CN106032542B - Method for producing ethanol by fermenting cellulose hydrolysate - Google Patents
Method for producing ethanol by fermenting cellulose hydrolysate Download PDFInfo
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 195
- 239000000413 hydrolysate Substances 0.000 title claims abstract description 73
- 239000001913 cellulose Substances 0.000 title claims abstract description 63
- 229920002678 cellulose Polymers 0.000 title claims abstract description 63
- 238000004519 manufacturing process Methods 0.000 title claims description 31
- 238000000855 fermentation Methods 0.000 claims abstract description 68
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- 238000000034 method Methods 0.000 claims abstract description 43
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 39
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 30
- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical compound O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 claims abstract description 24
- 235000000346 sugar Nutrition 0.000 claims abstract description 22
- 239000003112 inhibitor Substances 0.000 claims abstract description 20
- NOEGNKMFWQHSLB-UHFFFAOYSA-N 5-hydroxymethylfurfural Chemical compound OCC1=CC=C(C=O)O1 NOEGNKMFWQHSLB-UHFFFAOYSA-N 0.000 claims abstract description 12
- RJGBSYZFOCAGQY-UHFFFAOYSA-N hydroxymethylfurfural Natural products COC1=CC=C(C=O)O1 RJGBSYZFOCAGQY-UHFFFAOYSA-N 0.000 claims abstract description 12
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a process for fermenting a cellulosic hydrolysate to produce ethanol. The invention discloses a method for preparing ethanol, which comprises the following steps: providing a cellulosic hydrolysate comprising fermentable sugars and at least one fermentation inhibitor selected from the group consisting of: acetic acid, hydroxymethylfurfural, furfural, and phenolic compounds; adding an aqueous solution containing ammonia to the cellulose hydrolysate to form a mixed solution having a pH value falling within a range of 5.5 to 7.0; and adding xylose-utilizing saccharomyces cerevisiae to the mixed liquor and allowing the saccharomyces cerevisiae to ferment the mixed liquor such that ethanol is produced.
Description
Technical Field
The invention relates to a method for producing ethanol, comprising the following steps: providing a cellulosic hydrolysate comprising fermentable sugars (sugars) and at least one fermentation inhibitor selected from the group consisting of: acetic acid, hydroxymethylfurfural (hydroxymethy furfuran), furfural (furfurfurfuran), and phenolic compounds (phenolic compounds); adding an aqueous solution (aqueous solution containing ammonia) to the cellulose hydrolysate to form a mixed liquor having a pH value falling within the range of 5.5 to 7.0; and adding xylose-utilizing Saccharomyces cerevisiae to the mixed liquor and allowing the Saccharomyces cerevisiae to ferment the mixed liquor such that ethanol is produced.
Background
Lignocellulosic biomass (lignocellulosic biomass) is a renewable energy resource (renewable energy resources) that is produced in large quantities via industrial and agroforestric operations. Chemical or biological methods have been widely studied and discussed for converting lignocellulosic biomass into biomass energy, i.e., cellulosic ethanol (ethanol). Biological processes are particularly appreciated because of their ecological benefits and lower energy requirements compared to chemical processes.
Since lignocellulose has a specific crystalline structure, which mainly contains cellulose (cellulose), hemicellulose (hemicellulose), and lignin (lignin), the following 2 steps must be performed in the process of producing cellulosic ethanol: (1) subjecting lignocellulose to appropriate pretreatment (pretreatment) and hydrolysis (hydrolysis process) to release six-carbon sugar (mainly glucose) and five-carbon sugar (mainly xylose) from cellulose and hemicellulose; and (2) microbial fermentation (microbiological fermentation) of the resulting six and five carbon sugars to produce ethanol. In the process of producing ethanol from lignocellulosic biomass, a cellulose hydrolysate containing pentoses, and the like, and also fermentation inhibitors (for example, acetic acid, furfural, Hydroxymethylfurfural (HMF), and phenolic compounds) released in part by degradation of pentoses, hexoses, and lignin due to the above pretreatment is obtained.
Saccharomyces cerevisiae (Saccharomyces cerevisiae) has a metabolic capability of converting six-carbon sugars (e.g., glucose) in cellulose hydrolysate into ethanol, and thus has been widely used in the industrial fermentation industry. However, saccharomyces cerevisiae cannot effectively utilize a large amount of pentoses (e.g., xylose, arabinose, etc.) present in the cellulose hydrolysate. Therefore, in recent years, many studies have been made to improve the above problems by means of genetic metabolic engineering (genetic metabolic engineering). For example, xylose-fermenting Saccharomyces cerevisiae (xylose-fermenting Saccharomyces cerevisiae) obtained by introducing genes related to xylose metabolic pathway in xylose-fermenting bacteria into Saccharomyces cerevisiae can efficiently co-ferment pentose and hexose, thereby increasing the yield of ethanol (B).
