BRPI0802559A2 - pretreatment process and hydrolysis of lignocellulosic plant biomass, and product for the industrial production of alcohols - Google Patents

Abstract

PRE-TREATMENT AND HYDROOLYSIS PROCESS OF LIGNOCELLULOSTIC VEGETABLE BIOMASS, AND PRODUCT FOR INDUSTRIAL PRODUCTION OF ALCOHOLS. The present invention relates to pretreatment and hydrolysis processes of plant biomass. The product obtained by said process is substantially useful as an input in the industrial production of ethanol and / or other alcohols. In a preferred aspect, the process of the invention provides a process comprising the steps of: a) pre-treating the lignocellulosic plant biomass with an alkaline hydrogen peroxide solution; and b) Enzymatic hydrolysis of the pretreated biomass using mixtures comprising cellulases, β-glycosidases, hemicellulases, or combinations thereof. Optionally, said process further comprises a fermentation step of the carbohydrates produced in the previous steps, providing the production of ethanol and / or other alcohols.

Description

Patent Invention Descriptive Report

Pretreatment and hydrolysis process of Lignocellulosic Plant Biomass, and Product for the Industrial Production of Alcohols

Field of the Invention

The present invention relates to pretreatment and hydrolysis processes of plant biomass, as well as the products thus obtained. More specifically, the process of the invention comprises pretreatment steps of lignocellulosic plant biomass, in which alkaline hydrogen peroxide is used, followed by hydrolysis of the pretreated biomass and fermentation of the broth obtained by this process.

The pretreatment process of the invention provides a reduction in cellulose crystallinity, thereby facilitating further hydrolysis with increased glucose yield. The present work provides mild conditions of temperature and pressure, and absence of acids. This invention provides less sugar degradation than in acidic processes, so this process has great commercial potential. Preferably, the vegetable biomass is sugarcane bagasse and / or straw and the fermented product is ethanol.

Background of the Invention

The possibility of producing ethanol from lignocellulosic materials has received much attention due to the high availability of these materials, especially in the national territory, and also to the fact that the ethanol-obtaining route is considered "green" when compared to that employed in the production of petroleum-derived fuels, as agasoline.

Cellulose is a linear D-glucose polymer composed of p-1,4 glycosidic bonds with repeated units of cellobiose, forming a water-insoluble high crystallinity material. The degree of polymerization is in the range of 7500-15000 glucose molecules present in the cellulosic chain. Cellulose is organized into fibers with a diameter of 2.0-4.0nm. These fibers are joined by hydrogen and vander Waals bonds, forming a rigid molecular structure (microfibrils) with diameters from 10 to 30 nm. The crystalline fraction constitutes between 50 and 90% of cellulose. In this fraction, the capillaries are small, which makes it difficult for enzymes to penetrate the matrix (average size 5 nm). Therefore, enzymatic hydrolysis processes require a preliminary treatment (pretreatment) of the lignocellulosic biomass, aiming to "open" the cellulosic matrix. to the action of enzymes. The regions of low crystallinity (amorphous) in the microfibrils are susceptible to enzymatic action, without pretreatment of biomass.

Unlike cellulose, hemicelluloses are branched polymeric heterogeneous from different carbohydrates joined by different chemical bonds. Several substituents, for example, uronic acid acetyl groups and groups, are associated with the main chain or its branches in polymerization structures ranging from 20 to 300. Hemicelluloses do not show the degree of crystallinity in a microfibrillary oriented cellulose structure. so that it does not exert effective influence on the structural properties of plant tissue. Thus, they are more susceptible to acid and enzymatic hydrolysis, as well as higher solubility in aqueous-alkaline solutions. Generally, the term holocellulose is used to refer to the total saccharide fraction (cellulose ehemicelluloses) of the extractives-free plant tissue.

