TWI551688B - Process for the production of ethanol by the fermentation of cellulosic hydrolysates - Google Patents

Description

Method for fermenting cellulose hydrolyzate to produce ethanol

The present invention relates to a process for the preparation of ethanol comprising the steps of providing a cellulosic hydrolysate comprising fermentable sugars and at least one selected from the group consisting of Fermentation inhibitors in the group: acetic acid, hydroxymethyl furfural, furfural, and phenolic compounds; adding an aqueous solution containing ammonia to the a cellulose hydrolyzate to form a mixed solution having a pH falling within 5.5 to 7.0; and a xylose-utilizing Saccharomyces cerevisiae added to the mixture and allowing The Saccharomyces cerevisiae is used to ferment the mixture, so that ethanol is produced.

Lignocellulosic biomass is a renewable energy resource that is produced in large quantities through industrial and agroforestry operations. Chemical or biological methods to convert lignocellulosic biomass into biomass (ie, cellulose alcohol) (cellulosic ethanol)] has been extensively studied and explored. Compared to chemical methods, biological methods are particularly valued because they are more environmentally friendly and have lower energy requirements.

Since lignocellulose has a special crystalline structure, which mainly contains cellulose, hemicellulose, and lignin, it must pass the following in the process of producing cellulose alcohol. Steps: (1) The lignocellulose is subjected to appropriate pretreatment and hydrolysis processes to release cellulose and hemicellulose into six-carbon sugars (mainly glucose) and five-carbon sugars (mainly Xylose); and (2) microbial fermentation of the obtained hexose and five carbon sugars to produce ethanol. In the process of using lignocellulosic biomass to produce ethanol, a cellulose hydrolyzate is obtained. In addition to the six-carbon sugar and the five-carbon sugar, the cellulose hydrolyzate has a five-carbon sugar due to the above pretreatment. Or a fermentation inhibitor such as acetic acid, furfural, hydroxymethane aldehyde (HMF), and a phenolic compound released by hexose carbon and lignin degradation.

Saccharomyces cerevisiae (Saccharomyces cerevisiae) has a six-carbon sugars (e.g. glucose) a cellulose hydrolyzate into ethanol metabolism, and thus have been widely used for industrial fermentation industry location. However, Saccharomyces cerevisiae cannot effectively utilize a large amount of five-carbon sugars (for example, xylose, arabinose, etc.) present in the cellulose hydrolyzate. Therefore, in recent years, many studies have improved the above problems by means of genetic metabolic engineering. For example, a gene associated with the xylose metabolic pathway in xylose-fermentation bacteria is introduced into Saccharomyces cerevisiae, and the resulting xylose-fermenting Saccharomyces cerevisiae is obtained . It is possible to co-ferment five-carbon sugars and six-carbon sugars efficiently, thereby increasing the yield of ethanol (B. Hahn-Hägerdal et al . (2007), Appl. Microbiol. Biotechnol. , 74: 937-953).

In addition, among TW I450963 (corresponding to US 20140087438 A1 and CN 103695329 A), the applicant also discloses a xylose reductase gene, xylitol dehydrogenase gene and xylone. Saccharomyces cerevisiae of the xylulokinase gene, deposited under the accession number DSM 25508 in the German Collection of Microorganisms and Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ), and deposited in the Hsinchu Food Industry under the registration number BCRC 920077 Development Research Institute's Center for Biological Resources Conservation and Research (BCRC of FIRDI). The entire disclosure of this prior patent is incorporated herein by reference.

Although it has been disclosed in the literature that the production of ethanol is improved by genetic modification of Saccharomyces cerevisiae, the fermentation inhibitor contained in the cellulose hydrolyzate inhibits the growth and fermentation of Saccharomyces cerevisiae, and makes five carbon sugars and six carbons. The utilization of sugar is reduced, which in turn affects the yield of ethanol.

