WO1996034840A1

WO1996034840A1 - Bacterial fertilizer and production process

Info

Publication number: WO1996034840A1
Application number: PCT/ES1996/000097
Authority: WO
Inventors: Antonio Martin Gonzalez; Tilman Sanchez Elsner
Original assignee: Consejo Superior Investigaciones Científicas; Applicaciones Magneticas, S.L.
Priority date: 1995-05-04
Filing date: 1996-04-29
Publication date: 1996-11-07
Also published as: ES2093559A1; ES2093559B1

Abstract

The present invention relates to a bacterial fertilizer (biofertilizer) comprised of a suspension which contains cells of new strains of the micro-organisms Azotobacter vinelandii 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 these new strains of micro-organisms are their higher tolerance to very alcaline media and a better binding of N2, as well as a higher capacity or assimilation of compounds formed by the exudates of the plant roots.

Description

DESCRIPTIVE MEMORY Bacterial fertilizer and procedure for obtaining it

OBJECT OF THE INVENTION

The object of the present invention is a bacterial fertilizer consisting of a suspension containing cells of new strains of the microorganisms Rzotobacter and Azoεplrillum. Said microorganisms and the procedure for obtaining said bacterial fertilizer also constitute another object of the present invention.

STATE OF THE ART

The main atmospheric nitrogen-fixing microorganisms that have been used as bacterial fertilizers (biofertilizers) are Rzotobacter and Azoεpirllum.

Rzotobacter has been used as a nitrogenous fertilizer in some plants of agricultural interest such as corn, wheat, potato, beetroot, tobacco, barley, flax, oats, carrots, cabbage and tomato. Inoculation with Rzotobacter as a bacterial fertilizer has been developed for many years (Rubenchic L.I., "Azotobacter and Its Use in Agriculture". Academy of Sciences of the Ukrainian SSR. Microbiological Institute im. D.K. Zabolotnyi, 1963). Of all the strains, the most used as inoculant has been R. chroococcum, as it is the most frequent and abundant in the soil (Krieg N.R, Holt, J.G., Williams and Wilkins Co., Baltimore, 1984).

On the other hand, the ability of these microorganisms to synthesize plant growth activating substances, such as phytohormones (cytokinins, auxins,...) is of great interest (González López et al., Soil Biol. Biochem. 18:119-120 , 1986) since it is the most remarkable in their interaction with the plants. Among the auxins studied in Rzotobacter vinelandii are 3-indole-acetic acid and 3-indole-lactic acid (García Bilbao, J.; Tabares F. et al., 1. of Appl. Microbiol. Biotechnol. 25:502- 506, 1987). Significant densities of R . chroococcum (Martínez Toledo, FEMS Microbiol. Ecol. 31:197-203, 1985) demonstrating an association between AzotoJ acter and maize roots. The inoculation of Zea mayε with Rzotobacter represents a significant increase in the dry weight of the plant, although the size of the leaves is not affected. The application of 40 Kg/Ha of nitrogen plus Rzotobacter originates the highest grain yields. On the other hand, the inoculation of corn with R. chroococcum, isolated directly from its roots, represents a favorable effect, giving rise to better results in small doses (González López et al., Soil Biol. Biochem. 18:119-120, 1986). .

Using urea with Rzotobacter and Rzoεpirillum, [Tilak K. V. et al., "Azospirillum brasilense and Azotobacter chroococum: Effect on the yield of maize (Zea mays) and sorgum (Sorgum bicolor), 1982] similar results were obtained in Zea mays. Inoculation with Azotoiacter produces important economic benefits by applied together with small doses of nitrogen fertilizer, not only for its significant increase in agricultural production, but, at the same time, for savings in nitrogen fertilizers (Meshram; Shend; Plant and Soil 69:265-273, 1982). On many occasions, Rzotobacter and Rzoεpirillum have been used together as inoculants, obtaining more satisfactory results than when using only AzotoJbacter chroococcum (Tilak et al., see reference above).However, some crops such as wheat show higher yields with the inoculation of a microorganism only (Zambre et al., Plant and Soil 82:61-67, 1984) The first Azotobacter mutants showing increased N ₂ fixing capacity and its use as biofertilizers.

