WO2013163703A1

WO2013163703A1 - Integrated process for producing enzyme formulations from agro-industrial waste and biofuel production

Info

Publication number: WO2013163703A1
Application number: PCT/BR2012/000123
Authority: WO
Inventors: Aline Machado De Castro; Mariana MARTINS PEREIRA TEIXEIRA; Daniele FERNANDES CARVALHO; Carolina DA COSTA LÁZARO; Denise Maria GUIMARÃES FREIRE; Leda Dos Reis Castilho; Lidia Maria Melo Santa Anna; Danuza Nogueira Moyses; Absai DA CONCEIÇÃO GOMES; Claudia Julia Groposo Silveira; Vinicius Azevedo DE ARAUJO; Bernardo ALVES CINELLI; Jimmy Andrés LÓPEZ JIMÉNEZ
Original assignee: Petróleo Brasileiro S.A. - Petrobras; Universidade Federal Do Rio De Janeiro - Ufrj
Priority date: 2012-05-03
Filing date: 2012-05-03
Publication date: 2013-11-07
Also published as: BR112013005916A2; BR112013005916B1

Abstract

The invention relates to the production and use of enzyme formulations, and is aimed at the hydrolysis of starch granules from agro-industrial waste. These formulations are obtained by an economical process and after being produced are used for co-processing raw materials having different compositions for converting biomass having a high starch content and also lignocellulose-containing biomass into sugar-rich streams, it being possible to ferment said biomass subsequently in order to produce biofuels, in particular ethanol, and "green" chemical products, such as organic acids, biopolymers, antibiotics and polyols. The process for producing these formulations is conducted in an integrated manner, with the objective of decreasing the energy-related costs involved in such processes.

Description

INTEGRATED ENERGY FORMULATION PROCESS FROM AGRICULTURAL WASTE AND BIOFUEL PRODUCTION FIELD OF THE INVENTION

It is the present invention to obtain enzymatic formulations for the hydrolysis of granular starch from agro-industrial residues. Such formulations find application for converting starch-rich biomasses, as well as those containing lignocellulose, into sugar-rich streams that can then be fermented to produce biofuels, especially ethanol, and "green" chemicals such as organic acids, biopolymers, antibiotics and polyols. The process of obtaining such formulations is conducted in an integrated manner, aiming to reduce the energy expenditure involved in the processes of this nature currently in use, since the formulations obtained have all the enzymes necessary for the granular starch hydrolysis process and conversion to ethanol and others. Chemicals.

BACKGROUND OF THE INVENTION

Current methods for ethanol production from the fermentation of starchy materials use steps that demand a high energy expenditure, without the full utilization of the raw material, generating co-products that still contain proteins, fibers and unconverted starch. On the other hand, the use of commercially available special enzymes that can be used to improve the yield of these processes further increases the costs for their industrial scale application.

According to one of these traditional technologies, at the beginning of the process ground corn kernels are subjected to a cooking step in which a suspension containing about 30 to 35% solid material is heated and kept at a temperature of at least 100 ° C for about 20 minutes in the presence of calcium hydroxide and ammonia - gelatinization step. The solubilization of the starch contained in the material makes it more exposed to the action of the enzymes that are used in the later stages, when starch hydrolysis occurs. Initially an endoamilolytic concentrate consisting of alpha-amylase enzymes produced by bacteria is employed. The reaction is conducted at a temperature in the range of 80 ° C to 90 ° C for 30 to 120 minutes. Starch polysaccharides (molecules with only linear bonds such as amylose and with linear and branched bonds such as amylopectin) are attacked and release dextrins (medium polymerized glucose oligosaccharides). Sulfuric acid solution is then added to the medium by changing the pH of the medium from 6.0 to 4.5 and introducing another group of enzymes, glucoamylases, generally produced by filamentous fungi, which have the function of catalyze the hydrolysis of dextrins, finally releasing glucose. The reaction is conducted at a temperature in the range of 60 ° C to 70 ° C for at least 5 hours. A high glucose syrup is obtained.

The glucose-rich syrup is then fed to a fermenter, where it is converted to ethanol by microbiological action. A yeast, Saccharomyces cerevisiae, or a bacterium, Zymomonas mobilis, is commonly employed. Fermentation is conducted at a temperature of about 32 ° C for a period of 48 to 72 hours, yielding 10 to 16% by volume of ethanol in the fermented mash, which corresponds on average to 389 liters of ethanol per tonne of grains. of processed maize, calculated on a dry basis. The main co-product containing protein, fiber and unconverted starch (Distillers Dried Grains with Solubles - DDGS) is used as animal feed.

