Bacterial strains, probiotic mixtures, formulation and method, for processing vinasse

Abstract

The invention relates to isolated strains of Bacillus altitudinis identified as PV2, Bacillus subtilis identified as PV4 and Bacillus mojavensis identified as PV44, which comprise the characteristics of the deposited strains CM-CNRG TB232, CM-5 CNRG TB231 and CM-CNRG TB230, respectively, wherein the strains exhibit vinasse-purifying activity. The invention also relates to a probiotic mixture for preparing a formulation useful in vinasse processing, which comprises at least two isolated bacterial strains according to the present invention. The invention additionally relates to a formulation for vinasse processing, which comprises: a probiotic mixture according to the present invention; at least one prebiotic substance; and at least one excipient. The invention further relates to a method for processing vinasse, which comprises applying an effective amount of the formulation of the invention.

Description

[0001] BACTERIAL STRAINS, PROBIOTIC MIXTURES, FORMULATION AND METHOD FOR THE TREATMENT OF VINASES

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to the technical field of biotechnology and is applicable to the agroindustry, as it provides bacterial strains with vinasse-purifying activity; probiotic mixtures comprising the bacterial strains; and a formulation and method for vinasse treatment.

[0004] BACKGROUND OF THE INVENTION

[0005] Tequila is one of the most consumed beverages worldwide, according to the CRT. In 2008, it reached a peak of 313 million liters of tequila. Since 2018, it has continued to grow above that mark: 309 million in that year; 351.7 million in 2019; and 374 million liters in 2020. The production of this beverage is highly polluting, as 10 to 14 liters of vinasse are generated for every liter of tequila produced. Vinases are the wastewater produced by the distillation of tequila; they have a high organic matter content, with a chemical oxygen demand (COD) greater than 38,215 mg / L and total solids (TS) greater than 21,883 mg / L, and have a low pH of 3.5–3.9 (Íñiguez and Peraza 2007). They are discharged at a temperature of 90°C, which makes them a thermal pollutant.The high organic matter content and color reduce light penetration in rivers, lakes, and lagoons, decreasing the photosynthetic activity of aquatic organisms. This lowers the concentration of dissolved oxygen, which causes a deterioration of aquatic life. Furthermore, using soils for the final disposal of tequila vinasse without any treatment can favor the presence of pathogens for some crops (Iñiguez and Hernández, 2010).

[0006] The use of aerobic and anaerobic microorganisms for the bioremediation of this waste has been reported, obtaining good results in the analyses required according to the standard. Patent document CN114437988A discloses a bacterium isolated from the black soldier fly, which degrades cellulose, the bacterium that degrades cellulose is P15, classified and named as Bacillus subtilis, and deposited in the China Center for Type Culture Collection, on August 30, 2021 (CCTCC), with deposit number CCTCC M20211094. It also includes a bacterial agent and method for the degradation of distillery fiber, through the use of the strain B. subtillis CCTCC M20211094, which is used to degrade cellulose, more specifically for the degradation of fibers generated in a plant that distills alcohol, but does not suggest that they are useful for treating vinasse.

[0007] For its part, patent document CN104151042A refers to a type of organic fertilizer obtained from distillery fibers, and its production method. The organic fertilizer, whose fermentation material comprises in parts of mass: 100 parts of fibers generated by distilleries, 3-5 parts of straw, 1-2 parts of sawdust, 2-2.5 parts of quicklime and 0.2-0.3 parts of bacteria. The main components of the decomposition inoculum are N1 strains and TL-ZZ strains, and the number of effective viable bacteria in the decomposition inoculum is >2x10 8 cfu / g.The described bacterial strain N1 is Bacillus amyloliquefaciens conserved in General Center of Microbiology of Microorganism Culture Collection Management Committee of China, the accession number is CGMCC8143 and the preservation date is September 9, 2013; the strain TL-ZZ is Bacillus mojavensis conserved in General Center of Microbiology of Microbial Culture Collection Management Committee of China, the accession number is CGMCC8268, and the preservation date is September 25, 2013. As can be seen, said strains in the document are a fertilizer, so its purpose is to nourish vegetable plants, but it is not mentioned that it is useful to treat vinasse.

