BRPI0802153A2 - fermentative process copending pre-treatment step, enzymatic hydrolysis and fermented product using lignocellulosic plant biomass - Google Patents

Abstract

FERMENTATIVE PROCESS UNDERSTANDING PRE-TREATMENT STEP, ENZYMATIC HYDROLYSIS AND FERMENTED PRODUCT USING LIGNOCELLULOSIC VEGETABLE BIOMASS. The present invention relates to a fermentative process comprising a step of pre-treatment and enzymatic hydrolysis of lignocellulosic plant biomass, where such pre-treatment includes the use of steam, opejonally in the presence of catalysts, an enzymatic pretreatment and a product fermented by this process. In particular, vegetable biomass is sugarcane bagasse and the product is ethanol.

Description

Patent Invention Descriptive Report

Fermentation Process Comprising Pretreatment Step, Enzymatic Hydrolysis and Fermented Product Using Lignocellulosic Plant Biomass

Field of the Invention

The present invention relates to a fermentation process comprising lignocellulosic plant biomass pretreatment steps in which steam is used in the presence or absence of chemical and / or biochemical catalysts, enzymatic hydrolysis of said biomass and fermentation of the hydrolyzed broth obtained therefrom. enzymatic process. In particular, the vegetable biomass is sugarcane bagasse and / or straw and the product is ethanol.

Background of the Invention

The possibility of producing ethanol from lignocellulosic materials has received a lot of attention due to the high availability of these materials, especially in the national territory, and also to the fact that the ethanol production route is considered "green" when compared to that used in fuel production. petroleum products, such as gasoline.

Cellulose is a linear D-glucose polymer composed of β-1,4 glycosidic bonds, with repeated cellobiose units, 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 2.0-4.0 nm. These fibers are associated with hydrogen bridges and van der Waals bonds, forming a rigid molecular structure (microfibrils) with diameters from 10 to 30 nm. The crystalline fraction constitutes between 50 and 90% of the 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 pre-treatment (pre-treatment) of the lignocellulosic biomass, aiming to "open" the cellulosic matrix to the enzymes action. The regions of low crystallinity (amorphous) in the microfibrils are susceptible to enzymatic action, eliminating pretreatment of biomass.

Unlike cellulose, hemicelluloses are branched heterogeneous polymers of different carbohydrates joined through different chemical bonds. Various substituents, e.g., acetyl groups and uronic acids, are associated with the main chain or their respective branches in polymerization grade structures ranging from 20 to 300.

Hemicelluloses do not have the degree of crystallinity or the oriented microfibrillary structure of cellulose, so they do not exert an effective influence on the structural properties of plant tissue. Thus, they are more susceptible to acid and enzymatic hydrolysis, as well as greater solubility in aqueous-alkaline solutions. Generally, the term holocellulose is used to refer to the total saccharide fraction (cellulose and hemicelluloses) of the extractive-free plant tissue.

Hemicelluloses are associated to the phenolic fraction (Iignin) through covalent bonds and to cellulose via hydrogen binder. Its composition varies according to the lignocellulosic material. Softwoods such as Pinus radiata contain higher glycane content, while hardwoods such as birch have higher glycoxyl content. The sugarcane bagasse hemicelluloses are predominantly made up of xylans, although they also have lower amounts of glycoxylans and arabinoxylans.

Along with cellulose, Iignin is one of the most abundant organic polymers in the plant kingdom. Iignin is usually seen as a "cement" or "fouling substance" in plant tissue, contributing significantly 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 have 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 commonly associated with cellulose and hemicelluloses through ether and carbon-carbon bonds. 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, access to the various chemical (eg acids and alkalis) and biochemical (eg enzymes, microorganisms) agents used in the production processes of ethanol from lignocellulosic biomass through fermentation becomes quite restricted.

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

The efficient conversion of cellulose from lignocellulosic material into fermentable sugars and their subsequent fermentation into ethanol certainly pose a huge challenge. Cellulose is the main constituent of lignocellulosic fibers and consists of a very break-resistant crystal structure, as is hemicellulose, the second component in abundance. The conversion of cellulose / hemicellulose to ethanol requires separation of these components from lignin and / or increased enzyme access to these materials, ie, it is necessary to promote a depolymerization of these components into free sugars for subsequent fermentation of these sugars to ethanol.

Acid hydrolysis is a common method for converting cellulose to glucose, a fermentable sugar. It usually involves the use of concentrated or dilute acids. This process generates moderate glucose yields, with cost problems associated with the need for acid recovery and the use of special corrosion equipment construction materials.