There are different ways of inoculating different types of plants with Rzotobacter, either with lyophilized cells (Meshram and Shende, see previous reference) or preparation in the form of suspensions inoculated by peristaltic pump (Smith R.L., Appl. Environ. Microbiol. 47:1331- 1336, 1984). According to González López (see previous reference), double inoculation allows a more effective and prolonged action of the inoculant, achieving the highest yields.

The amount of nitrogen accumulated in the field cultivated with wheat, due to the effect caused by Rzoεpirillum and Rzotobacter, is comparable to the amounts of nitrogen commonly applied as fertilizer. The inoculation with Rzoεpirillum of naturally nodulated peas caused a significant increase in the number of nodules with respect to the controls and an increase in the harvest was produced.

The inoculation method in sugarcane, bathing the root in Rzotobacter , resulted in an increase of 24.3% compared to the control without inoculation.

In field trials conducted with rice, doses of nitrogen (as urea) of 0, 50, 75 or 100% of the recommended amounts were used. The use of biofertilizers (Rzoεpirillum lipoferum ) plus 50% of the recommended dose in each type of land is equivalent to the use of 100% urea.

Inoculating the following plants with AzotoJbacter: Reacia auriculiformiε , Gmelina arborea, Tectona grandiε , Dalbergia εiεεo , it was possible to increase survival from 15 to 75%. In studies with mulberry cuttings, a higher yield in wood production, tree height, size and length of the leaves was achieved when inoculating with Rzotobacter.

Studies in maize, inoculated with Rzoεpirillum braεilenεe, achieved high yields in plant development.

The increase in oak seeds, in various soils inoculated with AzotoJ acter, was 70.7% in the dry weight of the roots and 45% in that of the branches, at 90 days of growth.

The application of Rzotobacter and a phosphate solubilizer

(Bacillum megaterium) (phosphobacterin) increased all the mean parameters at 8 months over the controls: height 20.2%, rhizosphere diameter 37.7%, fresh weight 34.6% and weight 44%. dry.

Inoculating millet seeds with six mimics of Rzoεpirillum, using different doses of nitrogen, an average increase in production of 67% in the dry weight of the straw and 56.36% in the dry weight of the grain was achieved. This inoculation represents a saving of 20 kg of nitrogen per ha and an increase of 50% in the application of this element.

Inoculating with Rzoεpirillum braεilenεe rice and sorghum seeds and grown in soil without nitrogen fertilizers, an increase in production was obtained on fertilized land with 40 Kg of nitrogen per Ha, of 28.14% in rice cultivation.

Inoculating wheat with Rzoεpirillum lipoferum , an increase in grain production per Ha of 64.3% was achieved in unfertilized land, 31.22% in crops with 40 Kg of nitrogen per Ha and 40.22% with 80 Kg of this fertilizer. The increase in straw production was 18.54%,

17.90% and 32.52%, respectively. The increase in rice production, inoculating the seeds with Rzoεpirillum braεilenεe, is due to the synthesis of growth factors and N ₂ fixation.

Inoculating Rzotobacter and Rhizobium, both together and separately, in Piεum εativum L for 10 minutes. , an increase was observed in the height of the plants, the number of branches per plant, the production and in the protein content of the seeds. When replacing Fe ⁺² with micronized Fe ⁰ in the culture medium, an increase in N ₂ fixation by Rzotobacter and in the dry weight of the cells is observed (patent published under the number ES-2041219).

In the European patent with publication number EP-0570079 strains of Azospirillum are described by means of which it is possible to stimulate the growth of cereal plants.