Et al (2007), appl. Microbiol. Biotechnol.,74: 937-.
In addition, among TW I450963 (corresponding to US 20140087438a1 and CN 103695329a), the applicant also disclosed a saccharomyces cerevisiae having xylose reductase gene (xylose reductase gene), xylitol dehydrogenase gene (xylotol dehydrogenase gene) and xylulokinase gene (xylokinase gene), which was deposited at the german collection of microorganisms and cell cultures (DSMZ) under the accession number DSM 25508, and deposited at the bio-resources preservation and research center of the new bamboo food industry development institute (BCRC of FIRDI) under the accession number BCRC 920077. The entire disclosure of this prior patent is incorporated herein by reference.
Although the literature discloses that the yield of ethanol is improved by genetically modifying saccharomyces cerevisiae, a fermentation inhibitor contained in the cellulose hydrolysate inhibits the growth and fermentation of saccharomyces cerevisiae, so that the utilization rate of pentose and hexose is reduced, and the yield of ethanol is affected.
In order to reduce the adverse effects caused by fermentation inhibitors, studies have been made to improve the tolerance of saccharomyces cerevisiae to these fermentation inhibitors by using domestication (inoculation) or genetic modification (genetic modification) of the strain, thereby increasing the yield of ethanol. For example, in Carlos Mart i n et al (2007), Biosource Technology,98: 1767-. The resulting adapted strain can convert furfural and HMF at a faster rate and with higher ethanol production compared to the unadapted original strain.
In Petersson A. et al (2006), Yeast,23: 455-.
In addition, in Gorsich SW et al (2006), applied Microbiol Biotechnol.,71:339-349, Gorsich SW et al found that a Saccharomyces cerevisiae strain overexpressing ZWF-1 could grow in an artificial glucose medium supplemented with a toxic concentration of furfural, indicating that the strain overexpressing ZWF-1 was tolerant to furfural and more efficiently converted lignocellulose to ethanol.
Although the above studies related to genetic engineering have improved the adaptability of saccharomyces cerevisiae to fermentation inhibitors, the stability of the transgene should be tracked and confirmed for a long time, and if it is applied to the fermentation industry on a large scale for the purpose of producing ethanol by mass fermentation, it still has limitations and high risks.
For this reason, it has been proposed to remove the fermentation inhibitors in the cellulose hydrolysate by detoxification treatment (detoxification) without using a strain having a special transgene to reduce the adverse effects of the fermentation inhibitors. Common detoxification treatments include: (1) physical detoxification treatments such as evaporation (evaporation) and membrane-mediated detoxification treatments (membrane-mediated detoxification); (2) chemical detoxification (neutralization), such as calcium hydroxide super-liming (neutralization), activated carbon treatment (activated carbon treatment), and ion exchange resins (ion exchange resins); and (3) biological detoxification, such as the use of laccase (laccase) or lignin peroxidase (lignin peroxidase), and the like. However, these detoxification treatments make the cellulosic ethanol process more complicated and the cost required is relatively high, and the reducing sugar in the cellulosic hydrolysate may be lost.
Therefore, it is expected to achieve the goal of developing a method for directly fermenting a cellulose hydrolysate to produce ethanol without removing fermentation inhibitors, thereby simplifying the operation procedure, reducing the cost and energy consumption, and effectively utilizing organic wastes to produce ethanol as a clean biomass energy source.
Disclosure of Invention
Accordingly, in a first aspect, the present invention provides a process for the production of ethanol, comprising:
providing a cellulosic hydrolysate comprising fermentable sugars and at least one fermentation inhibitor selected from the group consisting of: acetic acid, hydroxymethylfurfural, furfural, and phenolic compounds;
adding an aqueous solution containing ammonia to the cellulose hydrolysate to form a mixed solution having a pH value falling within a range of 5.5 to 7.0; and
xylose-utilizing saccharomyces cerevisiae is added to the mixed liquor and allowed to ferment the mixed liquor such that ethanol is produced.
The method of the present invention, during fermentation of the mixed liquor, maintains the pH of the mixed liquor within the range of 5.5 to 7.0 by adding the aqueous solution containing ammonia.
In the method of the present invention, the mixed solution has a pH value falling within a range of 5.5 to 6.5.
In the method of the present invention, the mixed solution has a pH value falling within a range of 5.8 to 6.2.
The process of the invention, the aqueous ammonia-containing solution is selected from the group consisting of: aqueous ammonium hydroxide, aqueous ammonium sulfate, aqueous ammonium chloride, and combinations thereof.
In the process of the present invention, the fermentable sugar comprises a five carbon sugar and a six carbon sugar.