[0008] Patent document CN108046555A refers to a method for preparing and applying Bacillus highlandii, deposited under accession number CGMCC 18639, to remediate contamination of contaminated sediments in ponds, lakes, rivers, and the like. One example of remediation was in aquarium sludge, where an effect on restoring total phosphorus was observed. To begin with, there is no certainty that this bacteria can degrade or treat vinasse.

[0009] Currently, the isolation of microorganism strains for various purposes is common. Due to the endemic characteristics of geographic regions, countries, and / or territories, strategies are required to employ microorganisms with specific and efficient activity and / or functions in those regions, countries, and / or territories where they will be applied, as an advantage for the utilization of such isolated microorganisms. Therefore, to counteract the aforementioned disadvantages, endemic bacterial strains were isolated from vinasse that demonstrated vinasse purification activity. Therefore, mixtures, formulations, and methods for treating vinasse were developed.

[0010] The characteristic details of the present invention are clearly shown in the following detailed description supported by examples and figures attached for illustrative purposes, with the purpose of illustrating the conception of said invention and some of its preferred embodiments, where:

[0011] Figure 1 is a graph of the percentage discoloration of the vinasse formulation scaling.

[0012] Figure 2 is a graph of the turbidity scale of the stillage formulation.

[0013] DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention relates to an isolated strain of Bacillus altitudinis identified as PV2, which has the characteristics of the strain deposited under deposit number CM-CNRG TB232. Another object of the present invention is an isolated strain of Bacillus subtilis identified as PV4, which has the characteristics of the strain deposited under accession No. CM-CNRG TB231.

[0015] Another object provided by the present invention is an isolated strain of Bacillus mojavensis identified as PV44, which comprises the characteristics of the strain deposited with accession number CM-CNRG TB230.

[0016] The strains isolated and deposited from the present invention were isolated from vinasse and demonstrated vinasse-purifying activity; therefore, they may be useful in vinasse treatment.

[0017] Therefore, this invention also has as its object a probiotic mixture for preparing a formulation useful in the treatment of vinasse, which comprises: at least two bacterial strains isolated in accordance with the present invention.

[0018] Being a preferred embodiment of the probiotic mixture of the present invention, when said mixture comprises the bacterial strains CM-CNRG TB232 and CM-CNRG TB231.

[0019] It is also a preferred embodiment of the probiotic mixture according to the present invention when it comprises the bacterial strains: CM-CNRG TB232; CM-CNRG TB231; and CM-CNRG TB230.

[0020] Another object of the present invention is a formulation for the treatment of vinasse, characterized in that it comprises: a probiotic mixture, according to claim 5; at least one prebiotic substance; and at least one excipient. Wherein the probiotic mixture is present in a preferred amount of 0.00032 to 0.00036%.

[0021] This invention also relates to a method for treating vinasse, which comprises applying an effective amount of the vinasse treatment formulation proposed by the present invention. The effective amount of the formulation is preferably 0.001% relative to the volume of vinasse to be treated.

[0022] EXAMPLES

[0023] The following examples are included to illustrate the conception of the present invention and some preferred embodiments thereof, so such examples should not be considered as a limitation for the scope of protection of the present invention.

[0024] Example 1. Obtaining microorganisms that help in the treatment of vinasse.

[0025] With the aim of selecting microorganisms that help improve the treatment of vinasse, sampling was carried out from different ponds of an industrial waste treatment plant, located in Tototlán, Jalisco (20°28'56.3”N 102°44'33.7”W), Mexico, between the months of October 2021 to July 2022, to carry out an extraction of microorganisms.

[0026] Materials and methods

[0027] The samples were collected with gloves and in sterile jars, ensuring a representative sample of each pond (the sample was mixed as thoroughly as possible before collection). The vinasse samples collected were as follows:

[0028] - “Pure vinasses”, obtained from vinasses freshly taken from a truck that transports them.

[0029] - “Open pond vinasses” are vinasses from the pond that receives the effluent from the samples that are deposited in the receiving pond.

[0030] - "Biodigester stillage," effluent stillage from the tank with the train of an anaerobic biodigester. - "WWTP: stillage pond 1," samples from a mixture of approximately 80:20 WWTP (dairy industry waste): stillage, and were collected from a first WWTP reception pond.

[0031] - “WWTP:vinasse pond 2” samples from a mixture of approximately 80:20 WWTP:vinasse and were collected from a second closed WWTP reception pond.