The favorable effect of the inoculation with Azotoiacter and Rzoεpirillum cells on different agricultural crops is the product of a multiple action because it represents an alteration of the suppressive microbial balance of pathogenic microorganisms, mobilization of phosphates, assimilation of exudates from the roots of the plants (acid p -hydroxybenzoic, ... ) and production of metabolites such as phytohormones that stimulate growth before and after germination, also representing a very considerable saving in nitrogen fertilizers due to the fixation of N ₂ in the soil.

The beneficial effects of Rzotobacter and Rzoεpirillum are related not only to their ability to fix N ₂ but also to the ability to produce antibacterial and antifungal compounds, growth regulators, and siderophores (Pandey, A.; "Potential of Azotobacters and Azospirilla as biofertilizers for upland agriculture: a review"; Journal of Scientific and Industrial Research 48.3, 134-144, 1989). The saving of nitrogenous fertilizers supposes a parallel saving of fossil fuels. It is evident that this relationship implies an enormous interest in obtaining bacterial fertilizers that can partially replace the nitrogen from ammonia synthesis and, on the other hand, from an ecological point of view it is also necessary to replace these nitrogenous fertilizers with biofertilizers that prevent soil degradation.

EXPLANATION OF THE INVENTION

Unlike the state of the art, the object of the present invention is a bacterial fertilizer that contains cells of new strains of the microorganisms Rzotobacter and Rzoεpirillum specifically developed to improve N ₂ fixation and potentiate alginate biosynthesis. . Said bacterial fertilizer consists of a suspension of microorganisms in an aqueous medium (Burk medium) of pH=7.4 containing 1.6 grams/liter of sucrose; 0.8 g/1 of P0 ₄ HK ₂ ; 0.2 g/1 of P0 ₄ H ₂ K; 0.2 g/1 of S0 ₄ Mg.7H ₂ O; 0.05 g/1 of S0 ₄ Ca; 0.05 g/1 of S0 ₄ Fe and 0.001 g/1 of sodium molybdate. The microorganisms suspended in said medium are a strain of Rzotobacter vinelandii deposited in the Spanish Type Culture Collection (CECT) with the number CECT 4534 and a strain of Rzoεpirillum braεilenεe deposited in the Spanish Collection of Type Cultures with the number CECT 4533.

The microorganism AzotoJbacter vinelandii CECT 4534 is also the object of the invention and is obtained by means of a procedure that comprises: a) culture of the original lineage Rzotobacter vinelandii 382 DSM (German Collection of Microorganisms) in Burk medium with D-glucose at 1 0.6% (w/v) in place of sucrose for 72 hours. b) treatment of the colonies from the previous culture with a Burk's culture medium containing 0~05% DL glutamic acid and 1.6% (w/v) mannitol instead of sucrose by plating for a period of time between 72 and 96 hours. c) culturing the colonies isolated from the previous step with a medium containing 2% glutamine and 0.05% DL glutamic acid at pH=7.8 for 72 hours.

The microorganism Rzoεpirillum braεilenεe CECT 4533 is also the object of the present invention and is obtained by means of a procedure that comprises: a) culturing the original strain Rzoεpirillum braεilenεe 590 CECT, in Burk medium with 1.6% D-Ribose (weight/volume ) instead of sucrose for 72 hours. b) treatment of the colonies from the previous culture with a culture medium containing 0.05% DL glutamic acid and 1.6% (w/v) mannitol instead of sucrose by plating for a period of time between 72 and 96 hours. c) culturing the colonies isolated from the previous step with a medium containing 2% glutamine and 0.05% DL-glutamic acid at pH =7.8 for 72 hours.