In order to reduce the adverse effects of fermentation inhibitors, it has been studied to improve the tolerance of Saccharomyces cerevisiae to these fermentation inhibitors by acclimation or genetic modification of the strains. The production of ethanol. For example, Carlos Martín et al (2007), Biosource Technology, 98:. 1767-1773 in, Carlos Martín, who will be a recombinant xylose - utilizing Saccharomyces cerevisiae in a culture added has bagasse hydrolyzate (Sugarcane Bagasse hydrolysates), and a bagasse hydrolyzate containing various inhibitors [including phenolic compounds, furfural and adiphatic acid] is added to the medium in a continuous feed to enhance the inhibitor The concentration, whereby the xylose-utilized Saccharomyces cerevisiae can be adapted. The resulting adapted strain can convert furfural to HMF at a faster rate and has a higher ethanol yield than the unadapted original strain.

In Petersson A. et al . (2006), Yeast , 23: 455-464, Petersson A. et al . allow S. cerevisiae to express a large number of NADPH-dependent alcohol dehydrogenation responsible for the reduction of HMF by gene transfer. Enzyme (NADPH-dependant alcohol dehydrogenase) ADH6 gene, the resulting yeast strain overexpressing the ADH6 gene has been experimentally confirmed to be able to effectively utilize the glucose contained in the artificial medium in the presence of furfural, and has a high HMF conversion rate. (conversion rate) [It is also possible to reduce more HMF to 5-hydroxymethylfurfuryl alcohol], which means that the strain overexpressing ADH6 is more tolerant to HMF.

In addition, in Gorsich SW et al . (2006), Appl Microbiol Biotechnol ., 71: 339-349, Gorsich SW et al . found that S. cerevisiae strains overexpressing ZWF-1 were in artificial glucose supplemented with toxic concentrations of furfural. It can grow in the medium, which means that the strain which overexpresses ZWF-1 is resistant to furfural and can more efficiently convert lignocellulose to ethanol.

Although the above studies involving genetic engineering have improved the adaptability of Saccharomyces cerevisiae to fermentation inhibitors, the stability of the transgenic genes needs to be tracked and confirmed for a long time. If it is to be applied to the fermentation industry on a large scale, a large amount of fermentation can be achieved. For the purpose of producing ethanol, there are still limitations and high risks.

For this reason, it has been proposed to remove the fermentation inhibitor in the cellulose hydrolyzate by detoxification without using a strain transfected with a specific gene to reduce the adverse effects of the fermentation inhibitor. Common detoxification treatments include: (1) physical detoxification, such as evaporation and membrane mediated detoxification; (2) chemical detoxification, such as Neutralization, calcium hydroxide overliming, activated charcoal treatment, and ion exchange resins; and (3) biological detoxification, such as the use of shellac Enzyme (laccase) or lignin peroxidase (lignin peroxidase). However, these detoxification treatments can make the process of cellulose alcohol more complicated, and the cost required is relatively increased, and at the same time, the reducing sugar in the cellulose hydrolyzate may be lost.

Therefore, a method for producing ethanol without directly removing the fermentation inhibitor and directly fermenting the cellulose hydrolyzate has been developed, thereby simplifying the operation procedure, reducing the cost and energy consumption, and efficiently utilizing the organic waste to produce ethanol. For use as a clean biomass energy, it will be our business. Hope to achieve.

Summary of invention

Thus, in a first aspect, the present invention provides a method for producing ethanol, comprising: providing a cellulose hydrolyzate comprising a fermentable sugar and at least one selected from the group consisting of a fermentation inhibitor: acetic acid, hydroxymethane aldehyde, furfural, and a phenolic compound; adding an aqueous solution containing ammonia to the cellulose hydrolyzate to form a mixed solution having a pH falling within 5.5 to 7.0; A xylose-utilized Saccharomyces cerevisiae is added to the mixture, and the Saccharomyces cerevisiae is allowed to ferment the mixture, so that ethanol is produced.

The above and other objects, features and advantages of the present invention will become apparent from

Detailed description of the invention

All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains, unless otherwise defined. A person skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used in the practice of the present invention. invention. Of course, the invention is in no way limited by the methods and materials described.

In order to effectively solve the problem of energy depletion and to avoid the continued destruction of the ecological environment, researchers in the technical field are committed to the development of clean biomass. The use of biomass waste as a fermentation raw material for yeast production of ethanol not only solves the environmental problems caused by waste, but also achieves the purpose of recycling waste, and thus has become the most important research direction.