[0032] The samples were taken to the Microendo laboratory located in Atotonilco el Alto, Jalisco, Mexico; their pH was measured before the microorganisms were extracted, showing the following results (Table 1):

[0033] Table 1. pH value of the vinasse samples collected at the industrial waste treatment plant in Tototlán, Jalisco, Mexico.

[0034] Subsequently, each sample was diluted up to 9 times. From each dilution, as well as from the direct sample, 100 pL were taken and plated in a Petri dish with the widely known Nutrient Agar. These dishes were incubated at 37°C for 24 to 96 h. After the incubation time, a CFU / mL count was performed in each dish, obtaining the following results shown in Table 2.

[0035] From each box that showed bacterial growth, colonies with different morphology were selected, in relation to the others.

[0036] Each bacterial colony with a different morphology (size, color, texture) was removed with a sterile loop and streaked onto a Petri dish containing trypticase soy agar. The dishes were then incubated at 37°C for 24 h. After the incubation period, the dishes were examined against the light to determine whether the isolated strain was pure or not, as is conventionally done.

[0037] Table 2. Bacterial yield obtained from the different vinasse samples.

[0038] A total of 103 bacterial strains were isolated: 9 strains from the "pure vinasse" sample; 34 strains from the "WWTP: vinasse pond 1" sample; 22 strains from "WWTP: vinasse pond 2" sample; 12 isolates from "open pond vinasse"; and 26 from "biodigester vinasse."

[0039] Pre-selection of microorganisms

[0040] To eliminate strains, a pre-selection was performed using a 50% vinasse-based culture medium, supplemented with essential salts for healthy microbial growth. The salts and concentrations were as follows: 0.5% dextrose, 0.05% magnesium sulfate, 0.1% monobasic potassium phosphate, and 0.05% potassium chloride.

[0041] The isolated strains were reactivated from the vial where they were stored in a Petri dish containing trypticase soy agar (CASOY) and incubated at 37°C for 24 h. Subsequently, a small amount of bacterial biomass was removed and reseeded by streaking onto the vinasse medium if the samples came from “pure vinasse,” “open pond vinasse,” or “biodigester vinasse”; or onto both media if the samples came from “WWTP:vinasse pond 1” or “WWTP:vinasse pond 2,” and incubated at 37°C for 48 h. The dishes were then examined to determine if the colony was able to grow in the medium. Of the 103 bacterial strains isolated, 37 strains showed optimal growth in the WWTP:vinasse media, 48 strains showed poor growth in the media, and 18 did not grow in these media.

[0042] Hydrolysis tests

[0043] To perform the hydrolysis tests, 66 strains were selected for their ability to grow in the vinasse-based medium. The enzymes tested were: lipases, proteases, esterases, cellulases, and amilases.

[0044] The strains were reactivated from the vial where they were stored into a Petri dish containing trypticase soy agar (CASOY) and incubated at 37°C for 24 h; subsequently, a small amount of bacterial biomass was removed and reseeded in the form of a circular dot three times in the different media to be tested for the determination of lipases, proteases, esterases, cellulases, and vinasses.

[0045] The results of the hydrolytic tests of the 66 strains tested are shown in Table 3.

[0046] Of the 66 strains tested, 11 strains were selected: PV1, PV2, PV3, PV4, PV18, PV21, PV22, PV23, PV24, PV32 and PV44, since these strains produced a good amount of proteases, lipases, cellulases, amylases and esterases, in addition to having good growth in the medium made from vinasse.

[0047] These 11 strains advanced to the next round of testing (lignocellulase production, medium decolorization, and turbidity). Table 3. Results of hydrolytic tests for the 66 bacterial strains tested. Table 3. Continued... Table 3. Continued...

[0048] (-) negative test, (lig+) slightly positive, (+) low production, (++) medium production, and (+++) high production.

[0049] Example 2. Laccase production, percentage of discoloration and turbidity of the medium, of the 11 selected strains.

[0050] Laccase production tests were performed on the 11 strains selected in Example 1, and it was also determined whether these strains were capable of decolorizing the medium and improving the turbidity of the vinasses. Liquid media made from vinasses with the aforementioned salts were prepared. The strains were incubated in the medium, and the flasks were shaken at 90 to 98 rpm at 32°C, and sampled according to the test to be performed.

[0051] Laccase production

[0052] For laccase determination, the samples were left for 3 days under constant stirring at 100 rpm; then, 600 pL of broth was removed and mixed with a reaction indicator and citrate buffer. The samples were read at 525 nm, yielding the results shown in Table 4.