The process for preparing the bacterial fertilizer is also the object of the invention and comprises: a) plate culture of the strains Rzotobacter vinelandii CECT 4534 and Rzoεpirillum braεilenεe CECT 4533 in Burk's medium pH=7.4 containing 1.6 grams/ liter of sucrose; 0.8 g/1 of P0 ₄ HK ₂ ; 0.2 g/1 of P0 ₄ H ₂ K; 0.2 g/1 of S0 ₄ Mg.7H ₂ 0; 0.05 g/1 of S0 ₄ Ca; 0.05 g/1 of S0 ₄ Fe and 0.001 g/1 of sodium molybdate and incubation at 30°C for a period of time between 72 and 96 hours. b) growth of the previous culture by inoculating flasks with a capacity of 300 ml with 100 ml of the culture medium. culture used in the previous step and incubation in an orbital shaker at 175 revolutions per minute for 120 hours at a temperature between 30 and 32°C. c) inoculation in a fermenter at 1% inoculum by volume in Burk medium, with a minimum titer of Rzotobacter vinelandii and Rzospirillum braεilenεe of 109 bacteria/ml for each microorganism, and culture at a temperature between 30 and 32°C for 52 hours under aeration conditions by spraying air into the medium through a porous glass filter at a flow of 300 liters of air per minute previously sterilized through a 0.45 μm filter at a pressure of 1 Kg/cm ² .

DETAILED DESCRIPTION AND MODE OF CARRYING OUT THE INVENTION

By subjecting Rzotobacter and Rzoεpirillum cells in liquid and solid cultures (with agar-agar) to different substrates, variants of these strains capable of fixing more atmospheric nitrogen, synthesizing more phytohormones (cytokinins, auxins, . . . ) and metabolites such as alginates and essential amino acids, such as usine and methionine, in higher concentration. These strains also present a greater facility for using waste products as substrates, for example, the exudates of the roots of the plants (ortho-, meta- and parahydroxybenzoic acids,...) and agricultural residues such as molasses, vine shoots, stillage, etc The biosynthesis of gelling polysaccharides (alginates) allows these Rzotobacter and Rzoεpirillum cells to better adhere to plant roots and at the same time have this capsular substrate available for growth and reproduction in the soil.

Rzotobacter vinelandii culture

Starting from the original strain of Rzotobacter vinelandii 382 DSM (German Collection of Microorganisms), cultivated for 72 hours in Burk's medium, with 1.6% (weight/volume) D-glucose instead of sucrose, in Petri dishes and in liquid cultures, is achieved by purification and isolation of colonies, AzotoLacter cells that produce 1.7 g/1 of alginate instead of 0.25 g/1 and manage to fix a greater quantity of N ₂ . These cells are called by the initials 383 I _? of Azotobacter vinelandii.

Subsequently, they are treated and subjected to a culture medium for a period of time between 72 and 96 hours.

Burk containing 0.05% DL glutamic acid and 1.6% mannitol as a substrate instead of sucrose, as this is a precursor of alginic acid. The contribution of DL glutamic acid is intended to inhibit the nitrogenase enzyme to enhance alginate biosynthesis. In this way, cells are obtained, isolated on plates, which in subsequent standardized cultures, with sucrose as a substrate, reach a production of 2.4 g/1 of alginate with a high index of uronic acids (the basis for good gelation). These cells are designated by the initials 382 12 _E of R . vinelandii . At the same time that they synthesize a greater amount of alginate, they fix a higher N ₂ concentration than the original strains.

To further enhance N ₂ fixation and alginate biosynthesis, cells from the colonies are again subjected to

382 I2 _P of R. vinelandii for 72 hours in a culture medium with 2% glutamine and 0.05% DL-glutamic acid at pH=7.8.

In the same way as in the previous step, it is possible to isolate colonies that, in normalized cultures with 1.6% sucrose as a substrate, give rise to greater alginate biosynthesis (2.7 g/1) and greater N ₂ fixation (77 % more in the form of NH4+ ion in the culture medium). This strain is called with the initials 24APV of

Rzotobacter vinelandii and has been deposited in the Collection

Española de Cultivos Tipo (CECT), having been assigned the number CECT 4534. Cultivation of Rzoεpirillum brasilense

The strains of Rzoεpirillum braεilenεe obtained from the 590 CECT strain, are prepared by the same procedure as in the case of Rzotobacter , but with D-Ribose as the only substrate in all steps, obtaining a strain called Rzoεpirillum with the acronym 40AM. braεilenεe that has been deposited in the CECT having been assigned the number CECT 4533.

