbial consortiums I that improve the process of nitrogen fixation and the productivity of agricultural crops. i BACKGROUND OF THE INVENTION The food requirements for humanity have increased dramatically, for which the productivity of agricultural crops has improved with the use of chemical fertilizers promoting various physiological aspects such as the mobilization of phosphorus, the ability to decompose organic matter, plant growth, nitrogen fixation, etc. However, the indiscriminate use of these fertilizers has led to problems such as soil erosion and contamination of aquifers. Due to this, biofertilizers have turned out to be a better alternative to increase production in the field avoiding the harmful effects of chemical fertilizers.

The biofertilizers or fertilizers of microbial inocula are artificial cultures of certain i soil microorganisms such as Rhizobium, Mycorrhiza, Azotobacter, Azospirillum, Cyanophytes (Cyanobacteria) and Azolla, which can improve soil fertility and productivity of agricultural crops. 1 The first biofertilizer used was Rhizobium cultures in 1895, however, the properties of Azotobacter and Cyanobacteria were later discovered. In this sense, the first biofertilizing composition of commercial Rhizobium was described in a patent of 18961, starting several researches related to biofertilizer technology, including the one based on genetic engineering for i optimize them At the present time there are several commercial biofertilizing systems based on microorganisms such as Azospirillum brasilense '' ', Rhizobium etli and also those based on certain nitrogen fixing cyanobacteria and some types of Mycorrhizae, being the most important and with the greatest commercial impact the biofertilizers whose component active are the nitrogen-fixing microorganisms of the genera Azospirillum and Rhizobium.

The biological fixation of nitrogen is one of the most important phenomena in the ecosystem, since it is one of the main components of living matter (nucleic acids and proteins); Atmospheric nitrogen is not very reactive and only certain prokaryotic microorganisms are able to fix nitrogen and make it available to other living beings. The process of nitrogen fixation consists of highly endergonic reactions and invariably all nitrogen-fixing microorganisms use ATP as an electron donor to activate the nitrogen molecule.

Nitrogen-fixing microorganisms have certain characteristics in common, the most notorious for reducing nitrogen to ammonium (NH4 +), the presence of the nitrogenase enzyme complex, which has a structure highly conserved in tetrameric conformation. Two subunits of the enzymatic complex correspond to a protein with an iron-molybdenum core and two more to an iron-sulfur protein. Due to its metallic nuclei, the enzyme is highly sensitive to oxygen, since in its presence it completely loses its activity, so it is active only at low oxygen tensions. Ammonium or nitrate (N03 +), are chemical nitrogen species suitable for use by autotrophic organisms and thus pass to heterotrophs.

The enzymatic activity of nitrogenase can be measured by its ability to reduce compounds with triple bonds, using this technique since the late 60s. For this, it is enough to place the bacterial culture in microaerobiosis and replace 10% of its atmosphere with acetylene. . After one week, a sample is taken and injected into a gas chromatograph under the appropriate conditions for the correct resolution between acetylene and ethylene; if there is nitrogenase activity, the peaks will appear corresponding to acetylene and ejylene. Nitrogenase activity is proportional to the ethylene produced and by making determinations at various times the kinetics of the reduction can be known, as well as the specific growth activity expressed per mg of protein, number of cells, etc.

Cyanobacteria comprise a large and heterogeneous group of microorganisms 7 i oxygenic phototrophs of prokaryotic nature; Of these, the filamentous cyanobacteria are the ones that are essential to carry out the nitrogen fixation thanks to the heterocytes, which are differentiated cells and are the main sites of nitrogen fixation. The heterocytes are distributed individually along a filament or at the end of it, they have intercellular connections with the vegetative cells 1 adjacent, there is an exchange of materials product of photosynthesis between these cells, which move from the vegetative cells to the heterocytes and the nitrogen-fixing products formed in them, pass to the vegetative cells. The heterocytes have a low concentration of phycobilin and lack photosystem II, which is responsible for the production of oxygen. Due to the reductive nature of nitrogen fixation and the sensitivity of the nitrogenase enzyme to oxygen, it seems likely that the heterocyte, by maintaining an anaerobic environment, makes it possible to stabilize the nitrogen fixation system in organisms that are not only aerobic, but also producers of oxygen. It is also possible that some filamentous cyanobacteria without heterocytes, produce nitrogenase and fix nitrogen in normal vegetative cells if they grow anaerobically7. | Due to its great capacity for nitrogen fixation, cyanobacteria have been used as biofertilizers for the production of plants of commercial and food interest, as for example in the production of rice in tropical countries. In this sehtido, the products obtained from the fixation of nitrogen by cyanobacteria, such as ammonium or nitrites are captured in rice plants by exudation or autolysis or through microbial decomposition. However, this biofertilization technique using cyanobacteria is still limited, due to the inability to produce inoculants of good quality, coupled with the difficulty to effectively re-establish strains inoculated in the field. In the case of biofertilization of rice crops, advances have recently been made in the production of cyanobacteria isolated from rice fields, in open-system reactors8,9. The application of this biomass (produced in any type of open system) to the field, needs to be harvested and dried, which reduces the viability of the cells and increases the production cost10,1 1. One way to solve some of the problems before mentioned, is to cultivate the cyanobacteria in a reactor that guarantees a high yield; which in turn allows to obtain a high cell density to consider its direct application in the field. The option of using a closed reactor to solve the problems of low productivity, caused by 12 ' the suboptimal conditions present in the open system.

Although the problems associated with the yield in the production of cyanobacteria can be solved, their biofertilizing capacity depends to a large extent on their nitrogen fixation capacity, which is directly related to the type and quantity of microorganisms with which they are related in their microenvironment and with which they can form photosynthetic microbial consortiums. On the other hand, it has been observed that the main problems associated with the use of microbial consortiums (CMs) are basically their great lack of stability before their propagation, which decreases their efficiency in nitrogen fixation, as well as their great lability before procedures of conservation, such as drying.

In this sense, for example the proteobacteria of types a, ß, and or d are. chemolithrophic, phototrophic and chemoorganotropic bacteria with nitrifying activity, that is, they are able to oxidize ammonium to nitrite and to oxidize nitrite to nitrate, being very abundant in soils and waters with high levels of ammonium and alkaline pH11, so their interaction with cyanobacteria as part of microbial consortiums, is essential to close the nitrogen cycle, since nitrate is the most assimilable chemical species by plants.

A microbial consortium (CM) can be defined as a complex system of at least two different microorganisms, where each of the participants that constitute it it exerts an influence on others in a synergistic way and in which each microorganism does something for the benefit of the other12. One of these synergistic effects is the formation of biofilms that are not easily colonized by other microorganisms.

The use of CMs to elaborate biotechnological products is varied, however, the main difficulty for its use lies in maintaining a balance in the diversity of species present in it, always seeking to maintain the global functioning of the system at adequate levels.

To study the functions and interactions of cyanobacteria with other microorganisms, it is necessary to know the composition and dynamics of their populations. It is also important to relate the isolated microbial strains with their counterparts in nature, to finally extrapolate the results of physiological experiments carried out I out in the laboratory, to the natural conditions. The comparison of laboratory microbial cultures and natural populations often presents problems due to the inherent selectivity of the crop and the morphological changes that occur after propagation.