The method of the present invention, the fermentable sugar comprises glucose and xylose.
The method of the present invention comprises the steps of sequentially pretreating and hydrolyzing a cellulosic biomass selected from the group consisting of: bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper making, yard waste, waste and forestry waste, and combinations thereof.
In the method of the present invention, the cellulosic biomass is selected from the group consisting of: miscanthus, softwood, hardwood, corn cobs, crop residues, corn stover, grasses, wheat straw, barley straw, hay, rice straw, switchgrass, waste paper, bagasse, milo plant material, soybean plant material, ground components derived from grain, trees, branches, roots, leaves, wood chips, shrubs and bushes, vegetables, fruits and flowers, and combinations thereof.
The pretreatment is selected from the group consisting of: steam explosion, thermal pre-chemical treatment, mechanical comminution, acid treatment, organic dissolution, sulfite pretreatment, and combinations thereof.
Detailed Description
The foregoing and other objects, features and advantages of the invention will be apparent from the following detailed description and preferred embodiments.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Those of skill 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. Of course, the present invention is in no way limited to the methods and materials described.
In order to effectively solve the problem of energy exhaustion and avoid the continuous destruction of the ecological environment, researchers in the field are dedicated to developing clean biomass energy. The method for producing ethanol by using biomass waste as the fermentation raw material of yeast not only can solve the environmental protection problem caused by the waste, but also can achieve the purpose of recycling the waste, thereby becoming the most important research direction at present.
Plant biomass contains a large amount of cellulose, hemicellulose and lignin, which are intertwined and coated to form a complex and tough network structure, which is limited in the process of producing ethanol from plant biomass, and thus the plant biomass is usually subjected to a proper pretreatment such as thermo-chemical decomposition (thermochemical decomposition) to help the network structure to open, and then hydrolyzed into pentose and hexose by cellulolytic enzyme. However, fermentation inhibitors (e.g., acetic acid, furfural, and hydroxymethylfurfural, etc.) produced by the plant biomass after the above treatment affect the ability of yeast to ferment to produce ethanol. To solve this problem, the current method used more often is to genetically modify or acclimate the yeast strains or further remove the fermentation inhibitors, thereby increasing the overall yield of ethanol. However, the genetically modified or acclimatized yeast strain needs to be periodically traced to ensure that its characteristic is still present, and removal of the inhibitor complicates the process and causes a drastic increase in cost.
Based on the above, the applicant has been working on developing a method for more effectively utilizing cellulose and producing ethanol on a large scale for the industrial fermentation industry, and particularly, the method does not require detoxification treatment of cellulose hydrolysate and does not require domestication or gene recombination to reduce the influence of fermentation inhibitors on saccharomyces cerevisiae strains, and can improve xylose utilization rate of xylose-utilizing saccharomyces cerevisiae on cellulose hydrolysate, and reduce accumulation of xylitol, thereby increasing ethanol yield.
Accordingly, the applicant has studied in various ways and provided in the present invention a process for the preparation of ethanol, comprising the steps of:
providing a cellulosic hydrolysate comprising fermentable sugars and at least one fermentation inhibitor selected from the group consisting of: acetic acid, hydroxymethylfurfural, furfural, and phenolic compounds;
adding an aqueous solution containing ammonia to the cellulose hydrolysate to form a mixed solution having a pH value falling within a range of 5.5 to 7.0; and
xylose-utilizing saccharomyces cerevisiae is added to the mixed liquor and allowed to ferment the mixed liquor such that ethanol is produced.
According to the present invention, the pH of the mixed liquor is maintained within the range of 5.5 to 7.0 by adding the aqueous solution containing ammonia during the fermentation of the mixed liquor.
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-utilizing Saccharomyces cerevisiae" and "xylose-fermenting Saccharomyces cerevisiae" are used interchangeably and are intended to encompass all strains of Saccharomyces cerevisiae that have xylose fermentation capability, including those readily available to those skilled in the art (e.g., from domestic or foreign depositories) or produced by transgenesis of natural strains of Saccharomyces cerevisiae that do not have xylose fermentation capability using genetic engineering methods customary in the art. See, but not limited to, Saccharomyces cerevisiae strains for xylose-utilization: carlos Marcii n et al (2002), Enzyme and Microbial Technology,31: 274-; miroslav Sedlak and Nancy W.Y.Ho (2004), Applied Biochemistry and Biotechnology, 113-; B.
et al (2007) (supra); zhang A. et al (2007), Letters in Applied Microbiology,44: 212-; hubmann G. et al (2011), Applied and Environmental Microbiology,77: 5857-; US 20130196399a 1; and TW I450963.