Plant-based biomass contains a large amount of components such as cellulose, hemicellulose and lignin, which are intertwined to form a complex and tough network structure, which makes the use of plant-based biomass to produce ethanol. Restricted, so the plant biomass is usually first subjected to a suitable pretreatment [such as thermochemical degradation] to help its network structure open, and then hydrolyzed into five carbon sugars via cellulolytic enzymes. And six carbon sugar. However, the fermentation inhibitors (for example, acetic acid, furfural, and hydroxymethane aldehyde, etc.) produced by the plant biomass after the above treatment affect the ability of the yeast to ferment ethanol. In order to solve this problem, the currently more commonly used method is to genetically modify or acclimate the yeast strain, or to further remove the fermentation inhibitor to increase the overall yield of ethanol. However, genetically engineered or domesticated yeast strains need to be regularly tracked to ensure that their properties remain, and removal of inhibitors can complicate the process and result in cost increases.

Based on the above, the Applicant is committed to developing a method for more efficient use of cellulose in the industrial fermentation industry and large-scale production of ethanol. In particular, the method does not require detoxification of the cellulose hydrolyzate. There is no need to reduce the effect of fermentation inhibitors on S. cerevisiae strains by domestication or genetic recombination, while at the same time improving xylose utilization of xylose-utilized Saccharomyces cerevisiae for cellulose hydrolysate and reducing xylitol Accumulation, which in turn increases ethanol production.

Thus, after a plurality of studies by the applicant, the present invention provides a method for preparing ethanol comprising the steps of: providing a cellulose hydrolyzate comprising a fermentable sugar and at least one selected from the group consisting of Fermentation inhibitors in the group: acetic acid, hydroxymethylfurfural, furfural, and phenolic compounds; adding an aqueous solution containing ammonia to the cellulose hydrolyzate to form a pH having a pH of 5.5 to 7.0 a mixture; and a xylose-utilized Saccharomyces cerevisiae is added to the mixture, and the Saccharomyces cerevisiae is allowed to ferment the mixture, so that ethanol is produced.

According to the present invention, the pH of the mixed solution is maintained to fall within the range of 5.5 to 7.0 by adding the aqueous solution containing ammonia during the fermentation of the mixed solution.

Preferably, the pH of the mixture is maintained in the range of 5.5 to 6.5. More preferably, the pH of the mixture is maintained in the range of 5.8 to 6.2. In a preferred embodiment of the invention, the pH of the mixture is maintained at 6.0.

As used herein, the terms "xylose-utilized Saccharomyces cerevisiae" and "xylose-fermented Saccharomyces cerevisiae" can be used interchangeably, and it is intended to encompass all S. cerevisiae strains having xylose fermentative ability, These include those that are readily available to those skilled in the art (for example, those available from domestic or foreign hosting agencies), or those that do not have the ability to ferment xylose using genetic modification methods commonly used in the art. Yeast strains are produced by gene transfer. Saccharomyces cerevisiae strains for xylose-utilization can be found, but are not limited to: Carlos Marín et al . (2002), Enzyme and Microbial Technology , 31: 274-282; Miroslav Sedlak and Nancy WYHo (2004), Applied Biochemistry and Biotechnology , 113-116: 405-416; B. Hahn-Hägerdal et al . (2007) (same as above); Zhang A. et al . (2007), Letters in Applied Microbiology , 44: 212-217; Hubmann G. et Al . (2011), Applied and Environmental Microbiology , 77: 5857-5867; US 20130196399 A1; and TW I450963 and the like.

In a preferred embodiment of the present invention, the xylose-fermented Saccharomyces cerevisiae is a Saccharomyces cerevisiae strain in which both the fps1 gene and the gpd2 gene are deleted or destroyed. In a more preferred embodiment of the present invention, the xylose-fermented Saccharomyces cerevisiae is deposited at the Center for Bioresource Conservation and Research (BCRC of FIRDI) by the Food Industry Development Institute under the registration number BCRC 920077. The fps1 gene and the gpd2 gene in the genomic DNA of Saccharomyces cerevisiae are deleted or destroyed.