The size and morphological simplicity of the microorganisms has made it difficult to know their diversity, which is necessary to determine in studies of microbial ecology. The comparison of the percentage of culturable bacteria with the total of cells present in different habitats shows a huge discrepancy; One of the reasons for this may be the lack of knowledge of the actual conditions under which many bacteria grow in their natural environment. Currently, for the detection and identification of microorganisms, as well as for the exploration of microbial diversity and the analysis of the structure of microbial communities in open ecosystems, techniques of molecular biology and certain molecular markers are used, such as rRNA 16S or its coding sequence in DNA (rDNAl6S) 13.

The 16S rRNA is a polyribonucleotide of approximately 1500 bp, encoded by the rrs gene, also called 16S ribosomal DNA (rDNA 16S), from which sequence phylogenetic and taxonomic information can be obtained. The fragments of rRNA are they are highly conserved and present regions common to all prokaryotic organisms, which is why they are very useful for analyzing the composition of microbial communities in environmental samples14.

Denaturing gradient gel electrophoresis (DGGE) is a separation technique for PCR products, which can be used to evaluate the genotypic diversity of isolated strains in samples from the environment and judge their purity and uniqueness. The microorganisms isolated from field samples, can be assigned to natural populations based on the comparison of the profiles of bands obtained by DGGE and its sequencing, to be later characterized. Nubel and collaborators16 developed primers for the specific amplification of a 16S rRNA segment of a cyanobacterial gene, thereby allowing DGGE analysis of cyanobacterial populations. Therefore, it is necessary to have improved biofertilizers based on CMs that allow a better fertilization of the soil and that can be used in a repeatable manner, which will improve the production of plants of commercial and food interest.

Objectives of the invention.

One of the objects of the present invention is to provide effective biofertilizing compositions from nitrogen-fixing CMs that allow a better distribution of nutrients and nitrogen in agricultural cropping soils, for example in rice crops. ! Another objective of the invention is to provide biofertilizing compositions from CMs, where each of the biological components of the CM acts in an interrelated manner such that the CM presents a high biological performance to improve the process of nitrogen fixation and growth of the crops where it is applied, for example at the greenhouse level. , Another objective of the invention is to provide more effective CMs for fixing nitrogen in crop soils, where each of the biological components of the CM acts in an interrelated manner so that the CM presents a high performance biological to improve the process of nitrogen fixation and the growth of the crops where it is applied, it is also stable once it is applied and with a favorable cost-benefit balance.

Another object of the invention is to provide alternative methods for obtaining effective biofertilizing compositions from CMs, with increased capacity for nitrogen fixation and growth, when applied to a variety of culture soils. ' } Another object of the invention is to provide methods for increasing nitrogen fixation in culture soils by administering the biofertilizing compositions from CMs described herein. '' Brief description of the figures. ' Figure 1. Fragments of genomic DNA of the photosynthetic CM of the invention are shown.

(Al) fragments of genomic DNA obtained by extraction are observed using acetone17, (MI) genomic DNA fragments obtained by extraction using methanol17, (A2k) genomic DNA fragments obtained by extraction with a commercial kit using acetone, (cmk) ) genomic DNA fragments obtained by extraction with a commercial kit without previous treatment, (M2k) fragments of genomic DNA obtained by extraction with a commercial kit using methanol and (MW) molecular weight marker 1Kb (Invitrogen).

Figure 2. The amplification of the 16S rDNA of the CM of the invention is shown by PCR (1500 bp). It is observed (cmk) amplification of genomic DNA obtained by extraction with a commercial kit without previous treatment, (A2k) amplification of genomic DNA obtained by extraction with a commercial kit using acetone, (MI) amplification of genomic DNA obtained by extraction using methanol and (PM) 100 bp molecular weight marker (Invitrogen).

Figure 3. Amplification of the 16S rDNA of the CM of the invention is shown by PCR (500 bp). Observed (cmk) amplification of genomic DNA obtained by extraction with a commercial kit without previous treatment, (A2k) amplification of genomic DNA obtained by extraction with a commercial kit using acetone, (MI) amplification of genomic DNA obtained by extraction using methanol and (PM) molecular weight marker 100 bp (Invitrogen).

Figure 4. The band profile obtained from samples A2k and MI of Figure 3 is shown, 35-45% Figure 5. The amplification of the 16S rDNA is shown by PCR of the sample A2k (1500 bp); (PM) molecular size marker? (Styl) (Invitrogen).

Figure 6. A diagram of the pDRIVE vector is shown.

Figure 7. The plasmid DNA of various clones (1-14) of the CM of the invention is shown in the pDRIVE vector. Lanes 9, 11 and 13 show positive clones, while lane (-) shows a negative control of plasmid without fragment of interest; (PM) molecular size marker? (Styl) (Invitrogen).

Figure 8. The restriction analysis with the Eco Rl enzyme (ERI) is shown for the positive clones 17, 19, 23, 26, 27 and 29; is observed (PM) molecular size marker? (Styl) (Invitrogen).; Figure 9.. An outline of the phylogenetic relationship between the 16S rDNA sequences obtained from the CM of the invention grown in an 11L photoreactor (tree 1) is shown. The various DNA sequences of the clones that were positive for CM are indicated by a 4-character code (for example, 1F02).

Figure 10. A diagram of the phylogenetic relationship between the 16S rDNA sequences obtained from the CM grown in an 11L photoreactor (tree 2) is shown. The various DNA sequences of the clones that tested positive for CM are indicated by a 4-character code (for example, 1E08).

Figure 11. The growth of the CM of the invention is shown in culture per batch in a 52 L photobioreactor.

Figure 12. The amount of chlorophyll a (Chl a) of the CM of the invention is shown in a batch in a 52 L photobioreactor.

Figure 13. The nitrogenase activity of the CM is shown during the batch culture in a 52 L photobioreactor.

Figure 14. An amplification of fragments of approximately bp of the 16S rDNA by PCR from t0-t7 of a culture batch in a 52 L photobioreactor. (MW) 100 bp molecular weight marker is observed (Invitrogen) Figure 15. Fragment amplification of approximately 500 bp of the 16S rDNA is shown by PCR from t0-t7 of a culture batch in a 52 L FBR. (MW) 100 bp molecular weight marker (Invitrogen) is observed.

Figure 16. The band profile obtained from samples t0-t7 is shown by DGGE using the linear gradient (urea-formamide) DGGE 35-45%.

Figure 17. The monitoring of the growth of rice plants treated with the CM of the invention is shown.

Detailed description of the invention.

The present invention provides effective biofertilizing compositions from CMs, particularly isolated from rice cultivars, whose constituents have been elucidated from molecular biology techniques. The CM described here has excellent properties to be used as a biofertilizer, especially to promote nitrogen fixation when applied to crops, resulting in biofertilizing compositions with a favorable cost-benefit balance.