The prior art has several processes already described for converting lignocellulosic raw material into glucose. US 4,461,648 and US 7,198,925 describe methods of dilute acid vapor explosion pretreatment. In these documents the biomass is loaded into the steam gun and acid is added to the biomass. Steam is then rapidly injected into the tank, which remains under high pressure for a certain period of time. Finally, the tank is suddenly depressurized, thus generating the "blown up" biomass. This biomass can then be subjected to the hydrolytic action of enzymes (particularly cellulases), which convert cellulose to glucose and other sugars.

The present invention differs fundamentally from the above documents in that it addresses from the pretreatment of lignocellulosic biomass with steam to the final fermentation step of sugars provided in the enzymatic hydrolysis step, particularly glucose from cellulose. Additionally, the present invention further addresses the use of a "ροοΓ" enzymes (hemicellulases and cellulases) in the process of making sugars, particularly glucose, in SHF (Separate Hydrolysis and Fermentation) and SSF (Simultaneous Saccharification and Fermentation) systems. The present invention encompasses pretreatments using non-catalytic and autocatalyzed conditions, as well as catalytic systems using acids, metal salts (eg Lewis acids), alkaline (eg calcium salts) and neutral or slightly acidic (eg carbonates) in oxidative systems. It also addresses the enzymatic treatment of residual hemicelluloses present in the pretreated biomass through the physicochemical processes of steam pretreatment. This sequence sets up a two-stage pretreatment, the first being physicochemical and the second. enzymatic

US 6,423,145 describes a cellulose production process comprising a step of hydrolysis of a lignocellulosic material in a steam blast reactor (continuous or batch) in the presence of a catalyst mixture, where the catalysts are inorganic acids (eg H2SO4). , SO2, HCI and HNO3) and dilute acid salts selected from ferrous sulphate, ferric sulphate, iron chloride, aluminum sulphate, aluminum chloride and magnesium sulphate.

In the present invention, the pretreatment step employs not only inorganic acids and acidic metal salts as chemical catalysts, but also organic acids (eg acetic acid), carbonic acid (H2CO3), alkali metal salts (eg sodium or calcium carbonate). ) and organic compounds (eg acetic acid). In addition to these, the present invention also employs hemicellulases as biochemical (enzymatic) catalysts. Another aspect that differentiates the present process from the aforementioned is its global character, since besides the pretreatment it also contemplates the enzymatic hydrolysis steps of the pretreated biomass using cellulases and the fermentation of the sugars produced in the two previous steps, both in SHF systems as well as SSF systems.

US 7,189,306 describes a process for treating lignocellulosic material comprising the steps of milling, two-step alkaline steam pretreatment (pH> 8), whether or not present with an oxidizing agent (eg 02 and / or H2O2), followed by of a cellulose fractionation step and subsequent carbohydrate fermentation through the SSCF (Separated Saccharification and Co-Fermentation) process.

The present invention differs from this document in that pretreatment can be carried out in acidic (organic and inorganic), neutral and alkaline media using metal catalysts (acidic and alkaline salts) with the possibility of using an enzymatic step (hemicellulases) for removal. of residual hemicelluloses. In addition, it comprises steps of cellulose enzymatic hydrolysis and fermentation of the generated hydrolyzed broth, using Saccharomices cerevisae yeasts, for example, for the production of cellulosic ethanol in SHF and SSF processes. US 4,880,473 describes a process for producing fermentable sugars from cellulosic biomass comprising a pretreatment (dilute acid hydrolysis) step followed by separation of the cellulose-containing solid phase, with subsequent steps of rapid pyrolysis of the pretreated material and separation. sugars present in the aqueous phase.

The present invention differs from the aforementioned document in that it does not cover any step related to the pretreated biomass pyrolysis process, among other reasons already presented and discussed throughout the present text.

Therefore, based on the above, it is observed that the current state of the art does not anticipate or suggest the teachings of the present invention, and there is also a need for a lignocellulosic material pretreatment process for the production of sugars and also ethanol that provides a high yield.

Summary of the Invention

An object of the present invention is a fermentative process comprising the steps of:

(a) pre-treatment of lignocellulosic plant biomass in one (steam blast) or two (steam blast followed by hemicellulase treatment);

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

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

Preferably, the sugarcane bagasse that is part of the process proposed herein is derived from a diffuser, but it is possible to use bagasse from milling or a mixture of diffuser and milling.

Optionally, chemical catalysts and oxidizing agents may be used in the steam pre-treatment step. Optionally, the pretreatment process comprises a step of enzymatic hydrolysis of hemicelluloses present in the steam pretreated biomass using a mixture of hemicellulases.

