INDUSTRIAL PROCESS FOR OBTAINING AN AGRICULTURAL COMPOSITION CONSTITUTED BY SOLUBILIZING AND PHOSPHORUS MINERALIZING MICROORGANISMS, AND USE IN THE PRODUCTION AND OPTIMIZATION OF MINERAL, ORGANOMINERAL AND ORGANIC FERTILIZERS

Abstract

The present invention refers to an industrial process for obtaining an agricultural composition formed by association of one or more species of Bacillus spp., of Lactobacillus spp. and of Pseudomonas, the process for induction of exudates/metabolites, as well as the application thereof in the manufacture and in the increase of the efficiency of phosphate fertilizers of mineral, organomineral and organic origin. Surprisingly, the agricultural composition used in the fertilization industry increases the availability of macro and micronutrients to the plants with agricultural interest, such as soy and corn. Finally, both the use of the agricultural composition in the fertilizers industry as the application of the product in the field potentialize the greater availability of essential nutrients for the development of the cultures.

Claims

1.-29. (canceled)

30. An industrial process for obtaining an agricultural composition employed in the manufacture and optimization of phosphate fertilizers having mineral, organomineral or organic origin, comprising: (a) culturing followed by fermenting at least three microorganisms selected from the group consisting of Bacillus, Lactobacillus and Pseudomonas, wherein each microorganism is cultured and fermented at a specific condition comprising one or more parameters selected from the group consisting of a temperature, an agitation speed, an aeration, a pressure and any combination thereof, (b) inducing the biosynthesis of metabolites by the at least three microorganisms; wherein the metabolites are capable of solubilizing and mineralizing insoluble phosphates in soluble phosphate compounds; wherein each microorganism is fermented at a specific condition comprising one or more parameters selected from the group consisting of a temperature, an agitation speed, an aeration, a pressure and any combination thereof to biosynthesize the metabolites; and (c) admixing the at least three microorganisms and the biosynthesized metabolites and bottling the agricultural composition, wherein the biosynthesized metabolites by Pseudomonas and Lactobacillus can act instantaneously on inorganic phosphates during the manufacturing process of the phosphate fertilizers, and wherein the biosynthesized metabolites by Bacillus act on inorganic phosphates applied to an agricultural field.

31. The industrial process of claim 30, wherein the induction of the biosynthesis of metabolites of step (b) by Bacillus and Pseudomonas occurs at a temperature of about 22° C.-38° C. and an air flow of about 3.0-10.0 Nm.sup.3/h or about 0.25-about 0.83 vvm in a tank; and the inducing of the biosynthesis of metabolites by Lactobacillus occurs at a temperature of about 22° C.-38° C. and an air flow of about 3.0-about 10.0 Nm.sup.3/h or about 0.25-about 0.83 vvm in a fermenter.

32. The industrial process of claim 30, wherein culturing of step (a) of the three microorganisms comprises inoculating each microorganism into a separate culture.

33. The industrial process of claim 30, wherein culturing of the three microorganisms comprises expanding each culture in a volume from 100 mL to 1 L, then to 10 L, then to 180 L, and then up to 2000 L.

34. The industrial process of claim 30, wherein the culturing of step (a) of the Bacillus and the Pseudomonas in a culture medium volume ranging from 1 L-10 L is by agitation in an orbital shaker at about 80-about 200 rpm for about 8 hours-about 48 hours, wherein the 1 L culture medium is in a glass flask and the 10 L culture medium is in a stainless-steel balloon.

35. The industrial process of claim 34, wherein the culturing of the Bacillus is with an airflow of about 0.25-about 1.0 Nm.sup.3/h or about 4.16-about 16.67 vvm for a 1 L culture; and wherein the serially expanding of the Bacillus and Pseudomonas is with an airflow of about 0.25-about 1.5 Nm.sup.3/h or about 0.41-about 1.67 vvm for a 10 L of culture.

36. The industrial process of claim 34, wherein two stainless-steel balloons both comprising 10 L of culture medium and either Bacillus, Pseudomonas or Lactobacillus are inoculated into a fermenter tank comprising 180 L of culture medium.

