CN101665770B - Microbial compound inoculum for production of cellulosic ethanol and preparation method thereof - Google Patents

Microbial compound inoculum for production of cellulosic ethanol and preparation method thereof Download PDF

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CN101665770B
CN101665770B CN200910072967XA CN200910072967A CN101665770B CN 101665770 B CN101665770 B CN 101665770B CN 200910072967X A CN200910072967X A CN 200910072967XA CN 200910072967 A CN200910072967 A CN 200910072967A CN 101665770 B CN101665770 B CN 101665770B
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spore
spore suspension
microbial inoculum
complex microbial
aspergillus oryzae
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CN101665770A (en
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冯玉杰
于艳玲
刘嘉
李冬梅
徐琛
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JIANGSU HAYI ENVIRONMENTAL PROTECTION RESEARCH INSTITUTE
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Harbin Institute of Technology
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Abstract

The invention discloses a microbial compound inoculum for the production of cellulosic ethanol and a preparation method thereof, relating to a compound inoculum and a preparation method thereof, and solving the problems that the cost of fermentation production of cellulosic ethanol is high due to the adoption of a rinsing and detoxification method, and the ultimate yield of the ethanol is low because the rinsing and detoxification method loses the nutrition elements in the lignocellulose. The microbial compound inoculum is mainly prepared from a trichoderma koningii spore suspension, an aspergillus oryzae spore suspension and a chaetomium cupreum spore suspension. The preparation method comprises the following steps: firstly, mixing the trichoderma koningii spore suspension, the aspergillus oryzae spore suspension and the chaetomium cupreum spore suspension; and secondly, mixing and inoculating the mixed spore suspension in a culture medium for cultivation to obtain the microbial compound inoculum. The microbial compound bacterium has few dosage and reduces the cost of the fermentation production of cellulosic ethanol, and the ethanol prepared from the microbial compound inoculum has high ultimate yield.

Description

A kind of complex microbial inoculum that is used for cellulosic ethanol production and preparation method thereof
Technical field
The present invention relates to a kind of complex microbial inoculum and preparation method thereof.
Background technology
Whole world agricultural crop straw annual production surpasses 2,000,000,000 tons, and Chinese annual production reaches about 700,000,000 tons, and wherein maize straw output is about 2.5 hundred million tons, these maize straws are used for animal-feed and rural area fuel except small part, major part is all directly burnt, and has both wasted resource, again severe contamination environment.And on the other hand, along with fossil energy is petered out, energy dilemma is being aggravated, and ethanol is considered to the best regeneratable liquors energy.
Existing cellulose alcoholic fermentation technology is to utilize the Mierocrystalline cellulose in the agricultural crop straw to ferment; this zymotechnique is the research that raw material carries out alcohol production with reproducible cellulose resource; both alleviate energy dilemma, alleviated environmental pollution again, helped Sustainable development.
In cellulose alcoholic fermentation is produced, special construction based on natural cellulose, before the enzymolysis Mierocrystalline cellulose, carrying out suitable pre-treatment is the necessary technology means with the molecular structure of opening lignocellulose, wherein, the steam explosion pre-treatment is present most widely used a kind of Mierocrystalline cellulose pretreatment process, but maize straw through this method processing, the formic acid that in preprocessing process, produces, acetate, by product such as oxysuccinic acid and phenolic compound all has had strong inhibitory effects to cellulase hydrolysis saccharification and ethanol fermentation, therefore is necessary to remove in advance these poisonous substances or reduces its toxic action.At present, the detoxification that is most widely used in the cellulose alcoholic fermentation production is the washing disintoxication, though this method detoxifying effect in cellulose alcoholic fermentation is produced is good, but washing toxicide water loss is big, the burden of follow-up water treatment is heavy, cause the cellulose alcoholic fermentation production cost too high, and washing disintoxication nutritive ingredients such as wood sugar in the also easy loss lignocellulose in the toxicide process, the whole productive rate of the ethanol that causes cellulose alcoholic fermentation production to obtain is low.