In a preferred embodiment of the invention, the xylose-fermenting Saccharomyces cerevisiae is a strain of Saccharomyces cerevisiae in which both the fps1 gene and the gpd2 gene have been deleted or disrupted. In a more preferred embodiment of the present invention, the xylose-fermented Saccharomyces cerevisiae was prepared by deleting or disrupting the fps1 gene and the gpd2 gene from the genomic DNA of a strain of Saccharomyces cerevisiae deposited under accession number BCRC 920077 at the center for the conservation and research of biological resources of the institute of food industry development (BCRC of FIRDI).
As used herein, the term "aqueous solution containing ammonia (ammonia)" means that the following are added to an aqueous medium: ammonia gas (ammonia gas) (NH)3) Comprising ammonium ions (NH)4) Such as ammonium hydroxide, ammonium chloride or ammonium sulfate (a)mmonium sulfate)]Compounds that release ammonia upon 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 ammonium chloride solution. In a preferred embodiment of the invention, the aqueous ammonia-containing solution is aqueous ammonia.
According to the present invention, the cellulose hydrolysate is prepared by sequentially performing pretreatment and hydrolysis treatment on cellulose biomass (cellulose biomass).
As used herein, the terms "cellulosic hydrolysate" and "lignocellulosic hydrolysate" are used interchangeably and refer to the product resulting from saccharification of biomass, wherein the term "saccharification" refers to the production of fermentable sugars from polysaccharides.
As used herein, the terms "cellulosic biomass" and "lignocellulosic biomass (lignocellulosic biomass)" are used interchangeably and refer to any cellulosic material that includes cellulose, hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides.
According to the present invention, the cellulosic biomass may be derived from a single source, or the cellulosic biomass may comprise a mixture derived from multiple sources. For example, the cellulosic biomass may be a mixture of corn stover (corn stover) and corn cobs (corn cobs), or a mixture of grass (grass) and leaves.
Cellulosic biomass suitable for use in the present invention includes, but is not limited to: biological energy crops (bioenergy crops), agricultural residues (agricultural residues), municipal solid waste (municipal solid waste), industrial solid waste (industrial solid waste), sludge from paper manufacture (sludge from paper manufacture), yard waste (yard waste), wood waste (wood waste) and forestry waste (forest waste), and combinations thereof.
Preferably, the cellulosic biomass is selected from the group consisting of: miscanthus (miscanthus), softwood (softwood), hardwood (hardwood), corncobs (corn cobs), crop residues (crop residues) [ such as corn husks (corn husks) ], corn stover (corn stover), grasses (grasses), wheat straw (straw), barley straw (barley straw), hay (hay), rice straw (rice straw), switchgrass (switchgrass), waste paper (water paper), bagasse (bagasse), millet plant material (sorghum plant), soybean plant material (sorobean plant), ground components obtained from (grasses), trees, branches, roots, leaves, wood chips (sawdusts), shrubs (shrubs), shrubs, flowers, and fruits, and combinations thereof.
Suitable pretreatments for use in the present invention include, but are not limited to: steam explosion (steam expansion), thermal chemical pretreatment (thermal chemical pretreatment), mechanical comminution, acid treatment, organic dissolution (organosolve), sulfite pretreatment (sulfite pretreatment), and combinations thereof. In a preferred embodiment of the invention, the cellulosic biomass is subjected to a steam explosion treatment prior to being subjected to the hydrolysis treatment.
According to the invention, the cellulose hydrolysate can be further mixed with a nutrient salt composition before being added to the aqueous ammonia-containing solution. In a preferred embodiment of the invention, the nutrient salt composition comprises the following salts: (NH)4)2SO4、MgSO4·7H2O and KH2PO4。
< example >
The invention will be further described with respect to the following examples, but it should be understood that these examples are for illustration only and are not to be construed as limitations on the practice of the invention.
General experimental materials:
1. preparation of a Δ fps1 Δ gpd2 double mutant Saccharomyces cerevisiae inoculum (inoculum of the Δ fps1 Δ gpd2 double mutant of Saccharomyces cerevisiae):
the saccharomyces cerevisiae strain used in the following experiments was a Δ fps1 Δ gpd2 double mutant saccharomyces cerevisiae, prepared substantially according to the methods described in Zhang a. et al (2007) (supra) and Hubmann g. et al (2011) (supra). Briefly, first, the fps1 gene of saccharomyces cerevisiae BCRC 920077 (obtained from applicant's prior patent application TW I450963 and deposited at DSMZ under the accession number DSM 25508) which can co-ferment pentoses and hexoses was deleted according to the method described in Zhang a. et al (2007) (same as above), and then the resulting Δ fps1 mutant strain was further deleted for the gpd2 gene according to the method disclosed in Hubmann g. et al (2011) (same as above), thereby obtaining a Δ fps1 Δ gpd2 double-mutated saccharomyces cerevisiae strain (hereinafter referred to as "double-mutated saccharomyces cerevisiae").
