WO2013022332A1

WO2013022332A1 - Mixture containing microorganisms for bioremediation and bioaugmentation, production method and use of same

Info

Publication number: WO2013022332A1
Application number: PCT/MX2012/000076
Authority: WO
Inventors: Elva Teresa ARÉCHIGA CARVAJAL; Juan Manuel ADAME RODRÍGUEZ; Raúl Alejandro AGUIRRE GAMBOA; Gustavo VINIEGRA GONZÁLEZ; Ernesto FAVELA TORRES
Original assignee: Universidad Autónoma De Nuevo León; Universidad Autónoma Metropolitana
Priority date: 2011-08-05
Filing date: 2012-08-30
Publication date: 2013-02-14
Also published as: MX351319B; MX2011008329A

Abstract

The invention relates to a method for producing a mixture for bioremediation, containing nixtamalized maize flour and a composition based on bacteria of the Bacillus genus. This mixture is used for bioremediation and bioagumentation involving degradation, for the decontamination of compounds, as well as eliminating bad odours and faecal coliforms and reducing the concentration of lipids, starch and proteins present in contaminated water or solid surfaces exposed to the air and affected by moisture, as well as for the degradation of cellulose in soils and fertiliser additive.

Description

MIXTURE CONTAINING MICROORGANISMS FOR BIORREMEDIATION AND

BIOAUMENTATION, ELABORATION PROCESS AND ITS USE

OBJECT OF THE INVENTION

The object of the present invention is to provide a process for the preparation of a mixture based on microorganisms for use in bioremediation and bio-augmentation of wastewater or contaminated surfaces. This mixture degrades contaminating organic matter, eliminating odors, fecal coliforms and decreasing the content of lipids, starch and proteins present in contaminated water or solid surfaces exposed to the air and provided with moisture.

This mixture for bioremediation and bio-augmentation represents a natural and very economical way of degrading waste organic matter using saprophytic bacteria in high concentrations and easy production and handling.

BACKGROUND

The present invention belongs to the field of compositions for bioremediation and bio-augmentation of wastewater and surfaces contaminated with putrescible materials. Mainly for urban or mixed wastewater (small industry) that includes: sewage, oily water and washing water.

In general, it is the sewage and fats that cause the greatest discomfort to the communities that coexist day by day in areas with insufficient drainage systems or in semi-urban areas where septic tanks, latrines, microplants or plants are used instead of oxidation In both areas the efficient and safe treatment of wastewater becomes indispensable since there is a risk that the wastewater will contaminate groundwater, which in some cases are a source of drinking water; they can cause diseases and infections in people or animals; In the same way, a system with failure or lack of maintenance can emit bad odors or obstruct pipelines and pipes, in addition the water treatment will not be the most efficient. After the partial treatment of wastewater, these are discharged back to the soils, rivers and seas.

Currently, many systems used for wastewater treatment depend on microorganisms that live in the sediments of wastewater treatment plants. These microorganisms disperse organic solids and break down several of their components.

The bioaugmentation process consists in the proliferation of the inoculants so that their action is prolonged, but that depends on the presence of adequate nutrients for that purpose. That is why nutritional supplementation is required, along with the addition of the inoculants; especially if the microorganisms are attached to a support that acts as a dispersing and nutritive agent at the same time.

There are multiple antecedents of the culture of bacteria of the genus Bacillus obtained by solid phase fermentation in order to produce enzymes and / or metabolites that must then be purified from the fermentation system. For example: Bruno eí al. in 1995 they protected in the US patent 5,464,766, the use of a product composed of saprophytic bacteria of this genus, which were obtained by means of the submerged culture and were subsequently lyophilized. The dried product was mixed with salts and reinforced with commercial enzymes of bacterial or fungal origin for the treatment of water bodies contaminated with organic waste. Compared with the present invention, the lyophilization process of this product represents a high cost in equipment and energy. Vandenberg et. to the. in 2000, they describe in US Pat. No. 6,668,774, the use of a supernatant from the culture in liquid medium of Pseudomonas sp. in suspension for the control of fecal odor generated in chicken breeding farms. This invention is based on the production of biosurfactants by Pseudomonas grown in liquid medium that at the end of the fermentation is centrifuged and mixed with salts and urea. This liquid mixture therefore requires a greater number of steps in the process of obtaining, as well as components to enrich the microorganisms unlike the present invention.