[0053] According to the results shown in Table 4, the strain with the highest laccase production was strain PV2, with a production of 0.168 ll / min, followed by strains PV1 and PV21 with production of 0.0357 and 0.0234 ll / min, respectively. This enzyme is part of the lignocellulase family, making it important due to the solid material residues that may be present in the waste. Although the production of this enzyme is not as high as reported in the literature, it can be observed that all strains produce small amounts of laccase, but they also produce other types of enzymes, which is why it is believed that they will have a synergistic effect.

[0054] Table 4. Production of laccase enzymes of the 11 selected bacterial strains.

[0055] Discoloration

[0056] To determine the discoloration produced by the 11 bacterial strains, the liquid media were left in place for a total of 15 days, with samples taken every 3 days. The vinasse samples were filtered and diluted with distilled water at a 1:2 ratio. The samples were read at 475 nm, and the percentage of discoloration was calculated. The discoloration graphs for the strains tested can be seen in Table 5.

[0057] As can be seen in Table 5, all strains showed a percentage of discoloration of the vinasse medium. The strain with the highest percentage was strain PV18 with 88.65% on day 12 of sampling, followed by strain PV21 with 86.78% after 12 days of incubation. Strain PV32 had the highest percentages during the first 12 days, as did strain PV44, reaching percentages of 86.33 and 79.62% respectively.

[0058] Table 5. Percentage of discoloration of the 11 bacterial strains grown in vinasse medium.

[0059] Turbidity

[0060] For turbidity and decolorization measurements, the media were incubated for 15 days, with samples taken every 3 days. This determination was performed using a turbidimeter. 1 mL of all samples was taken for measurement and diluted with 9 mL of distilled water. The results of this test can be seen in Table 6.

[0061] Table 6 shows the turbidity results obtained, strains PV1, PV3, PV4, PV18 and PV21 show their lowest turbidity point at 6 days of incubation, while there are other strains where turbidity increases, as in the case of strains PV32, PV44, PV24, PV2 and PV23. The strain that managed to reduce turbidity the greatest amount was strain PV4, achieving a turbidity of 39.8 nephelometic turbidity units (NTU) at 6 days of incubation, followed by strain PV3 with 225.2 NTU at 15 days post inoculation.

[0062] Table 6. Turbidity of bacteria grown in vinasse medium

[0063] An interesting detail that we observed when carrying out these tests was that some of the strains inoculated in vinasse managed to flocculate the solids dissolved in the medium 6 days after inoculation, as is the case with strains PV4 and PV21.

[0064] Analyzing the results obtained from the tests carried out, six strains of bacteria were selected for the determination of BOD: PV2, PV3, PV4, PV18, PV21 and PV44.

[0065] Example 3. Determination of biochemical oxygen demand and strain mixtures that improve vinasse treatment.

[0066] Biochemical oxygen demand (BOD)

[0067] To determine the biochemical demand produced by the six bacterial strains selected in Example 2, they were reseeded in vinasse medium. The samples were shaken at 100 rpm, at approximately 20°C, and the oxygen in the culture was measured with the aid of a sensor. The initial reading and readings after 1, 2, 3, 4, and 5 days of inoculation were taken. The results are shown in Table 7.

[0068] Table 7. Readings obtained from the sensors for determining BOD.

[0069] Table 7 shows that almost all strains exceed 250 ppm of oxygen after two days, due to the sensitivity of the sensors used. The strain for stillage with the highest oxygen concentration is PV2, with 187 ppm of oxygen, followed by PV21, with 140 ppm.

[0070] Based on the data obtained from the BOD measurements, the following three bacterial strains PV2, PV4 and PV21 were selected, as they presented the best results, and were mixed to see their behavior in the treatment of vinasse.

[0071] Growth tests in liquid medium

[0072] The selected strains were grown in 100 mL of liquid medium made from vinasse, using salts at the following concentrations: 0.5% glucose, 0.05% anhydrous magnesium sulfate, 0.1% monobasic potassium phosphate, and 0.05% potassium chloride. The media were sterilized and then individually inoculated. These media were subsequently incubated at 37°C for 24 h, with plate counts performed after the incubation period. The results obtained are shown in Table 8. Table 8. CFU / mL on plate of the selected bacterial strains in vinasse media incubated at 37°C for 24 h.