Hemicelluloses are associated to the phenolic fraction (lignin) through covalent bonds and to cellulose via hydrogen bonds. Its composition varies by lignocellulosic material. Softwoods such as Pinus radiata contain higher glycane levels, while hardwoods such as birch have a higher glycoxyl content. The sugarcane bagasse hemicelluloses are predominantly made up of xylans, although they also have smaller amounts of glycoxylans and arabinoxylans. Along with cellulose, lignin is one of the most abundant organic polymers in the plant kingdom. Usually, lignin is seen as a "cement" or "fouling substance" in plant tissue, significantly contributing to its mechanical strength. On the other hand, while a relatively large number of microorganisms are capable of decomposing and converting cellulose and hemicelluloses, only a very small number has the ability to effectively decompose lignin, which justifies the high resistance of plants to deterioration.

Lignin is a complex, high molecular weight three-dimensional amorphous polymer, generally associated with cellulose and hemicelluloses through ether and carbon-carbon deletions. Its "in natura" chemical structure is strongly aromatic, phenolic in nature, composed of phenyl propane units associated with methoxyl groups, phenolic and aliphatic hydroxyls.

Due to the association of cellulose with hemicelluloses and lignin, the access of various chemical (eg acids and alkalis) and biochemical (eg enzymes, microorganisms) agents used in hydrolysis processes and ethanol production from fermentative lignocellulosic biomass becomes quite restricted.

Therefore, there is a need for pre-treatment (pre-treatment) of lignocellulosic biomass, aiming to remove non-cellulosic components, predominantly lignin, and eventually part of hemicellulose, in order to favor the accessibility of enzymes to cellulose.

Much research has been done on pretreatment of lignocellulosic materials to facilitate their hydrolysis with subsequent fermentation of sugars obtained. A good part of them, however, require carose equipment. The developed process ends up generating inhibitory products that affect the fermentation stage. Pretreatment with alkaline hydrogen peroxide has been studied by some authors, however the influence of process variables on glucose yield has not been established. In addition, it has not determined the conditions or the viability of the process when the bagasse is used as it is available in the mills without being milled or sieved. The pre-treatment process we have proposed has high glucose yields, high hemicellulose removal and lignin, and there is no need to sift or previously grind the bagasse. Other important factors in the proposed process are the low times and temperature of the process, which minimizes process costs, as well as the use of mild reagents that do not generate toxic waste after pretreatment, thus minimizing environmental problems, minimizing the inhibition in the fermentation step and the degradation of sugars.

Some technologies similar to the technology of the present invention were found in previous searches, however, presenting some striking differences in relation to our proposal. Gould (US4,806,475 and US 4,649,113) proposed the pretreatment of lignocellulosic material with alkaline hydrogen peroxide, but did not present studies using sugarcane dust as an alternative to biomass, besides working with small particle size (2 mm ). The present invention differs from such a document, among other factors, by working with sugarcane bagasse, study of different combinations of process variables and studies related to particle size variation, all of which were evaluated in our study.

US 4,314,854 (Takagi) discloses the use of a hydrogen peroxide combination and the addition of different metal salts to observe the effect on pretreatment of agricultural residues such as wood, rice straw, sugarcane bagasse. , corn husk and paper waste. The present invention differs from said document among other factors in that it does not use metal salts in the pretreatment step of the material.

In the Mitrani et al. Patent application (BR PI 9201830 A), the pretreatment agent used for sugarcane bagasse is a solution of sodium hydroxide at a concentration between 4-20 g / l and possibly hydrogen peroxide or other brightening agents at a temperature between 60-95 ° C. The objective was to use 60-85% pulp in the formulation of newsprint pulp and other paper and paper. The present invention differs from said document among other factors in that it uses room temperature and does not require heating. Moreover, in this case hydrogen peroxide is the main agent and not only a bleaching agent, besides the desired end product being ethanol and not paper.

In US patent application US 2006/207734 A1 (Day eChung), the agent used for pretreatment was an oxidative solution of sodium hypochlorite and hydrogen peroxide (5: 1), used to remove lignin and hemicellulose from bagasse. of sugar cane. The present invention differs from said document, among other factors, in that it does not require the use of sodium hypochlorite.