In document USWO2003 / 071874A1 Zahn et al. in 2005, they establish a mixture of bacteria of the genus Bacillus produced by solid phase fermentation to whose matrix (rye bran) they add urea, yeasts and phosphates, obtaining, at most, 2 x 10 ⁹ colony forming units per gram of substrate (CFU / g). This product is used to control algae in lakes and artificial lagoons. It is noted that said mixture has a lower concentration than that obtained in the mixture of the present invention.

In all the aforementioned works related to the cultivation of bacteria at low cost, nitrogen sources, salts and other components were added to support the growth of microorganisms. The FMS (Solid Medium Fermentation) technique has been used by numerous researchers to enrich the protein content of agricultural substrates intended for the production of other primary and secondary metabolites (Ruossos S. and Perraud-Gaime, I. 1996 Physiology and Biochemistry of Microorganisms used in fermentation processes in solid medium). The main disadvantages of the current compositions previously patented and produced by FMS of bacteria are: a) That sometimes, it is necessary to separate by various methods the enzymes or metabolites of the substrate where the fermentation was carried out and then mixed with the supports end of the microbial product; and b) In other cases, it must be supplement the substrate with salts and various sources of nitrogen to support microbial growth.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a diagram with the different stages of the process of obtaining the composition with high bacteriological and enzymatic content that contains a biodegradable and porous organic substrate, saprophytic bacteria of the genus BacHIus in vegetative state, lithic enzymes and spores.

Figure 2 shows a diagram of the dilution or mixing process of the Composition with high bacteriological and enzymatic content in the diluent to obtain a commercial product useful for direct application in bioremediation and bioaugmentation. Figure 3 shows the bottom view of a petri dish with a culture of the mixture useful for bioremediation and bioaugmentation, subjected to growth in agar supplemented with starch, where the results obtained from the biochemical amylase test are observed. The dyed or dark area corresponds to the area of the residual starch while the light halo indicates the enzymatic activity (amylase) on the starch contained in the culture medium.

Figure 4 shows the bottom view of a petri dish with a culture of the mixture useful for bioremediation and bioaugmentation, subjected to growth in agar supplemented with milk, where the results obtained from the biochemical protease test are observed. Colorful is the result of the enzymatic activity of (protease) on the protein contained in the culture medium.

Figure 5 shows the bottom view of a petri dish with a culture of the mixture useful for bioremediation and bioaugmentation, subjected to agar growth supplemented with oil olive, where the results obtained from the biochemical lipase test are observed. The fluorescent halo (slightly colorful) is the result of enzymatic activity (lipase) on the fat contained in the culture medium. Figure 6 shows, by way of example, the scheme of a system where the mixture is used for bioremediation and bio-augmentation. The number (1) in the figure indicates the pipe of the residence that connects to a water tank, marked with the number (2), in which the bioremediation and bio-augmentation process will be carried out. DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to a mixture useful for bioremediation and bio-augmentation that, through the bioremediation process, improves the quality of wastewater and solid surfaces contaminated with putrescible materials, that is, material that rots and produces a bad smell. Specifically, this mixture degrades starch, proteins and lipids, also reduces the bad smell and the number of fecal coliforms and generally reduces the content of volatile solids.