37. The industrial process of claim 36, wherein the culturing of step (a) of either Bacillus, Pseudomonas or Lactobacillus in a fermenter tank comprising 180 L of culture medium is for about 24-about 168 hours.

38. The industrial process of claim 37, wherein the culturing of step (a) of the Bacillus or Pseudomonas is with an airflow of about 1.0-about 15.0 Nm.sup.3/h or about 0.16-about 0.25 vvm.

39. The industrial process of claim 34, wherein the culturing of step (a) of the Lactobacillus is at a temperature of about 22° C.-about 38° C. and without agitation or aeration.

40. The industrial process of claim 30, wherein the fermenting of step (a) is by batch fermentation.

41. The industrial process of claim 30, wherein the fermenting of step (a) is at a pressure ranging from about 1.0-about 2.0 kgf/cm.sup.2 in a 2000 L fermenter.

42. The industrial process of claim 30, wherein the fermenting of step (a) is with agitation of about 40 hz-about 45 hz, at a temperature of about 22° C.-about 30° C., and with an airflow of about 1.0-about 2.5 Nm.sup.3/h or about 0.0085-about 0.021 vvm.

43. The industrial process of claim 30, wherein the admixing is in a 2000 L fermenter for about 30-about 120 minutes.

44. A method for using the agricultural composition of claim 30 selected from the group consisting of: fertilizer production, conversion of by-products generated in the industrialization of fertilizers into compounds with agricultural applications, broadcasting and sowing furrow together with a phosphate fertilizer; directly applying to seeds or a sowing furrow; and increasing the availability of macronutrients and micronutrients in the plants with agricultural interest.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0054] For a more complete understanding of the invention, reference must be made now to the embodiments of the invention illustrated in more detail in the figures accompanied and described by means of the embodiments of the invention.

[0055] FIGS. 1A-1C illustrate the phosphorus analysis in a fertilizer sample during the production process (ground stage). A. P-CNA; B. P-Total; C. P-CNA/P-Total Conversion Rate. Biologic 1—Lactobacillus plantarum and Lactobacillus buchneri; Biologic 2—Pseudomonas fluorescens; Biologic 3—Lactobacillus plantarum, Lactobacillus buchneri and Pseudomonas fluorescens.

[0056] FIGS. 2A-2C illustrate the phosphorus analysis in fertilizer sample during the production process (final product—granulated). A. P-CNA; B. P-Total; C. P-CNA/P-Total Conversion Rate. Biologic 1—Lactobacillus plantarum and Lactobacillus buchneri; Biologic 2—Pseudomonas fluorescens; Biologic 3—Lactobacillus plantarum, Lactobacillus buchneri and Pseudomonas fluorescens; Biologic 4—Lactobacillus plantarum, Lactobacillus buchneri, Bacillus subtilis and Bacillus licheniformis.

[0057] FIGS. 3A-3C illustrate the phosphorus analysis in a fertilizer sample during the production process (process subproduct). A. P-CNA; B. P-Total; C. P-CNA/P-Total Conversion Rate. Biologic 1—Lactobacillus plantarum and Lactobacillus buchneri; Biologic 2—Pseudomonas fluorescens; Biologic 3—Lactobacillus plantarum, Lactobacillus buchneri and Pseudomonas fluorescens; Biologic 4—Lactobacillus plantarum, Lactobacillus bucnheri, Bacillus subtilis and Bacillus licheniformis.

[0058] FIG. 4 illustrates the average productivity of the soy bean culture carried out in 8 different regions. The treatments were inoculated with the microorganisms on their own or mixed and 25% reduction of phosphate fertilizing.

[0059] FIG. 5 illustrates the average productivity of the corn culture carried out in 8 different regions. The treatments were inoculated with the microorganisms on their own or mixed and 25% reduction of phosphate fertilizing.

DETAILED DESCRIPTION OF THE INVENTION

[0060] In a preferred embodiment, according to the present invention, the fermentation (step (a)) of the different Bacillus, Lactobacillus and Pseudomonas by batch occurs for approximately 24-168 hours.