Optionally the enzymatic hydrolysis processes of hemicelluloses and cellulose occur simultaneously with carbohydrate fermentation.

A further object of the present invention is a fermented product obtained by the fermentation process described above. In particular, the fermented product is ethanol.

Brief Description of the Figures

Figure 1 shows a process for ethanol production from lignocellulosic biomass in which the pretreatment step can be carried out by physicochemical and enzymatic route (hemicellulases), and the subsequent steps of enzymatic hydrolysis of cellulose (cellulases) and fermentation (Saccharomyces cerevisiae) occur separately (Process SHF).

Figure 2 shows a process for ethanol production in which the pretreatment of lignocellulosic biomass can be performed in three steps: physicochemical treatment, enzymatic hydrolysis (hemicellulases) for removal of residual hemicellulose and washing of the final pretreated bagasse. Subsequently, the pretreated bagasse is subjected to the simultaneous process of enzymatic hydrolysis of cellulose (cellulases) and fermentation (Saccharomyees cerevisiae) - (SSF process).

Figure 3 shows a process for ethanol production from lignocellulosic biomass in which the pretreatment step is performed in two steps: physical-chemical treatment and washing of the final pretreated bagasse. Subsequently, the pretreated bagasse is subjected to a simultaneous process of enzymatic hydrolysis of cellulose (cellulases) and residual hemicellulose (hemicellulases) and fermentation (Saccharomyees cerevisiae) - (SSF process).

Figure 4 shows the chromatogram for the liquid fraction after steam pretreatment of the biomass. The presence of xylose shows partial removal of hemicelluloses from biomass after the process. Glucose from sucrose from sugarcane milling and bagasse pulp.

Figure 5 shows the chromatogram for compositional analysis of steam pretreated biomass (BPT). The presence of xylans (characterized as xylose) in the biomass is evident, indicating non-total removal of hemicelluloses.

Figure 6 shows the chromatogram for the SHF experiment. Enzyme hydrolyzate from pre-treated biomass (BPT) washed using only cellulases. Lower glucose production is observed in relation to the condition presented in Figure 4, due to the recalcitrance exerted by the residual hemicelluloses present in the biomass.

Figure 7 shows the chromatogram for the SHF experiment. Enzyme hydrolyzate from pre-treated biomass (BPT) washed using mixture of cellulases and hemicellulases. Higher glucose production is observed in relation to the condition presented in Figure 3, due to the action of hemicellulases, showing synergy between the different enzymes. The presence of sorbitol in the enzymatic hydrolyzate characterizes a potential activation of glucose in the presence of hemicellulases, which remove hemicelluloses from biomass.

Figure 8 shows the fermentation-broth chromatogram of the SSF1 experiments using only hemicellulases (without cellulases) and washed pretreated biomass (BPT). Significant xylose production and selective removal of residual hemicelluloses present in PTB are observed (Cxiiose: CgijCOse> 100). No glucose production or consumption was observed during the process, as evidenced by the almost zero production of ethanol by the yeast present in the reaction medium. Here, the efficiency of the enzymatic pretreatment (using hemicellulases) of the biomass supplementary to the steam pretreatment process in the selective removal of residual hemicelluloses with minimal cellulosic loss is configured.

Figure 9 shows the chromatogram of the fermented broth from the SSF experiments using only cellulases (without hemicellulases) and washed pretreated biomass (BPT). Minimal glucose concentration is observed, but with high production and simultaneous consumption, as evidenced by ethanol production by the yeasts present in the reaction medium (Cetanei> 0.5% w / w). The presence of xylose (Cxylose = 1.20 g / kg) in the fermented liquid shows moderate xylanolytic activity of cellulases, indicating the potential of enzymatic pretreatment of biomass with hemicellulases, supplemental to the steam pretreatment process, in selective removal. of residual hemicelluloses with minimal cellulosic loss.

Figure 10 shows the chromatogram of the fermented broth from SSF experiments of washed and hemicellular treated BPT. Minimal glucose concentration is observed, but with high production and simultaneous consumption, evidenced by the production of ethanol (0.5% w / w) by the yeasts present in the reaction medium. The presence of xylose in the broth at a concentration of about 0.49 g / kg is observed, but with high production and consumption, evidenced by the production of products distinct from ethanol, characterizing potential synergy between hemicellulases and yeast. Ethanol production shows that there was no inhibition of yeast activity due to the presence of hemicellulases in the medium.