As used herein, the term "aqueous solution comprising ammonia" means the addition of the following substances in an aqueous medium: ammonia gas (NH ₃ ), containing ammonium ions ( A compound of NH ₄ ) [such as ammonium hydroxide, ammonium chloride or ammonium sulfate], a compound which releases ammonia after decomposition (such as urea), and combinations thereof.

According to the invention, the aqueous ammonia-containing solution is an aqueous ammonium hydroxide solution (i.e., aqueous ammonia) or an aqueous solution of ammonium chloride. In a preferred embodiment of the invention, the aqueous ammonia-containing solution is aqueous ammonia.

According to the present invention, the cellulose hydrolyzate is prepared by sequentially subjecting a cellulosic biomass to a pretreatment and a hydrolysis treatment.

As used herein, the terms "cellulose hydrolysate" and "lignocellulosic hydrolysate" and "biomass hydrolysate" may be used interchangeably and mean saccharification by biomass. The product produced, wherein the term "saccharification", refers to the production of fermentable sugars from polysaccharides.

As used herein, the terms "cellulosic biomass" and "lignocellulosic biomass" are used interchangeably and mean any including cellulose, hemicellulose, lignin, starch, oligo. Cellulose material of sugar and/or monosaccharide.

In accordance with the present invention, the cellulosic biomass can be derived from a single source, or the cellulosic biomass can comprise a mixture derived from a variety of sources. For example, the cellulosic biomass can be a mixture of corn stover and corn cobs, or a mixture of grass and leaves.

Cellulosic biomass suitable for use in the present invention includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial Industrial solid waste, sludge from paper manufacture, yard waste, wood waste, and forestry waste, and combinations thereof.

Preferably, the cellulosic biomass is selected from the group consisting of: miscanthus, softwood, hardwood, corn cobs, crop residues [ Such as corn husks, corn stover, grasses, wheat straw, barley straw, hay, rice straw, switchgrass Switchgrass), waste paper, sugarcane bagasse, sorghum plant material, soybean plant material, ground components from grains, trees, Branches, roots, leaves, sawdust, shrubs and bushes, vegetables, fruits, and flowers, and combinations thereof.

Pretreatments suitable for use in the present invention include, but are not limited to, steam explosion, thermal chemical pretreatment, mechanical comminution, acid treatment, organosolve, sulfite pretreatment (sulfite) Pretreatment), and combinations thereof. In a preferred embodiment of the invention, the cellulosic biomass is subjected to a steam burst treatment prior to being subjected to the hydrolysis treatment.

According to the present invention, the cellulose hydrolyzate may be further mixed with a nutrient salt composition prior to the addition of the aqueous ammonia-containing solution. In a preferred embodiment of the invention, the nutrient salt composition comprises the following salts: (NH ₄ ) ₂ SO ₄ , MgSO ₄ ‧7H ₂ O, and KH ₂ PO ₄ .

Example

The invention is further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting.

General experimental materials: 1. Preparation of Δ fps1 △ gpd2 double mutant S. cerevisiae inoculation source (inoculum of the Δ fps1 △ gpd2 double mutant of Saccharomyces cerevisiae ):

Saccharomyces cerevisiae strains used in the following experiments is a double mutant △ fps1 △ gpd2 Saccharomyces cerevisiae, which is substantially based on Zhang A. et al. (2007) ( same as above) and Hubmann G. et al. (2011 ) (as described above) was prepared as described in the above. In short, first, according to the method described in Zhang A. et al . (2007) (same above), a Saccharomyces cerevisiae BCRC 920077 that can co-ferment five- and six-carbon sugars (from the applicant) The prior patent TW I450963 is also deleted by the fps1 gene deposited in DSMZ with the accession number DSM 25508, and the resulting Δ fps1 mutant strain is further based on the disclosure of Hubmann G. et al . (2011) (supra). a method to remove gpd2 gene, thereby to obtain S. △ fps1 △ gpd2 double mutant yeast strain (hereinafter referred to as "double-mutant yeast was Saccharomyces").