In one of its embodiments, the present invention provides a nitrogen-fixing photosynthetic CM, whose microorganisms that comprise it have been identified using molecular biology techniques. The biological performance of the CM of the invention has been tested experimentally, finding a high capacity of fixation of nitrogen in soil and a great stability of its microbial population, which are preserved approximately for at least 7 days in liquid culture. Likewise, its cost-benefit balance has also been assessed by forming a biofertilizer for nitrogen fixation in rice crops, for example, being able to extend its benefit by using it in crops of other plants.

Here we have already described the importance of cyanobacteria for their nitrogen-fixing capacity, however, the presence of all the groups of microorganisms found in a CM is determinant for the biological performance of the consortium, which gives it value as a biofertilizer.

The presence of proteobacteria species of subclasses a, β, y, and d each play a specific role in the microbial ecology of the CM of the invention, as well as the presence of bacteriodetes. The so-called satellite microflora, that is, on which the nitrogen fixers depend, which in this case can be considered to be the species of proteobacteria and bacteriodetes described as part of the CM, help in the process of ecological succession of the microflora. native soil where the CM is applied as biofertilizer; said succession is a difficult characteristic to achieve with the biofertilizers known in the art, since it is reported that most of these present problems of functionality precisely because of the competition with the microflora of the soil where it is applied. The CM of the invention used as biofertilizer has proved resistant to invasion by other microorganisms as shown below.

For effects of the invention, the CM has been characterized according to the type and proportion of microorganisms that are in it, which allows it to have an excellent nitrogen fixing capacity, be stable during its cultivation and propagation as well as be resistant to colonization by other microorganisms.

The CM of the invention comprises, with respect to its total population of microorganisms: • From 38 to 42% of phylum microorganisms Cyanobacteria, selected from the group comprising Aphanizomenon, Leptolyngbya, Anabaena and mixtures thereof, preferably Aphanizomenon aphanizomenoides, Leptolyngbya so., Anabaena Oscillarioides and mixtures thereof, which have as main characteristics that they are filamentous and two of them fix nitrogen. • 30 to 33% of microorganisms of phylum Proteobacteria, Class alpha proteobacteria, selected from the group comprising Rhodobacter, Devosia, Pedomicrobium and mixtures thereof, preferably Rhodobacter sp, Devosia insulae, Pedomicrobium americanum and mixtures thereof.

• From 5 to 7% of microorganisms of phylum Proteobacteria, Class beta proteobacteria, selected from the group comprising Methylibium, Aquamonas and mixtures thereof, preferably Methylibium sp, Aquamonas sp and mixtures thereof. · From 3 to 6% of phylum microorganisms Proteobacteria, Class gamma proteobacteria, selected from the group comprising the family Xanthomonadaceae, • From 11 to 14% of the microorganisms of the phylum Proteobacteria, delta class proteobacteria, selected from the group comprising Nannocystis, order Acidobacteriales, order Myxococcales and mixtures thereof, and · From 3 to 6% of microorganisms of the phylum Bacteriodetes, Class Flavobacteria belonging to the genus Flavobacterium.

As can be seen, the CM of the invention contains 3 important groups of microorganisms involved in nitrogen fixation, such as those belonging to phylum Cyanobacteria, Proteobacteria and Bacteriodetes.

Regarding the microorganisms that make up the CM of the invention, a brief description of these is included below. i I I I. Cyanobacteria.

The cyanobacteria make up a large and heterogeneous group of prokaryotic microorganisms of great ecological and economic importance, because they carry out oxygenic photosynthesis and, in addition, a large number of them are capable of fixing nitrogen in aquatic and terrestrial environments19, which is why they are widely used as 20 biofertilizers in tropical countries, particularly in rice crops; . They can be found living in free form, in the form of symbiotes with some type of plant or forming consortiums11. Likewise, cyanobacteria possess the ability to synthesize chlorophyll a, phycobilin and phycocyanin as pigments21.

Cyanobacteria are divided into non-filamentous (Chroococcales and Pleurocapsales orders) and filamentous (Oscillatoriales, Nostocales and Stigonematales) 22.

Leptolyngbya is a filamentous cyanobacterium belonging to the order Oscillatoriales. Form long filaments wrapped by a very thin and colorless sheath, trichomes of slightly elongated cells of approximately 0.5 to 3.2 μ? T? in width and 2.2-3 μ? t? ^ long, while its cellular morphology is isodiametric, without heterocytes; It contains granules of cyanophycin at the junction of the septa and is divided by binary fission. It grows in a pH range of 7.0-8.5 and a temperature of 10-30C, contains more phycocyanin than phycoerythrin. It has a G + C content in its DNA of 41.3-49.4% and live in coastal areas of Antarctica, lakes, glacial streams, humid soils, wet rocks, in fresh water and there are halophilic biotypes.

Aphanizomenon is a filamentous cyanobacterium belonging to the Nostocales order, family Nostocaceae. It forms filaments floating free, solitary, in observations the colonies with trichomes are oriented in parallel; the trichomes are straight, slightly curved, cylindrical (subsymetric structure) and do not form pods; several species produce a mucilaginous layer. While the vegetative cells are cylindrical, isodiametric, they divide transversely until they reach their original size before dividing again and the terminal cells do not divide. They consist of one to three heterocytes per filament, they are cylindrical rounded at the end, they can disappear if the cyanobacterium is in a medium with high nitrogen content and can form aquinetos. It lives in lagoons and lakes.

Aphanizomenon apahanizomenoides belongs to the order Nostocales, family Nostocaceae, genus Aphanizomenon. It is a nitrogen-fixing filamentous cyanobacterium; has trichomes alone, straight, approximately 350 μ? t? long, not tapered towards the ends; the vegetative cells are cylindrical, blue-green in color, the terminal cells are rounded; forms one or two spherical or cylindrical heterocytes per filament, with a thick wall. They can be found located between aquinetos or only along the filament; it forms aquinetos that are larger than the vegetative cells, only one per filament, regularly spherical or oval. It lives in eutrophic waters in tropical and subtropical zones24. i Anabaene is a filamentous cyanobacterium belonging to the Nostocales order, family Nostocaceae. Several species produce a colorless mucilaginous layer; they form three to nine heterocytes per filament, oval or cylindrical, slightly elongated, usually slightly larger than vegetative cells. It grows at a pH of 6 to 10 with an optimum of 8; grows well with low levels of C02 (0.03% present in the air) and under certain conditions 25 of culture produce exopolysaccharide. It lives in lagoons, lakes, submerged woods, stones, soils and in saline habitats. It fixes atmospheric nitrogen by establishing a symbiotic relationship, for example with the Azolla aquatic fern, and this characteristic has been used extensively in biofertilization of rice crops20.

II. Proteobacteria a) Alpha proteobacteria.

The a-proteobacteria are phototrophic purple non-sulfur bacteria, capable of anoxigenic photosynthesis. They contain various photosynthetic pigments such as baeteriochlorophylls a, b and some carotenoids, which are located in the cytoplasmic membrane and in the internal membrane. They are able to grow photoorgano-heterotrophically, but some species can grow photolyte-autotrophically with molecular hydrogen, sulfur or thiosulfate as donors of photosynthetic electrons; some can also grow chemotrophically with oxygen and in the dark.