[0061] In a preferred embodiment, the method of the present invention comprises the sequencing expansion (scaling-up) of the culture of Bacillus, Lactobacillus and Pseudomonas for inoculation of the fermentation culture. Preferably, the sequencing expansion starts in volumes of 100 mL, which serves to inoculate 1 L. This, in its turn, is inoculated in 10 L, which, then are inoculated two balloons in 180 L tanks and which, finally, are transferred to reactors containing 2,000 L.

[0062] In a preferred embodiment, the species of Bacillus and Pseudomonas are expanded in 100 mL flasks by incubation in orbital shaker of 80 rpm to 200 rpm, and without shaking when cultivated the species of Lactobacillus. The incubation time is of, preferably, 8 hours to 48 hours. Preferably, the species of Bacillus are then cultivated in stainless-steel balloons containing 1 L of culture medium. The incubation time is preferably of 8 to 48 hours with air flow of 0.25 Nm.sup.3/h to 1.0 Nm.sup.3/h (=4.16-16.67 vvm). Preferably, the species of Pseudomonas are cultivated in flasks of around 1 L of culture medium by incubation in orbital shaker at 80 rpm to 200 rpm. Preferably the species of Lactobacillus are cultivated without shaking.

[0063] In a preferred embodiment, the air flow of the stainless-steel balloons containing 10 L for the cultivation containing the species Bacillus and Pseudomonas is of 0.25 to 1.5 Nm.sup.3/h (=0.41-2.5 vvm), and the incubation time is preferably of about 8 hours to about 48 hours.

[0064] In a preferred embodiment, stainless-steel balloons containing 10 L for the cultivation containing the species of Lactobacillus are cultivated without the need for aeration.

[0065] In a preferred embodiment, the incubation temperature for multiplication of the species of Bacillus, Lactobacillus and Pseudomonas according to the present invention is from 22° C. to 38° C.

[0066] In a preferred embodiment, the species of Bacillus, Lactobacillus and Pseudomonas are inoculated separately in the scaling-up process up to 180 L and mixed in the 2.000 L fermenters as described for the present invention. For this, in a preferred embodiment, after the cultivation of Bacillus in two stainless-steel balloons with 1 L of culture medium, the referred balloons are inoculated in two other stainless-steel balloons of 10 L and then transferred in tanks containing 180 L of specific culture medium for each microorganism, whereby Table 2 shows the specific culture medium for the B. licheniformis; and Table 3 the specific culture medium for the B. subtilis with the addition of a stainless-steel balloon containing 5 L of the Endospore formation inductor salt solution for the Bacillus spp. (Table 4), incubated for 24 to 168 hours. The air flow is, preferably of 1.0 to 15.0 Nm.sup.3/h (=0.16-1.25 vvm).

[0067] In a preferred embodiment, after the cultivation of two flasks containing 1 L of Lactobacillus and Pseudomonas, the referred cultivations are inoculated in two other stainless-steel balloons of 10 L and then transferred to tanks containing 180 L of specific culture medium for each microorganism, whereby Table 5 shows the specific culture medium for the species of Lactobacillus; and Table 6 for the specific culture medium for Pseudomonas, incubated for 24 to 168 hours. The air flow for the Pseudomonas is, preferably, from 1.0 to 15.0 Nm.sup.3/h (=0.16-1.25 vvm) and for the cultivation of the Lactobacillus there is no need for aeration during the incubation.

[0068] In a preferred embodiment, the step of mixing of the Bacillus, Lactobacillus and Pseudomonas is carried out with temperature from 22° C. to 38° C. The air flow is preferably of 1.0 Nm.sup.3/h a 2.5 Nm.sup.3/h (=0.0085-0.021 vvm). The pressure is preferably of 0.5 to 1.2 kgf/cm.sup.3. The shaking is preferably from 40 hz to 45 hz.