[0073] As observed in Table 8, some bacteria did not grow at 24 h, so they were plated at 48 h, but the result was the same: no bacterial growth. For this reason, it was decided to change a parameter: the bacteria were grown in the same media, but now they were incubated with shaking (100 rpm) at 32°C for 24 h, with plate counts performed after the incubation period. The results are shown in Table 9.

[0074] Table 9. CFU / mL on plate, of the strains in the vinasse media incubated with shaking at 32°C, at 24 h.

[0075] In Table 9 we observe that the PV4 strain requires agitation instead of temperature to grow at 24 h, but the PV21 strain did not show growth at 24 h with agitation, which is why it was plated at 48 h after inoculation, but the result was the same, there was no bacterial growth. Due to this situation it was decided to test the PV44 strain under both conditions, temperature and agitation in order to replace the PV21 strain, the results of these tests are observed in Table 10.

[0076] Table 10. CFU / mL of strain PV44 incubated with shaking and temperature. As observed in Table 10, strain PV44 behaves similarly to strain PV4, requiring agitation rather than temperature for growth. Overall, the three selected strains grew well in the salt-based stillage medium, reaching up to 14 logarithms. Therefore, the bacterial strains ultimately selected were as follows:

[0077] - Bacillus altitudinis PV2 (Firm cutes), was selected because it produces lipases and proteases, it is the strain with the highest production of laccases, it reaches up to 79% decolorization and decreases turbidity up to 620 NUT.

[0078] - Bacillus subtilis PV4 (Firm cutes), was selected because it produces lipases, proteases and laccases, reaches up to 74% decolorization and decreases turbidity up to 39.8 NTU.

[0079] - Bacillus mojavensis PV44 (Firm cutes), was selected because it produces lipases, proteases, cellulases and laccases, reaches up to 79% decolorization and decreases turbidity up to 620 NTU.

[0080] Considering the results of the growth tests of the bacterial strains in 100 mL, it was decided to make the following selection of strains, to make mixtures or combinations between said bacterial strains: PV2, PV4 and PV44.

[0081] Scaling to 10 liters, pure cultures

[0082] Subsequently, the individual strains were scaled up to 10 L. For the vinasse medium, the aforementioned salts were used, and the pH was adjusted to 6.5–7 with 50% sodium hydroxide. This procedure was carried out liter by liter until reaching 9 L. The media were then sterilized at 120°C and 1.05 kg / cm 2 for 40 min to ensure that the medium was sterile. To inoculate these media, 1 L of each selected bacterium was prepared under the conditions described above and incubated at 32°C, at 100 rpm, for 24 h. The inoculated 10 L jars were incubated at 32°C with aeration as a means of agitation (500 cm 3 / min). After 24 and 48 h of incubation, a sample was taken to perform a plate count, the results of said counts are shown in table 11.

[0083] The results from the scale-up of pure cultures showed better growth results than the 100 mL cultures. Strains PV2 and PV44 are the strains that exhibit the best bacterial growth, presenting 1.15x10 14 and 2.05x10 14 CFU / mL respectively. PV2 increased one log relative to the 100 mL culture, while strain PV44 remained at the same CFU as the 100 mL culture. On the other hand, strain PV4 presented 5x10 11 CFU / mL in the scaling, increasing 3 logarithms with respect to the 100 mL culture.

[0084] Table 11. CFU / mL on plate in the vinasse media incubated with shaking and

[0085] 32°C of 10 L.

[0086] Scaling to 10 liters, mixed cultures

[0087] Once it was determined that the microorganisms were growing individually, they were grown together in a 10 L jar. As described above, vinasse media was prepared with the salts described above, 1 L per bacterium was inoculated and incubated at 32°C with 100 rpm for 24 h. Each 10 L jar was inoculated with 1 L of each bacterium, incubated at 32°C with aeration, and after 24 and 48 h, samples were taken to be plated and counted. The results are shown in Table 12.

[0088] Table 12. CFU / mL on plate in the media of vinasse and WWTP incubated with shaking and 32°C of 10 L of mixed cultures. As observed in Table 12, the formulation yields a high bacterial concentration of up to 14 logs after 48 h of incubation. The vinasse formulation shows the three morphologies of the three different bacteria. This indicates that the three strains are capable of growing simultaneously, so growing the strains together for reproduction would be no problem.