The energy balance of the process, equal to 1, 3 (ratio between the energy obtained in the process and the energy demanded in the industrial plant), is considered unfavorable (McAloon and collaborators - NREL Techincal Report 580-28893, 2000), since the The energy balance for ethanol production from sugarcane in Brazil is 8.9 (Moreira - Energy Sust. Develop, 4:43, 2000). More recent study finds very promising use of a technology for converting starch materials whose hydrolysis process is conducted at a low temperature - below the starch solubilization temperature - called "cold hydrolysis", which provides a reduction of up to 17%. of energy demand (Mueller - An analysis of the projected energy use of future dry mill corn ethanol plants 2010/2030).

In this process the starch, in its granular form, is subjected to a previous saccharification step in order to become more susceptible to hydrolysis. The reaction is conducted at 57 ° C for 2 hours at acid pH and in the presence of enzyme preparations containing endoamylases and accessory enzymes such as proteases and cellulases, which aid in the degradation of starch-associated proteins and polysaccharides. The suspension, further containing granular starch and some free glucose content, is conveyed to a fermenter and receives the addition of an enzyme preparation containing endoamylases and exoamylases, capable of hydrolyzing the starch in its native form, and a yeast such that saccharification of dextrins and fermentation of released sugars occur simultaneously.

Although cold hydrolysis technology provides energy savings, enzyme preparations, consisting of one or a few enzyme groups, still represent a significant portion of the process costs. In addition, research is often directed to processing a specific type of biomass. Wang et al. (2010) studied the production of FAN (Free Amino Nitrogen) by hydrolysis of rapeseed protein, a high protein feedstock, using proteases. An assimilable nitrogen concentration in the culture medium is required to supply the cells.

The specialized technical literature presents several studies and patent publications that report the developments achieved in this technological field. In general the enzymes employed are specific to a particular type of substrate. When substrate mixing occurs in the same reaction system, specific enzymes are added separately during the various processing steps. The following patent documents may be cited as examples:

US2009 / 0017511 describes a process for producing ethanol from granular starch comprising a first step in which a slurry of water and solubilized starch is obtained which is pre-treated at elevated temperature in the presence of a acid alpha-amylase and a glycoamylase at a temperature between 0 ° C and 20 ° C below the initial gelatinization temperature for a period of 5 minutes to 12 hours. In a second step, the sludge is fermented in the presence of an alpha-amylase, a glycoamylase and a yeast at a temperature in the range of 10 ° C to 35 ° C for 20 to 250 hours to produce alcohol, and is preferable. for the reaction to occur in the presence of a phytase. In this process various raw materials can be used, such as corn, wheat, oats, rye, barley, cassava, sorghum, among others. The enzymes used in the process are commercial enzymes obtained from strains of bacteria and fungi that have undergone some kind of genetic modification.

US6667066 teaches the production of a multi-enzyme product with glycoamylase, protease and xylanase obtained by fermentation of wheat bran with an Aspergillus niger strain. units per gram of dry material. The product has application to produce ethanol or monogastric animal feed.

US2008 / 0003344 describes a process for producing ethanol from the hydrolysis of starch contained in ground vegetable waste using a combination of enzymes. such as phytase, glucoamylase and alpha-amylases of microbiological origin, aiming to achieve hydrolysis at temperatures below starch gelatinization. However, to carry out the complete process, it is necessary to add beta-glucosity in combination with other enzymes. The final toxin free residue can be used as animal feed.

US201 1/0097446 describes a method for producing high ethanol contents from starch-containing plant materials (corn, wheat, oats, sorghum, among others). Generally speaking, the method consists of milling the plant material to achieve a particle size of between 0.1 and 0.5 mm, subjecting the material to saccharification without starch cooking in the presence of specific enzymes, fermenting the incubated starch in the presence of a yeast. and recover the ethanol from the fermented medium. During fermentation the temperature is varied. The method allows to recover at least 18% v / v ethanol. The process makes use of commercial enzymes with specific activities for starch hydrolysis.

WO20 / 0 7093 teaches the use of alpha-amylase mixtures in the liquefaction and saccharification of starch for ethanol production. The mixture employs enzymes used in the ratio of 1 unit of low pH thermostable alpha-amylase for every 5 Modified Wohlgemuth Units (MWU) of Bacillus licheniforms.

As observed, the attempt to improve the processes is directed towards obtaining enzymes with higher enzymatic activity, due to genetic modifications, and when applied together, a maximum of two or three types of enzymes are used, usually from sources. different.

The present invention is concerned with obtaining enzyme formulations containing enzymes produced by solid state fermentation, a low cost process, so that said formulations have characteristics that allow application to different especially aimed at improving ethanol production from different sources of properly balanced biomass. The presence of free amino acids easily assimilated by cells is an important differential of the invention.