Development of 24APV Rzotobacter vinelandii and 40AM Rzoεpirillum braεilenεe cells

The ideal culture medium for the best development of Rzotobacter and Rzoεpirillum cells is the following:

substrate: Sucrose 1.6 g/1 salts: P0 ₄ HK ₂ 0.8 g/1

P0 ₄ H ₂ K 0.2 g/1 S0 ₄ Mg . 7H ₂ 0 0 , 2g/1

S0 ₄ Ca 0 , 05 g/1

Sodium molybdate ... 0.001 g/1 trace elements: S0 ₄ Fe 0.05 g/1

The final pH of the culture medium is adjusted to 7.4.

Using this culture medium, the strains of AzotoJ acter vinelandii and Rzoεpirillum braεilenεe are seeded on solid media in Petri dishes and incubated at 30°C for a period of time between 72 and 96 hours. These freshly grown, pure cultures are used to inoculate 300 ml flasks with 100 ml of culture medium, which are incubated at a temperature between 30 and 32°C for 120 hours at 175 revolutions per minute in an orbital incubator. Once grown, they are inoculated in a fermentor reactor, at a temperature between 30 and 32°C, adding 1% of inoculum, expressed by volume, in a Burk medium with a minimum titer of 109 for Rzotobacter vinelandii and Rzoεpirillum J rasilense. bacteria/ml for each microorganism. The cultivation is carried out under aerated conditions, passing through said reactor 300 liters of air per minute previously sterilized through a 0.45 μm filter at a pressure of 1 Kg/cm ² . The air is sprayed into the medium through a fritted glass filter. The optimum time for this submerged culture is 52 hours.

The cell biomass of the mixture of Rzotobacter and Rzoεpirillum is then available to be inoculated into the seeds and subsequently into the soil.

The advantages of using the Rzotobacter and Rzoεpirillum strains object of the present invention over the known strains are the following: 1.- The obtained strains of Rzotobacter and Rzoεpirillum reproduce well at a greater pH range and better than the known strains, supporting very alkaline media (pH=10), thus avoiding sterilization problems and possible contamination. 2.- Said strains are capable of fixing more N ₂ and more easily eliminating the NH ₄ ⁺ ion from the culture medium, while excreting polysaccharides with higher concentrations than known strains. These polysaccharides will serve the Rzotobacter and Rzoεpirillum cells as an energy reserve for their subsequent development in the soil, at the same time that they will allow them to adhere more easily to the roots of the plants. 3.- These variants of Rzotobacter and Rzoεpirillum also have a greater capacity to assimilate the compounds formed by the exudates of the roots of the plants, such as p-hydroxybenzoic acid, which is hardly used by other strains.

4.- The speed of reproduction and assimilation of the substrates is also higher in the Rzotobacter and Rzoεpirillum variants than in the known strains, since the maximum dry weight reached by them is up to 3 times higher after 52 hours of culture. submerged at about 28-30°C. 5.- The performance and effectiveness of these Rzotobacter variants is much higher than the known strains and the initial strain 382 DSM, also synthesizing polysaccharides with a high gelling index (alginates). The cell biomass production of the Rzotobacter strain 24 APV is of the order of 4 g/1 dry weight of lyophilized cells. Starting with sucrose as the only substrate, at a concentration of 1.6%, a yield of 25% is obtained, that is, for every 16 grams of sucrose, 4 grams of dry weight of cells of the microorganism that is cultivated are obtained. Bearing in mind that, according to Rubenchick (see reference in STATE OF THE ART), the amount of Azotoibacter cells needed to fertilize one Ha is of the order of 25 to 40 g, this amount would be equivalent to approximately 7.51 of cell culture. That is to say, that with 120 g of sucrose as the only substrate, the Rzotobacter cells necessary for the fertilization of one Ha are obtained.