[0089] For the vinasse formulation, an additional test was performed to reduce the concentration of salts used and thus reduce plant costs. Three salt concentrations were tested: 0%, 1%, and 10%. 50 mL of vinasse broth was prepared with the original salt concentration described above, and each bacterium was individually inoculated. They were incubated with shaking at 32°C for 24 h. They were then inoculated into a flask containing 350 mL of vinasse broth with the concentrations to be tested, reaching a final volume of 500 mL. The flasks were incubated with shaking at 32°C for 24 h and subsequently plated to determine the colony-forming units. The results are shown in Table 13.

[0090] Table 13. CFU / mL on plate in the vinasse media incubated with shaking and 32°C of 10 L of mixed cultures, in three salt concentrations.

[0091] In Table 13 we observe the bacterial concentration obtained with the different salt concentrations. The best salt concentration is 10% with 3.1x10 14 CFU / mL, followed by 1% concentration of salts with 7x10 13 CFU / mL. Although the 10% concentration is one log higher than the 1% concentration, the 1% salt concentration is close to reaching a 14th log, and at this concentration, all three bacterial morphologies were observed. Therefore, we recommend using the 250 ml tank. 3 1% salt concentration. Example 4. Identification of the three best bacterial strains selected in Example 3.

[0092] Once the best strains were selected, they were identified. Bacterial identification was carried out using the MALDI-TOF technique, which consists of obtaining a protein profile of the microorganism to be analyzed and comparing it with a database. The result is a score that indicates the accuracy of the identification. Scores range from 0 to 3, where a value of 0-1,599 indicates an unreliable identification; a value of 1,600-1,999 indicates a high probability that the identified genus is correct; a value of 2,000-2,299 indicates a 100% probability of the identified genus, while a value of 2,300-3,000 indicates a 100% probability of the identified genus and species. The identification results are shown in Table 14.

[0093] Table 14. MALDI-TOF identification of the selected strains.

[0094] Example 5. Determination of discoloration, turbidity and BOD in 10 L.

[0095] Discoloration and turbidity

[0096] As described above, the scaling up of the selected strains to 10 L was carried out. The vinasse media were pH adjusted to 6.5 to 7, with 50% sodium hydroxide. The vinasse media were then sterilized at 120°C and 0.05 kg / cm 2 for 40 min. The vinasse medium was inoculated with 14 microliters of the three selected bacteria (this was to simulate the actual dilution that would be performed in the plant) and incubated at 32°C with aeration as a means of agitation (500 cm 3 / min) and were left to incubate for 15 days, taking samples every 3 days. As described above, to determine discoloration, 1 mL was taken and filtered to subsequently be read at 475 nm. To determine turbidity, 1 mL was taken and diluted with 9 mL of distilled water, the sample was read in a turbidimeter. The results obtained are shown in Figures 1 and 2, where the scaling showed good behavior both in decolorization of the medium and in its turbidity, achieving a decolorization percentage of up to 64.67% on day 12, while, for turbidity, it managed to reduce the turbidity of the medium to 499.6 NTU on day 6 of inoculation.

[0097] BOD

[0098] To determine BOD, 250 mL were taken from the 10 L flask corresponding to the scale-up 2 days post-inoculation to simulate what would happen in the plant. The sample was stirred at a temperature of approximately 20°C, and a sensor measured the oxygen in the crop. The initial reading and readings after 1, 2, 3, 4, and 5 days of inoculation were taken. The results are shown in Table 15.

[0099] Table 15. Readings obtained from the sensor for determining BOD.

[0100] In Table 15, we can see that, compared to individual strains, the scaling showed an increase above 250 ppm O2 after 3 days, reaching 248 ppm O2 after two days of agitation. This indicates that the proposed formulation can help reduce oxygen demand.

[0101] Example 6. Determining the best combinations or mixtures of the three selected strains.

[0102] An experiment was conducted to assess the behavior of the three selected strains, both alone and in all possible combinations, on the discoloration and turbidity of vinasse. Ten treatments were chosen, and two controls were included, as shown in Table 16.

[0103] Table 16. Suggested treatments.

[0104] For each treatment and control, 250 mL of vinasse was prepared with the salts described above at 1%, the pH was adjusted to 7 and sterilized, subsequently inoculated with the quantities described in Table 16, these quantities represent the inoculum that will be made in the field. The flasks were shaken at 100 rpm at 32 ° C for 15 days, taking samples every third day to measure turbidity and discoloration of the samples, the results are observed in Tables 17 and 18.