Then, the double-mutated Saccharomyces cerevisiae obtained above was inoculated into YPD medium, and cultured in a constant temperature shaking incubator (30 ℃, 200rpm) until OD600The value reached 20. Subsequently, the resulting culture was centrifuged, and then cell pellets were collected and washed several times with sterile water, followed by sufficiently suspending the cells with sterile water, whereby the resulting cell suspension was taken as a source of the double-mutated strain of Saccharomyces cerevisiae in the following examples.
2. Preparation of cellulose hydrolysate (cellulose hydrolysate):
cellulosic biomass used in the following examples includes: rice straw (from the great agriculture commercial business) and miscanthus sinensis (from the great agriculture and reform farm of jiayi). Firstly, cutting the miscanthus or the rice straw into proper sizes, then crushing the miscanthus or the rice straw by a crusher, then adding 3 wt% of sulfuric acid solution to mix the materials evenly, and reacting the materials at 60 ℃ for 60 minutes. Thereafter, the resulting mixture was placed in a vertical cylindrical autoclave (available from seven-fu industrial products, ltd.), followed by introduction of steam and heating at a temperature of 190 to 200 ℃ for 7 minutes. Next, the pH of the cooking solution obtained by the acid-catalyzed steam explosion pretreatment (acid-catalyzed steam explosion pretreatment) was adjusted to 5.0 with NaOH, and a mixture of cellulase (cellulose) and hemicellulase (hemicellulase) (Novozymes) was added thereto
CTec3 in an amount of 0.12 g enzyme mixture/g cellulosic biomass) and subjected to a cellulolytic treatment (cellulolytic processes) at a temperature of 50 ℃ and a stirring rate of 120rpm for 72 hours, thereby obtaining an acid catalyzed steam exploded rice straw or miscanthus cellulose hydrolysate.
In addition, steam exploded straw or miscanthus cellulose hydrolysate was prepared substantially as described above, except that: in the pretreatment, water is mixed with the pulverized cellulosic biomass instead of the sulfuric acid solution.
3. In the examples below, the nutrient salt composition for addition to the cellulose hydrolysate had the formulation shown in table 1 below.
TABLE 1 formulation of nutrient salt composition
| Composition (I) | Concentration (g/L) |
| (NH4)2SO4 | 5 |
| MgSO4·7H2O | 0.05 |
| KH2PO4 | 3 |
General experimental methods:
1. high Performance Liquid Chromatography (HPLC) analysis:
in the following examples, the components contained in cellulose hydrolysate and fermentation metabolites (fermentation metabolites) and their concentrations (g/L) were determined by reference to Laboratory Analytical Procedures (LAPs) for standard biomass analysis, issued by the National Renewable Energy Laboratory (NREL), using a high performance liquid chromatograph (DIONEX Umat 3000) equipped with a Refractive Index (RI) detector, using columns and operating conditions as follows: the analytical column was an Aminex HPX-87H column (BioRad); the mobile phase is 5mM sulfuric acid (prepared in water); the flow rate was controlled to 0.6 mL/min; the sample injection volume was 20 μ L; the RI temperature was controlled at 65 ℃.
In addition, for comparison, different concentrations of glucose (1.2-24mg/mL), xylose (1.2-24mg/mL), xylitol (0.2-6mg/mL), glycerol (0.2-8mg/mL), Hydroxymethylfurfural (HMF) (0.2-8mg/mL), acetic acid (0.2-12mg/mL), ethanol (1.0-15mg/mL), furfural (0.2-8mg/mL) and phenolic compounds (0.2-8mg/mL), which were purchased from Sigma, were used as calibration standards (contystrand), respectively, and the same analysis was performed.
Example 1 addition of NH to a hydrolysate of cellulose of rice straw Using acid catalyzed steam explosion as a substrate4Influence of OH on ethanol production by fermentation of double-mutated Saccharomyces cerevisiae
In this example, preparation of cellulose hydrolyzate according to item 2 of the above "general Experimental Material]The obtained acid-catalyzed steam-exploded rice straw cellulose hydrolysate was used as a substrate and studied for NH during fermentation4And (3) whether the OH alkaline solution is added or not influences the double-mutation saccharomyces cerevisiae to produce the ethanol by fermenting the glucose and the xylose.
