AGRICULTURAL COMPOSITION COMPOSED BY CONSORTIUM BETWEEN Azospirillum sp. and Pseudomonas sp., PRODUCTION PROCESS AND INCREASED STABILITY THEREOF, AND USE AS PROMOTOR OF PLANT GROWTH FOR AGRICULTURAL APPLICATION

Abstract

The present invention refers to an agricultural composition that comprises a mixture of Azospirillum brasilense and Pseudomonas fluorescens in a single commercial inoculant product that promotes increased stability and cellular viability due to the synergism between these microorganisms, and which surprisingly increases the shelf-life achieved by a differentiated industrial process. The industrial process of the mixture of Azospirillum and Pseudomonas as product inoculant plant growth promoting is composed by steps of: (a) fermentation of the microorganisms to obtain an agricultural composition with plant growth promotion action through biological mechanisms to fix nitrogen, biosynthesis of auxins, siderophores and solubilization of phosphorus; (b) stabilization of the product biotechnological inoculant composed by the mixture of Azospirillum and Pseudomonas in a technical solution that enables the mixture of the parts, presented in a single package. The synergism demonstrated is mainly connected to (i) the high production of EPS by Azospirillum which is consumed by Pseudomonas as energy source and (ii) to the production of organic acids by Pseudomonas which enables the maintenance of cellular viability of the Azospirillum.

Claims

1.-34. (canceled)

35. An agricultural composition composed of a mixture of Azospirillum and Pseudomonas microorganisms as an inoculant product promoting plant growth, comprising: Azospirillum brasilense in a concentration of 1.010.sup.8 UFC/mL to 3.010.sup.9 UFC/mL and Pseudomonas fluorescens in a concentration of 1.010.sup.7 to 5.010.sup.8 UFC/mL of inoculant; wherein the microorganisms propagate at low temperatures, below 20 C., in soils with low pH, below 6.0; the agricultural composition contains water, sucrose, carboxymethyl cellulose, gelatin and mannitol as carrier; and the mixture promotes prolonged storage time.

36. The agricultural composition of claim 35, wherein the prolonged storage time is 18 months, with an initial count of 110.sup.9 to 310.sup.9 numbers of cells of Azospirillum brasilense and 110.sup.7 to 510.sup.7 number of cells of Pseudomonas fluorescens, and after 18 months, reaches 110.sup.8 to 110.sup.9 number of cells of Azospirillum brasilense and 910.sup.7 to 510.sup.8 number of cells of Pseudomonas fluorescens, in a temperature range of 5-40 C.

37. Industrial process of the mixture of Azospirillum and Pseudomonas as inoculant product promoting plant growth, comprising the steps of: (a) formulation of a commercial biotechnological inoculant product composed of two genera of microorganisms in a technical solution that enables the mixture of the parts with high stability, presented in a single package; and (b) stabilization of a biotechnological inoculant product composed by the mixture of Azospirillum and Pseudomonas in a technical solution that enables the mixture of the parts, presented in a single package.

38. The industrial process of the mixture of Azospirillum and Pseudomonas of claim 37, wherein the stabilization process of the product of step (b) is carried out for approximately 1 to 2 hours.

39. The industrial process of the mixture of Azospirillum and Pseudomonas of claim 37, wherein the two genera are selected from the group consisting of Azospirillum brasilense and Pseudomonas fluorescens.

40. The industrial process of the mixture of Azospirillum and Pseudomonas of claim 37, wherein the microorganisms are mixed in fermenters, and the step of mixture of Azospirillum and Pseudomonas is carried out during about 1 to about 2 hours.

41. The industrial process of the mixture of Azospirillum and Pseudomonas of claim 37, wherein the fermentation of the culture is by batch; the fermentation process is carried out at a temperature of approximately 22 C. to 38 C.; the fermentation process is carried out at an air flow of approximately 1.0 Nm.sup.3/h to approximately 2.5 Nm.sup.3/h or 0.0085-0.021 vvm.

42. The industrial process of the mixture of Azospirillum and Pseudomonas of claim 37, further comprising the sequential expansion of the culture of different genera of bacteria for inoculating the fermentation culture, and the sequential expansion is made in volumes of about 100 mL, to about 10 L, about 180 L to about 2000 L.