The ecological niches of a-proteobacteria are very diverse as they can. to be found in lakes, coastal lagoons, wastewater, sediments, wet soils, rice fields, etc. They have various applications such as water treatment residuals, biomass production, biopolymers, molecular hydrogen and biotin, among others26.

The a-proteobacteria class is subdivided into seven orders Caulobacterales, Parvularculares, Rhizobiales, Rhodobacterales, Rhodospirillales, Rickettsiales and Sphingomonadales.

The a-proteobacteria can be divided into three large groups: a-1 proteobacteria that is represented by Rhodospirillum, a-2 proteobacteria that is represented by Rhodopseudomonas and oc-3 proteobacteria that is represented by Rhodobacter26.

Devosia insulae belongs to the order Rhizobiales, family Hyphomicrobiaceae; Gram negative bacterium, aerobic, does not form spores, its microscopic morphology is in the form of a rod (bacillus) or oval, its optimum pH of growth is 6.5-7.5 and its optimum temperature is 25, mobile by means of a single flagellum, its optimal growth occurs in the presence of 0.5% (w / v) sodium chloride (NaCl) 27.

Rhodobacter belongs to the order Rhodobacteriales, family Rhodobacteraceae; Gram negative bacteria that can obtain energy through photosynthesis; The best growth conditions are anaerobic phototrophic and aerobic chemotrophic in the absence of light. Some species of Rhodobacter are capable of fixing nitrogen (eg Rhodobacter sphaeroides); Rhodospirillum rubrum and Rhodobacter sphaeroides are capable of assimilating nitrate as nitrogen source; these species can be isolated from deep lakes and standing water, and are widely used for wastewater treatment26. Pedomicrobium americanum belongs to the order Rhizobiales, family Hyphomicrobiaceae; is a ubiquitous bacterium dominant in biofilms of man-made aquatic environments such as water distribution systems and bioreactors; has the ability to accumulate and oxidize Mn, reproduces in a dimorphic way and can be isolated from aquatic and terrestrial environments ! b) Beta-proteobacteria. : The β-proteobacteria also conform to the CM of the invention; These microorganisms are phototropic purple non-sulfur bacteria, capable of anoxigenic photosynthesis with bacteriochlorophylls and carotenoids as photosynthetic pigments. They are able to grow photoheterotrophically under anoxic conditions, and many of the species can grow photoautotrophically with hydrogen as a photosynthetic electron donor; some can also grow chemotrophically with oxygen and in the dark. The ecological niches of ß-proteobacteria are very diverse and can be found in fresh water, stagnant water exposed to light that is enriched with organic compounds and nutrients, activated sludge, soil, etc. They grow adequately at a temperature of 12-20 ° C, some species can also grow in a temperature range of 0-2.5 ° C29. They have various applications such as water and waste treatment, production of proteins from soybean residues and using agricultural bioproducts as wheat bran as a carbon source for Rubrivivax gelatinosus30. j The ß-proteobacteria class is subdivided into seven orders: Burkhqlderiales, Hydrogenophilales, Methylophilales, Neisseriales, Nitrosomonadales, Rhodocyclales and Procabacteriales. j Methylibium petroleiphilum belongs to the order Burkholderiales, is a gram negative bacteria, not mobile, its microscopic morphology is in the form of a rod (bacillus), hydrolyses urea I and it reduces nitrate or nitrite, possesses granules of PHB (???? - ß-hydroxybutyrate) as reserve material, reproduces by binary fission and grows heterotrophically under aerobic conditions. It is considered optional methylotrophic because it is capable of using as a carbon source methanol, ethanol, toluene, benzene, ethylbenzene and dihydroxybenzoates.

Its optimum pH and growth temperature are 6.5 and 30QC respectively. The content of G + C in their DNA is 69%. It inhabits soils highly contaminated with MTBE (methyl-terbutyl ether). Methylotrophs have been widely studied due to their potential for use in biotechnology and bioremediation31. | Aquaspirillum delicatum belongs to the order Burkholderiales, family Comamonadaceae: It is a Gram negative, mobile bacterium (it has one to two flagella), grows chemoreganotrophically with oxygen, has the ability to reduce nitrate to nitrite, has granules of PHB (??? - ß -hydroxybutyrate) as a reserve material; some species of Aquaspirillum grow anaerobically with nitrate and can also use ammonium as a nitrogen source. The content of G + C in its DNA is 63% and it mainly lives in fresh water32. c) Gamma-proteobacteria.

The? -proteobacteria encompass several families of bacteria important for science and medicine such as Enterobacteriaceae, Vibrionaceae and Pseudomonadaceae. Members of Chromatium are photosynthetic and oxidize hydrogen sulfide. Some y-proteobacteria can oxidize methane11. , The class y-proteobacteria is subdivided into 14 orders: Acidithiobacillales, Aeromonadales, Alteromonadales, Cardiobacteriales, Chromatiales, Enterobacteriales, Legionellales, Methylococcales, Oceanospirillales, Pasteurellales, Pseudomonadales, Thidtrichales, Vibrionales and Xanthomonadales. d) Delta-proteobacteria.

The d-proteobacteria encompass a group of predominantly aerobic genera, myxobacteria, which form fruiting bodies and a group of strictly anaerobic genera that contain most of the sulfate-reducing bacteria [Desllfovibrio, Desulfobacter, Desulfococcus, Desulfonema, etc.) and sulfur-reducing bacteria (for example, Desulfuromonas) together with other anaerobic bacteria with different physiology (Geobacter and the genera Pelobacter and Syntrophus) 11.

The class d-proteobacteria is subdivided into seven orders: Bdellovibrional, Desulfobacterales, Desulfovibrinoal, Desulfurhal, Desulfuromonadales, Myxococcales and Syntrophobacterales. i Nannocystis belongs to the order Myxococcales, family Nannocystaceae, is a Gram negative bacterium that has the ability to form fruitful bodies when food is scarce; in these fruiting bodies the cells start as vegetative cells of the bacillus type, but then develop into spherical myxospores (6 x 3.5 μ ?? the smallest and 110 x 40 μ ?? the largest) with thick cell walls. Inhabits large 33 caverns, tunnels, deep wells and soil.

III. Bacteriodetes.

They are a phyium with wide distribution in the environment, including the soil, sediments, sea water and the digestive tract of animals. The group includes three classes: Bacteriodetes, Flavobacteria and Sphingobacteria. ' Flavobacterium belongs to the order Flavobacteriales, family Flavobacteriaceae; it is a Gram negative, aerobic, immobile bacterium and grows chemoorganotrophically, from 20 to 309C; some species grow chemoheterotrophically and in aquatic environments play an important role in the mineralization of various types of organic matter (carbohydrates, amino acids, proteins and polysaccharides). The content of G + C in their DNA is 32 to 37%. They inhabit freshwater, soil, sewage, marine sediments, marshes, compost, rhizosphere of the tomato plant, etc.