Therefore, based on the analysis of the antecedents found so far, it is observed that there was no anticipation or suggestion of the teachings of the present invention, as well as the need for a pretreatment process of lignocellulosic material for the production of sugars and also ethanol that provides a high yield.

Summary of the Invention

It is an object of the invention to determine a new industrial process for pretreatment of plant biomass so that it can be hydrolyzed and subsequently fermented.

In a preferred aspect, a process is developed comprising the steps of:

(a) Pretreatment of lignocellulosic plant biomass with a solution

of alkaline hydrogen peroxide; and

b) Enzymatic hydrolysis of the pretreated biomass using mixtures comprising cellulases, p-glycosidases, hemicellulases, or combinations thereof.

The product obtained by said process is substantially useful as input in the industrial production of ethanol and / or other alcohols. It is therefore other objects of the invention to provide an input comprising lignocellulosic biomassavegetal pretreated with an alkaline hydrogen peroxide solution, and enzymatically hydrolyzed by cellulases, p-glycosidases, hemicellulases, or combinations thereof.

In a preferred aspect, thus being another of the objects of the invention, a fermentative process is provided which further comprises a step of:

c) Fermentation of carbohydrates produced during the enzymatic hydrolysis step of the pretreated biomass.

These and other objects of the invention will be appreciated by those skilled in the art, and will be better understood from the following detailed description.

Brief Description of the Figures

Figure 1 shows the glucose yield after hydrolysis of the non-sifted and sifted bagasse in each of the assays.

Figure 2 shows the response surface of the glucose mass (g / g gross mass) obtained from the sifted bagasse as a function of peroxide concentration and temperature when the pretreatment reaction time is fixed at 6 h.

Figure 3 shows the glucose yield after unshielded bagasse hydrolysis in each of the assays performed in the optimization step.

Figure 4 shows the response surface for glucose mass (g / g gross biomass) obtained from unshielded bagasse as a function of peroxide concentration and temperature in the pretreatment optimization step.

Figure 5 illustrates the hydrolysis profile for optimum untreated bagasse pretreatment conditions (1 h reaction time, 25 ° C temperature and 7.35% peroxide concentration).

Detailed Description of the Invention The present invention provides an industrial process for pretreating plant biomass so that it can be hydrolyzed and subsequently fermented. In a preferred aspect, the invention process comprises the steps of:

(a) Pretreatment of lignocellulosic plant biomass with a solution

of alkaline hydrogen peroxide; and

b) Enzymatic hydrolysis of the pretreated biomass using mixtures comprising cellulases, (3-glycosidases, hemicellulases, or combinations thereof.

The product obtained by said process is substantially useful as an input in the industrial production of ethanol and is therefore another object of the invention.

The examples described herein are intended solely to exemplify the objects of the invention, and not to limit their application or scope.

Lignocellulosic Plant Biomass

In the present invention, the term "lignocellulosic plant biomass" includes any type of plant, namely: herbaceous biomass; cultivars as C4 plants - belonging to the genera Lolium, Spartina, Panicum, Miscanthus, and combinations thereof; sugarcane bagasse (from milling and / or diffuser); cereal straws such as wheat, rice, rye, barley, oats, corn and the like (e.g. switchgrass elephant grass); wood; ethereal banana tree trunks; cactus plants and combinations thereof. In addition, lignocellulosic materials may further comprise cardboard, sawdust, agro-industrial waste or similar municipal waste.

Vegetable biomasses of different origins may have particular differences although they have relatively similar overall chemical composition. Some variations in composition between different species and within the same species are due to environmental and genetic variability, in addition to the location of plant tissue in different parts of the plant. Typically, about 35-50% consists of cellulose, 20-35% dehemicelluloses and about 20-30% lignin. The remainder consists of smaller quantities of ashes, soluble phenolic compounds and fatty acids, as well as other constituents called extractive. Cellulose and plant tissue ashemicelluloses consist of structural carbohydrates (e.g. glycans, xylans, mananas) and are generally referred to as saccharide fractions. Lignin is the phenolic fraction of plant biomass.