Thereafter, the double mutant S. cerevisiae obtained above was inoculated into YPD medium, and cultured in a constant temperature shaking incubator (30 ° C, 200 rpm) until the OD ₆₀₀ value reached 20. Next, the formed culture was centrifuged, and then the cell pellet was collected and washed several times with sterile water, and then the cells were sufficiently suspended with sterile water, whereby the obtained cell suspension was taken as the following The double-mutated Saccharomyces cerevisiae inoculation source in the case.

2. Preparation of cellulosic hydrolysate:

The cellulose biomass used in the following examples includes: rice straw (available from Hongyuan Agricultural Products Co., Ltd.) and Miscanthus (available from the Chiayi University Agricultural Reformation Field). First, the grass or rice straw was cut into an appropriate size, and then pulverized by a pulverizer, followed by adding a 3 wt% sulfuric acid solution to be uniformly mixed, and the reaction was carried out at 60 ° C for 60 minutes. Thereafter, the obtained mixture was placed in a vertical cylindrical high pressure cooking tank (purchased from Qifu Industrial Co., Ltd.), and then steam was introduced and heated at a temperature of 190 to 200 ° C for 7 minutes. . Next, the cooking liquid obtained by the acid-catalyzed steam explosion steam explosion pretreatment is adjusted to pH 5.0 with NaOH, and then a cellulase and hemicellulose are added. a mixture of enzymes (hemicellulase) (Novozymes Cellic ^® CTec3, used in an amount of 0.12 g of enzyme mixture per gram of cellulosic biomass), and cellulolytic at a temperature of 50 ° C and a stirring rate of 120 rpm The cellulolytic processes lasted for 72 hours, thereby obtaining an acid-catalyzed vapor bursting of rice straw or Miscanthus cellulose hydrolyzate.

In addition, a steam cracked rice straw or Miscanthus cellulose hydrolysate is generally prepared in accordance with the above manner, except that: When the pretreatment is carried out, water is used instead of the sulfuric acid solution to mix with the pulverized cellulose biomass.

3. In the following examples, the nutrient salt composition for addition to the cellulose hydrolyzate has the formulation shown in Table 1 below.

General experimental method: 1. High performance liquid chromatography (HPLC) analysis:

In the following examples, the components contained in the cellulose hydrolyzate and the fermentation metabolites and their concentrations (g/L) are referred to the National Renewable Energy Laboratory (NREL). Laboratory analytical procedures (LAPs) for standard biomass analysis, and by using a high performance liquid chromatography (DIONEX) equipped with a refractive index detector (refractive index detector) Ultimate 3000) was used for the measurement. The column used and the operating conditions were as follows: the analytical column was Aminex HPX-87H column (BioRad); the mobile phase was 5 mM sulfuric acid (in water); the flow rate was controlled to 0.6 mL/ Minutes; sample injection volume was 20 μL; RI temperature was controlled at 65 °C.

In addition, for comparison, different concentrations of glucose are used. (1.2-24 mg/mL), xylose (1.2-24 mg/mL), xylitol (0.2-6 mg/mL), glycerol (0.2-8 mg/mL), hydroxymethane aldehyde (HMF) (0.2-8 mg/mL) ), acetic acid (0.2-12mg/mL), ethanol (1.0-15mg/mL), furfural (0.2-8mg/mL) and phenolic compounds (0.2-8mg/mL) as the control standard For the same analysis, these chemicals were purchased from Sigma.

Example 1. Effect of NH ₄ OH on the production of ethanol by double-mutated Saccharomyces cerevisiae using an acid-catalyzed steam explosion of rice straw cellulose hydrolysate as a substrate

In the present embodiment, one of the rice stem cellulose hydrolyzates which have been acid-catalyzed by steam explosion according to the second item "Preparation of Cellulose Hydrolyzate" of the above "General Experimental Materials" is taken as a substrate and discussed in fermentation. The addition of NH ₄ OH alkaline solution during the process affects the ethanol production by fermentation of glucose and xylose by double-mutated Saccharomyces cerevisiae.