Figure 11 shows chromatograms of fermented broth from the washed BPT SSF experiments with cellulases in medium supplemented with saccharide solution (sugarcane broth). (A) Time 0 (zero), (B) Time 24 h. Almost integral consumption of glucose is observed, evidencing that the addition of a solution rich in fermentable sugars ("booster") does not inhibit the fermentative activity of yeast. From the values observed in Fig. 10 for ethanol concentrations, a positive synergy between the enzymatic hydrolyzate (produced during SSF) and the sucrose-rich solution used as a booster is evident. This evidence enhances the use of molasses or sugarcane juice as a booster in SSF processes.

Figure 12 shows chromatograms pertaining to: (A) SSF experiment using steam pretreated bagasse [ECa = 0.531% w / w] in the presence of cellulolytic enzymes. (B) Fermentation of sugarcane juice [CEb = 4.341% w / w]. (C) SSF experiment using steam pretreated bagasse and medium supplemented with sucrose booster juice in the presence of a mixture of cellulolytics [ECc = 4,885% w / w],

Cei = ethanol concentration from Experiment i.

Detailed Description of the Invention

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

Liqnocellulosic Plant Biomass

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, with the preferred diffuser bagasse); cereal straws such as wheat, rice, rye, barley, oats, corn and the like (e.g. switchgrass elephant grass); wood; banana tree stalks and stalks; cactus plants and combinations thereof. In addition, lignocellulosic materials may further comprise cardboard, sawdust, newspaper and similar agroindustrial or 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% hemicelluloses and about 20-30% lignin. The remainder consists of smaller amounts of ashes, soluble phenolic compounds and fatty acids, as well as other constituents called extractives. Cellulose and plant tissue hemicelluloses consist of structural carbohydrates (e.g., glycans, xylans, mananas) and are generally referred to as the saccharide fraction. Iignin is the phenolic fraction of plant biomass.

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

(a) pre-treatment of lignocellulosic plant biomass with a steam blast step; whether or not followed by a second biomass pretreatment step using hemicellulases (enzymes);

(b) enzymatic hydrolysis of the pretreated biomass using mixtures containing cellulases and / or β-glycosidases and / or hemicellulases;

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

Pretreatment Step

The removal of hemicelluloses from lignocellulosic biomass such as sugarcane bagasse and maize straw increases the accessibility of cellulose to chemical (eg acid or alkali) or biochemical (eg enzymes) agents that will convert it into fermentable sugars, particularly glucose. . The combination of chemical (e.g. steam treatment) and biochemical (e.g. hemicellular hydrolysis) processes enables fragmentation and subsequent removal of hemicelluloses present in high selectivity biomass, making it a particularly efficient pre-treatment alternative. In the present invention, the concept of pre-treatment is the set of operational steps that follow the preparation of the biomass and the reactor feed and precede the enzymatic hydrolysis of the cellulose.

The pretreatment of lignocellulosic biomass with saturated steam or pressurized water at different temperature and process time levels promotes partial removal of hemicelluloses, increasing the accessibility of the cellulosic matrix to cellulolytic enzymes (cellulases). The steam used can be generated in the chamber itself or introduced into it. Pretreatment processes can be performed under the steam explosion (STEX or wet explosion - WEX) configuration, in which rapid decompression occurs in material discharge. A "cooking" configuration can also be used in which no rapid decompression is used after the process time. Generally, steam blasting processes tend to produce greater fragmentation of hemicelluloses, making cellulose more accessible to chemical and enzymatic agents.

In principle, harsher process conditions lead to higher hemicellulose extraction, however such conditions often result in high cellulose losses due to their fragmentation. In addition, degradation of the carbohydrates available in the process may occur in compounds such as furfural and hydroxy methyl furfural (HMF).

The use of catalysts and adjuvants such as oxidants (e.g. O2 and H2O2) in acidic, alkaline or neutral systems makes it possible to remove high amounts of hemicelluloses under less severe process conditions.

In addition, high selectivity can be evidenced in terms of predominant extraction of hemicelluloses, preserving the cellulosic content of the pretreated biomass. Depending on the availability of water and operating conditions, the deacetylation of hemicelluloses can be intensified to produce acetic acid, which acts in this context as a "native catalyst" of hemicelluloses, characterizing an autocatalytic process.

The pretreatment disclosed herein may comprise one or more catalysts, including, but not limited to, inorganic acids such as H 2 SO 4, HCl, HNO 3, H 3 PO 4 or combinations thereof, organic acids such as acetic, formic acid and carbonic (H2CO3), oxides (SO2, MnO2, CO2), sulfates (FeSO4, AI2 (SO4) 3), carbonates (FeCO3, CaCO3, Na2CO3) and metal chlorides (FeCI3, ZnCl2, MnCI2, CaCl2, AICI3).