The present invention comprises a fermentative process having technical and operational advantages as compared to processes present in the state of the art and involves the following steps:

(a) pre-treatment of lignocellulosic plant biomass with an alkaline hydrogen peroxide solution;

b) Enzymatic hydrolysis of the pretreated biomass using mixtures containing cellulases and / or p-glycosidases and / or hemicellulases; and

c) Fermentation of carbohydrates produced from the enzymatic hydrolysis of pretreated biomass.

Pretreatment Step

The pretreatment step of the present invention comprises the use of alkaline hydrogen peroxide in a suitable lignocellulosic material, preferably unground sugarcane bagasse.

In this invention, hydrogen peroxide is used in concentrations determined by statistical designs ranging from 1 to 9.83% v / v and it is concluded that this variable is the one that most influences the glycoside yield after hydrolysis, being its optimal value 7,35 % v / v. Sodium hydroxide is used to raise the pH of hydrogen peroxide solution to 11.5, which makes the solution an effective agent in the delignification and solubilization of hemicellulose.

Time and temperature were also studied process variables, but they did not show significant influence on glucose yield. At the end of the optimization process, it was concluded that the pretreatment can be carried out at room temperature and with a reaction time of 1 h, which are much lower values than those obtained in previous patents. Cellulosic material obtained under the conditions above can be hydrolysed by cellulase enzymes or enzyme cocktails, requiring low enzyme load (values much lower than those usually found in literature) to achieve high glucose yields.

The pre-treatment conditions determined by us result in a biomass with almost 80% dry weight cellulose. The significant improvements in hydrolysis of the pretreated material under the conditions determined by the invention can be recognized by the fact that 0.3740 g / g crude glucose biomass was released under optimal conditions, which corresponds to a glucose yield of 84.07%. Biomass without pretreatment provided 0.004 g / g dry glucose biomass with a yield of 0.907% glucose.

With this procedure it is possible to obtain high yields of sugar and sugar for ethanol production from residues of sugarcane industries (sugarcane bagasse), which increases the production of fuel without increasing the planted area. What's more, it results in better use of the raw material. The sugars obtained may be used not only for the production of alcohol but also for other fermentation products. The need for a reduction in bagasse particle size to facilitate pretreatment / hydrolysis is assessed.

Fermentation Process

The fermentation step can be performed after enzymatic hydrolysis via a process known as Separated Hydrolysis and Fermentation (SHF), or concomitantly with hydrolysis in a process known as Simultaneous Saccharification and Fermentation (SSF). (Simultaneous Saccharification and Fermentation). Depending on the concentration of sugars produced in enzymatic hydrolysis, a concentrated saccharide solution (e.g. molasses or sugarcane juice) may be added to the reaction medium.

The present invention can also be used in the CBP (Consolidated Bioprocessing) process, which combines the four biological events required for the conversion of plant biomass to ethanol: production of cellulolytic enzyme, hydrolysis of polysaccharides present in pretreated biomass, fermentation of hexoses and fermentation of pentoses in the mesmoreator.

Example 1. Material Preparation

The sugarcane bagasse (Saccharum officinarum) used in the experiments was dried at 45 ° C for 48 h and left at room temperature for a further 24 h and then stored in the freezer in tightly closed bags. The moisture content determined was 5%.

Tests were carried out using the bagasse from the last plant mill (heterogeneous material), called non-sifted bagasse and sifted bagasse in the range of -12 + 60 mesh (particles between 0.248 and 1.397 mm). The smaller particles were discarded because they correspond mainly to sand. The objective was to evaluate the influence of particle size on the release of fermentable sugars.

Example 2. Chemical Characterization of Material

After dividing the bagasse into two parts, they were analyzed for extractives, ashes, total lignin, glycan, xylan and acetyl groups according to the methodology described by the National Renewable Energy Laboratory.

Example 3. Pretreatment

Pretreatment with alkaline hydrogen peroxide (alkaline H2O2) was studied. The influence of pretreatment time (h), temperature (° C) and H2O2 concentration (%) on the release of fermentable sugars was evaluated through experimental designs aiming at optimizing pretreatment conditions.