43. The industrial process of the mixture of Azospirillum and Pseudomonas of claim 37, wherein the bacteria genera are inoculated separately.

44. The industrial process of the mixture of Azospirillum and Pseudomonas of claim 37, wherein the genera of bacteria are expanded by incubation in an orbital agitator to about 80 rpm to about 200 rpm; the genera of bacteria are expanded by incubation for about 18 hours to about 96 hours; the genera of bacteria are expanded in inox flasks containing about 10 L of culture medium; the genera of bacteria are incubated for approximately about 18 to about 96 hours; the genera of bacteria are incubated with air flow of about 0.25 Nm.sup.3/h to about 1.0 Nm.sup.3/h or 0.45-2.5 vvm for 10 L of culture medium, for approximately 18-96 hours and temperature approximately 22-38 C.

45. The industrial process of the mixture of Azospirillum and Pseudomonas of claim 44, wherein the genera of bacteria after the culture segregated in two inox flasks of about 10 L, said two flasks are inoculated in tanks containing about 180 L of culture medium; the strains are incubated for about 24 to about 168 hours; the genera of bacteria are incubated with air flow of about 1.0-10.0 Nm.sup.3/h or about 0.1-0.83 vvm; and the incubation temperature is from about 22 C. to about 38 C.

46. The industrial process of the mixture of Azospirillum and Pseudomonas of claim 37, wherein the fermentation step is carried out with pressure of about 1.0 to about 1.2 kgf/cm.sup.3; the fermentation step is carried out with agitation of about 40 hz to about 45 hz; the fermentation step is carried out at a temperature of about 22 C. to about 38 C.; the fermentation step is carried out with air flow of about 1.0 Nm.sup.3/h to about 2.5 Nm.sup.3/h or about 0.0085-0.021 vvm.

47. The industrial process of the mixture of Azospirillum and Pseudomonas of claim 37, wherein production occurs of exopolysaccharides (EPS) in Azospirillum, and of the biosynthesis of organic acids by Pseudomonas.

48. The industrial process of the mixture of Azospirillum and Pseudomonas of claim 37, wherein a mixture of microorganisms is applied in amounts of 150 mL/ha via seed, 300 to 1000 mL/ha via plantation furrow and 500 to 1000 mL/ha via leaf spraying.

49. Use of agricultural composition composed by a mixture of Azospirillum and Pseudomonas as inoculant product promoting plant growth, as defined in claim 35, wherein it is used for application in various agricultural crops, for application via seed, furrow and post-emergence spraying, and for promoting plant growth and solubilizing phosphorus.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0027] For improved understanding of the invention, reference should now be made to the embodiments of the invention illustrated in greater detail in the accompanying drawings and described by means of the embodiments of the invention.

[0028] FIG. 1 illustrates the synergism of Azospirillum and Pseudomonas in relation to the concentration of exopolysaccharides (EPS) and concentration of organic acids in different storage times for products composed by the microorganisms bottled in consortium.

[0029] The FIG. 2 illustrates the standard calibration curve with different concentrations of propionic acid (0.5-4 mM) as an indirect method of quantifying the production of organic acids produced by microorganisms.

[0030] The FIG. 3 illustrates the effect of the microorganisms bottled individually relative to the concentration of exopolysaccharides (EPS) and concentration of organic acids at different storage times.

DETAILED DESCRIPTION OF THE INVENTION

[0031] In a preferred embodiment, according to the present invention, in step (a), the fermentation of the Azospirillum brasilense and Pseudomonas fluorescens per batch occurs for approximately 18 to 120 hours.

[0032] In a preferred embodiment, the method of the present invention comprises the sequential expansion (grading) of the culture of A. brasilense and P. fluorescens for inoculating the fermentation culture. Preferably, the sequential expansion is initiated in volumes of 100 mL, which serves as inoculum for about two flasks with volumes of 10 L. This, in turn, is inoculated in tanks with about 180 L of culture, which, ultimately, are transferred to reactors containing about 1600 L.