In addition, for comparison, detoxified straw cellulose hydrolysate (hereinafter referred to as "detoxified hydrolysate") was taken together to perform the same fermentation experiment. Detoxification treatment of cellulose hydrolysate of rice straw was carried out according to the ultra-liming (overliming) described in Jing-Ping Ge et al (2011), African Journal of microbiological Research,5:1163-Activated charcoal (activated charcoal) and slightly modified. Briefly, the preparation of cellulose hydrolyzate according to item 2 of the above "general Experimental materials]The resulting acid-catalyzed steam exploded straw cellulose hydrolysate was treated with an appropriate amount of Ca (OH)2To adjust the pH to 10, then left to react at room temperature for 120 minutes, followed by centrifugation at 10000rpm for 5 minutes, then the supernatant was collected and washed with H2SO4The solution was adjusted to pH 5.0. Then, 5% activated carbon (available from Hetao corporation, model MAX-703) was added and reacted at 40 ℃ for 60 minutes, and then insoluble materials such as activated carbon filter cake (filter cake) were removed by filtration and centrifugation, and the obtained filtrate was the detoxified hydrolysate.
Before the ethanol production by fermentation, the acid-catalyzed steam-exploded rice straw cellulose hydrolysate and the detoxified hydrolysate were each measured for the concentration of each of saccharides and inhibitors according to the method described in item 1 [ HPLC analysis ] of the above "general Experimental methods", and the measured results are shown in the following Table 2.
TABLE 2 concentration of saccharides and inhibitors in the respective hydrolysates
As can be seen from table 2, the detoxified hydrolysate contained no HMF, furfural and phenolic compounds, and only a small amount of acetic acid, compared to the acid-catalyzed steam-exploded straw cellulose hydrolysate. In addition, the concentration of glucose in the detoxified hydrolysate is also low. It is known that the detoxification treatment removes most of the inhibitors, but at the same time the glucose content is reduced.
The experimental method comprises the following steps:
dividing the acid-catalyzed steam exploded straw cellulose hydrolysate into 2 groups (including control group and NH)4OH group) followed by addition of nutrient salt composition as shown in table 1 to each group of hydrolysates followed by NH separately4OH to adjust the pH to 6. Thereafter, will be according to the above "Item 1 of general Experimental materials [ preparation of inoculum of Saccharomyces cerevisiae doubly mutated at Δ fps1 Δ gpd2 ]]The obtained saccharomyces cerevisiae inoculation source subjected to double mutation is inoculated into each culture bottle in an inoculation amount of 1:20(v/v) and uniformly mixed, then the mixture is placed in a constant-temperature shaking culture chamber, and fermentation reaction is carried out for 72 hours at the temperature of 30 ℃ and the shaking speed of 150 rpm. For NH during the whole fermentation period4Timely addition of NH to OH group4OH so that its pH is maintained at 6. The control group was not treated at all.
In addition, the detoxified hydrolysate was substantially as described above with reference to NH4The same experiment was performed in the OH group mode, except that NaOH was used instead of NH4And (5) OH. Thereafter, the fermentation cultures of each group were centrifuged at 12000rpm for 10 minutes, and the resulting fermentation metabolites were analyzed by high performance liquid chromatography according to item 1 of the above "general Experimental methods]The method described therein was used to perform the content analysis of glucose, xylose, xylitol and ethanol. The utilization of glucose or xylose in question is expressed in percent (%) of the total amount of glucose or xylose measured relative to the respective total amount measured before fermentation. In addition, the total amount of xylitol measured after fermentation was compared to the total xylose utilization during fermentation (i.e., the total amount of xylose measured before fermentation minus that measured after fermentation) to obtain a ratio (i.e., xylitol production) that is lower, indicating that more xylose is converted to ethanol, rather than being accumulated as xylitol. As for the ethanol yield (yield), it was calculated by comparing the total amount of ethanol produced after fermentation with the total amount of glucose and xylose utilization during fermentation (i.e., the total amount of glucose and xylose measured before fermentation minus that measured after fermentation).
As a result:
the results of this experiment are shown in table 3 below.
TABLE 3 carbohydrate utilization, xylitol production and ethanol production measured after fermentation of each hydrolysate group inoculated with double-mutated Saccharomyces cerevisiae
As can be seen from Table 3, when the acid-catalyzed steam-exploded rice straw cellulose hydrolysate was used as the substrate (regardless of whether NH was added during the fermentation process or not)4OH) or directly using detoxified hydrolysate as matrix, wherein the glucose utilization rate measured by each group of fermentation metabolites is 100%, and the xylose utilization rate, ethanol yield and NH4The OH groups were all significantly better than the control group. Thus, NH was added during the fermentation4OH can obviously improve the utilization rate of xylose, thereby increasing the yield of ethanol.
In addition, NH of the acid-catalyzed steam-exploded straw cellulose hydrolysate4Comparison of OH groups with detoxified hydrolysate revealed that the acid-catalyzed steam-exploded rice straw cellulose hydrolysate was used as the matrix and NH4OH to adjust the composition of the hydrolysate or NH is added during the fermentation process4OH can result in better xylose utilization and ethanol yield. The experimental results show that the technology disclosed by the invention can effectively improve the utilization rate of xylose and the yield of ethanol without further detoxification treatment of hydrolysate.