Compounds that are generated during pretreatment can also act as catalysts, being called autocatalysts. Examples of these compounds include organic acids such as acetic acid and carbohydrate degrading acids, as well as lignin-derived phenolic species. Catalysts may be present in a concentration ranging from 0.10% to 8%, relative to the dry mass of the biomass, but should preferably be in the range of 0.25% to 6%.

Removal of Residual Hemicelluloses

Despite the benefits associated with the use of catalysts in the pre-treatment stage, residual hemicelluloses are often evident in the pretreated biomass. In this context, the selective removal of these hemicelluloses under moderate process conditions results in pretreated biomasses with greater accessibility to cellulose, enabling the hydrolysis reactor to be loaded with greater amounts of this component.

Selective enzymatic treatment of the pretreated biomass by physicochemical process (eg STEX, WEX) under moderate temperature conditions provides a high cellulosic material in addition to the high accessibility of cellulose to enzymes, eg mixtures of cellulases and β -glycosidases. The present invention provides complementary enzymatic pretreatment of lignocellulosic biomass (pretreated by physicochemical processes) based on the use of hemicellulases (enzymes that break down hemicelluloses) under temperature conditions ranging from 35 ° C to 60 ° C, preferably between 45 ° C and 55 ° C with reaction times ranging from 6h to 48h, preferably from 12h to 24h. Such enzymes may be employed in SHF and SSF systems as a residual hemicellulose pretreatment agent. Hemicellulases are in proportions ranging from 5.5% to 30%, preferably from 12% to 22%, in relation to the amount of pretreated biomass (dry basis).

The data presented in Table 1 allow us to observe, as previously mentioned, the beneficial effects that the presence of auxiliary enzymes such as β-glycosidases and, above all, hemicellulases bring to the hydrolytic system in question. Auxiliary hemicellulase present in the reaction medium acts by removing the remaining hemicelluloses from the steam blast process, thereby conferring greater cellulase accessibility to cellulose. With this, higher conversions of cellulose to glucose are observed and, therefore, higher concentrations of glucose can be detected in the final hydrolyzate that will be destined for fermentation. It is evident, therefore, that the enzymatic treatment of steam-blown bagasse with auxiliary enzymes is indeed efficient.

TABLE 1 - Enzymatic hydrolysis of pretreated biomass. Glucose concentration in the liquid fraction (hydrolyzed).

<table> table see original document page 15 </column> </row> <table>

Substrate: Pre-steamed sugarcane bagasse.

Fermentation Process

The fermentation step can be performed after enzymatic hydrolysis via a process known as Separated Hydrolysis and Fermentation (SHF), or concurrently 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 ranging from 80 g / L to 820 g / L, preferably from 120 g / L to 200 g / L, may be added to the reaction medium. molasses or sugarcane juice).

The present invention also contemplates the possibility of simultaneously carrying out enzymatic pretreatment of hemicelluloses, enzymatic hydrolysis of cellulose and fermentation simultaneously, characterizing a Consolidated BioProcess (CBP) using steam pretreated biomass or other agents as substrate.

Figures 1, 2 and 3 show the flow charts concerning the different configurations contemplated in the present invention.

Fig. 1 presents a typical SHF process consisting of three distinct steps (pretreatment, enzymatic hydrolysis and fermentation) performed separately. Cellulosic biomass is subjected to a physicochemical pretreatment (e.g. STEX, WEX, etc.) in the presence or absence of catalysts, oxidizers and other inputs. The catalysts are present in a concentration ranging from 0.10% to 8% relative to the dry mass of biomass, preferably between 0.25% and 6%. The solids charge varies between 5% and 60%, preferably between 15% and 50% relative to the total mass.

The biomass remains in the reactor for a reaction time between 30 s and 60 min, preferably between 5 min and 15 min, at a process temperature ranging from 1100 ° C to 240 ° C, preferably 180 ° C to 2100 ° C. After the reaction time has elapsed, the reactor is preferably discharged through sudden decompression and optionally without sudden decompression.

Depending on the amount of remaining or residual hemicelluloses, the pretreated biomass may be subjected to additional pretreatment using specific enzymes (hemicellulases). Hemicellulases are in proportions ranging from 5.5% to 30%, preferably from 12% to 22%, relative to the pretreated biomass mass (dry basis). The biomass remains in the reactor for a reaction time of 6h to 48h, preferably between 12h and 24h, at a process temperature ranging from 35 ° C to 60 ° C, preferably between 45 ° C and 55 ° C.