[0033] In a preferred embodiment, the A. brasilense and the P. fluorescens are expanded in flasks of about 100 mL of the NFb medium (Dobereiner, 1995) and 4.1 medium (Table 1), respectively, by incubation in orbital agitator to about 80 rpm to about 200 rpm. The incubation time is preferably about 18 hours to about 96 hours. Preferably, the microorganisms are then inoculated in inox flasks containing about 10 L of culture medium 4.4 (Table 2) and 4.1 (Table 1) for the A. brasilense and P. fluorescens, respectively. The incubation time is preferably about 18 to about 96 hours with air flow of about 0.25 Nm.sup.3/h to about 1.5 Nm.sup.3/h (=0.45-2.5 vvm).

TABLE-US-00001 TABLE 1 CULTURE MEDIUM USED FOR GROWTH OF P. FLUORESCENS UP TO THE SCALE OF 200 L. Reagents 01 K.sub.2HPO.sub.4 0.4-4 g 02 KH.sub.2PO.sub.4 0.4-4 g 03 (NH.sub.4).sub.3PO.sub.4 0.1-2 g 04 MgSO.sub.47H.sub.2O 0.1-2 g 05 NaCl 0.001-1 g 06 KNO.sub.3 0.5-5 g 07 Yeast Extract 0.5-5 g 08 Solution MnSO4 (10%) 10-100 L 10 Solution FeCl.sub.3 (10%) 10-100 L 11 Glycerol 1-20 mL 12 Water q.s. 1 L q.s.: quantum sufficit

TABLE-US-00002 TABLE 2 CULTURE MEDIUM USED FOR GROWTH OF A. BRASILENSE UP TO SCALES OF 180 L. Reagents 01 K.sub.2HPO.sub.4 1-10 g 02 KH.sub.2PO.sub.4 0.5-5 g 03 CaCl.sub.22H.sub.2O 0.01-1 g 04 MgSO.sub.47H.sub.2O 0.2-2 g 05 NaCl 0.01-1 g 06 NH.sub.4Cl 1-10 g 07 Yeast Extract 1-10 g 09 KOH 30-100 g 10 Solution of micronutrients*.sup.1 1-10 mL 11 Fe-EDTA 0.01-1 g 12 Malic acid 1-10 g 13 Water q.s. q.s.: quantum sufficit *.sup.1see table 4.

[0034] In a preferred embodiment, the culture temperature for multiplication according to the present invention is about 22 C. to about 38 C.

[0035] In a preferred embodiment, A. brasilense and P. fluorescens are inoculated separately in the process of grading up to 180 L and mixed in the fermenters of 2000 L as described for the present invention. Accordingly, in a preferred embodiment, said inocula of 100 mL are inoculated in two flasks made of inox of about 10 L and then transferred in tanks containing about 180 L of specific culture medium for each microorganism, incubated for about 24 to about 168 hours. The air flow is, preferably, about 0.25 to about 1.0 Nm.sup.3/h (=0.45-2.5 vvm).

[0036] In a preferred embodiment, the tank of the A. brasilense is then inoculated in fermenters of 2000 L and the culture is carried out at a temperature of about 22 C. to about 38 C. The air flow is preferably about 1.0 Nm.sup.3/h to about 2.5 Nm.sup.3/h (=0.0085-0.021 vvm). The fermentation time is preferably about 18 to about 120 hours. The pressure is preferably about 0.5 to about 1.2 kgf/cm 2. Agitation is preferably from about 30 hz to about 45 hz. Preferably, the A. brasilense is conducted for a period of 18 to 120 h.

[0037] In a preferred embodiment, at the end of the fermentation process of the A. brasilense, a tank containing 200 L of P. fluorescens is then inoculated and mixed to the fermenter of 2000 L.

[0038] In a preferred embodiment, according to the present invention, in step (b), the formulation used for the agricultural composition that enables the mixture of two microorganisms in fermenters of 2000 L is described as per Table 3.

TABLE-US-00003 TABLE 3 CULTURE MEDIUM USED FOR GROWTH OF MICRORGANISMS IN FERMENTER 2000 L. Reagents 01 K.sub.2HPO.sub.4 1-10 g 02 KH.sub.2PO.sub.4 0.5-5 g 03 CaCl.sub.22H.sub.2O 0.01-1 g 04 MgSO.sub.47H.sub.2O 0.2-2 g 05 NaCl 0.01-1 g 06 NH.sub.4Cl 1-10 g 07 Yeast Extract 1-10 g 09 KOH 30-100 g 10 Solution of micronutrients*.sup.1 1-10 mL 11 Fe-EDTA 0.01-1 g 12 Carboxymethyl cellulose (CMC) 1-10 g 13 Saccharose 10-100 g 14 Gelatin 0.1-1 g 15 Mannitol 1-10 g 16 Glycerol 5-10 mL 17 Water q.s. q.s.: quantum sufficit *.sup.1see table 4.