Further, for comparison, once detoxified rice straw cellulose hydrolyzate (hereinafter referred to as "detoxified hydrolyzate") was taken together for the same fermentation experiment. The detoxification treatment of the rice straw cellulose hydrolyzate is based on the overliming and the use of activated carbon as described in Jing-Ping Ge et al . (2011), African Journal of Microbiology Research , 5: 1163-1168. Charcoal) to carry out, and slightly modified. In short, according to the "General Experimental Materials" item 2 "Preparation of Cellulose Hydrolyzate", one of the acid-catalyzed steam-exploded rice straw cellulose hydrolyzates is adjusted with an appropriate amount of Ca(OH) ₂ to adjust the pH. The value was changed to 10, and then the reaction was allowed to stand at room temperature for 120 minutes, followed by centrifugation at 10,000 rpm for 5 minutes, and then the supernatant was collected and the pH was adjusted to 5.0 with a H ₂ SO ₄ solution. Thereafter, 5% activated carbon (purchased from Hetai Co., Ltd., model MAX-703) was added and reacted at 40 ° C for 60 minutes, followed by filtration and centrifugation to remove the activated carbon filter cake. The solvent is dissolved, and the obtained filtrate is the detoxified hydrolyzate.

Before the fermentation to produce ethanol, the acid-catalyzed vapor bursting of the rice straw cellulose hydrolyzate and the detoxified hydrolyzate are respectively described in the first item "High Performance Liquid Chromatography Analysis" of the "General Experimental Method" above. Methods were used to measure the concentrations of various sugars and inhibitors, and the measured results are shown in Table 2 below.

It can be seen from Table 2 that the detoxified hydrolyzate does not contain HMF, furfural and phenolic compounds, and contains only a small amount of acetic acid, as compared with the acid-catalyzed vapor bursting of the rice straw cellulose hydrolyzed liquid. In addition, the concentration of glucose in the detoxified hydrolysate is also low. It can be seen that the detoxification treatment removes most of the inhibitors, but at the same time, the glucose content is lowered.

experimental method:

The acid-catalyzed steam-exploded rice straw cellulose hydrolyzate was divided into two groups (including a control group and a NH ₄ OH group), and then a nutrient salt composition as shown in Table 1 was added to each group of hydrolyzate. The pH was then adjusted to 6 with NH ₄ OH, respectively. Thereafter, the above obtained based on the "General Experimental Materials" item 1 "prepared △ fps1 △ gpd2 double mutant of Saccharomyces cerevisiae inoculum" double mutant was inoculated with Saccharomyces cerevisiae to a source of 1:20 (v The inoculum amount of /v) was inoculated into each flask and mixed uniformly, and then placed in a constant temperature shaking culture chamber, and subjected to a fermentation reaction at a temperature of 30 ° C and an oscillation rate of 150 rpm for 72 hours. Throughout the fermentation, the group of NH ₄ OH NH ₄ OH was added timely to be maintained at a pH of 6 to make. As for the control group, no treatment was done.

In addition, the detoxified hydrolyzate was subjected to the same experiment generally in the manner of the above NH ₄ OH group, except that NaOH was used instead of NH ₄ OH. Thereafter, the fermentation cultures of each group were centrifuged at 12,000 rpm for 10 minutes, and the obtained fermentation metabolites were respectively subjected to the method described in the first item "High Performance Liquid Chromatography Analysis" of the "General Experimental Method" above. To analyze the content of glucose, xylose, xylitol and ethanol. The utilization rate of glucose or xylose is expressed as a percentage value (%) of the measured total amount of glucose or xylose relative to the total amount measured before fermentation. In addition, the total amount of xylitol measured after fermentation is compared with the total amount of xylose used during fermentation (ie, the total amount of xylose measured before fermentation minus the one measured after fermentation). A ratio (i.e., xylitol production) is obtained, and if the ratio is lower, the more xylose is converted to ethanol instead of being accumulated in the form of xylitol. As for the yield of ethanol, the total amount of ethanol produced after fermentation and the total amount of glucose and xylose during fermentation (ie, the total amount of glucose and xylose measured before fermentation are subtracted from the fermentation). The measured one is calculated by comparison.

result:

The results measured in this experiment are shown in Table 3 below.