The solids charge varies between 2% and 20%, preferably between 7.5% and 15% relative to the total mass. After pretreatment, the biomass may optionally be washed with water or acidic or alkaline solutions at room temperature.

Subsequently, the pretreated biomass is subjected to enzymatic hydrolysis and sent to a preferably discontinuous and optionally continuous reactor together with specific enzymes (cellulases and β-glycosidases). The cellulases are in proportions ranging from 5.5% to 30%, preferably between 12% and 22%, relative to the pretreated biomass mass (dry basis). The β-glycosidases are in proportions ranging from 2.5% to 15%, preferably from 5.5% to 7.5%, in relation to the pretreated biomass mass (dry basis). The solids charge varies between 2% and 20%, preferably between 7.5% and 15% relative to the total mass.

The biomass remains in the reactor for a reaction time ranging from 12h to 72h, preferably between 24h and 48h, at a process temperature ranging from 35 ° C to 60 ° C, preferably between 45 ° C and 55 ° C. After the reaction time, the reactor is discharged and the material is subjected to a separation process, preferably filtration and optionally centrifugation or ultrafiltration, yielding a liquid fraction (enzymatic hydrolyzate) and a solid fraction (lignocellulosic or cellulignin residue).

The enzymatic hydrolyzate is then subjected to an ethanolic fermentation preferably using Saccharomices cerevisae yeasts and optionally using Zymomonas mobilis bacteria.

The enzymatic hydrolyzate is preferably mixed with molasses or sugarcane juice and optionally fermented individually without any mixture with molasses or juice.

As shown in Fig. 1, the enzymatic hydrolysis and fermentation steps occur separately.

Fig. 2 presents a typical SSF process in which the enzymatic hydrolysis and fermentation steps are performed simultaneously. Cellulosic biomass is subjected to a physicochemical pretreatment (e.g. STEX, WEX, etc.) in the presence or absence of catalysts, oxidizers and other inputs. Where pretreatment includes catalysts, they will be present in concentrations ranging from 0.10% to 8% relative to the dry mass of the biomass, preferably 0.25% to 6%. The solids charge varies between 5% and 60%, preferably between 15% and 50% relative to the total mass.

The biomass remains in the reactor for a reaction time of 30 s to 60 min, preferably 5 min to 15 min, at a process temperature ranging from 110 ° C to 240 ° C, preferably 180 ° C to 210 ° C. After the reaction time has elapsed, the reactor is preferably discharged through sudden decompression and optionally without sudden decompression.

Depending on the amount of remaining or residual hemicelluloses, the pretreated biomass may be subjected to additional pretreatment using specific enzymes (hemicellulases). Hemicellulases are in proportions ranging from 5.5% to 30%, preferably from 12% to 22%, relative to the pretreated biomass mass (dry basis). The biomass remains in the reactor for a reaction time ranging from 6h to 48h, preferably from 12h to 24h, at a process temperature ranging from 35 ° C to 60 ° C, preferably from 45 ° C to 55 ° C. The solids charge varies between 2% and 20%, preferably between 7.5% and 15% relative to the total mass. After pretreatment, the biomass may optionally be washed with water or acidic or alkaline solutions at room temperature.

The pretreated biomass is subjected to enzymatic hydrolysis and is sent to a fermentor-type reactor, preferably discontinuous and optionally continuous, together with specific enzymes (cellulases and β-glycosidases). The cellulases are in proportions ranging from 5.5% to 30%, preferably from 12% to 22%, relative to the pretreated biomass mass (dry basis). The β-glycosidases are in proportions ranging from 2.5% to 15%, preferably from 5.5% to 7.5%, in relation to the pretreated biomass mass (dry basis). The solids charge varies between 2% and 20%, preferably between 7.5% and 15% relative to the total mass. As enzymatic hydrolysis is carried out sugars, preferably glucose, are produced, which are subjected to an ethanolic fermentation preferably using Saccharomices cerevisae yeast and, optionally, Zymomonas mobilis bacteria.

Preferably, a concentrated saccharide solution (booster), preferably molasses and optionally sugarcane juice, is added to the fermenter at the beginning or during the enzymatic hydrolysis process, but may be operated without the addition of saccharide solution. The saccharide solution (booster) has a sugar concentration ranging from 80 g / l to 820 g / l, preferably from 120 g / l to 200 g / l.

The biomass remains in the reactor for a reaction time ranging from 12h to 72h, preferably from 24h to 48h, at a process temperature ranging from 30 ° C to 50 ° C, preferably from 37 ° C to 40 ° C with a pH of the medium. reaction range from 4.2 to 5.5, preferably from 4.8 to 5.0.