SUMMARY OF THE INVENTION

In a first aspect the invention relates to the production of enzyme formulations produced from agro-industrial waste, in particular waste from the vegetable oil and brewery industries. More particularly, it is also intended to prepare well-balanced microorganism culture medium formulations for simultaneous production via solid state fermentation of all enzyme groups required for granular starch hydrolysis. The obtained enzymatic formulations are intended for the conversion of starch-rich biomasses or those containing lignocellulose, with a view to generating streams rich in fermentable sugars for later production of biofuels, particularly ethanol, and "green chemicals" (organic acids, biopolymers, antibiotics and polyols, among others).

In a second aspect the invention aims at the application of such enzyme formulations containing amylases (alpha-amylases, glucoamylases, isoamylases), cellulases (endoglucanases, beta-glucosidases, cellobiohydrolases), hemicellulases (xylanases, beta-xylosidases) and proteases (endopeptidases), exopeptidases, exopeptidases, exopeptidases, exopeptidases. hydrolysis of granular starch present in residues and other agro-industrial materials, in particular biomass from the processing of properly balanced oilseeds and starch-containing grains, for the final conversion into ethanol and green chemicals by means of an integrated process, with reduced energy cost.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows a block diagram of the process. of the present invention.

Figure 2 shows profiles of glucose consumption (hollow squares) and ethanol production (filled squares) obtained from the babassu flour fermentation process, as well as glycerol production (filled triangles). The concentrations of the molecules are expressed in g / l.

DETAILED DESCRIPTION OF THE INVENTION

The enzymatic conversion performed at low temperatures of starch from biomass has been pointed as one of the main trends for ethanol production from starchy materials. However, provided that the starch is not previously gelatinized, it is essential to use several enzymatic preparations to meet the needs of different raw materials, ie preparations containing at least enzymes such as amylases, proteases, cellulases and xylanases. .

The integrated process of the present invention aims to establish balanced formulations of solid culture medium for microorganisms in order to simultaneously produce all enzyme groups necessary for granular starch hydrolysis. To this end, agroindustrial residues are used for the production of these enzyme formulations, particularly residues originating from the vegetable oil and brewery industry, so that their production cost becomes greatly reduced.

The culture media preparation process should initially consider the characteristics of the different agroindustrial residues to be used and then acclimate the microorganisms to produce the desired enzymes.

Basically the integrated process comprises the following steps:

- feed into a first reactor (100) for enzyme production, the agroindustrial residue (10), with particle size in the range of 0.05 - 2.00mm, a first stream of water (15), to adjust and maintain the moisture content in the range of 40 - 80%, and previously propagated filamentous fungi (20) with an initial concentration of 10 ³ - 10 ⁷ / ml, selected from fungal strains, for example Aspergillus, to form a solid matrix (30); maintain fermentation (F1) in solid state (FES) for 12 - 168h at a temperature in the range 20 ° C - 55 ° C;

Extract the produced enzymes from the solid matrix (30) into a liquid phase by adding a second stream of water (35) to a mechanically rotating stirred reactor (F2) of 50-1500 rpm in the liquid ratio. : solid between 5 and 15 (L / kg); separating the solid matrix (30) from the liquid containing the enzyme extract (40) by a filtration and / or sedimentation and / or centrifugation step (S) to remove the fermentation residue (50);

concentrating the enzyme extract-containing liquid stream (40) by membrane ultrafiltration or lyophilization (C) to obtain a concentrated enzyme extract stream comprising the enzyme formulation (45) and residual water (50 ') of the extraction step. In some circumstances, depending on the fermentation yield (F1), this step may not be required and the enzyme extract (40) may be sent directly to the enzymatic hydrolysis and submerged fermentation reactor (F3);

lead a new stream (M) of raw material formed from residue and / or starch-rich agroindustrial material (10) such as babassu flour, corn kernels, wheat kernels, sorghum kernels, castor cake and formulation enzyme (45) to a second reactor (200), to undergo enzymatic hydrolysis and submerged fermentation (F3), in the presence of the enzyme formulation (45) obtained, using microbial cells (60) suitable for the production of the desired product (70), said microbial cells selected from bacteria, actinomycetes, yeast and filamentous fungi.