It can be seen from Table 3 that when the rice straw cellulose hydrolyzate which is catalyzed by acid-catalyzed vapor is used as a substrate (whether or not NH ₄ OH is added during the fermentation), or directly using a detoxified hydrolyzate as a substrate, each group of fermentation metabolites The measured glucose utilization rate was 100%. As for the xylose utilization rate and ethanol production, the NH ₄ OH group was significantly better than the control group. It can be seen that the addition of NH ₄ OH during the fermentation process can significantly increase the utilization of xylose, thereby increasing the yield of ethanol.

In addition, the NH ₄ OH group of the rice straw cellulose hydrolyzate which was catalyzed by the acid-catalyzed vapor was compared with the detoxified hydrolyzate, and it was found that the acid-catalyzed vapor bursting of the rice straw cellulose hydrolyzate was used as the substrate and NH _{4 was used.} OH to adjust the composition of the hydrolyzate or to add NH ₄ OH during the fermentation can result in better xylose utilization and ethanol production. The results of this experiment show that the technique disclosed in the present invention can effectively improve the utilization of xylose and the yield of ethanol without further passing through the detoxification treatment step of the hydrolyzate.

Example 2. Effect of adding different alkaline solutions on the ethanol production by double-mutation of Saccharomyces cerevisiae under the use of acid-catalyzed steam explosion of rice straw and Miscanthus cellulose hydrolyzate as matrix

In this embodiment, an acid-catalyzed steam-exploded rice straw or Miscanthus cellulose hydrolyzate prepared according to the second item "Preparation of Cellulose Hydrolysate" of the above "General Experimental Materials" is used as a substrate, and different Alkaline solution (including NH ₄ OH, KOH and NaOH solution) to adjust the pH of the hydrolyzate, and add these alkaline solutions during fermentation to ferment the double-mutated Saccharomyces cerevisiae using glucose and xylose The effect of ethanol.

Before the fermentation to produce ethanol, the acid-catalyzed steam-exploded rice straw or Miscanthus cellulose hydrolyzate is measured according to the method described in the above-mentioned "General Experimental Method", Item 1 "High Performance Liquid Chromatography Analysis". The concentrations of sugars and acetic acid were measured and the results obtained are shown in Table 4 below.

experimental method:

First, the acid-catalyzed steam-exploded rice straw and Miscanthus cellulose hydrolyzate were each divided into three groups (including KOH group, NaOH group and NH ₄ OH group), and then the pH was adjusted with KOH, NaOH and NH ₄ OH, respectively. Up to 6, the obtained rice straw and Miscanthus cellulose hydrolysate are generally subjected to the fermentation method of the double-mutated Saccharomyces cerevisiae in accordance with the experimental method described in Example 1 above, except that: During the whole fermentation period, NaOH, KOH, and NH ₄ OH were added to the KOH group, the NaOH group, and the NH ₄ OH group, respectively, so that their pH values were maintained at 6.0.

Thereafter, the fermentation metabolites of each group were analyzed for the contents of glucose, xylose, xylitol, and ethanol according to the method described in the first item "High Performance Liquid Chromatography Analysis" of the "General Experimental Method" above. Then, the sugar utilization rate, the xylitol production amount, and the ethanol production were respectively calculated according to the methods described in Example 1.

result:

The results measured in this experiment are shown in Table 5 below.

It can be seen from Table 5 that whether the rice straw or Miscanthus cellulose hydrolyzate is used as the substrate, the glucose utilization rate measured by each group of fermentation metabolites is 100%. As for the xylose utilization rate and ethanol production, the NH ₄ OH group Significantly better than the NaOH group or the KOH group. It can be seen that, compared with NaOH and KOH, adjusting the pH of the hydrolyzate with NH ₄ OH and adding NH ₄ OH during the fermentation process can significantly improve the utilization of xylose and reduce the accumulation of xylitol. This in turn increases the yield of ethanol.