Summary of the invention
The objective of the invention is to adopt the washing disintoxication to cause the nutritive ingredient in high problem of cellulose alcoholic fermentation production cost and the washing disintoxication loss lignocellulose to cause the low problem of the whole productive rate of ethanol, and a kind of complex microbial inoculum that is used for cellulosic ethanol production and preparation method thereof is provided in order to solve.
Complex microbial inoculum of the present invention is 10 by concentration 8The mould spore suspension of healthy and free from worry wood of individual/mL, concentration are 10 8The aspergillus oryzae spore suspension of individual/mL and concentration are 10 8The angle chaetomium spore suspension of individual/mL is formed according to 1: 1: 1 volume ratio.
The making method of complex microbial inoculum of the present invention is carried out according to following steps: be 10 with concentration one, 8The mould spore suspension of healthy and free from worry wood of individual/mL, concentration are 10 8The aspergillus oryzae spore suspension of individual/mL and concentration are 10 8The angle chaetomium spore suspension of individual/mL obtains mixing spore liquid according to 1: 1: 1 volume ratio mixing; Two, under aseptic condition, in the mixing spore liquid access substratum with step 1, static cultivation 48～72h under 30～40 ℃ of conditions promptly makes and obtains complex microbial inoculum, and the volume ratio of wherein mixing spore liquid and substratum is 2～3: 25.
Utilize complex microbial inoculum of the present invention to carry out cellulosic ethanol production technology, do not need the washing detoxifcation, just can carry out the fermentation of cellulosic ethanol, greatly reduce production costs of cellulosic ethanol, compare with the cellulosic ethanol production technology of existing employing washing detoxification, the water consumption of utilizing complex microbial inoculum of the present invention to carry out cellulosic ethanol production reduces 40%～50%, and production cost has reduced by 5%～10%.Utilize complex microbial inoculum of the present invention to carry out in the cellulosic ethanol production technology that cellulosic alcohol concn can reach about 33g/L in the lignocellulosic material, the cellulosic ethanol transformation efficiency can reach more than 85%, and do not lose the hydrolysate wood sugar of hemicellulose, these wood sugars also can further be fermented into ethanol, utilize complex microbial inoculum of the present invention to carry out the whole productive rate height of cellulosic ethanol production ethanol.
Description of drawings
Fig. 1 is first group of change in concentration figure that tests furfural, phenol and hydroxymethylfurfural in the fermenting process in the simultaneous test, and wherein a represents the concentration of furfural, the concentration that b represents phenol, the concentration that c represents hydroxymethylfurfural; Fig. 2 is first group of change in concentration figure that tests formic acid, acetate and oxysuccinic acid in the fermenting process in the simultaneous test, and wherein d represents the concentration of formic acid, the concentration that e represents acetate, the concentration that f represents oxysuccinic acid; Fig. 3 is first group of test and the second group of comparison diagram of testing cellulase activity variation in the fermenting process in the simultaneous test, wherein
Figure DEST_PATH_GSB00000349676100021
The detected result of representing first group of test,
Figure DEST_PATH_GSB00000349676100022
The detected result of representing second group of test; Fig. 4 is a glucose concn variation comparison diagram in the simultaneous test first group test and the second group of test fermenting process, wherein
Figure DEST_PATH_GSB00000349676100023
The detected result of representing first group of test,
Figure DEST_PATH_GSB00000349676100024
The detected result of representing second group of test; Fig. 5 is an alcohol concn variation comparison diagram in the fermenting process of first group of test and second group of experiment in the simultaneous test, wherein
Figure DEST_PATH_GSB00000349676100025
The detected result of representing first group of experiment
Figure DEST_PATH_GSB00000349676100026
The detected result of second group of experiment.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the complex microbial inoculum that present embodiment is used for cellulosic ethanol production is 10 by concentration mainly 8The mould spore suspension of healthy and free from worry wood of individual/mL, concentration are 10 8The aspergillus oryzae spore suspension of individual/mL and concentration are 10 8The angle chaetomium spore suspension of individual/mL is made according to 1: 1: 1 volume ratio.