Samples of approximately 4.0 g of unshielded and sieved dry bagasse were treated with 100 mL of the solution containing alkaline H2O2 at a predetermined concentration by design and immediately elevated to an incubator where they were maintained at a rotation of 150 rpm with pre-set reaction temperature and time. also determined by experimental planning. Example 4. Enzymatic Hydrolysis

At the end of each reaction, the reaction liquid was discarded and the remaining residues washed with distilled water until all staining disappeared. The samples were then dried at a temperature of 50 ° C and weighed to determine mass loss.

One gram of the pretreated bagasse was hydrolyzed with 300 ml of cellulase-containing solution and (3-glycosidase with the pH of the medium adjusted to 4.8. For hydrolysis 3.50 FPU / g of pre-treated dry cellulase biomass from Trichoderma reesei ( Sigma) and 1.00 CBU / g Aspergillus niger P-glycosidase pretreated dry biomass (Sigma) Cellulase activity was determined as paper filter units per milliliter (FPU / mL) as recommended by the International Chemical Union. Pure and Applied (IUPAC) P-glycosidase activity was determined by a 15 mmol / L decelobiose solution and expressed in units per milliliter (CBU / mL).

Experiments were performed in 500 ml conical flasks in incubators with orbital agitation at 100 rpm at 50 ° C. Aliquots were taken at predetermined times, boiled for 15 minutes to inactivate the enzymes and analyzed for glucose content. Glucose yield values used for statistical analysis were chosen at the time after which the masses released into the reaction medium remained unchanged.

Example 5. Analytical Methods

Glucose concentration was determined according to the Glucose GOD-PAP enzyme method described by Henry (1974). It is a enzymatic kit based on enzymatic oxidation of glucose through enzymatic glucose oxidase (GOD) resulting in hydrogen peroxide, which is subsequently used in the generation of peroxidase pink coloration (PAP). The concentration of total reducing sugars (ART) was determined according to the dinitro-3,5-salicylic acid (DNS) method described by Miller (1959). Example 6. Effects of Glucose-Pretreatment Process Variables

Initial experiments were conducted for unshielded (N) and screened (P) bagasse according to a complete two-level statistical design, which has a combination of process variables; time (6.15 and 24 h); temperature (20, 40 and 60 ° C) and peroxide concentration (1, 3 and 5%). Responses were obtained from the mass of ART and glucose, expressed as g / g dry crude biomass (without pretreatment), and the percentage glycoside-expressed yield (%) after the cellulose hydrolysis was performed under the conditions already described.

It was observed that under the studied operating conditions the maximum deglucose was obtained for the unshredded bagasse in 24 hours of pretreatment at a temperature of 20 ° C using 5% alkaline hydrogen peroxide, which corresponds to a glucose yield of 69.427%. (test 6) as shown in Figure 1. Thus, it can be concluded that decreasing particle size was not important in the release of fermentable sugars after the pretreatment and hydrolysis step. Unsifted sugarcane bagasse was then used for the later stage of process optimization.

This study was of paramount importance, as no previous work had made such a comparison. By eliminating the need to reduce particle size, greater use was made of the raw material, which can be fully utilized, and to exclude joint operation of the process, which can greatly reduce its complexity and costs.

For the pre-treatment optimization stage, the variable "time" was eliminated from the new planning, as its effect was not statistically significant during the first planned trials. As the study had been performed at 6, 15 and 24 hours of pre-treatment, the time of 6 hours was set for all trials in the optimization step, thus varying only the concentration of H202 and the temperature. 7. Optimization of pretreatment conditions

In the second stage, a new experimental design was used, taking as factors the temperature and the concentration of alkaline hydrogen peroxide (%). The pretreatment time was eliminated as it was not significant in the previous step and remained fixed at the minimum point (6 h). It was observed that under the studied operating conditions the maximum glucose was obtained at 50 ° C at a concentration of 7% hydrogen peroxide, leading to a glucose yield of 0.8131% (run 5) as shown in Figure 3.