EXAMPLES

Example 1—Scaling Up of Culture

[0069] The different species of Bacillus, Pseudomonas and Lactobacillus are inoculated separately in flasks containing 100 mL of the culture medium as described in Table 1, 6 and 5, respectively, being incubated in orbital shaker of 80-200 rpm, at 22-38° C. for approximately 8-48 hours, with exception of the species of Lactobacillus which do not require shaking, only incubation at the temperature of 22-38° C. The next step in the scaling-up for the Bacillus consists in the inoculation of stainless-steel balloons containing 1 L of culture medium (Table 1), wherein the species are separately cultivated and incubated for approximately 8-48 hours, with air flow of 0.25-1.0 Nm.sup.3/h (=4.16-16.67 vvm) and temperature approximately of 22-38° C. For the Pseudomonas, the inoculum of 100 mL is then transferred to flasks containing 1 L of culture medium (Table 6), being incubated in orbital shaker of 80-200 rpm, at 22-38° C. for approximately 8-48 hours. In the same manner, 100 mL of each species of Lactobacillus are transferred to 1 L of culture medium (Table 5) and incubated at a temperature of 22-38° C. for approximately 8-48 hours.

[0070] After the incubation period, the species of Bacillus and Pseudomonas the cultivations are inoculated in stainless-steel balloons containing 10 L of specific culture medium for each microorganism and incubated for approximately 18-96 hours, with air flow 0.25-1.5 Nm.sup.3/h (=0.41-2.5 vvm) and temperature varying from 22-38° C. For the cultivation of Lactobacillus there is no need for aeration during the incubation time.

TABLE-US-00001 TABLE 1 CULTURE MEDIUM USED FOR THE GROWTH OF THE BACILLUS SPP. UP TO THE SCALE-UP OF 10 L. Reagents 01 K.sub.2HPO.sub.4 0.1-4 g 02 KH.sub.2PO.sub.4 0.1-4 g 03 MgSO.sub.4•7H.sub.2O 0.1-0.6 g 04 NaCl 0.05-0.3 g 05 Yeast Extract 0.1-4 g 06 Peptone 0.2-4 g 07 Solution FeCl.sub.3 10% 0.05-1 mL 08 Solution MnSO.sub.4 10% 0.05-1 mL 09 Saccharose 5-10 g 10 Water q.s.p. 1 L q.s.: quantum sufficit

[0071] After this time, each culture containing two stainless-steel balloons with 10 L of culture medium is inoculated in a tank containing 180 L of specific culture medium for each microorganism, being presented in Table 2 the specific culture medium for B. licheniformis; and in Table 3 the specific culture medium for B. subtilis with the addition of a stainless-steel balloon containing 5 L of the Endospore formation salt solution for the Bacillus spp. (Table 4), in Table 5 the specific culture medium for Lactobacillus and in Table 6 the culture medium for Pseudomonas and incubated for approximately 24-168 hours, with air flow 3.0-10.0 Nm.sup.3/h (=0.25-0.83 vvm) and temperature varying from 22-38° C., with exception of the species of Lactobacillus which are not incubated with aeration.

TABLE-US-00002 TABLE 2 CULTURE MEDIUM USED FOR THE GROWTH OF THE B. LICHENIFORMIS FOR 200 L TANKS. Reagents 01 Maize 2-20 g 02 Yeast extract 1-10 g 03 NaCl 1-10 g 04 Water q.s.p. 1 L q.s.: quantum sufficit

TABLE-US-00003 TABLE 3 CULTURE MEDIUM USED FOR THE GROWTH OF THE B. SUBTILIS FOR 200 L TANKS. Reagents 01 Sodium glutamate 5-20 g 02 Peptone 1-10 g 03 KCl 0.1-5 g 04 MgSO.sub.4•7H.sub.2O 0.1-2 g 05 Yeast Extract 0.1-5 g 06 Water q.s.p. 1 L q.s.: quantum sufficit