The making method of the mould spore suspension of healthy and free from worry wood in the present embodiment is: be inoculated on the potato dextrose agar inclined-plane healthy and free from worry wood is mould, under 28～32 ℃ of conditions, cultivate 48～72h and obtain the mould spore of healthy and free from worry wood, wash cultivating the mould spore of healthy and free from worry wood that obtains on the potato dextrose agar inclined-plane with sterilized water then, and sterilized water and the mould spore uniform mixing of healthy and free from worry wood promptly obtained the mould spore suspension of healthy and free from worry wood, mould can the purchase from the market of wherein said healthy and free from worry wood obtains.
The making method of the aspergillus oryzae spore suspension in the present embodiment is: aspergillus oryzae is inoculated on the potato dextrose agar inclined-plane, under 28～32 ℃ of conditions, cultivate 48～72h and obtain the aspergillus oryzae spore, wash cultivating the aspergillus oryzae spore that obtains on the potato dextrose agar inclined-plane with sterilized water then, and sterilized water and aspergillus oryzae spore uniform mixing promptly obtained the aspergillus oryzae spore suspension, wherein said aspergillus oryzae can be bought from the market and obtain.
The making method of the angle chaetomium spore suspension in the present embodiment is: the angle chaetomium is inoculated on the potato dextrose agar inclined-plane, under 28～32 ℃ of conditions, cultivate 48～72h and obtain angle chaetomium spore, wash cultivating the angle chaetomium spore that obtains on the potato dextrose agar inclined-plane with sterilized water then, and sterilized water and aspergillus oryzae spore uniform mixing promptly obtained angle chaetomium spore suspension, the wherein said angle chaetomium can be bought from the market obtains.
The complex microbial inoculum of present embodiment respectively with formic acid, acetate, oxysuccinic acid, hydroxymethylfurfural (HMF) and aldehydes matter react, reacted result shows composite fungus agent formic acid of the present invention, acetate, oxysuccinic acid, the degradation rate of hydroxymethylfurfural (HMF) and aldehydes matter is respectively 72.37%～81.69%, 50.61%～60.89%, 78.18%～89.12%, 98%～100% and 70.08%～86.23%, from these data as can be seen, composite fungus agent of the present invention is used for cellulosic ethanol production technology, to the formic acid that produces in the maize straw preprocessing process, acetate, the removal effect of oxysuccinic acid and phenolic compound is good, and the detoxifying effect in cellulosic ethanol production technology of the present invention is good.
It is as follows to utilize the complex microbial inoculum of present embodiment to carry out the step of cellulosic ethanol production technology:
One, sophisticated maize straw is cut into the segment of 4～6cm, steam explosion pre-treatment under 220～240 ℃, the condition of 1.6～1.8MPa promptly obtains steam puffed stalk then; Two, the distilled water of getting 100g steam puffed stalk and 250～350mL mixes and to obtain mixture, places under 121 ℃ of conditions moist heat sterilization 20min then; After the cooling, the pH that mixture is regulated in aseptic technique is 5.0～5.5, cellulase (the good benefit in Tianjin that adds 10～13g then in the mixture after sterilization, the CMC enzyme is lived and to be 55.6IU/g), 1.0～1.5g yeast saccharomyces cerevisiae (Angel Yeast, 84000024) and the complex microbial inoculum of 3.5～5g present embodiment production code member:, fully stirring evenly, is that 39 ℃, rotating speed are that diastatic fermentation 70～80h obtains fermented liquid under the 200r/min condition in temperature, has promptly finished cellulosic ethanol production technology; Wherein, the alcohol concn in the fermented liquid is 32～35g/L, and the turnover ratio of cellulosic ethanol is 81%～92% in process of production.