The mixture useful for bioremediation and bio-augmentation that is presented to protecció comprises: a diluent and a composition with high bacteriological and enzymatic content. The latter contains bacteria in the vegetative phase, its spores and some of its lytic enzymes adhered to an organic substrate (b). Being the organic substrate, a biodegradable material, with high fiber content, that does not gelatinize (low starch content), with a porous structure such as, but not limited to, husks or bran grains such as wheat, rice, sorghum, millet , oats, barley, etc., or, coconut fiber, etc. This substrate can be preferably but not excluded, wheat bran which is characterized by having approximately the following components per 100 g: Vitamin K 131 ug, vitamin B6 1.3 mg, B5 2.18 mg, B3 18.28 mg, B2 0.58 mg, B1 0.52 mg, B9 79 ug, 3.32 mg of vitamin E, magnesium 61 1 mg, Zinc 7.27 mg, potassium 1 182 mg, fiber 42.4 mg, iron 10.5 7mg, Among the nutritional properties of wheat bran, it should also be noted that it has the following nutrients: 15.55 g of protein, 73 mg of calcium, 21.72 g of carbohydrates, 2 mg of sodium, 4.25 g of fat, 0.09 g of sugar. The diluent, for its part, fulfills a triple function in the mixture useful for bioremediation and bioaugmentation, because in addition to serving to obtain the desired number of bacteria per gram of mixture for commercial use, due to its high starch content it serves as a flocculant and, finally, as a nutritional supplement or starter to sustain the growth of bacteria in poorly nutritious media and is selected, for illustrative purposes, that are not limiting, from flours such as nixtamalized corn, rice, wheat, barley, rye , oatmeal, cassava, potato, etc.

In the present invention, the composition with high bacteriological and enzymatic content is characterized in that it uses a solid substrate (b) of biodegradable type and in which saprophytic bacteria of the genus Bacillus are inoculated. The inoculated substrate is fermented under a conventional solid phase aerobic process. However, some common steps of this type of process are not required in this invention, such as: separation or purification, since they subtract essential elements from the composition with high final bacteriological and enzymatic content and increase the cost of the process. The bacteria transform the substrate during the fermentation process thus producing lithic enzymes and spores, which will be used later for the degradation of organic matter present in wastewater or contaminated surfaces. Finally, the composition with high bacteriological and enzymatic content manages to be more efficient when dried, ground and mixed with the biodegradable powder diluent that can be a flour, preferably nixtamalized cornmeal, which also serves as a starter of low cost and complement Nutritious to sustain the growth of bacteria. A mixture useful for bioremediation and bioaugmentation is presented for legal protection and its use for the treatment of water, soil and / or solid surfaces contaminated with putrescible wastes containing a composition with high bacteriological and enzymatic content with bacteria in a vegetative state, their spores, their enzymes and the solid substrate (b) in which they grew. The composition with high bacteriological and enzymatic content is mixed with a powder dispersant or diluent which may be a flour, preferably nixtamalized cornmeal. During fermentation, of the composition with high bacteriological and enzymatic content, bacteria use nutrients from a solid substrate of biological and biodegradable origin, and during their growth they secrete lithic enzymes, produce spores and increase their number. At the end of the fermentation, the said composition with high bacteriological and enzymatic content is dried, ground and mixed with the diluent. The components of the diluent serve so that the bacteria contained in the composition start their growth at the place of application and start the production of enzymes to degrade complex proteins present in the sites contaminated with putrescible materials that generate bad smell. A good dispersant or diluent is cornmeal cooked with lime (nixtamalized), which gives greater availability to the minerals, vitamins and proteins it possesses, than raw corn (Figueroa ef. Al. 1994) Technological Modernization of the Nixtamalization process. Advancement and Perspective. 13: 323-329), is an easily accessible and low cost material in Mexico and many other countries.