Thus, in the first instance, according to the process of the invention, enzyme production is achieved via solid state fermentation. Fungal strains are maintained for 3 to 7 days of static cultivation in solid culture medium containing polysaccharides as carbon source, aiming at the acclimatization of cells to produce enzymes capable of degrading such molecules. Then the spores, at a rate of 10 ³ to 10 ⁷ / ml, are inoculated in liquid medium for cell propagation at a temperature in the range of 20 ° C to 40 ° C under orbital agitation in the range of 100 to 300 ° C. rpm for a period of 10 to 48 hours. The grown cells are then inoculated into solid media which are formulated from agro-industrial wastes, generally wastes of low economic value, from the vegetable and brewing oil supply industries. These can be cited as examples of raw materials: babassu pie, obtained from pressing babassu almonds, babassu flour, obtained from pressing babassu mesocarp, castor pie, canola pie, sunflower pie, brewer bagasse, starch corn, sugarcane molasses, soybean molasses, corn (corn maceration residual water), soybean biodiesel crude glycerin, sugarcane juice, among others.

Agroindustrial residues are mixed in different proportions and combinations in order to induce, in a customized way, the synthesis of enzymatic formulations, with balanced activity between the enzymes of interest, for subsequent hydrolysis of different biomasses and release of fermentable sugars to be used. in the process of obtaining the final product.

Balanced culture medium formulations are studied, combining the simultaneous supplementation of solid and liquid materials with babassu cake to customize its composition.

The results presented in Table 1 prove that supplementation of babassu cake with babassu flour and corn starch increases the production of endoamylases by up to 60% after 120 hours of fermentation.

TABLE 1

For the conduction of the process, 1.62 cubic meters of suspension containing the grown cells are used for each ton of raw material and enough liquid volume to guarantee an initial humidity in the range of 40 to 80%, preferably 62%, in the cake. babassu with granulometry in the range between 0.05 and 2.00mm. Trays are incubated in an environment with about 95% humidity. Solid state fermentation (FES) takes place over a period of time ranging from 10 to 120 minutes at a temperature between 20 ° C and 55 ° C, preferably 23 ° C.

The microorganisms used are selected from Aspergillus fungi strains, particularly A. awamori, A. wentii and A. oryzae.

During FES, besides the production of several enzymes capable of hydrolysis of starch, protein hydrolysis of agroindustrial materials occurs, thus producing amino acids and small peptides (FAN - Free Amino Nitrogen), which are quantified by the colorimetric method proposed by European Brewery Convention in 1987 (Lie, J. Inst. Brew., 79: 37-41, 1973). Thus, in the extraction stage, at the end of the FES, the enzymes and free amino acids and peptides are obtained, as they are present in the soluble fraction.

After 120 hours of FES in babassu cake, the enzyme extraction step is promoted. This extraction is done with distilled water at a ratio of 5: 1 to 20: 1 in relation to the initial cake mass, followed by maceration of the fermentation residue. The material is then placed under agitation in the range of 100 to 300 rpm for a period of 10 to 120 min at a temperature in the range of 20 ° C to 55 ° C, preferably at 37 ° C. Then the material is centrifuged at 11,000 g and lyophilized. An enzymatic extract is obtained as a brown soluble powder which can be vacuum packed and stored at -20 ° C.

Analysis of the material reveals that in addition to the major enzyme groups such as amylases (alpha-amylases, glucoamylases, isoamylases), cellulases (endoglucanases, beta-glucosidases), hemicellulases (xylanases) and proteases (endopeptidases, exopeptidases), other enzymatic activities. are also observed in the preparations obtained, which makes These inputs are suitable for application in the co-processing of raw materials with different compositions. For example, the presence of invertases contributes to the rapid assimilation of sugarcane juice sucrose; Beta-galactosidase allows dairy industry residues such as whey to be added to starch and lignocellulosic materials in the same reaction environment. The presence of endoglucanase and beta-glucosidase indicates that the enzymatic preparation of the invention can act for the complete solubilization of cellulosic fibers until the release of glucose, its fermentable unit.

Hydrolysis testing experiments are performed at different temperatures, ranging from 30 ° C to 60 ° C. The lyophilized enzyme extract is used to prepare solutions with amylolytic activity in the range of 2 to 30 U / ml. These solutions are adjusted to pH = 4.8 with sulfuric acid as needed (the initial pH of the enzyme extract is in the range of 4.8-5.5). Babassu flour is added in concentrations ranging from 3.33% to 25% (w / w), and hydrolysis is performed in small stirred reactors. Sodium azide (0.2 g / L) is added in all hydrolysis experiments to avoid contamination.

The results obtained in the experiments are compared with the results of a commercial preparation indicated for use in this same application: hydrolysis of ungelled starch. A strain of yeast Saccharomyces cerevisiae from the industrial strain JP1 is used for the simultaneous saccharification step.