Utilize the complex microbial inoculum of present embodiment to carry out cellulosic ethanol production technology, do not need the washing detoxifcation, just can carry out the fermentation of cellulosic ethanol, greatly reduce production costs of cellulosic ethanol, compare with the existing cellulosic ethanol production technology that washes detoxification with water that makes, utilize the complex microbial inoculum of present embodiment to carry out the total water amount minimizing 40%～50% of cellulosic ethanol production, production cost has reduced by 6%～10%.Utilize the complex microbial inoculum of present embodiment to carry out in the cellulosic ethanol production technology that cellulosic alcohol concn can reach about 33g/L in the lignocellulosic material, the cellulosic ethanol transformation efficiency can reach more than 85% and not lose the hydrolysate wood sugar of hemicellulose, these wood sugars also can be fermented into ethanol, utilize the complex microbial inoculum of present embodiment to carry out the whole productive rate height of cellulosic ethanol production ethanol.
Embodiment two: what present embodiment and embodiment one were different is that the complex microbial inoculum that is used for cellulosic ethanol production also comprises substratum, and substratum is made up of 7～9 parts wheat bran, 11～13 parts Microcrystalline Cellulose and 14～16 parts inorganic salt solution according to parts by weight.Other are identical with embodiment one.
The making method of present embodiment substratum is: according to ratio of weight and number 7～9 parts wheat bran, 11～13 parts Microcrystalline Cellulose and 14～16 parts inorganic salt solution are mixed, then mixture is placed under 121 ℃ of conditions, sterilization 20min promptly obtains substratum.
Embodiment three: that present embodiment and embodiment two are different is KH in the inorganic salt solution 2PO 4Mass concentration be 0.7%, (NH 4) 2SO 4Mass concentration be 3.3%, K 2HPO 4Mass concentration be 0.7% and MgSO 4Mass concentration be 0.1% the aqueous solution, the pH value of inorganic salt solution is 5～7.Other are identical with embodiment one.
Embodiment four: present embodiment is used for the making method of the complex microbial inoculum of cellulosic ethanol production and carries out according to following steps: be 10 with concentration one, 8The mould spore suspension of healthy and free from worry wood of individual/mL, concentration are 10 8The aspergillus oryzae spore suspension of individual/mL and concentration are 10 8The angle chaetomium spore suspension of individual/mL obtains mixing spore liquid according to 1: 1: 1 volume ratio mixing; Two, under aseptic condition, in the mixing spore liquid access substratum with step 1, static cultivation 48～72h under 30～40 ℃ of conditions promptly makes and obtains complex microbial inoculum, and the volume ratio of wherein mixing spore liquid and substratum is 2～3: 25.
Embodiment five: present embodiment and embodiment four are different is that the making method of the mould spore suspension of healthy and free from worry wood in the step 1 is: be inoculated on the potato dextrose agar inclined-plane healthy and free from worry wood is mould, under 28～32 ℃ of conditions, cultivate 48～72h and obtain the mould spore of healthy and free from worry wood, wash cultivating the mould spore of healthy and free from worry wood that obtains on the potato dextrose agar inclined-plane with sterilized water then, and sterilized water and the mould spore uniform mixing of healthy and free from worry wood are promptly obtained the mould spore suspension of healthy and free from worry wood.Other steps and parameter are identical with embodiment four.
Embodiment six: present embodiment is different with embodiment four or five is that the making method of aspergillus oryzae spore suspension in the step 1 is: aspergillus oryzae is inoculated on the potato dextrose agar inclined-plane, under 28～32 ℃ of conditions, cultivate 48～72h and obtain the aspergillus oryzae spore, wash cultivating the aspergillus oryzae spore that obtains on the potato dextrose agar inclined-plane with sterilized water then, and sterilized water and aspergillus oryzae spore uniform mixing are promptly obtained the aspergillus oryzae spore suspension.Other steps and parameter are identical with embodiment four or five.