TABLE-US-00004 TABLE 4 ENDOSPORE FORMATION SOLUTION FOR THE TWO SPECIES OF BACILLUS. Reagents 01 Ca (NO.sub.3).sub.2 50-400 g 02 MnCl.sub.2 1.0-10 g 03 FeSO.sub.4 0.1-0.8 g 04 Water q.s.p. 1 L q.s.: quantum sufficit

TABLE-US-00005 TABLE 5 CULTURE MEDIUM USED FOR THE GROWTH OF THE LACTOBACILLUS SPP. UP TO THE SCALE OF 2000 L. Reagents 01 K.sub.2HPO.sub.4 0.1-4 g 02 Na.sub.2HPO.sub.4 2-10 g 03 MgSO.sub.4•7H.sub.2O 0.1-3 g 04 NaCl 0.1-1 g 05 Yeast Extract 10-35 g 06 Peptone 0.2-2 g 07 KNO.sub.3 0.1-1 g 08 Soy flour 5-20 g 09 Saccharose 5-20 g 10 Water q.s.p. 1 L q.s.: quantum sufficit

TABLE-US-00006 TABLE 6 CULTURE MEDIUM USED FOR THE GROWTH OF THE PSEUDOMONAS SPP. UP TO THE SCALE OF 180 L. Reagents 01 K.sub.2HPO.sub.4 0.1-1 g 02 KH.sub.2PO.sub.4 0.1-1 g 03 MgSO.sub.4•7H.sub.2O 0.1-3 g 04 NaCl 0.1-1 g 05 Yeast Extract 0.3-3 g 06 (NH.sub.4).sub.3PO.sub.4 0.3-3 g 07 KNO.sub.3 0.5-5 g 08 Solution MnSO.sub.4 10% 0.05-0.1 mL 09 Solution FeCl.sub.3 0.05-0.1 mL 10 Glycerol 1-10 mL 11 Saccharose 1-10 g 11 Water q.s.p. 1 L q.s.: quantum sufficit

Example 2—Mix of Bacillus, Lactobacillus and Pseudomonas in Bioreactor

[0072] For the mix of the species of Bacillus, Lactobacillus and Pseudomonas in fermenter of 2,000 L, preferably there is used 1,200 L of the formulation of the Lactobacillus spp. (Table 5), which passes through a sterilization process for approximately 60 to 120 minutes, at a temperature of approximately 121° C. to approximately 130° C. Preferably, the sterilization is carried out at a pressure of approximately 1.0-2.0 Kgf/cm.sup.2.

[0073] After the sterilization and cooling period, the tank containing the species of Lactobacillus spp. are then inoculated in the 2000 L fermenter, containing 1.200 L of the sterile cultivation medium, starting the fermenting process, which is of, preferably 24 to 72 hours at a temperature of 22° C.-38° C. The air flow is preferably of 1.0 Nm.sup.3/h to 2.5 Nm.sup.3/h (=0.0085-0.021 vvm). The pressure is preferably from 1.0 to 2.0 kgf/cm.sup.3. The shaking is preferably from 40 hz to 45 hz.

[0074] Preferably, after the fermentation time of the Lactobacillus spp., the mix of the tanks of B. licheniformis, B. subtilis and Pseudomonas spp. are inoculated and mixed to the 2.000 L fermenter. Preferably, the mixing time comprises from 30 to 120 minutes. Preferably, the product is bottled in gallons, in which packaging the product is stored.

Example 3—the Induction of Metabolites Enables the Use in Industries for Phosphate Fertilizers and their by-Products

[0075] There were made applications of different combinations and proportions of microorganisms to verify the action in the phosphate fertilizers in different steps of the productive process of the industries of phosphate fertilizers. In FIG. 1, ground samples of the fertilizer were treated with the biologicals and passed through a process which simulates the production steps of the fertilizers, 90° C. for 10 minutes and 200° C. for 30 minutes. The treatment with the biological 4 (mix of Lactobacillus and Bacillus) presented higher conversion rate, increasing in 8% the availability of P relative to the control without the biological. The conversion rate is the P-CNA relation contained in the P-Total sample; the higher the value the better is the conversion rate, since in the extraction by the neutral citrate method there is simulated the absorption potential by the plant, while the P-total is all the phosphorus contained in the fertilizer.