The conversion of granular starch using the obtained enzyme formulations allows a so-called "Very High Gravity" (VHG) fermentation of a medium with high soluble solids content. Thus there is a significant reduction in osmotic stress on yeast. The decrease in osmotic pressure exerted on the yeast also results in a lower production of undesirable products such as glycerol, and lower glycerol production enables greater conversion of glucose to ethanol.

In order that the invention may be better understood and evaluated, examples are set forth below, but they are merely illustrative and in no way to be construed as limiting the invention.

EXAMPLES

Exemplol - Employment of Commercial Enzyme

A- Preparation of the inoculum for submerged fermentation (FS)

JP1 yeast cells (yeast strain from the Japungu -Santa Rita -Paraiba plant) are kept in Petri dishes on YPD agar medium (10g / L yeast extract, 20g / L peptone, 20g / L glucose, 20 g / L agar). After 48 or 72 hours in this medium at 30 ° C, the cells are aseptically transferred to a plastic tube with sterile distilled water, and thus the cell mass concentration calculated. With the cellular concentration, the YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose) is inoculated and the pH adjusted to 5 with sulfuric acid.

B. Solid State Fermentation and Enzyme Extract According to the process of the invention, the grown cells are inoculated into solid media, suitably formulated from agro-industrial materials.

Thus, in the fermentation medium used in the experiments: babassu cake containing 62% of initial moisture with supplements, the enzymes are generated, and these are extracted by adding water and stirring the system.

C-Enzymatic Hydrolysis and Submerged Fermentation

By adding babassu flour to the enzyme extract at 50 ° C for 4 hours, fermentable sugars and free amino acids are released into the reaction medium. This fermentation medium is previously propagated cells are added. The inoculum concentration is 10% relative to the volume of the fermentation medium (corresponding to 0.5 g / l cells). The submerged fermentation (FS) step is conducted at 32 ° C and under constant agitation for up to 72 hours.

The various enzymes present in the enzyme extract obtained act together in the hydrolysis of starch in granular form, releasing glucose. Simultaneously, Saccharomyces cerevisiae yeast cells consume glucose producing ethanol. Furthermore, in the enzymatic extract obtained according to the present invention, there are accessory enzymes such as xylanases, proteases and cellulases, which represent considerable technical and economic advantages.

Example 2 - Use of the Enzyme Extract of the Invention

As already described above, after solid state fermentation

(FES) in a custom culture medium based on babassu cake, the extraction step begins, obtaining the enzymes and free amino acids and peptides.

Due to the extraction of nitrogenous compounds from FES, a culture medium suitable for various fermentative processes is formulated, containing all the basic nutritional sources necessary for the growth of different microorganisms. The enzymes are used directly in liquid form, or the liquid extract is lyophilized for later redissolution, or the fermented solid mass undergoes proper drying and conditioning treatment.

In this example, the lyophilized enzyme extract is solubilized in water providing an exoamylolytic activity of 20 U / mL. A suspension with 19% solids from babassu flour is prepared. The pH is adjusted to 4.8 with sulfuric acid and the medium is thermostated at 50 ° C. for 4 hours, and then kept at 32 ° C for up to 72 hours. During incubation, the suspension is stirred continuously.

Comparative results of babassu flour hydrolysis experiments using the enzyme formulation produced by solid state fermentation with Aspergillus awamori IOC-3914 and using a commercial enzyme preparation are presented in Table 2. Table 2 shows the observed glucose and ANA contents during the experiments.

TABLE 2

Enzymatic formulation of

Commercial Product

Weather A. awamori IOC-3914

(hours) FAN Glucose FAN Glucose

(g / l) (mg / l) (g / l) (mg / l)

0 0.6 832.5 0.3 0

4 43.2 879.8 36.8 18.2

24 66.2 888.7 51, 2 22.3

72 110.9 106.4 74.5 51.6

The concentration of assimilable nitrogen present in the hydrolyzed medium can meet all nutritional needs of yeast cells, confirming that it can be used as a fermentation medium for subsequent bioprocesses aimed at producing ethanol and other chemicals. It is further found that a FAN concentration between 300 and 400 mg / L is consumed during such subsequent fermentations.

The enzymes used in the process of the invention at low temperature act on the starch in granular form, thus eliminating the need for high energy demand for starch processing, providing higher glucose production for conversion to ethanol and other bioproducts. of higher added value.

Example 3 - Use of low temperature enzyme extract In an alternative way of conducting the process, a second experiment is performed under the same conditions as described in the previous Example, but changing the temperature.

Babassu flour is also used, varying only the temperature, which is kept from the beginning at 32 ° C, lasting up to 24 hours.