Example 2 Effect of adding different alkaline solutions on ethanol production by fermentation of double mutant Saccharomyces cerevisiae Using Rice straw cracked by acid catalyzed steam and Mangifera indica cellulose hydrolysate as matrix
This example used the preparation of cellulose hydrolyzate according to item 2 of the above "general Experimental Material]The obtained hydrolyzed solution of rice straw or Chinese silvergrass cellulose cracked by acid-catalyzed steam is used as matrix, and different alkaline solutions (including NH) are discussed4OH, KOH and NaOH solution) to adjust the pH value of the hydrolysate, and adding the alkaline solution in the fermentation process to produce the ethanol by fermenting the saccharomyces cerevisiae subjected to double mutation by using glucose and xylose.
The acid-catalyzed steam exploded rice straw or miscanthus fiber hydrolysate was subjected to the measurement of the concentrations of various sugars and acetic acid according to the method described in item 1 [ HPLC analysis ] of the above "general Experimental method", respectively, before the fermentation for producing ethanol, and the measured results are shown in the following Table 4.
TABLE 4 concentration of glucose, xylose and acetic acid in acid catalyzed steam exploded miscanthus and rice straw cellulose hydrolysate
| Composition of | Rice straw cellulose hydrolysate | Chinese silvergrass cellulose hydrolysate |
| Acetic acid (g/L) | 6.2 | 8.7 |
| Glucose (g/L) | 79.2 | 80 |
| Xylose (g/L) | 36.9 | 55.9 |
The experimental method comprises the following steps:
first, the rice straw and the Chinese silvergrass cellulose hydrolysate which are cracked by acid-catalyzed steam are respectively divided into 3 groups (including KOH group, NaOH group and NH group)4OH group), followed by KOH, NaOH, and NH, respectively4OH to adjust the pH to 6, the resulting rice straw and miscanthus cellulose hydrolysate were subjected to ethanol production by fermentation of double mutant Saccharomyces cerevisiae substantially according to the experimental method described in example 1 above, except that: during the whole fermentation period, respectively for KOH group, NaOH group and NH4Timely adding NaOH, KOH and NH to OH group4OH, so that their pH values are 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 item 1 [ HPLC analysis ] of the above "general Experimental methods", and then the sugar utilization rate, xylitol production amount and ethanol production amount were calculated respectively in the manner described in example 1.
As a result:
the results of this experiment are shown in table 5 below.
TABLE 5 sugar utilization, xylitol production and ethanol production measured after each group of rice straw and miscanthus cellulose hydrolysate was inoculated with double-mutated saccharomyces cerevisiae and fermented
As can be seen from Table 5, the glucose utilization rates measured for the various fermentation metabolites were all 100% regardless of whether the rice straw or the cellulose hydrolysate of miscanthus was used as the substrate, with respect to xylose utilization rate and ethanol yield, NH4The OH group is significantly better than either the NaOH or KOH group. Thus, the ratio of NH to NaOH and KOH is shown4OH is used for adjusting the pH value of the hydrolysate and NH is added in the fermentation process4OH can more obviously improve the utilization rate of xylose, reduce the accumulation of xylitol and further increase the yield of ethanol.
Example 3 Using steam exploded cellulose hydrolysate of miscanthus as substrate, by adding NH4Effect of OH to adjust pH on ethanol production by fermentation of double-mutated Saccharomyces cerevisiae
This example used the preparation of cellulose hydrolyzate according to item 2 of the above "general Experimental Material]The obtained steam-exploded Chinese silvergrass cellulose hydrolysate is used as matrix and discussed by adding NH during fermentation4OH dissolving the miscanthus celluloseThe pH value of the hydrolysate is respectively maintained at 5, 6 or 7, and the double-mutation saccharomyces cerevisiae utilizes glucose and xylose to ferment to produce ethanol.
Prior to ethanol production by fermentation, the steam exploded miscanthus fiber hydrolysate was measured to contain 7.3g/L acetic acid, 81.8g/L glucose and 34.7g/L xylose according to the method described in item 1 [ HPLC analysis ] of general Experimental methods above.
The experimental method comprises the following steps:
first, the steam exploded cellulose hydrolysate of miscanthus was divided into 3 groups (including pH 5 group, pH 6 group and pH 7 group), and then treated with NH4OH to adjust pH of pH 5, pH 6 and pH 7 to 5, 6 and 7 respectively, the obtained mangrove grass cellulose hydrolysate was subjected to the ethanol production fermentation test of saccharomyces cerevisiae by double mutation substantially according to the test method described in example 1 above, except that: adding NH to each group in time during the whole fermentation period4OH, so that their pH is maintained at the initially established value.