TABLE-US-00004 TABLE 4 STOCK SOLUTION OF MICRONUTRIENTS AND QUANTITY NEEDED FOR 1 L OF FINAL SOLUTION OF MICRONUTRIENTS. 1 L 1 L Reagents (stock solution) (micronutrients) 01 (NH.sub.4).sub.6Mo.sub.7O.sub.24 100-200 g 84 mL 02 MnSO.sub.4 100-300 g 84 mL 03 H.sub.3BO.sub.3 50-150 g 648 mL 04 CuSO.sub.4 1-10 g 84 mL 05 ZnSO.sub.4 1-10 g 84 mL

EXAMPLES

Example 1Grading of Culture

[0039] The A. brasilense and P. fluorescens are inoculated separately in flasks containing 100 mL of culture medium NFb (Dobereiner, 1995) and 4.1 as described in Table 1, being incubated in orbital agitator of 80-200 rpm, at 22 to 38 C. for approximately 18 to 96 hours. The following step of grading consists of the inoculation of inox flasks containing 10 L of culture medium as described in Table 2 and Table 1, for the microorganisms A. brasilense and P. fluorescens, respectively, the species are cultivated separately and incubated for approximately 18 to 96 hours, with air flow 0.25 to 1.0 Nm.sup.3/h (=0.45-2.5 vvm) and temperature approximately 22-38 C.

[0040] Once this time has expired, each culture containing two inox flasks with 10 L of culture medium is inoculated in a tank containing about 180 L of specific culture medium for each microorganism, using the culture medium in Table 2 the specific culture medium for the A. brasilense; and in Table 3 the specific culture medium for P. fluorescens and incubated for approximately 24 to 168 hours, with air flow 1.0 to 10.0 Nm.sup.3/h (=0.1-0.83 vvm) and temperature varying from 22 to 38 C.

Example 2Mixture of Azospirillum and Pseudomonas in Bioreactor

[0041] For the mixture of the microorganisms in a fermenter of 2000 L, preferably the process of sterilization uses a volume of 1600 L of culture medium (Table 3) and is carried out for approximately 60 to 120 minutes, at a temperature of approximately 121 C. to approximately 130 C. Preferably, sterilization is carried out at a pressure of approximately 1.0-2.0 Kgf/cm 2. Preferably, after the sterilization process, a tank of A. brasilense is then inoculated and fermented for about 18 to about 120 hours. The air flow is preferably about 1.0 Nm.sup.3/h to about 2.5 Nm.sup.3/h (=0.0085-0.021 vvm). The pressure is preferably about 1.0 to about 1.2 kgf/cm 2. The agitation is preferably about 40 hz to about 45 hz. The stabilization process of the inoculant product that enables the mixture of the microorganisms is preferably carried out with a tank containing 200 L of the culture of P. fluorescens is inoculated to the fermenter for approximately about 1 h to 2 h. Preferably, the final product is bottled in sterile bags.

Example 3the Mixture of Different Genera of Microorganisms Enables Increased Shelf-Life of the Agricultural Composition

[0042] Although the microorganisms generally grow in pH near to neutral, they present different characteristics when cultivated in different sources of carbon (C). When cultivated using malic acid as main source of carbon (Dobereiner, 1995), Azospirillum causes an increase in the pH of the medium that assumes an alkaline content, whereas strains of Pseudomonas, when cultivated with sucrose or glycerol as sources of carbon, they acidify the culture medium. This imbalance in the pH of the culture medium may anticipate the cellular death phase of these microorganisms cultivated in isolation.