Example 3. Under the use of a steam-exploded Miscanthus cellulose hydrolyzate as a substrate, by adding NH ₄ Effect of OH to adjust pH value for ethanol production by double-mutation Saccharomyces cerevisiae

In this embodiment, a steam-exploded Miscanthus cellulose hydrolyzate prepared according to the second item "Preparation of Cellulose Hydrolyzate" of the above "General Experimental Materials" is used as a substrate, and the addition of NH by fermentation is discussed. ₄ OH was used to maintain the pH of the Miscanthus cellulose hydrolyzate at 5, 6 or 7 respectively, and the effect of fermentation on ethanol production by using glucose and xylose for the double-mutated Saccharomyces cerevisiae.

The steam-exploded Miscanthus cellulose hydrolyzate was measured to contain 7.3 g/L according to the method described in the above-mentioned "General Experimental Method", "High Performance Liquid Chromatography" before the fermentation to produce ethanol. Acetic acid, 81.8 g/L glucose, and 34.7 g/L xylose.

experimental method:

First, the steam-exploded Miscanthus cellulose hydrolyzate was divided into three groups (including pH 5 group, pH 6 group, and pH 7 group), followed by NH ₄ OH to pH 5 group, pH 6 group, and pH 7 group. The pH values were adjusted to 5, 6, and 7, respectively, and the obtained groups of Miscanthus cellulose hydrolysate were generally subjected to the fermentation method of the double-mutated Saccharomyces cerevisiae ethanol production experiment according to the experimental method described in Example 1 above. The difference was that NH ₄ OH was added to each group at a time during the entire fermentation so that their pH values were maintained at the initial values.

Thereafter, the fermentation metabolites of each group were analyzed for the contents of glucose, xylose, xylitol, and ethanol according to the method described in the first item "High Performance Liquid Chromatography Analysis" of the "General Experimental Method" above. Then, the sugar utilization rate, the xylitol production amount, and the ethanol production were respectively calculated according to the methods described in Example 1.

result:

The results measured in this experiment are shown in Table 6 below.

It can be seen from Table 6 that regardless of the pH of the steam-exploded Miscanthus cellulose hydrolysate maintained at 5, 6, or 7, the glucose utilization rate measured by each group of fermentation metabolites is 100%, as for the utilization of xylose. The amount of xylitol and ethanol production were significantly better in the pH 6 group than in the pH 7 and pH 5 groups. Applicants believe that during the fermentation process, by adding NH ₄ OH to control the pH of the cellulose hydrolysate at 6.0, the optimal xylose utilization can be achieved, and the accumulation of xylitol can be effectively reduced. Increase the production of ethanol.

All of the patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of a conflict, the detailed description of the case (including definitions) will prevail.

While the invention has been described with respect to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only by the scope of the appended claims.

Claims (7)

A method for preparing ethanol, comprising the steps of: providing a cellulose hydrolyzate comprising a fermentable sugar and at least two fermentation inhibitors selected from the group consisting of: acetic acid, hydroxyl a furfural, a furfural, and a phenolic compound; adding an aqueous solution containing ammonia to the cellulose hydrolyzate to form a mixed solution having a pH falling within 5.5 to 7.0; and using a xylose-utilizing wine Yeast is added to the mixture, and the Saccharomyces cerevisiae is allowed to ferment the mixture, so that ethanol is produced. The method of claim 1, wherein the pH of the mixed solution is maintained to fall within a range of 5.5 to 7.0 by adding the aqueous solution containing ammonia during the fermentation of the mixed solution. The method of claim 1, wherein the mixture has a pH falling within the range of 5.5 to 6.5. The method of claim 1, wherein the mixture has a pH falling within the range of 5.8 to 6.2. The method of claim 1, wherein the aqueous ammonia-containing solution is selected from the group consisting of aqueous ammonium hydroxide solution, aqueous ammonium sulfate solution, aqueous ammonium chloride solution, and combinations thereof. The method of claim 1, wherein the fermentable sugar comprises a five carbon sugar and a six carbon sugar. The method of claim 6, wherein the fermentable sugar comprises glucose and xylose.