As can be observed even at low temperature, after 24 hours there is still significant release of glucose corresponding to conversion above 40% of the starch contained in the material. Results are presented in Table 3 below.

TABLE 3

Example 4 - Use of Enzyme Extract in Corn Raw Material

In this example the process is conducted under the same conditions as described in Example 2, but using as a source of starch corn flakes smaller than 0.6 mm (28 mesh Tyler).

Glucose and ANA contents are shown in Table 4 below. TABLE 4

Enzymatic formulation of

Commercial Product

A. awamori IOC-3914

(hours) FAN Glucose FAN Glucose

(g / l) (mg / l) (g / l) (mg / l)

0 2.4 927.8 2.4 0

4 93.7 1273,1 85.7 9.8

24 134.9 1481, 8 126.4 18.9

48 150.6 1712.4 135.7 29.6

72 182.6 1699.4 161, 4 23.6

Example 5 - Use of Enzyme Extract with Enzyme Load Variation

The experiment is carried out under the same conditions as the previous Example with corn flakes, but the enzymatic load used in the enzyme formulation produced by solid state fermentation with Aspergillus awamori IOC-3914 is 4.5U / mL. The glucose levels obtained are presented in Table 5.

TABLE 5

Thus, it is verified that the enzymatic formulation obtained from solid state fermentation, after separation of solids and without undergoing lyophilization process, with final activity five times lower, is also capable of producing good results. Example 6 - Application of Enzyme Extract Employing Batch-Fed Process

In this experiment the hydrolysis process has its procedure changed. The starch source feedstock is added at different times of the reaction.

Babassu flour content, in the range of 5% to 20% (by mass, relative to the total mass of the system), are added after 2, 4 and 24 hours of saccharification, totaling an addition between 10 and 50% of the solids contained in the system. initial step.

The experiment is conducted under the same conditions described in

Example 2, using the enzyme formulation produced by solid state fermentation with Aspergillus awamori IOC-3914 in the presence of babassu flour, with 10% of the initial solids content added after 2, 4 and 24 h saccharification, totaling addition 30% of the solids contained at the beginning

Results are shown in Table 6 below.

TABLE 6

Example 7 - Production of Ethanol from Babass Flour Using the Enzyme Formulation of the Invention

In this experiment the enzymatic extract obtained for fermentation of babassu flour aiming at ethanol production is applied. A suspension with 19% babassu flour is prepared. The pH is adjusted to 4.8 with 7M sulfuric acid solution when required. The medium is placed at 50 ° C for 4 hours, and then kept at 32 ° C for up to 72 hours.

Fermentation is conducted without any nitrogen source addition as the material from the biomasses themselves used to produce the enzyme formulation is hydrolyzed by the enzymes themselves and contains all the enzymes necessary for the final fermentation, including amylases (alpha-amylases, glucoamylases). , isoamylases), cellulases (endoglucanases, beta-glycosidases), hemicellulases (xylanases) and proteases (endopeptidases, exopeptidases).

The graph shown in Figure 2 shows the result of the kinetic profile of ethanol production during fermentation.

The conversion of granular starch using the enzymatic formulation allows a fermentation called "Very High Gravity" (VHG) of a medium with high soluble solids content.

Table 7 below shows the result of fermentation experiments with Saccharomyces cerevisae strain JP1.

TABLE 7

Glucose initial FAN initial ^FAN _f! ^L ^{"o1 and} _f ^tan ^ce ° ^'Pr ° ductivity maximum end end end

(g / l) (mg / l) _{(mg / l) (g / l) (g / l) (g /} _h)

45.75 847.31 534.77 1, 68 55.49 1.16

It is evident that the enzyme formulations of the invention, besides being produced at low cost, have the advantage of having application in the simultaneous conversion of oligosaccharides and polysaccharides of different materials aiming to obtain biofuels (ethanol, butanol) and other green chemicals (acids biopolymers, antibiotics and polyols, among others), enabling an integrated processing of industrial plants and, consequently, reducing operating costs.

Example 8 - Ethanol Production from Babassu Flour Using the Enzymatic Formulation of the Invention

This experiment is conducted similarly to that described in example 7, but with some changes, since hydrolysis occurs in a separate fermentation stage.

A suspension with 19% babassu flour is prepared. The pH is adjusted to 4.8 with sulfuric acid when required. The middle is. It is placed at 50 ° C for 24 hours and then the residual solid is separated by filtration and / or centrifugation. The liquid from this separation is then added to the yeast cells and the fermentation process is conducted for up to 48 hours at 32 ° C.

Table 8 below shows the result of fermentation experiments with Saccharomyces cerevisae strain JP1.