A common disadvantage of bacterial digestion in the disposal of wastewater is that it is a very slow process, however, the diluent in addition to potentiating the bioremediating effect supports the growth of bacteria. Thus, the mixture useful for bioremediation and bioaugmentation, object of the invention, does not require the addition of salts and complex and expensive additives to sustain bacterial growth, once it has been applied for bioremediation treatments since the substrate and the Diluent, are sufficient for the activity of bacteria to start efficiently. What makes this mixture useful for bioremediation and bioaugmentation, very easy to produce and low cost. The shelf life of this mixture useful for bioremediation and bioaugmentation, if the product is kept closed under vacuum and without exposure to light, is at least one year, due to its drying process which does not happen with liquid products. The organic fermentation substrate is steam sterilized in an autoclave at a pressure of 10 to 22 Ib, with 15 Ib being preferred, for 10 to 20 minutes, preferably 15 min. It is then inoculated with the selected saprophytic bacteria of the genus Bacillus. This composition (B?) Comprises mixtures of the subtilis, cereus, licheniformis, megaterium, polyimixa, thuringensis species, but the use of other species of the same genus and other related genera is not ruled out. A biodegradable organic substrate that contains large amounts of fiber and without large amounts of starch is used so that it does not gelatinize. In addition, it must have a porous structure that facilitates forced aeration that maintains a constant flow of oxygen to the microorganisms and be a source of nutrients in the medium for solid fermentation. The growth and / or production phase is carried out on this solid substrate by placing it in a tray reactor, in a 1 to 4 cm layer of the organic substrate and proceeding to heat in a temperature range of 28 ° C until 40 ° C, preferably at 37 ° C for 16 hours in the case of species such as B. cereus up to 24 hours in less accelerated growth species such as B. polimixa depending on the species. The proportion of the organic substrate should be 20 to 40% with a humidity of 50-75% with the optimum humidity being 60% and 2 to 10% inoculum. In this phase of growth, bacteria produce lytic enzymes, spores, and also increase in number. In this phase the addition of no nutrient is not required since the organic substrate provides all the necessary nutrients to sustain growth. After the bacterial growth and / or production phase, the substrate contains the bacteria, spores and lytic enzymes that secrete during the process. The composition with high bacteriological and enzymatic content is subjected to a drying process for a period of 10 to 14 hours by injection of hot air at a temperature range of 45 ° C to 75 ° C, the optimum being 60 ° C. The composition with high bacteriological and dry enzymatic content is ground to generate a highly active granulate in enzymes and with a large beneficial microbial load ranging from 1x10 ⁹ to 1x10 ¹¹ CFU per gram of substrate, the optimum being 1x10 ¹¹ CFU per gram.

In the present invention, the composition with high bacteriological and enzymatic content obtained at the end of the fermentation is preferably applied to the solid substrate. used during fermentation, that is, no processes of purification or separation of bacteria and / or enzymes are required since even the fermented material becomes a suitable material for storage and for the application of bacteria during the treatment of bioremediation and bioaugmentation. Another advantage is that the formulation of the fermentation medium of this invention does not require the subsequent addition of complementary sources of nitrogen or mineral salts for its production or to improve the effectiveness of the composition with high bacteriological and enzymatic content.

Preferably, an amount of diluent is added to the composition with a high bacteriological and enzymatic content that does not reduce the concentration of bacteria to less than 1x10 ^to CFU per gram of the mixture useful for bioremediation and bioaugmentation, since Below this concentration, the functionality of the resulting mixtures that are difficult to homogenize is compromised. The maximum concentration for the resulting mixture for bioremediation and bioaugmentation is 1 x10 ⁹ , which corresponds to a dilution of 99 parts of diluent and a part of the composition already described, and may vary depending on the application and requirements in terms of microbial concentration, without affecting the expected result. This allows the mixture of bacteria and enzymes to be distributed evenly at the bottom and surface of the tank to be treated. Preferred mode of realization

In the first step for the preparation of the composition with high bacteriological and enzymatic content to be protected, the inoculum of the consortium of bacteria is planted in Petri dishes from the growth of isolated colonies on each plate of the species to produce in 50 to 100 ml of a nutrient-rich medium or broth, which contains 0.3 to 0.75% gelatin peptone, 0.15 to 0.5% meat extract in water and is maintained at a temperature range between 30 ° C to 38 ° C being the optimum 34 ° C, for a period between 16 to 30 h. The organic substrate is sterilized, with steam, in an autoclave, at a pressure of 15 Ib / ft for 15 minutes.