After the reaction time has elapsed, the reactor is discharged and the material is subjected to a separation process, preferably filtration and optionally centrifugation, producing a liquid fraction (wort) and a solid fraction (lignocellulosic or cellulignin residue).

As shown in Fig. 2, the enzymatic hydrolysis and fermentation steps occur simultaneously.

Fig. 3 presents an advanced SSF process in which the enzymatic pretreatment with hemicellulases, enzymatic hydrolysis with cellulases and fermentation steps are performed simultaneously. Cellulosic biomass is subjected to a physicochemical pretreatment (e.g. STEX, WEX, etc.) in the presence or absence of catalysts, oxidizers and other inputs. The catalysts are present in a concentration ranging from 0.10% to 8% relative to the dry mass of the biomass, preferably between 0.25% and 6%. The solids charge varies between 5% and 60%, preferably between 15% and 50% relative to the total mass. The biomass remains in the reactor for a reaction time between 30 s and 60 min, preferably between 5 min and 15 min, at a process temperature ranging from 1100 ° C to 240 ° C, preferably 180 ° C to 2100 ° C. After the reaction time has elapsed, the reactor is preferably discharged through sudden decompression and optionally without sudden decompression.

The pretreated biomass is subjected to additional pretreatment using specific enzymes (hemicellulases). The pre-treated biomass by physicochemical processes is routed to a preferably discontinuous and optionally continuous fermenter-type reactor together with specific enzymes (hemicellulases). Hemicellulases are in proportions ranging from 5.5% to 30%, preferably from 12% to 22%, relative to the pretreated biomass mass (dry basis). The biomass remains in the reactor for a reaction time ranging from 6h to 48h, preferably from 12h to 24h, at a process temperature ranging from 35 ° C to 60 ° C, preferably from 45 ° C to 55 ° C. The solids charge varies between 2% and 20%, preferably between 7.5% and 15% relative to the total mass.

Concomitantly with the pre-treatment process with hemicellulases or after 1h to 6h, preferably 4h, from the beginning of this step, the enzymatic hydrolysis and cellulose hydrolysis step is initiated in the fermenter, characterizing an SSF process. The cellulases are in proportions ranging from 5.5% to 30%, preferably from 12% to 22%, relative to the pretreated biomass mass (dry basis). The β-glycosidases are in proportions ranging from 2.5% to 15%, preferably from 5.5% to 7.5%, in relation to the pretreated biomass mass (dry basis). The solids charge varies between 2% and 20%, preferably between 7.5% and 15% relative to the total mass. As the enzymatic hydrolysis reaction takes place sugars, preferably glucose, are produced, which are subjected to an ethanolic fermentation preferably using Saccharomices cerevisae yeast and optionally using Zymomonas mobilis bacteria. Preferably, a concentrated saccharide solution (booster), preferably molasses and optionally sugar cane juice, is added to the fermenter at the beginning or during the enzymatic hydrolysis process, but may be operated without addition of saccharide solution. The saccharide solution ("oosfer") has sugar concentrations ranging from 80 g / l to 820 g / l, preferably from 120 g / l to 200 g / l.

The biomass remains in the reactor for a reaction time ranging from 12h to 72h, preferably from 24h to 48h, at a process temperature ranging from 30 ° C to 50 ° C, preferably from 37 ° C to 40 ° C with a pH of the medium. reaction range from 4.2 to 5.5, preferably from -4.8 to 5.0.

After the reaction time has elapsed, the reactor is discharged and the material is subjected to a separation process, preferably filtration and optionally centrifugation, producing a liquid fraction (wort) and a solid fraction (lignocellulosic or cellulignin residue).

As shown in Fig. 3, the hemicellulose pretreatment, enzymatic hydrolysis and fermentation steps occur simultaneously.

Claims (35)