The results of this new planning showed that, in the new study range, the temperature is also not significant at the 90% confidence level. A significant statistical model was proposed that allowed the determination of the H202 concentration value that leads to the maximum release of glucose after hydrolysis, which was 7.35% peroxide as shown by the surface response of Figure 4.

Since pretreatment time and temperature were not significant in the studied range, further studies were conducted to determine whether pretreatment could be performed at room temperature and the shortest time required. As a result it was found that maximal glucose release can be achieved around 25 ° C, at a hydrogen peroxide concentration of 7.35% for 1 h. The results of ART eglucose mass after 48 h of hydrolysis at 50 ° C and pH 4.8 were 0.4899 g / g dry biomass and 0.3740 g / g dry gross biomass, respectively, as shown in Figure 5. The concentration The cellulase used for hydrolysis was 3.50 FPU / g of pre-treated dry mass and 3-glycosidase 1.00 CBU / g of pre-treated dry biomass. The glucose mass corresponds to a yield of 84.07%.

Those skilled in the art will appreciate the process of the present invention as an excellent alternative to existing counterparts. The examples described in the present invention should not be construed as limiting the spirit and scope of the present invention as defined in the appended claims.

Claims (17)

Pre-treatment and hydrolysis process of Vegetalllgnocellulosic Biomass, and product for the industrial production of alcohols 1. Pretreatment and hydrolysis process of vegetallignocellulosic biomass characterized by comprising the steps of: i. Pretreatment of lignocellulosic plant biomass with alkaline hydrogen peroxide solution; hey. Enzymatic hydrolysis of the pretreated biomass using mixtures comprising cellulases, β-glycosidases, hemicellulases, or combinations thereof. Process according to Claim 1, characterized in that the vegetable fiber is selected from the group comprising: i. herbaceous biomass; ii. plants belonging to the genera Lolium, Spartina, Panicum, Miscanthus, and combinations thereof iii. sugarcane bagasse from milled and / or diffuser iv. cereal straws such as wheat, rice, rye, barley, oats, corn and mixtures thereof; v. wood vi. banana tree trunks and / or stalks vii. cardboard, sawdust, newspaper and similar agri-industrial or municipal waste; eviii. combinations of items i to vii. Process according to Claim 2, characterized in that the vegetable pulp is chosen from the group comprising sugarcane bagasse. Process according to Claim 1, characterized in that the peroxide solution has a concentration within a range from 1% v / v to 9.83% v / v. Process according to Claim 4, characterized in that the peroxide solution has a concentration of approximately 7.35% v / v. Process according to Claim 1, characterized in that the alkaline peroxide solution has a pH value of approximately 11.5. Process according to Claim 6, characterized in that the pH is adjusted by a sodium hydroxide solution. Process according to Claim 1, characterized in that the step) is carried out at a temperature of from 15 ° C to 50 ° C. Process according to Claim 1, characterized in that the step is carried out in a time of up to approximately 24 hours. Process according to Claim 1, characterized in that the bagasse: alkaline peroxide solution ratio ranges from 1g: 20ml to 1g: 30mL. Process according to Claim 1, characterized in that the hydrolysis step comprises the use of p-glycosidases from Aspergillus niger. Process according to Claim 1, characterized in that the hydrolysis step comprises the use of Trichodermareesei cellulases. Process according to Claim 1, characterized in that the hydrolysis step occurs at a pH of approximately 4.8. Process according to Claim 1, characterized in that the hydrolysis step occurs at a temperature of approximately 50 ° C. Process according to claim 1, characterized in that the bagasse: enzymes ratio is in the range from 1g: 3 FPU to 1g: 5FPU for cellulase and 1g: 1 CBU to 1g: 10 CBU for p-glycosidase. Process according to Claim 1, characterized in that it comprises an additional fermentation step of the carbohydrates produced from the enzymatic hydrolysis of the pretreated biomass. 17. A product for the industrial production of alcohols comprising lignocellulosic plant biomass pretreated with an alkaline hydrogen peroxide solution and hydrolyzed with cellulases, p-glycosidases, hemicellulases, or combinations thereof.