Flavobacterium species produce a wide variety of enzymes that have great biotological potential in applications that involve the degradation of biomacromolecules such as agar, alginate, chitin, xylan, pectin, etc; production of biosurfactants, bioremediation of hydrocarbons in soil, treatment of effluents from paper mills, production of antifungal products, etc.34. ! Once the phylogenetic analysis was performed and knowing the components that make up the C of the invention, several parameters were determined that are illustrated in the examples, which refer to kinetic parameters and to the behavior of the consortium in experimental crops at greenhouse level, such as nitrogen fixing capacity, scaling and its biofertilization potential.

In one of its embodiments, the invention also provides biofertilizing compositions for application in crops, for example rice, which include the CM described herein. The biofertilizer can be presented in various forms, such as solid, liquid and those forms and presentations known in the art.

The solid presentation of the biofertilizer of the invention, can be made as a granulated powder containing an inert carrier or base, which can be lignite or peat, with a granule size of 0.15 to 0.2 mm, with a pH in a range of 6.5 to 7.5 and humidity in a range of 30% to 60% by weight. In this case, the CM of the invention is added to the composition in a concentration of 1 to 20% w / w of biomass. The product can be packed in bags from 1 to 25 Kg to facilitate its handling. In this case, the CM of the invention is added to the soil in an amount of 10 to 20 g of biomass per hectare of soil to be fertilized, which represents a reduction of 3 to 6 times the necessary amount of other biofertilizers to achieve the same biofertilizing effect.

As for the liquid presentation of the biofertilizer of the invention, this can be formulated to support the microbial population of the CM, which is stable for at least 7 days as long as it is stored at 4 ° C.

In the case of rice cultivation, approximately 150 kg of nitrogen is needed for each hectare of crop, which is traditionally added as 322 kg of urea per hectare. Using other biofertilizers, it would be necessary to add at least 58 g of biomass per hectare, however with the biofertilizer of the present invention, it is only necessary to add from 12 to 18 grams of CM biomass per hectare, which represents a 5-fold decrease the amount of biomass that should be added with other biofertilizers. Consequently, the biofertilizer of the invention can fix 150 Kg of atmospheric nitrogen per hectare of crop with smaller amounts of biomass of the CM of the invention, which results in an increase in the productivity of the crop, a greater efficiency in fixing nitrogen in the soil and lower costs of production, transport and application of the biofertilizer.

Due to the ability of the CM of the invention to remain viable after the drying process, for example by stove for 24 h at 70 ° C, it is possible to manipulate it in a manner constant and homogeneous to obtain biofertilizers that retain their activity even during storage under suitable conditions for long periods.

The invention is also described in detail with the following examples which are illustrative of the manner in which it was obtained and its forms of application; These examples are not intended to limit the scope of the invention, so that specialists in the field will be able to appreciate certain modifications that can be applied to the invention without changing its scope and original spirit.

Example 1. Establishment of the CM of the invention and determination of its biological performance.

To establish the CM of the present invention, it was started by collecting in sterile containers with medium BG-11035 samples of microbial flora of the soil, water of flooded and stem of plants in contact with the ground, of a rice field in the State from Morelos, Mexico36,37. Cultures of 250 mL were established in the same culture medium with these samples, with an air flow of 1.5 vvm and incubation at two different temperatures (21 and 28? C) to subsequently proceed with the scaling of two cultures in volumes of 500 mL and 1 L, with an air flow of 1.5 vvm at both temperatures, constantly evaluating its growth and nitrogen fixation capacity. According to the results obtained from biomass production and nitrogen fixation, the culture incubated at 21SC was selected because it was the most suitable for the purposes pursued, so it was scalded in 2 L bubbling columns and photobioreactors of 11 L with an air flow of 1 vvm. Once the culture was established in a volume of 11 L, it was scalded in a 52 L air-lift flat-panel photobioreactor with an air flow of 0.6 vvm (30 L / min) 37, an amount of light of 80 μ ???? photons / m2 / s and the same mineral medium.

The evaluation of the biological performance of the CM was carried out by measuring the activity of nitrogenase and dry weight, carried out once a week, thus obtaining the concentration of biomass in a determined volume of culture38; likewise chlorophyll "a" was determined by spectrophotometric tests at 665, 666 and 750 nm11. The quantification of nitrogenase activity is an indirect measure of the nitrogen-binding capacity of CM19. The method is based on the fact that nitrogenase can also reduce acetylene to ethene, which is quantified by gas chromatography and related to the nitrogen fixation capacity of the biomass studied, being directly proportional to the concentration of ethene produced11 .

The determination of chlorophylls was used to indirectly quantify the amount of cyanobacteria in a sample as described by Whitton21. By having different pigments, two unrelated microorganisms can coexist in the same habitat, therefore, the diversity in pigmentation has ecological significance11.

The propagation of the CM of the invention was first performed in a photovoltaic reactor of 11 L of total capacity, bubble colmmna type, evaluating its biological performance during 8 replantings, with a duration of 14 days per crop batch, using a light intensity of 80 μ ???? photons / m2 / s and an aeration of 11 L / min (1 vvm). The results of the CM's biological performance are shown in Table 1.

Table 1 Results of the CM biological performance in the 11 L photobioreactor The culture of CM grown at a temperature of 21 C produced an extracellular matrix of polysaccharide; the production of similar substances by cyanobacteria has been previously reported39, where it is attributed as its main function, that of serving as protection against desiccation or predators. Due to the above, it is possible that the presence of Exopolysaccharide in the CM of the invention increases its resistance against the disturbances of the controlled environment in which it is found, since in the culture the presence of protozoa was observed; resulting in an improvement in their biological performance.

Once the CM was established in the 11 L photobioreactor, it was proceeded to its scaling illustrated in more detail in Example 3, in a 52 L photo-reactor of total air-lift flat-face volume, using 6 L of culture as inoculum and 44 L of mineral medium BG-110, with the air flow of 30 L / min and the amount of light 80 μ ???? photons / m2 / s; the MC was propagated during 8 reseeding with a duration of 14 days per crop lot, evaluating its biological performance during all replantings. The results obtained are shown in Table 2.

Table 2 Results of the CM biological performance in the 52 L air-lift photobioreactor The CM grown at a temperature of 212C in the 52 L photobioreactor again produced an extracellular polysaccharide matrix characteristic of this culture when it grows under the aforementioned conditions. On the other hand, when observing the optical microscope, it was possible to observe whole filaments containing 5 to 8 heterocytes each, which could contribute to the increase of nitrogenase activity; it was further confirmed that the air flow used (30 L / min) did not affect the production and development of the filamentous organisms that make up the consortium of the invention. From the data in Table 2 it can be deduced that the nitrogenase activity value was approximately 12 times higher than that obtained in the 11 L photobioreactor: (Table 1), probably due to the increase in the amount of filamentous cyanobacteria, nitrogen fixers, in addition to the amount of heterocytes present per filament.

Table 3 shows some nitrogenase activity values reported in the literature, which are approximately 1.5 to 2.3 times lower than the value obtained for the CM of the invention in the 11 L photobioreactor (5188.5 nmol ethene / g dry weight / h) and approximately 21 to 33 times lower than the value obtained in the 52 L photobioreactor (75211.32 nmol ethene / g dry weight / h). i Table 3 Nitrogenase activity for different strains of cyanobacteria Example 2. Identification of the components present in the photosynthetic CM of the invention.