Fermentation Process Comprising Pretreatment Step, Enzymatic Hydrolysis and Fermented Product Using Lignocellulosic Plant Biomass 1. A fermentative process comprising a pretreatment step comprising the following steps: (a) physicochemical (steam blast) pretreatment of plant biomass optionally comprising an additional hemicellulase biomass pretreatment step; (b) enzymatic hydrolysis of the pretreated biomass using cellulases or the cellulases + β-glycosidases mixture; and c) Fermentation of carbohydrates produced from enzymatic hydrolysis of pretreated biomass. Fermentation process according to Claim 1, characterized in that the plant biomass is chosen from the group comprising herbaceous biomass, cultivars, cardboard, sawdust, newspaper and / or a mixture thereof. Fermentation process according to Claim 2, characterized in that the cultivars are selected from the group comprising: (i) plants belonging to the genera Lolium, Spartina, Panicum, Miscanthus and combinations thereof; ii) sugarcane bagasse; (iii) cereal straws selected from the group comprising wheat, rice, rye, reed, barley, oats, maize, switchgrass elephant grass, wood, banana trunks and / or stalks and combinations thereof; iv) combinations of i), ii) and / or iii). Fermentation process according to Claim 3, characterized in that the sugarcane bagasse comes from mills and / or diffusers. Fermentation process according to claim 1, characterized in that the vegetable biomass comprises: i) about 35-50% cellulose; ii) about 20-35% hemicelluloses; and iii) about 20-30% lignin. Fermentation process according to Claim 1, characterized in that the steam is saturated or overheated and can be generated in and / or introduced into the chamber itself. Fermentation process according to Claim 1, characterized in that it comprises catalysts selected from the group comprising H2SO4, HCl1 HNO3, H3PO4, acetic, formic and carbonic acid (H2CO3), SO2, MnO2, CO2, FeSO4, AI2 (SO4). 3, FeCO3, CaCO3, Na2CO3, FeCl3, ZnCl2, MnCl2, CaCl21 AlCl3 and mixture thereof. A fermentative process according to claim 7, characterized in that the catalysts are generated during the pretreatment step. Fermentation process according to Claim 8, characterized in that the catalyst is acetic acid. Fermentation process according to claim 7, characterized in that the catalyst is present in a concentration ranging from 0.10% to 8% relative to the dry mass of biomass. A fermentative process according to claim 10, characterized in that the catalyst is present in a concentration ranging from 0.25% to 6% relative to the dry mass of biomass. Fermentation process according to Claim 1, characterized in that the temperature of the steam explosion process is in the range from 110 ° C to 240 ° C. Fermentation process according to Claim 12, characterized in that the temperature of the steam blasting process is in the range from 180 ° C to 210 ° C. A fermentative process according to claim 1, characterized in that the reaction time of the steam blast step is from 30s to 60mins. A fermentative process according to claim 14, characterized in that the reaction time of the steam blast step is from 5min to 15min. Fermentation process according to claim 1, characterized in that the hemicellulases are present in a concentration ranging from 5.5% to 30% with respect to the dry mass of pretreated biomass. Fermentation process according to claim 16, characterized in that the hemicellulases are present in a concentration ranging from 12% to 22% relative to the dry weight of pretreated biomass. Fermentation process according to Claim 1, characterized in that the hemicellulases are used at temperatures ranging from 35 ° C to 60 ° C. Fermentation process according to Claim 18, characterized in that the hemicellulases are used at temperatures in the range of 40 ° C to 50 ° C. Fermentation process according to claim 1, characterized in that the reaction time with hemicellulases ranges from 6h to 48h. Fermentation process according to Claim 20, characterized in that the reaction time with hemicellulases varies between 12h and 24h. Fermentation process according to claim 1, characterized in that the pretreatment step comprises from 5% to 60% w / w total solids charge. Fermentation process according to Claim 22, characterized in that the total solids charge is in the range from 3% to 25% w / w. Fermentation process according to claim 1, characterized in that it comprises a step of washing with water or acid or alkaline solutions at room temperature after step a) and before step b). Fermentation process according to claim 1, characterized in that the cellulases are in proportions ranging from 5.5% to 30% relative to the pretreated biomass mass (dry base). Fermentation process according to Claim 25, characterized in that the cellulases are in proportions ranging from 12% to 22% relative to the pretreated biomass mass (dry basis). Fermentation process according to Claim 1, characterized in that the β-glycosidases are in proportions ranging from 2.5% to -15% relative to the pretreated biomass mass (dry basis). Fermentation process according to claim 27, characterized in that the β-glycosidases are in proportions ranging from 5.5% to -7.5% relative to the pretreated biomass mass (dry base). A fermentative process according to claim 1, characterized in that the hydrolysis step comprises from 2% to 20% w / w total solids charge. Fermentation process according to Claim 29, characterized in that the total solids charge is in the range from 2.5% to 25% w / w. Fermentation process according to claim 1, characterized in that the fermentation is carried out with yeast and / or bacteria. Fermentation process according to Claim 31, characterized in that the yeast is Saccharomices cerevisae. A fermentative process according to claim 31, characterized in that the bacterium is Zymomonas mobilis. Fermentation process according to claim 1, characterized in that it comprises the addition of a concentrated saccharide solution with a concentration of 80 g / l to 820 g / l. A fermentative process according to claim 34, characterized in that the concentrated saccharide solution is molasses and / or sugarcane juice.