The already sterilized organic substrate is placed in a layered reactor of layers of 1 to 2.0 cm in height, the optimum being 1.5 cm, since within these ranges the metabolic heat does not increase much during fermentation and allows bacteria multiply.

Next, the substrate is inoculated with a roast of the bacteria culture obtained in the first step and prepared to have 30% v / v sterile wheat bran as the sole source of nutrients, 65% v / v water and 5 % v / v inoculum.

It is incubated for growth at a range of 28 ° C to 40 ° C, 37 ° C being the optimal condition for 16 to 28 h, with 24 hours being the most frequent optimal time. The humidity of the bed is maintained in ranges of 50 to 75% being the optimum of 60% and the pH of 6 to 8, the optimum being 7 with a Water Activity (Aw) of 0.99. Once the growth period is completed, a white film can be observed on the surface of the substrate, which corresponds to the bacteria obtained.

The product obtained from the previous step is subjected to a drying process in the same oven or incubator to avoid contamination by increasing the temperature and allowing the induction of hot air at a rate of 900-1000 ml per minute in a range of 55 ° C at 65 ° C for 12 hours. As a result of this process a composition is obtained that contains the organic substrate (wheat bran) / bacteria cells of the genus Bacillus in a vegetative state, its spores and secreted enzymes. The composition with high bacteriological and enzymatic content obtained is a preparation with high enzymatic activity that can be mixed with different amounts of a diluent that can be selected, but not limited, from nixtamalized cornmeal, rice, wheat, barley, flour rye, oatmeal, cassava, potato, etc. The diluent allows to obtain the desired number of bacteria per gram of mixture useful for bioremediation and bioaugmentation for commercial use, which may be one or more of the examples presented in the document. Another advantage that has been observed is that said diluent acts as a flocculant due to its high starch content and provides the microorganisms cultured, according to the process already described, with sufficient nutritive organic matter to start their growth in poorly nutritious media and thus a useful mixture for bioremediation and bioaugmentation can be obtained, depending on the degree of dilution, which can finally contain from 1X10 ¹⁰ to 1X10 ⁸ CFU per g of support, provided with a high content of lipase, amylase and protease enzymes, which are agents catalysts for bioremediation of bodies of water and / or contaminated soils such as septic tanks, latrines, microplants or oxidation plants, or in any other biotechnological activity to decontaminate solid surfaces or volumes contaminated with biodegradable materials.

Biodegradation capacities of the mixture useful for bioremediation and bioaugmentation.

To demonstrate the enzymatic action of the mixture useful for bioremediation and bioaugmentation, plaque activity tests of amylases, proteases and lipases were performed.

From these tests, the degradation rates of starch, protein and plate lipids that are obtained by dividing the diameter of the colony formed by the bacterial growth were determined by the sum of the diameter of the bacterial degradation halo and the diameter of the colony of growth, this index is known as the Pz index and the lower the Pz index the higher the activity is considered. The results obtained shown in Table 1, Table 2 and Table 3 were compared with the activity of a positive control corresponding to a commercial product available for wastewater bioremediation.

Amylase test In the test for amylase (Potato Dextrose Agar by its acronym in English PDA) yeast extract (10 g / L), bacteriological Agar (15 g / L), Peptone (20 g / L) and Starch (20 g /) were used L). It is heated to clarify and sterilize in an autoclave. The inoculant material is sown in the center of the plate and incubated for 24 to 48 hours at 37 ° C, then lugol iodine is added to stain the starch present in the plate, in case of amylase production a color halo is observed of the agar because the starch has been consumed by the action of the amylases, where there is still undigested starch, it is dyed dark blue. The halo around the product indicates enzymatic degradation of starch, which demonstrates the ability of the product to degrade starch. The more extensive the halo, the greater the ability of the product to degrade starch. See Figure 3. With these results it is observed that the product has a high amylase activity and falls within the same range of activity as the control product, however, the raw material used in the product of the present invention has specific characteristics. which have been defined above, as greater stability.