- DNA extraction.

A sample of the CM of the invention was taken in an 11 L photobioreactor (day 14) at 21 ° C and the genomic DNA extraction was carried out using the conventional technique of Cüllen17 as well as a commercial kit (QuickGene DNA tissue kit S, Model : QuickGene-810 / QiliickGene-Mini80) to assess the purity and performance of extraction by both techniques. Previously the samples were subjected to a treatment with methanol (99.96%) to remove pigments and with acetone (99.6%) to eliminate the exopolysaccharide matrix produced by the CM. After extraction of the DNA from the CM, electrophoresis was performed at 110 V on a 0.8% agarose gel using TAE as a buffer; IX (50X, 242 g of Tris base, 57.1 mL of glacial acetic acid, 100 mL of 0.5 M EDTA (pH 8), using ethidium bromide (0.5 μg / mL) to stain the gel. UV 2000 transilluminator (Gel Doc 2000, BIO-RAD Laboratories Inc., Carlsbad, CA) to visualize the obtained bands.Full and high molecular weight DNA fragments were obtained, with higher purity and yield using the commercial kit to make the extraction of genomic DNA from samples A2k, cmk and M2k Figure 1 shows the genomic DNA fragments obtained with both extraction techniques.

- Amplification of the 16S rDNA of the CM of the invention by PCR.

At all taxonomic levels of the species, analysis of the sequence of genes encoding the small subunit of the ribosome (30S) is currently the most promising method for the phylogenetic classification of cyanobacteria. further, the comparative analysis of the 16S rDNA gene sequences provides new means to investigate the difference between collections of strains and natural communities42. To analyze the 16S rDNA of the microbial components of the CM of the invention, it was amplified using the genomic DNA as an annealing. By PCR, fragments of approximately 1500 and 500 bp were obtained (Fig. 2 and 3). For this amplification a commercial kit was used, under the following conditions43: a) Denaturation for 10 min at 935C, b) 35 cycles of 1 min at 932C, 1 min at 575C, 2 min at 722C, and c) A final elongation time of 10 min at 722C.

The following universal initiators were used for the 16S rDNA region: 46F (5'-GCCTAACACATGCAACTC-3 ') 44 and 1540R (5'-AACGAGGTGATCCAGCCGCA-3 ') 45.

The amplification was carried out in a Touchgene Gradieñt FTGRAD2D (TECHNE DUXFORT, Cambridge, UK). The expected size of the amplified region of the 16S rDNA fragment was approximately 1500 bp (FIG. 2). Once the fragments of 1500 bp were obtained, a second amplification was carried out (figure 3) to obtain fragments of approximately 500 bp using the following primers: 46F (5'-GCCTAACACATGCAACTC-3 ') 44, with a sequence rich in GC (5'-CGCCCGGGGCGCGCCCCGGGCGGG GCGGGGGCACGGGGG) and 518 R (5'-ATT ACC GCG GCT GCT GG-3 ') 46 The amplification products were visualized in 0.8% agarose gels, with TAE IX and .110 V buffer, ethidium bromide (0.5 ml-1) and visualization in UV transilluminator (figures 2 and 3).

- Separation of the amplified rDNA by gel electrophoresis with denaturing gradient (DGGE).

The PCR products (amplified rDNA) were separated by DGGE. With this technique, 50% of the variants of the sequences can be detected in DNA fragments larger than 500 bp. This percentage can be increased up to 100% by the binding of a GC-rich sequence at the 5 'end of one of the PCR primers. The length of the GC-rich sequence can vary between 30 and 50 nucleotides47.

I For separation, a polyacrylamide gel (10% (v / v)), arcrylamide-bis-acrylamide (37.5: 1), with a denaturing linear gradient (urea-formamide) of 35-60% was used. Electrophoresis was carried out for 20 h at 45 V and SO ^ C in a DCODE (Bio-Rad Laboratories Inc., USA); The gel was then stained with ethidium bromide (0.5 μg ml-1) for 20 min48. The band profile obtained (Figure 4) was observed under a UV transilluminator, evidencing 15 different bands that could each correspond to a microbial component of the CM of the invention.

Nubel et al49 using DGGE carried out the analysis of segments amplified by PCR of the 16S rDNA region of cultures of pure strains, and obtained as a result a profile of 10 different bands corresponding to the same species, indicating their heterogeneity (sequences that differ by less 1% at the position of their nucleotides). It follows that the profiles obtained with the DGGE technique is indicative but not conclusive of the number of different microorganisms present in the sample, since it detects interspecific heterogeneity.

From the band profile obtained, the calculation of the Simpson index (D) was carried out, which allowed to quantify the existing diversity in the CM. The calculation was made using the Quantity One program and the following algorithm: where S is the number of species, N is the total number of organisms present (or square units) and n is the number of specimens per species.

The values obtained were D = 0.08137 and 1 / D = 12.28996, which indicated that the CM of the present invention is made up of approximately 12 species and that its diversity is low.

- Construction of a library from 16S rDNA fragments of the CM of the invention. The amplification of rDNA 16S was carried out according to what was mentioned above, obtaining the fragments shown in figure 5.

Once the amplified region was obtained, it was inserted into the cloning vector pDRIVE (figure 6), which has a size of 3851 bp, contains two genes that confer resistance to kanamycin and ampicillin as well as a gene that codes for a fragment of the β-galactosidase that allows a complementation a.

Once the ligation was carried out, competent cells of E. coli DH5-a were transformed in the vector by thermal shock. Subsequently, the clones were planted in LB medium with kanamycin and those that they contained the fragment of interest (figure 7), for its subsequent sequencing and bioinformatic analysis.

The positive clones were very numerous. Figure 8 shows a restriction analysis with the enzyme Eco Rl, which verified the release of the fragment of interest of the vector pDRIVE, releasing fragments of 1500 and 500 bp, which indicated that the sequences of interest have an intergenic site for EcoRI.

Positive clones were selected and sequencing of the cloned fragment was followed by a 3730X DNA Analyzer (Applied Biosystems, Foster City, CA). 47 sequences of approximately 980 nucleotides each of the 16S rDNA gene were obtained. ' - Identification of the microbial components of the CM through bioinformatic analysis. The programs that were used for the bioinformatic analysis of the sequences obtained from the 16S rDNA fragments of the CM of the invention were BLAST50, CLUSTAL X51, and MEGA452.

Figures 9 and 10 show the phylogenetic relationship of the 16S rDNA of the 47 clones obtained, using Pyrococcus abyssi (AY099167) as an external group. The phylogenetic tree was constructed with related sequences obtained from the NCBI Taxonomy Homepage (TaxBrowser) using the Neighbor-joining method and the Tamura-Nei distance analysis model, with 100 bootstrap-type randomizations 52. This phylogenetic analysis allowed us to identify 20 different organisms that make up the CM, of which the largest proportion corresponded to filamentous nitrogen fixing cyanobacteria.