[0076] In the same manner, when the biologicals are applied to the granulated fertilizer (FIG. 2), another form of presentation of the product, there was an increase of 25% in the availability of P.

[0077] When the treatment is carried out in the by-product, which is generated during the industrial process for obtaining the fertilizer, the conversion rate was even more surprising in the treatments with the biologicals 3 (Lactobacillus and Pseudomonas) and 4 (FIG. 3), increasing in 30 and 38% the availability of P in the product, respectively.

[0078] Independent of the step of the productive process for obtaining the phosphate fertilizer, the use of the microorganisms results in higher availability of the phosphorus.

Example 4—the Combination of the Microorganisms Enhances the Effect of the Phosphate Fertilizer

[0079] Field trials were carried out to validate the effect of the microorganisms in the soy and corn cultures with a reduction of 25% in the phosphate fertilization and the addition of the microorganism by itself or in a mixture. In FIG. 4, there is presented the average productivity of soy as from the 8 distinct regions, in a general manner the 25% reduction in fertilization did not result in loss of productivity when compared to the treatment with 100% of phosphate fertilization, except for the B. licheniformis by itself. It is possible to observe the potentiating effect on the availability of P which the microorganisms provide to the plants. In the same manner, when the trials were carried out for the corn culture (FIG. 5) the average productivity of 8 different edaphoclimatic areas with the application of the microorganisms on their own or in mixture provided a 25% reduction of the fertilization without resulting in loss of productivity. The greater productivity of both the cultures was only possible due to the use of the microorganisms which have the ability to make the P available to the plants, thus culminating in greater productivity. In this manner, the application of microorganisms in phosphate fertilizers, apart from acting in the greater availability of P in the fertilizers, also provided the residual effect in the field, since these microorganisms remain viable in the fertilizers, particularly the bacteria of the genus Bacillus which are industrially induced to the endospore formation, as mentioned in example 1.

[0080] Apart from the greater efficiency in the availability of P for the plants, the application of microorganisms in the cultures further presents greater availability of other nutrients such as, calcium (Ca), sulfur (S), copper (Cu) and iron (Fe) as presented in Table 7. It was possible to verify that the inoculation with the species of Bacillus presented greater absorption of the micronutrient Cu and Fe. These nutrients are essential for the plants, since they act as activator or component of enzymes, influence in the biological fixation of nitrogen, is essential to the balance of nutrients which regulate the plant transpiration, impacts in the photosynthesis and in the plant transpiration, among other benefits. Other advantages also with the application of the species of Pseudomonas is the availability of the macronutrients Ca and S, and the micronutrients Cu and Fe, presenting statistical difference when compared to the treatments with only 75% of P.

TABLE-US-00007 TABLE 7 ANALYSIS OF CHEMICAL ATTRIBUTES OF THE AERIAL PART OF THE SOY PLANTS 35 DAE. Ca S Cu Fe Ca Content S Content Cu Content Fe Content Treatments (g/Kg) (g/pl) (g/Kg) (g/pl) (mg/Kg) (mg/pl) (mg/Kg) (mg/pl) Witness 10.99 b 61.77 2.09 d  11.75 13.25 b 74.48 1203.50 c  6764.67 Inoculated 11.36 b 71.42 2.46 cd 15.47 18.00 b 113.17 2159.50 abc 13576.66 witness 75P 11.66 b 67.65  2.62 bcd 15.2 17.50 b 101.53 2296.50 ab  13324.17 100P 11.02 b 64.05  3.03 abc 17.61 16.50 b 95.9 2233.50 abc 12981.6 Pseudomonas + 13.79 a 84.93 3.75 a  23.09 29.25 a 180.14 3199.00 a  19701.4 Azospirillum B. subtilis + B. 10.21 b 62.88 3.36 ab 20.69 25.75 a 158.58 3149.00 ab  19393.47 licheniformis LSD averages test (FISCHER) 5%. DAE: days after the emergency.