Table 1

In table 1 R means repetition and AC means activity. Protease Test

In the protease test (Milk Agar), a common nitrogen based agar (YNB) (1.7 g / L), 1 M basic sodium phosphate (46 ml / L), Monobasic sodium phosphate (37) was used ml / L), Bacteriological agar (15 g / L) and Milk (500 ml / L). The first three ingredients are mixed, adjusted to pH 7 and the agar is added, set to 500 ml., Autoclaved, allowed to cool until the temperature is equalized with the milk (55 ° C), mixing and stirring. vigorously to sow on plate. To perform the test, it is sown in the center of the plate and incubated for 24 to 48 hours at 37 ° C, after that in case of protease production, a degradation halo will appear, which will be translucent and can be observed either with the naked eye or backlight. The translucent halo around the product indicates enzymatic degradation of casein. The larger the halo, the more extensive the product's ability to degrade proteins. See Figure 4.

Table 2

Lipase Test

In the lipase test (Rhodamine Oxidation Agar), LB Agar (37 g / L), Rhodamine B Solution (1 mg / ml) (10 ml / L) and Olive Oil (40) were used ml / L). The LB Agar is mixed with the rhodamine solution and autoclaved, then the olive oil is added and stirred vigorously to form a uniform mixture, quickly placed in plates to prevent oil from separating from the rest of the solution. It is sown in the center of the plate and incubated for 24 to 48 hours at 37 ° C, after that it is observed with light at 260 nm, the presence of fluorescence confirms the presence of lipases. The fluorescent halo indicates enzymatic degradation of fat (lipid). The more extensive the halo, the greater the ability of the product to degrade fat. See Figure 5

Table 3

Regarding the variability of the activity of proteases, lipases and amylases of the mixture useful for bioremediation and bioaugmentation to be protected, this was determined by calculating the Pz index, the degradation halo in a medium supplemented with skim milk, olive oil or starch; as appropriate, by repetitions (No. 1, No. 2 and No. 3) of each of the tests and their comparison with a commercial product based on bacteria (control +) used in bioremediation. As can be seen, in tables 1, 2 and 3, the protein degradation capacity of the mixture useful for bioremediation and bioaugmentation to be protected equals the capacity of the commercial product; in the case of fats, the mixture useful for bioremediation and bioaugmentation exceeds the commercial product and, in the case of starch, the capacity of the mixture useful for bioremediation and bioaugmentation is quite close to the capabilities of the commercial product.

For the best understanding of the invention, a description is made of some of the methods of use thereof, for illustrative purposes, but not limitation. Example 1 :

Residential water treatment. The mixture useful for bioremediation and bioaugmentation is used to eliminate fecal odor, eliminate fecal coliforms, fats and volatile suspended solids in residential wastewater when it does not have a drainage system. The equivalent of a range of 4 X 10 ⁹ to 5 X 10 ⁹ CFU / g of the mixture useful for bioremediation and bioaugmentation in the rate of the bathroom of the residence is dosed and the pipe (1) of the residence is connected to a tank of water (2) corresponding to three times the volume of water expenditure of the residence to ensure at least one day of water retention with the composition, as can be seen in Figure 6. The content of the tank compared to a untreated control does not show proliferation of faecal coliforms or a bad smell, and its suspended solids are reduced. The case, object of this example, has a year of operation and at the moment the extraction of mud from the septic tank has not been required, when previously it was required to clean it of sediments, every 4 months. The water obtained from the septic tank, after 2 days of residence, is a gray water with high nitrogen content and no bad odors. The dosage of the mixture useful for bioremediation and bioaugmentation already mentioned, is applied every two to four days considering a deposit of 2 m ³ , depending on the physical inspection made to the tank, so that the characteristics of gray water are maintained without bad smells. Example 2:

Restoration of sludge microflora in activated sludge treatment systems. In a case where, by chlorine spill, microorganisms were accidentally removed from a treatment plant with activated sludge, this mixture useful for bioremediation and bioaugmentation at 100 ppm was used and the natural oxidative function of the system was restored. Example 3:

Treatment of bad smell in latrines. We added the mixture useful for bioremediation and bioaugmentation at a rate of 100 g per week in a latrine for 6 people and the bad smell was eliminated in 8 days. In addition, the capacity of the latrine has doubled since the present bioremediation mixture liquefies the stool and accelerates its degradation naturally.

Claims

Having sufficiently described the invention, it is considered as a novelty and therefore, the content contained in the following claims is claimed as exclusive property: 1. Process for obtaining a composition with high bacteriological and enzymatic content useful for bioremediation and bioaugmentation which comprises the following stages:

to. Preparation of the inoculum.- Prepare an inoculum in a nutrient-rich medium from a roast of bacteria growing in colonies in isolated Petri dishes, using a temperature range from 30 ° C to 38 ° C for a period of 16 to 30 hours (h). b. Sterilization of the substrate.- The organic substrate is sterilized in an autoclave, with steam, at a pressure of 10 to 22 Ib / ft for a period of 10 to 20 minutes.

C. Fermentation.- The organic substrate is placed in a tray reactor, in a 1 to 4 cm layer of the organic substrate and inoculated with saprophytic bacteria in a vegetative state resulting from step a., In a temperature range between 28 ° C and 40 ° C for a time range from 16 h to 24 h.

d. Drying.- Dry the composition with high bacteriological and enzymatic content product of the previous step c. by induction of hot air at a rate of 900 ml to 1000 ml per minute in a temperature range from 45 ° C to 75 ° C for 10 to 14 hours.

and. Grinding.- Grind the composition with high bacteriological and enzymatic content product of the previous step d. until obtaining a homogeneously distributed composition.

2. Process according to claim 1 characterized in that the optimum temperature of step a. It is 34 ° C.

3. Process according to claim 1 characterized in that the fermentation of step c. It is carried out with an optimum height of the organic substrate layer of 1.5 cm, the optimum temperature is 37 ° C for an optimal time of 24 h.

4. Process according to claim 1 characterized in that the organic substrate of subsection c. It is a biodegradable material, with high fiber content, which does not gelatinize (low starch content), with a porous structure such as, but not limited to, husks or bran of cereals such as wheat, rice, sorghum, millet, oats, barley , etc., or, coconut fiber, etc .; preferably wheat bran is used.

5. Process according to claim 1 characterized in that the material used in the fermentation of subsection c. It has a ratio of 20-40% v / v of sterile organic substrate as the sole source of nutrients, 50-75% v / v of sterile water and 2-10% v / v of inoculum.

6. Process according to claim 1 characterized in that the material used in the fermentation of subsection c. It has an optimal ratio of 30% v / v of sterile organic substrate as the sole source of nutrients, 65% v / v of sterile water and 5% v / v of inoculum.

7. Process according to claim 1 characterized in that the optimal time for incubation of step c. It is 12 h.

8. Process according to claim 1 characterized in that the optimum temperature for drying of step d. It is 60 ° C.

9. A composition with high bacteriological and enzymatic content obtained by the method of claim 1 characterized in that it comprises:

i) A biodegradable and porous organic substrate;

ii) saprophytic bacteria of the genus Bacíllus in a vegetative state;

iii) lytic enzymes; Y

iv) spores.

10. The composition with high bacteriological and enzymatic content according to claim 9, characterized in that the organic substrate of item i) can be chosen, but not limited to, among the husks or bran of cereals such as wheat, rice, sorghum, millet, oats , barley, etc., or coconut fiber, etc .;

11. The composition with high bacteriological and enzymatic content according to claim 9, characterized in that the organic substrate of item i) is preferably composed of 100% wheat bran.