The similarity between the sequences was calculated from the following formula, where the total and variable nucleotides of the aligned sequences are considered: Similarity = (Total number of nucleotides) - (Number of variable nucleotides) (Number of total nucleotides) X 100 The taxonomic assignment to establish the level of species must have a similarity greater than 97.5%, while for identification at the level of gender is required, 95% with the best related sequence, and with a similarity lower than this value, it is placed on a level above the gender53. Table 4 shows the taxonomic assignment to [genus and species level of 32 sequences analyzed for the CM of the present invention, Table 4 Taxonomic assignment of the microorganisms of the CM of the invention Table 4 continued Of the 47 clones analyzed from the CM, it was found that: · 19 clones (40.4%) belonged to the phyium cyanobacteria, such as Aphanizomenon aphanizomenoides, Leptolyngbya sp. and Anabaena oscillarioides, which have as main characteristics that they are filamentous and two of them fix nitrogen, > • 15 clones (31.9%) belonged to the phyium proteobacteria, class alpha proteobacteria, such as Rhodobacter, Devosia insulae and Pedomicrobium americanum, among others. · 3 clones (6.3%) belonged to the phyium proteobacteria, class beta proteobacteria such as Methylibium and Aquamonas, • 2 clones (4.3%) belonged to the phyium proteobacteria, class gamma proteobacteria belonging to the family Xanthomonadaceae, • 6 clones (12.8%) belonged to the phylum proteobacteria, class delta proteobacteria such as Nannocystis and some belonging to the order Acidobacteriales and Myxococcales, and 1 • 2 clones (4.3%) belonged to the phylum bacteriodetes, class flavobacteria belonging to the genus Flavobacterium. i Example 3. Nitrogen binding activity of the photosynthetic CM of the invention i during its growth in lot of 52 L Once the CM was established in the 11 L photobioreactor, it was scaled in a 52 L54 air-lift flat-panel photobioreactor, using an inoculum of 6 μL with the following characteristics: 513.33 mg / L, 0.005127 mg / mg dry weight, 8181.7024 nmol ethene / g dry weight / h, and brought to a total volume of 50 L with BG-110 medium. For the growth an air flow of 30 L / min, an amount of light of 80 μ ???? photons / m2 / s and a temperature of 212C.

Samples were taken every third day during a culture batch, which lasted 16 days; For each sample, the nitrogenase activity test and the chlorophyll a determination were performed to analyze the growth and nitrogen-fixing activity of the photosynthetic CM of the invention, obtaining the results shown in Table 5 and Figures 11 to 13.

According to the data shown in table 5, the greatest amount of biomass was obtained at day 14 of culture with 280 mg / L, observing the start of growth with an adaptation phase (figure 11), followed by an exponential phase to from day 2 of cultivation and observing a slight decrease in biomass on days 7 and 9 that managed to recover on day 12 until reaching the maximum value; subsequently a considerable decrease in the amount of biomass was observed, which may indicate a phase of death at day 16. The maximum value of chlorophyll a was obtained at the beginning of the crop and was 0.016 mg mg dry weight, followed by a considerable decrease until reaching a point (day 5 of cultivation) where no significant change in chlorophyll a values was observed (figure 12), possibly due to the fact that the photosynthetic organisms present in the CM of the invention managed to adapt to the culture conditions from this point, so there was no considerable variation in the quantity of these organisms from day 5 until the end of the culture lot.

Table 5 V = 52 L; t = 16 d; () = standard deviation.

The maximum value of nitrogenase activity according to Table 5 was obtained at day 9 of the culture with a value of 64069.32 nmoles of ethene / g dry weight / h, which coincided with the decrease of biomass mentioned above at the same time of cultivation . This could be due to a direct relationship between the amount of nitrogen and carbon necessary for the optimal growth of the nitrogen-fixing cyanobacteria and the bacteria that make up the CM of the invention. This type of nitrogen and carbon interaction necessary for the growth of various consortiums has been reported for genera of cyanobacteria such as Anaboena and Aphanizomenon that rapidly transfer nitrogen from host cyanobacteria to epiphytic bacteria that do not fix nitrogen (approximately 1 h).; The magnitude and speed of the transfer from the host to the epiphyte apparently depends on the growth phase and the physiological state of the cyanobacterium. When the phototrophic growth of the host cyanobacterium is favored, the extracellular transfer and release of nitrogen to the epiphytic bacteria is limited. However, under conditions of carbon limitation, the nitrogen-fixing cyanobacterium releases a greater amount of nitrogen to the outside where the epiphytic bacteria are found, this due to an inadequate cellular supply of carbon compounds necessary to incorporate the recently fixed nitrogen. An alternative to help nitrogen fixation is to excrete excess nitrogen fixed as NH3 / NH4 +, which when excreted can then be assimilated by epiphytic bacteria increasing their growth, which results in an increase in organic matter, generating a greater amount of C02, which is used by the cyanobacteria that were limited by the amount of available carbon55. I This type of associations cyanobacterium-epiphytic bacteria can be considered CM because there is an exchange of metabolites and growth factors through the EC weather. Lange, for example, speculates that the C02 generated by the epiphyte bacteria plays an important role in optimizing the growth of cyanobacteria under carbon limiting conditions, in addition to the fact that it has been suggested that the growth factors supplied by associations of bacteria may be partially responsible for the optimal growth and development of blooms. On the other hand, Paerl and Pickney55 suggest I that the epiphytic bacteria help to reduce the tension of 02 near the cells that make up the filament of the cyanobacterium so that biochemical processes can be carried out that are sensitive to 02 such as photosynthesis and nitrogen fixation. . ' Example 4. Population variation of the CM of the invention.

Samples were taken every third day from time zero (inoculation) of the photosynthetic CM culture lot of the invention in the "air-lift" flat-face photobioreactor, whose duration was 16 days. Once the batch was finished, DNA extraction of the samples obtained was carried out using a commercial kit (QuickGene DNA tissue kit S, Model: QuickGene-810 / Qu¡ckGene-Mini80), obtaining genomic DNA in duplicate in each sample. Using the genomic DNA as a template, amplification was performed by PCR of the 16S rDNA region to obtain fragments of approximately 1500 and 500 bp. The results of the amplification of the 16S rDNA by PCR (about 1500 bp) of the samples taken along a culture lot (t0-t7) are shown in Figure 14.

Once the fragments of 1500 bp were obtained, a second amplification was performed to obtain fragments of approximately 500 bp. The amplification of the 16S rDNA by PCR (approximately 500 bp) is shown in Figure 15.

Once obtained the PCR products were separated by DGGE in a polyacrylamide gel (10% (v / v)), acrylamide-bisacrylamide (37.5: 1), with a denaturing linear gradient (urea-formamide) of 35-45% . Electrophoresis was carried out for 20 h at 45 V and 6Ó9C in a DCODE (Bio-Rad Laboratories Inc., USA). The band profile obtained by DGGE is shown in Figure 16.

As can be observed, there is no significant variation of the components of the consortium along a crop lot, since three bands are observed at t0 ,: while from t2 to t5 there are five bands that are constant to these time. In t7 only two bands were observed, which may be due to the fact that at this time the crop was in the death phase, which is possible to corroborate with the growth curve presented in figure 11 where the behavior is exemplified of the growth of the CM in a 52L reactor, being evident a stage of exponential growth followed by a stage of stability that refers an adaptation and a slight decrease on days 7 and 9, recovering on day 12; this kinetics was performed by providing the data of Table 5 described above together with the culture conditions. It is probable that each band corresponds to a different component that integrates the CM, which could be corroborated by means of a phylogenetic study in aliquots of each one of the times considered throughout a crop by lot.