Thereafter, the fermentation metabolites of each group were analyzed for the contents of glucose, xylose, xylitol and ethanol according to the method described in item 1 [ HPLC analysis ] of the above "general Experimental methods", and then the sugar utilization rate, xylitol production amount and ethanol production amount were calculated respectively in the manner described in example 1.
As a result:
the results of this experiment are shown in table 6 below.
TABLE 6 utilization rate of saccharides, xylitol production and ethanol production measured after fermentation of mango cellulose hydrolysate having different pH values inoculated with double-mutated Saccharomyces cerevisiae
| pH 5 group | pH 6 group | pH 7 group | |
| Glucose utilization (%) | 100 | 100 | 100 |
| Xylose utilization (%) | 35.1 | 99.3 | 94.1 |
| Xylitol production amount (g/g) | 0.18 | 0.09 | 0.11 |
| Ethanol yield (g/g) | 0.393 | 0.413 | 0.396 |
As can be seen from table 6, the glucose utilization rate measured by each set of fermentation metabolites was 100% regardless of whether the pH of the steam exploded miscanthus fiber hydrolysate was maintained at 5, 6 or 7, and the pH 6 set was significantly superior to the pH 7 and pH 5 sets with respect to xylose utilization rate, xylitol production and ethanol production. The Applicant believes that, during the fermentation, NH is added4OH can control the pH value of the cellulose hydrolysate to be 6.0 to reach the optimal valueThe utilization rate of sugar is reduced, the accumulation of xylitol is effectively reduced, and the yield of ethanol is improved.
All patents and publications cited in this specification are herein incorporated by reference in their entirety. In conflict, the present specification, including definitions, will control.
While the invention has been described with reference to the specific embodiments described above, it will be apparent that numerous modifications and variations can be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only as indicated by the claims appended hereto.
Biological material preservation information description
The preservation number is: DSM 25508
And (3) classification and naming: saccharomyces cerevisiae
The preservation date is as follows: 2011 12 months and 20 days
The preservation unit: german Collection of microorganisms GmbH
The address of the depository: yinhofensisto 7B D-38124 brenrelix.
Claims (8)
1. A process for the production of ethanol, characterized in that it comprises the following steps:
providing a cellulosic hydrolysate containing a fermentable sugar and at least one fermentation inhibitor selected from the group consisting of: acetic acid, hydroxymethylfurfural, furfural, and phenolic compounds;
adding an aqueous solution containing ammonia to the cellulose hydrolysate to form a mixed solution having a pH falling within a range of 5.5 to 7.0; and
adding a xylose-utilizing saccharomyces cerevisiae to the mixed liquor and allowing the saccharomyces cerevisiae to ferment the mixed liquor such that ethanol is produced, wherein the xylose-utilizing saccharomyces cerevisiae is a Δ fps1 Δ gpd2 double mutant saccharomyces cerevisiae DSM 25508, during fermentation of the mixed liquor the pH of the mixed liquor is maintained within the range of 5.5 to 7.0 by adding the aqueous ammonia-containing solution, and the aqueous ammonia-containing solution is an aqueous ammonium hydroxide solution.
2. The method of claim 1, wherein the mixed liquor has a pH value in the range of 5.5 to 6.5.
3. The method of claim 1, wherein the mixed liquor has a pH value in the range of 5.8 to 6.2.
4. The method of claim 1 wherein said fermentable sugars comprise a five carbon sugar and a six carbon sugar.
5. The method of claim 4, wherein the fermentable sugar comprises glucose and xylose.
6. The method of claim 1, wherein the cellulose hydrolysate is prepared by sequentially pretreating and hydrolyzing a cellulosic biomass selected from the group consisting of: bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper making, yard waste, waste and forestry waste, and combinations thereof.
7. The method of claim 6, wherein the cellulosic biomass is selected from the group consisting of: miscanthus, softwood, hardwood, corn cobs, corn stover, grasses, wheat straw, barley straw, hay, rice straw, switchgrass, waste paper, bagasse, milo plant material, soybean plant material, ground components derived from grain, trees, branches, roots, leaves, wood chips, shrubs and bushes, vegetables, fruits, and flowers, and combinations thereof.
8. The method of claim 6, wherein the pre-processing is selected from the group consisting of: steam explosion, thermal pre-chemical treatment, mechanical comminution, acid treatment, organic dissolution, sulfite pretreatment, and combinations thereof.
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| CN101638673A (en) * | 2009-08-26 | 2010-02-03 | 安徽丰原发酵技术工程研究有限公司 | Method for manufacturing alcohol by utilizing fermentation of plant straws |
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