TABLE 8

Initial Glucose initial FAN _f ^FAN ° ^{^{Ε ί 3η}} 'Productivity

end final maximum

(g / l) (mg / l) _{(mg / l) (g / l) (g / [} _ _h)

88.1 934.5 581, 9 32.9 0.69

Example 9 - Ethanol Production from Corn Starch Using the Enzyme Formulation of the Invention

This experiment is conducted under the same conditions as described in Example 7, replacing babassu flour with cornstarch.

Table 9 below shows the result of fermentation experiments with Saccharomyces cerevisae strain JP1.

TABLE 9

Glucose initial FAN initial ^FAN _f, ^and I ° ^an 'Productivity

(Q / LΙ \) / (m 'g / Li) \ _(m ^{* n} _q ^l _{_JL)} _"(q ^n, _L ^_a)' m _(G A, _G im x _h)

82.0 1228.6 980.0 82.6 3.25

Claims

1. PROCESS FOR OBTAINING ENZYMATIC FORMULATIONS FROM AGRO-INDUSTRIAL WASTE characterized by the following steps:

- feed into a first reactor (100) for production of enzymes the raw material agroindustrial residue (10), with particle size in the range of 0.05 - 2.00mm, a first stream of water (15), to adjust and maintain the moisture content in the range 40 - 80%, and grown filamentous fungal cells (20), between 1 and 7 grams of cells on dry basis per liter of suspension, selected from Aspergillus fungi strains to form a solid matrix (30); maintain fermentation (F1) in solid state (FES) for 12 - 168h at a temperature in the range 20 ° C - 55 ° C;

- extracting the produced enzymes from the solid matrix (30) into a liquid phase by the addition of a second stream of water (35) to a mechanically stirred reactor rotating between 150-1500 rpm in the liquid ratio: solid between 5 and 15 (L / kg) (F2);

separating the solid matrix (30) from the liquid containing the enzyme extract (40) by means of a filtration and / or sedimentation and / or centrifugation step (S) to remove the fermentation residue (50);

optionally concentrating the enzyme extract containing liquid stream (40) by membrane ultrafiltration or lyophilization (C) to obtain a concentrated enzyme extract stream comprising the enzyme formulation (45) and residual water (50). ') of extraction.

Process for obtaining enzymatic formulas from agro-industrial waste according to claim 1, characterized in that said agro-industrial raw material (10) comprises agro-industrial waste and materials. selected from babassu pie, babassu flour, castor bean pie; canola pie; sunflower pie; brewer's bagasse; maize starch; sugar cane molasses; soy molasses; millionocin; crude glycerin from soybean biodiesel; sugar cane juice.

Process for obtaining enzymatic formulations from agri-industrial waste according to claim 1, characterized in that the microorganism used to produce said enzyme extract (40) is selected from filamentous fungus strains of the genus Aspergillus, in particular Aspergillus awamori, Aspergillus. wentii and Aspergillus oryzae.

Process for obtaining enzymatic formulations from agroindustrial waste according to claim 1, characterized in that said enzyme extract (40) simultaneously contains enzymes capable of hydrolyzing granular starch from feedstocks of different compositions.

Process for obtaining enzymatic formulations from agroindustrial waste according to claim 1, characterized in that said enzyme extract (40) simultaneously contains enzymes such as amylases (alpha-amylases, glucoamylases, isoamylases), cellulases (endoglucanases, beta-glucosides). ), hemicellulases (xylanases) and proteases (endopeptidases, exopeptidases).

Process for obtaining enzymatic formulations from agro-industrial waste according to claim 1, characterized in that the filamentous fungus cells (20) added to the reactor (100) used as inoculum consist of previously grown cells.

A process for obtaining enzymatic formulations from agri-industrial waste according to claim 1, characterized in that said formulations are used. enzymatic materials directly in crude liquid form or concentrated by membrane processes or as lyophilized solids.

8. ENZYMATIC FORMULATIONS, characterized by being obtained from solid state fermentation of residues and agro-industrial materials selected from babassu cake; babassu flour; castor cake; canola pie; sunflower pie; brewer's bagasse; maize starch; sugar cane molasses; soy molasses; millionocin; crude glycerin from soybean biodiesel; sugar cane juice

ENZYMATIC FORMULATIONS according to claim 8, characterized in that said residues and agroindustrial materials are mixed in different proportions and combinations in order to induce, in a personalized manner, the synthesis of said enzyme formulation, with balanced activity between the enzymes of of interest for conversion of different biomasses and release of fermentable sugars.

ENZYMATIC FORMULATIONS according to Claim 8, characterized by the use, in the solid-state fermentation step, of filamentous fungi strains of the genus Aspergillus, in particular Aspergillus awamori, Aspergillus wentii and Aspergillus oryzae.