Various tests have been carried out in the greenhouse and in the field with different types of plants such as tomato, carrot, beetroot and cabbage, inoculating the seeds and irrigating the soil in which they were sown with a cell suspension of various strains of Rzotobacter and Rzoεpirillum. The results, both in the greenhouse and in the field, demonstrated a much higher effect of the strains object of the present invention over the known strains.

Claims

MODIFIED CLAIMS [received by International office on September 10, 1996 (09.10.96); claims 1-4 superseded by new claims 1-6;(2 pages)]

1.- Bacterial fertilizer consisting of a suspension of microorganisms in an aqueous medium (Burk medium) of pH=7.4 containing 1.6 grams/liter of sucrose; 0.8 5 g/1 of PO ₄ HK ₂ ; 0.2 g/1 of PO ₄ H ₂ K; 0.2 g/1 of SO ₄ Mg.7H ₂ O; 0.05 g/1 of SO ₄ Ca; 0.05 g/1 of SO ₄ Fe and 0.001 g/1 of sodium molybdate, characterized in that the microorganisms suspended in said medium are a strain of Azotobacter vinelandii deposited in the Spanish Collection of Type Cultures (CECT) with the number CECT 4534 and a strain of Azospirillum brasilense deposited in the 10 Spanish Collection of Type Cultures under the number CECT 4533.

2.- Microorganism that belongs to the Azotobacter vinelandii lineage, deposited in the CECT with the number 4534.

3. Procedure for obtaining the microorganism according to claim 2, characterized by the following stages: a) plate culture of the original strain Azotobacter vinelandii 382 DSM, in Burk medium with D-glucose at 1.6% (weight/volume) instead of sucrose for 72 hours. b) treatment of the colonies from the previous culture with a Burk's culture medium containing 0.05% DL glutamic acid and 1.6% (w/v) mannitol instead of sucrose by plating for a period of time between 72 and 96 hours. c) culturing the colonies isolated from the previous step with a medium containing 2% glutamine and 0.05% DL-glutamic acid at pH=7.8 for 72 hours.

25

4.- Microorganism that belongs to the genus Azospirillum brasilense, deposited in the CECT with the number 4533.

5. Procedure for obtaining the microorganism according to claim 3, characterized by the following stages: a) plate culture of the original lineage Azospirillum brasilense 590 CECT, in Burk medium with D-Ribose at 1.6% (weight/volume) instead of sucrose for 72 hours. b) treatment of the colonies from the previous culture with a Burk's culture medium containing 0.05% DL glutamic acid and 1.6% (w/v) mannitol instead of sucrose by plating for a period of time between 72 and 96 hours. c) culturing the colonies isolated from the previous step with a medium containing 2% glutamine and 0.05% DL glutamic acid at pH=7.8 for 72 hours.

6.- Procedure for the preparation of a bacterial fertilizer characterized in that said procedure comprises: a) plate culture of the strains Azotobacter vinelandii CECT 4534 and Azospirillum brasilense CECT 4533 in a Burk medium pH=7.4 containing 1.6 grams /liter of sucrose; 0.8 g/1 of PO ₄ HK ₂ ; 0.2 g/1 of PO ₄ H ₂ K; 0.2 g/1 of SO ₄ Mg.7H ₂ O; 0.05 g/1 of SO ₄ Ca; 0.05 g/1 of SO ₄ Fe and 0.001 g/1 of sodium molybdate and incubation at 30°C for a period of time between 72 and 96 hours. b) growth of the previous culture by inoculating 300 ml flasks with 100 ml of the culture medium used in the previous step and incubation in an orbital shaker at 175 revolutions per minute for 120 hours at a temperature between 30 and 32 °C. . c) inoculation in a fermentor at 1% inoculum by volume in Burk's medium, with a minimum titer of Azotobacter vinelandii and Azospirillum brasilense of 10 ⁹ bacteria/ml for each microorganism, and cultivation at a temperature between 30 and 32 °C for 52 hours under aeration conditions by spraying air into the medium through a porous glass filter at a flow of 300 liters of air per minute, previously sterilized through a 0.45 μm filter at a pressure of 1 Kg/cm ² .