Embodiment seven: present embodiment and embodiment six are different is that the making method of chaetomium spore suspension in angle in the step 1 is: the angle chaetomium is inoculated on the potato dextrose agar inclined-plane, under 28～32 ℃ of conditions, cultivate 48～72h and obtain angle chaetomium spore, wash cultivating the angle chaetomium spore that obtains on the potato dextrose agar inclined-plane with sterilized water then, and sterilized water and aspergillus oryzae spore uniform mixing are promptly obtained angle chaetomium spore suspension.Other steps and parameter are identical with embodiment six.
Embodiment eight: present embodiment and embodiment four, five or seven are different is that substratum in the step 2 is made up of 7～9 parts wheat bran, 11～13 parts Microcrystalline Cellulose and 14～16 parts inorganic salt solution according to parts by weight.Other steps and parameter are identical with embodiment four, five or seven.
The making method of present embodiment substratum is: according to ratio of weight and number 7～9 parts wheat bran, 11～13 parts Microcrystalline Cellulose and 14～16 parts inorganic salt solution are mixed, then mixture is placed under 121 ℃ of conditions, sterilization 20min promptly obtains substratum.
Embodiment nine: that present embodiment and embodiment eight are different is KH in the inorganic salt solution 2PO 4Mass concentration be 0.7%, (NH 4) 2SO 4Mass concentration be 3.3%, K 2HPO 4Mass concentration be 0.7% and MgSO 4Mass concentration be 0.1% the aqueous solution, the pH value of inorganic salt solution is 5～7.Other steps and parameter are identical with embodiment eight.
Embodiment ten: present embodiment and embodiment eight are different be in the step 2 under 40 ℃ of conditions static cultivation 62h.Other steps and parameter are identical with embodiment eight.
Embodiment 11: present embodiment is used for the making method of the complex microbial inoculum of cellulosic ethanol production and carries out according to following steps: be 10 with concentration one, 8The mould spore suspension of healthy and free from worry wood of individual/mL, concentration are 10 8The aspergillus oryzae spore suspension of individual/mL and concentration are 10 8The angle chaetomium spore suspension of individual/mL obtains mixing spore liquid according to 1: 1: 1 volume ratio mixing; Two, under aseptic condition, in the mixing spore liquid access substratum with step 1, static cultivation 62h under 35 ℃ of conditions promptly makes and obtains complex microbial inoculum, and the volume ratio of wherein mixing spore liquid and substratum is 2.5: 25.
The making method of the mould spore suspension of healthy and free from worry wood in the present embodiment step 1 is: be inoculated on the potato dextrose agar inclined-plane healthy and free from worry wood is mould, under 28～32 ℃ of conditions, cultivate 48～72h and obtain the mould spore of healthy and free from worry wood, wash cultivating the mould spore of healthy and free from worry wood that obtains on the potato dextrose agar inclined-plane with sterilized water then, and sterilized water and the mould spore uniform mixing of healthy and free from worry wood are promptly obtained the mould spore suspension of healthy and free from worry wood.
The making method of the aspergillus oryzae spore suspension in the present embodiment step 1 is: aspergillus oryzae is inoculated on the potato dextrose agar inclined-plane, under 28～32 ℃ of conditions, cultivate 48～72h and obtain the aspergillus oryzae spore, wash cultivating the aspergillus oryzae spore that obtains on the potato dextrose agar inclined-plane with sterilized water then, and sterilized water and aspergillus oryzae spore uniform mixing are promptly obtained the aspergillus oryzae spore suspension.