[0043] Preferably, the formulation of A. brasilense cultivated in isolation uses as main sources of carbon or mannitol, the glycerol and the sucrose, essential sources for the production of exopolysaccharides during the fermentative process. In this embodiment of culture, the average of the pHs of the batches stored is 7.1 (Table 5) and the viability of the cells, after 7 months of storage, drastically falls, increasing from 1.010 9 UFC/mL to about 3.310 7 UFC/mL, a reduction of 97% in cellular viability. In the same way, when the P. fluorescens is cultivated and bottled individually, after the same seven months of storage, the concentration of viable cells is reduced by 97.7%, increasing from 2.510 9 UFC/mL to about 5.610.sup.7 UFC/mL (Table 6), with the initial pH of about 6.92 acidified to 5.22. In contrast, when the product is made up of the consortium between Azospirillum and Pseudomonas, the average of the pH of the batches stored is 6.2 (Table 7). Unexpectedly, when the two microorganisms are mixed in a single product, synergy occurs, enabling the stability of the cells of the two genera employed for prolonged storage periods of up to 18 months as observed in Table 7; initial concentrations of about 1.010.sup.9 UFC/mL undergo a slight reduction to about 7.710.sup.8 UFC/mL after 12 months of storage, a reduction of just 33% in the viability of the cells in a longer storage period. The production of the organic acids by Pseudomonas fluorescens begins after the process of stabilizing the mixture of the microorganisms during the storage period, since Pseudomonas fluorescens consumes the exopolysaccharide produced by Azospirillum brasilense during the fermentative process.

TABLE-US-00005 TABLE 5 CONCENTRATION OF CELLS OF AGRICULTURAL COMPOSITIONS CONTAINING AZOSPIRILLUM STORED FOR DIFFERENT TIME PERIODS. CONCEN- INITIAL TRATION CONCEN- AFTER TRATION initial STORAGE pH AFTER STORAGE BATCH (UFC/mL) pH (UFC/mL) STORAGE TIME A 3.10 10.sup.9 6.82 6.0 10.sup.7 7.21 7 months B 2.39 10.sup.9 6.75 3.0 10.sup.7 7.10 7 months C 1.30 10.sup.9 7.30 2.1 10.sup.7 7.18 8 months D 1.52 10.sup.9 6.91 5.0 10.sup.7 7.26 9 months E 1.65 10.sup.9 6.91 2.8 10.sup.7 7.30 10 months F 1.82 10.sup.9 6.97 4.0 10.sup.7 7.95 10 months G 1.31 10.sup.9 7.04 1.3 10.sup.7 6.93 10 months H 2.00 10.sup.9 6.90 3.1 10.sup.7 7.10 10 months I 3.60 10.sup.9 6.98 2.0 10.sup.7 6.97 11 months J 1.51 10.sup.9 7.05 4.5 10.sup.7 6.96 12 months

TABLE-US-00006 TABLE 6 CONCENTRATION OF CELLS OF INOCULANTS CONTAINING PSEUDOMONAS STORED FOR DIFFERENT TIME PERIODS. INITIAL CONCEN- CONCEN- TRATION TRATION STORAGE BATCH (UFC/mL) pH (UFC/mL) pH TIME 1 3.1 10.sup.9 6.95 5.2 10.sup.7 5.22 8 months 2 2.5 10.sup.9 6.97 5.0 10.sup.7 5.12 7 months 3 1.9 10.sup.9 6.89 6.6 10.sup.7 5.34 7 months

TABLE-US-00007 TABLE 7 CONCENTRATION OF CELLS OF AGRICULTURAL COMPOSITIONS COMPOSED BY AZOSPIRILLUM AND PSEUDOMONAS IN A SINGLE PACKAGE STORED FOR DIFFERENT TIME PERIODS. INITIAL CONCENTRATION CONCENTRATION (UFC/mL) (UFC/mL) STORAGE BATCH Azospirillum Pseudomonas pH Azospirillum Pseudomonas pH TIME 1 1.9 10 4.1 10 6.89 6.6 10 1.8 10 6.11 6 months 2 1.4 10 1.3 10 7.04 8.5 10 1.5 10 6.47 10 months 3 2.3 10 1.8 10 7.05 6.6 10 3.5 10 6.35 10 months 4 1.6 10 1.6 10 6.94 9.5 10 1.0 10 6.25 10 months 5 1.4 10 1.8 10 6.95 7.4 10 1.0 10 6.10 10 months 6 2.2 10 5.4 10 6.72 1.05 10 2.2 10 6.04 10 months 7 1.6 10 5.0 10 6.87 8.3 10 1.7 10 6.12 12 months 8 2.3 10 1.1 10 6.95 3.0 10 9.0 10 6.22 18 months indicates data missing or illegible when filed

[0044] Example 4Synergy of the Azospirillum and Pseudomonas enables increased stability and cellular viability for longer storage periods.