12. The composition with high bacteriological and enzymatic content according to claim 9, characterized in that the organic substrate is inoculated and fermented with the saprophytic bacteria of item ii).

13. The composition with high bacteriological and enzymatic content according to claim 9, characterized in that the saprophytic bacteria selected from the genus Bacíllus for subparagraph ii) are preferably: subtilis, megaterum, cereus, polyimixa and thuringensis.

14. The composition with high bacteriological and enzymatic content according to claim 9, characterized in that the lithic enzymes and spores of subsections iii) and iv) refer to them obtained during the fermentation of the organic substrate of subsection i) with the saprophytic bacteria in part ii).

15. The composition with high bacteriological and enzymatic content according to claim 9, characterized in that the saprophytic bacteria of the genus Bacillus of subsection / ^' /) are in a concentration of 1x10 ⁹ to 1x10 ¹¹ colony forming units per gram of composition With high bacteriological and enzymatic content.

16. A mixture useful for bioremediation and bioaugmentation, characterized in that it comprises: a. An organic diluent; Y

b. A composition based on bacteria inoculated in an organic substrate.

17. The mixture useful for bioremediation and bioaugmentation according to claim 16, characterized in that the composition of part b. dilute with the organic diluent of part a. from a proportion of 9 parts of composition per 1 part of organic diluent to a proportion of 1 part of composition per 9 parts of organic diluent, where preferably, the optimum mixture is formed by 1 part of the composition with high bacteriological content and enzymatic and 9 parts of the organic diluent.

18. The mixture useful for bioremediation and bioaugmentation according to claim 16, characterized in that the organic diluent of part a. you can choose from flours such as nixtamalized corn, rice, wheat, barley, rye, oats, cassava, potatoes, etc; preferably, nixtamalized cornmeal is used.

19. The mixture useful for bioremediation and bioaugmentation according to claim 16, characterized in that the composition with high bacteriological and enzymatic content of part b) is that obtained by the method of claim 1.

20. The mixture useful for bioremediation and bioaugmentation according to claim 16, characterized in that this mixture has a high content of lipase enzymes, amylase enzymes and protease enzymes

21. The mixture useful for bioremediation and bio-augmentation according to claim 20, characterized in that the lipase enzymes are present in a range of the Pz index from 0.24 to 0.39.

22. The mixture useful for bioremediation and bio-augmentation according to claim 20, characterized in that the amylase enzymes are present in a range of the Pz index from 0.20 to 0.38.

23. The mixture useful for bioremediation and bio-augmentation according to claim 20, characterized in that the protease enzymes are present in a range of the Pz index from 0.55 to 0.65.

24. The use of the mixture useful for bioremediation and bio-augmentation according to claims 16 to 23, for the degradation of organic matter, the elimination of bad odors in wastewater, fecal coliforms and in the decrease of lipids, starch and proteins in contaminated water as well as in the degradation of cellulose in soils and fertilizer additive.

25. The use of the mixture useful for bioremediation and bio-augmentation according to claim 16 to 23, for bioremediation and bio-augmentation of wastewater in latrines, microplants and oxidation plants, grease traps, septic tanks and surfaces contaminated with organic matter.

26. The use of claim 25 wherein the mixture useful for bioremediation and bio-augmentation is applied to a septic tank of 2 m ³ in a range of 4 X 10 ⁹ to 5 X 10 ⁹ CFU / g.

27. The use of claim 25 wherein the mixture useful for bioremediation and bio-augmentation is administrable every two to four days, depending on the physical inspection that is made to the reservoir, so that the characteristics of gray water are maintained without odors.

28. The use of claim 25 wherein the mixture useful for bioremediation and bioaugmentation is applied in a treatment plant with activated sludge at a rate of 100 ppm to restore the natural oxidative function of the system.

29. The use of claim 25 wherein the mixture useful for bioremediation and bioaugmentation is applied to a latrine at a rate of 100 g per week.