[0105] In Table 17 we can observe the percentage of discoloration presented by the different treatments tested, the best treatment was 5 achieving 55.71% discoloration on the third day, followed by treatments 8, 4, 6 and 9 which presented a percentage of discoloration of 54.96, 44.6, 33.94 and 18.55% respectively, on the third day post inoculation. The treatment with the highest percentage of discoloration was treatment 3 with a percentage of discoloration of 61.68%, achieving this value until day 12 post inoculation. Although the mixture we are proposing is the mixture represented in treatment 9 was not the best, it had a constant discoloration throughout the sampling period, achieving a maximum of 34.22% on day 12. Treatments 11 and 12, which are the controls, showed no discoloration in the medium.

[0106] Table 17. Percentage of discoloration of the different treatments tested with the bacterial mixtures.

[0107] In Table 18 we can observe the NTU presented by the different treatments tested, the best treatment was treatment 9 achieving a value of 329.3 NTU on the third day, followed by treatments 3 and 1 which presented a turbidity of 358.9 NTU and 369.9 NTU respectively, on the third day post inoculation. The treatment with the lowest turbidity was treatment 4 with 167.9 NTU, achieving this value until day 15 post inoculation. The mixture we are proposing is the one that corresponds to treatment 9, although it does not present the lowest turbidity value, it is the one that decreases the turbidity value in a short time, this being a very valuable characteristic when used in the field. Treatments 11 and 12, which are the controls, are the treatments with the smallest decrease in turbidity with respect to the other treatments.

[0108] Table 18. Turbidity of the different treatments tested with the bacterial mixtures.

[0109] STORAGE OF ISOLATED AND SELECTED BACTERIAL STRAINS

[0110] Since the isolated bacterial strains PV2, PV4 and PV44 demonstrated agronomic potential, they were deposited on January 13, 2023, in the Microorganism Collection of the National Center for Genetic Resources, which belongs to the National Institute of Forestry, Agriculture and Livestock Research, with registration number 1006 before the World Federation of Culture Collection (CM-CNRG) and International Deposited Authority with notification 308 for the purposes of patent proceedings in accordance with the Budapest Treaty; and which has its address at Boulevard de la Biodiversidad 400, CP 47600, Tepatitlán de Morelos, Jalisco, Mexico. The bacterial strain PV2 Bacillus altitudinis was assigned deposit number CM-CNRG TB232, the strain PV4 Bacillus subtilis was assigned accession No. CM-CNRG TB231, and the strain PV44 Bacillus mojavensis was assigned accession No. CM-CNRG TB230.

Claims

CLAIMS 1. An isolated strain of Bacillus altitudinis identified as PV2, characterized in that it comprises the characteristics of the strain deposited under deposit number CM-CNRG TB232.

2. An isolated strain of Bacillus subtilis identified as PV4, characterized in that it comprises the characteristics of the strain deposited under accession No. CM-CNRG TB231.

3. An isolated strain of Bacillus mojavensis identified as PV44, characterized in that it comprises the characteristics of the strain deposited under accession number CM-CNRG TB230.

4. The strains of the preceding claims, wherein said strains exhibit vinasse purifying activity.

5. A probiotic mixture for preparing a formulation useful in the treatment of vinasse, characterized in that it comprises: at least two bacterial strains isolated in accordance with claims 1 to 3.

6. The mixture of the preceding claim, wherein said strains exhibit vinasse purifying activity.

7. The mixture of claim 5, wherein the strains are: CM-CNRG TB232 and CM-CNRG TB231.

8. The mixture according to claim 5, wherein the strains are: CM-CNRG TB232; CM-CNRG TB231; and CM-CNRG TB230.

9. A formulation for the treatment of vinasse, characterized in that it comprises: a probiotic mixture according to claim 5; at least one prebiotic substance; and at least one excipient.

10. The formulation of the preceding claim, wherein the probiotic mixture is present in an amount of 0.00032 to 0.00036%.

11. A method for treating vinasse, characterized in that it comprises: applying an effective amount of the formulation of the preceding claim to the vinasse.

12. The method of the preceding claim, wherein the effective amount is 0.001% with respect to the volume of vinasse to be treated.

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