[0045] The combination of the microorganisms of different genera in a single commercial product with different action mechanisms, such as promoting growth in plants by the biosynthesis of phytohormones or else by solubilization of phosphorus is of fundamental importance for more sustainable agriculture.

[0046] However, the major challenge of this combination in a single product with high stability and cellular viability is associated to growth metabolism, for example, the use of the different sources of carbon, culture time, pressure, temperature, aeration and, even the particular aspects possessed by each genus. For the bacteria of the genus Pseudomonas, as mentioned in example 3, the reduction in the pH during the culture and storage is related to the production of organic acids as demonstrated in FIG. 1.

[0047] For quantifying the biosynthesis of organic acids by Pseudomonas, an indirect colorimetric method was developed to evaluate the quantification of these compounds. In this context, a calibration curve was initially made using different gradients of propionic acid (0.5-4 mM) as an indirect method of quantifying the production of organic acids produced by microorganisms (FIG. 2).

[0048] Quantifying the production of organic acids was made by spectrophotometer at 620 nm, using the solution of bromothymol blue 0.5% in absolute alcohol as colorimetric method. This compound is an indicator of pH, that is, when added to a more acidic culture medium, the coloring becomes yellow whereas in the more alkaline culture medium the coloring is blue.

[0049] To evaluate the samples, the inoculants were centrifuged at 10,000 rpm for 10 minutes, in order to separate the microbial biomass, aliquots of 2 mL of each supernatant were added 20 L of the bromothymol blue solution and three-fold readings were taken.

[0050] The aliquots of the products containing just Pseudomonas stored for 1 to 2 months did not present detectable amounts of organic acids, whereas with longer storage periods (6 to 12 months), the presence of 3.45 mM/mL of organic acids was detected. This high quantity of organic acids caused a strong acidification of the medium, leading to a loss in the cellular viability of 97.7%.

[0051] Regarding the biosynthesis of EPS, Pseudomonas fluorescens did not demonstrate capacity to produce it during the course of storage studied.

[0052] Contrary to that which occurs with Pseudomonas, for Azospirillum it is possible to note (FIG. 1) the high concentration of EPS independent of the storage period and the absence of production of organic acids. Commonly recognized by the importance in protecting different embodiments of abiotic stress, the EPS produced by Azospirillum were not important for maintaining the cellular viability of this microorganism when bottled individually (Table 5).

[0053] Surprisingly, when the two microorganisms were mixed in a consortium agricultural composition, it was possible to establish the increased stability and cellular viability of both the microorganisms, Azospirillum and Pseudomonas (Table 7). The mixture of the two microorganisms enables the prolonged storage of 18 months with an initial count of 110.sup.9 to 310.sup.9 numbers of cells of Azospirillum brasilensis and from 110.sup.7 to 510.sup.7 numbers of cells of Pseudomonas fluorescens, and after 18 months, attains 110.sup.8 to 110.sup.9 number of cells of Azospirilum brasilensis and from 910.sup.7 to 510.sup.8 number of cells of Pseudomonas fluorescens, in a temperature range of 5-40 C.

[0054] Unexpectedly, synergism occurs between Azospirillum and Pseudomonas which is explained by; i. High biosynthesis of EPS by Azospirillum; ii. Consumption, by Pseudomonas, of the EPS produced by Azospirillum; biosynthesis of organic acids by Pseudomonas; consumption, by Azospirillum, of the organic acids synthesized by Pseudomonas (FIGS. 1 and 3). This biosynthesis and consumption dynamics is shown in FIGS. 1 and 2, where the EPS produced by Azospirillum is gradually consumed by Pseudomonas during the course of the storage time. It is noted in this case that the EPS are synthesized during fermentation. The organic acids synthesized during the course of storage by Pseudomonas tend to assume stable concentrations, since, in parallel to biosynthesis, it is consumed by Azospirillum, which is reaffirmed by the maintenance of the pH of the consortium agricultural composition over time.

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