According to the above results, the CM of the invention has the following characteristics, which are advantageous in comparison with other CMs: · Low biodiversity of components (according to the value of the Simpson index),, • Contains at least 20 different microorganisms, of which the highest proportion correspond to filamentous nitrogen-fixing cyanobacteria, • It is well established, so the number of components that make it they do not vary with respect to time, and · Its characteristics are such that the amount of components that make up the CM, and the balance between them, are necessary to carry out an efficient nitrogen fixation.

Example 5. Biofertilization tests with the CM of the invention and monitoring of rice crops.

Rice cultivation was carried out under laboratory conditions, determining the average height, root frequency and sowing at the depth of 2 cm recommended by the FAO. We worked a group to which was added an inoculum of the CM of the invention, another group to which urea was added and a control group, monitoring the height difference of the plant with respect to its initial height. At the end of the experiment, the following parameters were measured: percentage of survival, number of leaves, leaf color, plant growth, root growth and biomass produced.

Regarding the percentage of survival, it was observed that the control had a 58.3%, while the plants with urea (less than 5%) had a lower percentage than the control (16.67%). On the other hand, the plants that were inoculated with the CM of the invention (biofertilizer) both in the form of wet biomass (cake) and in suspension (2.5 mL), produced a 100% survival, which proves the positive effect of the CM Biofertilizer of the invention on plants to allow them to have a greater chance of survival, increasing the growth of the plant.

The positive effects were corroborated by a statistical analysis with the t-student test applied to the growth and biomass production results of the plant. The trial showed that there is a clear difference in net growth between the plants that were inoculated with the biofertilizer and the control plants (figure 17), finding a difference between the plants with biomass and the control with a level of significance of 99.5%, which confirms that the use of the CM of the invention as biofertilizer favors the growth of the plants. On the other hand, the plants with urea also had an increase of growth with significant differences in 95% with respect to the control, however between the plants with biomass of the CM and the plants with urea there is a difference that is 95% significant, which allows to conclude that the CM of the invention used as a biofertilizer produces a positive effect on the plant above the effect provided by the urea. During the experiment, soil colonization by the CM of the invention was evident, as a film constituted by CM microorganisms was formed. , Example 6. Establishment of the CM of the invention in rice cultivation and its stability and resistance to the invasion of other microorganisms.

A sampling of the water from the waterlogging was done in order to know what kind of microorganisms existed in the environment that could colonize the experimental cultures of rice made in the laboratory and if these microorganisms could cause a decrease of the microbial species of the CM of the invention that provide their beneficial effect observed for the plant.

I It was found that in the first month of the trial, the microbial genera found in the water of the waterlogging were practically the same as those found in the reactor; on the other hand, the control was colonized with diatoms, while the cultures treated with urea presented a great microbial diversity, observing diatoms, protozoa and bacteria. Two months after the start of the trial, it was observed that there was an ecological succession in the control cultures, since the presence of cyanobacteria and diatoms different from those found in the observation made in the first month was observed; on the other hand, the cultures inoculated with the CM of the invention and urea did not have an important change in their microflora.

Based on the foregoing, it is possible to say that the CM of the invention is well established and can be used to form a photosynthetic biofertilizer, since its microbial composition does not vary over time and rapid variations in microbial concentration are not observed.

Example 7. Stability of the CM of the invention upon drying.

On the other hand, in order to determine the ability of the CM of the invention to resist drying, oven drying was carried out for 24 h at 70 ° C on a substrate sample previously inoculated with the CM of the invention; the resulting sample was resuspended in BG110 medium and the remaining nitrogenase activity was determined, in order to I determine if this operation is technically feasible to use it as a method of conservation of the biomass of the CM of the invention. It was found that the dry biomass has a specific activity of 1027 nmol ethene / g biomass / h; that is to say, approximately 18% of the initial nitrogenase activity is recovered, and the good state of the biomass could be verified by observations under the optical microscope. The results suggest that there is an expected level of nitrogenase activity so this method can be used in the conservation of the CM of the invention without it undergoes alterations in its capacity as a biofertilizer.

I Example 8. Estimation of the amount needed of the CM of the invention to fertilize a normal rice crop.

Each rice crop needs approximately 150 kg of nitrogen per hectare of crop, that is, 322 kg of urea per hectare that must be distributed throughout the cultivation time. Rice cultivation needs approximately 75% of its nitrogen requirement from sowing to flowering, this occurs in a period of 56 days57, which means that approximately 107.8 mg of nitrogen / h are needed in each hectare of crop, this it is 144.88 kg of ammonium per hectare. If we take into account that the plant will only capture the nitrogen that is in soluble form, then we must base the calculations on the nitrogen excreted in the form of ammonium. According to this, 57.92 g of biomass per hectare would be needed, with an overestimation factor of 20%. However, the results obtained with the CM of the invention suggest that the amount of CM biomass necessary to obtain a positive effect is 14.4 grams per hectare, with an overestimation factor of 20%.

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Claims (1)

1. A biofertilizer composition comprising an agronomically effective amount of a nitrogen-fixing photosynthetic microbial consortium comprising: a. 40% of phylum cyanobacteria, species Aphanizomenon aphanizomenoides, Leptolyngbya sp. and Anabaena oscillarioides, b. 32% belong to phylum proteobacteria, alpha class Rhodobacter, Devosla insulae and Pedomicrobium americanum, c. 7% belong to phylum proteobacteria, beta class Methylibium petroleiphilum and Aquaspirillum delicatum (Aquamonas), d. 4% belong to the phylum proteobacteria, of gamma class Xanthomonadaceae, e. 13% belong to the phylum proteobacteria, class delta Nannocystis and some belong to the order Acidobacteriales and Myxococcales, and F. 4% belong to the phylum bacteriodetes, Flavobacterium aquatile. The biofertilizing composition of claim 1, characterized in that it is in the form of granules. The biofertilizing composition of claim 2 characterized in that it contains an inert carrier or carrier substrate with a granule size of 0.15 to 0.2 Mesh sieve, a pH in a range of 6.5 to 7.5, humidity in 30% to 60% in and the microbial consortium is inoculated and in a final concentration of 70 to 300 Plate Formers per gram of biofertilizer, The biofertilizing composition of claim 1 characterized in that it is in liquid form. The biofertilizing composition of claim 4 characterized in that it contains a liquid inert base or carrier substrate, a pH in a range of 6.5 to 7.5 and the microbial consortium is inoculated and in a final concentration of 70 to 300 Plate Formers per mL of biofertilizer , The biofertilizer composition according to any of claims 2 to 5, wherein said inoculum of microbial consortium comes from the culture performed in a reactor of 11L to 150L, preferably of 52L | A method for promoting nitrogen fixation in rice cultivars where 14 to 58 grams of the biofertilizing composition of claim 2 to 3 are added per hectare of culture. j