Enzymatic formulations according to Claim 8, characterized in that they contain enzymes such as amylases (alpha-amylases, glucoamylases, isoamylases), cellulases (endoglucanases, beta-glucosidases), hemicellulases (xylanases) and proteases (endopeptidases, exopeptidases).

Enzymatic formulations according to Claim 8, characterized in that they comprise an enzymatic extract (40) for use directly in the crude liquid form.

Enzymatic Formulations A according to Claim 8, characterized in that they comprise a concentrated enzyme extract (45) resulting from the concentration of a liquid stream. containing the enzyme extract (40), which was subjected to the process of separating excess water with membranes or drying and lyophilization treatment.

14. INTEGRATED PROCESS FOR BIOFUEL PRODUCTION characterized by using enzymatic formulations obtained from residues and agroindustrial materials to hydrolyze granular starch, comprising the steps of:

- feed into a first reactor (100) for production of enzymes the raw material agroindustrial residue (10), with particle size in the range of 0.05 - 2.00mm, a first stream of water (15), to adjust and maintain the moisture content in the range 40 - 80%, and grown filamentous fungus cells (20), with a concentration ranging from 1 to 7 grams of dry base cells per liter of suspension selected from Aspergillus fungi strains to form a solid matrix (30); keep fermentation (F1) in solid state (FES) for 12 - 168h at a temperature in the range 20 ° C - 55 ° C

- extracting the produced enzymes from the solid matrix (30) into a liquid phase by adding a second stream of water (35) to a mechanically stirred reactor (F2) between 150 and

1500 rpm, in the liquid: solid ratio between 5 and 15 (L / kg);

separating the solid matrix (30) from the liquid containing the enzyme extract (40) by means of a filtration (S) and / or sedimentation and / or centrifugation (S) step to remove the fermentation residue (50);

concentrating the stream of the enzyme extract-containing liquid (40) by membrane ultrafiltration or lyophilization (C) to obtain a concentrated stream of the enzyme extract comprising the enzyme formulation (45) and a concentrated residue (50). ') from fermentation;

- drive a new raw material stream (M) consisting of agroindustrial residue and / or material (10) and enzyme formulation (45) for a second reactor (200), to undergo enzymatic hydrolysis and submerged fermentation (F3), in the presence of the enzyme formulation (45) obtained using cells microbial (60) suitable for the production of biofuel or other desired chemical (70), said microbial cells selected from bacteria, actinomycetes, yeast and filamentous fungi.

An integrated process for the production of biofuels according to claim 14, characterized in that the raw material (10) comprises residues and agro-industrial materials selected from babassu cake; babassu flour; castor cake; canola pie; sunflower pie; brewer's bagasse; maize starch; sugar cane molasses; soy molasses; millionocin; crude glycerin from soybean biodiesel; sugar cane juice; corn grains, wheat grains, sorghum grains, among others.

An integrated process for the production of bio-fuels according to claim 14, characterized in that the microorganism used to produce said enzyme formulation (40, 45) is selected from filamentous fungus strains of the genus Aspergillus, in particular Aspergillus awamori, Aspergillus wentii and Aspergillus oryzae.

An integrated process for the production of biofuels according to claim 14, characterized in that it uses an industrial strain Saccharomyces cerevisiae yeast strain for the enzymatic hydrolysis and submerged fermentation (F3) process in the presence of said enzymatic formulation obtained.

An integrated process for the production of biofuels according to claim 14, characterized in that the concentration of solids added in the reaction medium during the hydrolysis and submerged fermentation (F2) phases are in the range of 15 to 35% as a suspension.

An integrated process for the production of biofuels according to claim 14, characterized in that said enzymatic formulation is used directly in the crude liquid form.

An integrated process for the production of biofuels according to claim 14, characterized in that said enzyme formulation is used as a concentrated enzyme extract (45) obtained by membrane processes and / or by dissolving the previously lyophilized enzyme preparation. .

INTEGRATED PROCESS FOR PRODUCTION OF BIOFUELS according to Claims 14 and 20, characterized in that the concentrated lyophilized enzyme extract (45) is used to prepare suspensions with amylolytic activity in the range of 2 to 30 U / mL.

An integrated process for the production of biofuels according to claims 14 and 8, characterized in that said enzymatic formulations are employed to hydrolyze granular starch for ethanol production and other biofuels.

An integrated process for the production of biofuels according to claim 14, characterized in that alternatively the starch source material is added at different reaction times, in the range of 5% to 20%, added after 2 to 48 hours of saccharification, totaling between 10 and 70% of the solids contained in the initial step.