The making method of the angle chaetomium spore suspension in the present embodiment step 1 is: the angle chaetomium is inoculated on the potato dextrose agar inclined-plane, under 28～32 ℃ of conditions, cultivate 48～72h and obtain angle chaetomium spore, wash cultivating the angle chaetomium spore that obtains on the potato dextrose agar inclined-plane with sterilized water then, and sterilized water and aspergillus oryzae spore uniform mixing are promptly obtained angle chaetomium spore suspension.
Substratum in the present embodiment step 2 is formed the aqueous solution wherein, KH in the inorganic salt solution according to parts by weight by 7～9 parts wheat bran, 11～13 parts Microcrystalline Cellulose and 14～16 parts inorganic salt solution 2PO 4Mass concentration be 0.7%, (NH 4) 2SO 4Mass concentration be 3.3%, K 2HPO 4Mass concentration be 0.7% and MgSO 4Mass concentration be 0.1% the aqueous solution, the pH value of inorganic salt solution is 5～7.
Simultaneous test:
First group of experiment is to utilize present embodiment to make the complex microbial inoculum that obtains to carry out cellulosic ethanol production, concrete steps are as follows: the segment that, sophisticated maize straw is cut into 4～6cm, steam explosion pre-treatment under 230 ℃, the condition of 1.7MPa promptly obtains steam puffed stalk then; Two, the distilled water of getting 100g steam puffed stalk and 300mL mixes and to obtain mixture, places under 121 ℃ of conditions moist heat sterilization 20min then; After the cooling, the pH that mixture is regulated in aseptic technique is 5.0, cellulase (the good benefit in Tianjin that adds 15g then in the mixture after sterilization, the CMC enzyme is lived and to be 55.6IU/g), 1.5g yeast saccharomyces cerevisiae (Angel Yeast, 84000024) and the complex microbial inoculum of 4g present embodiment production code member:, fully stirring evenly, is that 39 ℃, rotating speed are that diastatic fermentation 72h obtains fermented liquid under the 200r/min condition in temperature, has promptly finished cellulosic ethanol production technology.
Second group of experiment is to adopt the washing detoxification to carry out the method for cellulosic ethanol production, concrete steps are as follows: the segment that, sophisticated maize straw is cut into 4～6cm, then at 220～240 ℃, 1.7MPa condition under the steam explosion pre-treatment, promptly obtain steam puffed stalk, wherein, the water content of steam puffed stalk is about 20%, and the dry content of cellulose is about 40%; Two, the distilled water of getting 100g steam puffed stalk and 300mL mixes and to obtain mixture, places under 121 ℃ of conditions moist heat sterilization 20min then; After the cooling, the pH that mixture is regulated in aseptic technique is 5.0, cellulase (the good benefit in Tianjin that adds 20g then in the mixture after sterilization, the CMC enzyme is lived and is 55.6IU/g) and 2.0g yeast saccharomyces cerevisiae (Angel Yeast, production code member: 84000024), fully stirring evenly, is that 40 ℃, rotating speed are that diastatic fermentation 72h obtains fermented liquid under the 200r/min condition in temperature, has promptly finished cellulosic ethanol production technology.
The change in concentration of furfural, phenol, hydroxymethylfurfural, formic acid, acetate and oxysuccinic acid as depicted in figs. 1 and 2 in the fermenting process of first group of test; From Fig. 1 and Fig. 2 as can be seen the degradation rate of furfural, phenol, hydroxymethylfurfural, formic acid, acetate and oxysuccinic acid reached 76.23%, 74.08%, 100%, 75.37%, 53.61%, 80.18% respectively.Present embodiment is made the complex microbial inoculum obtain has good degradation effect to the by product of the inhibition cellulose alcoholic fermentation that produces in the steam puffed stalk, and present embodiment is made the complex microbial inoculum that obtains good detoxifying effect.
The variation comparison diagram that first group of test and second group are tested cellulase activity in the fermenting process as shown in Figure 3, as can be seen from Figure 3, the activity of cellulase can reach 7.5～10.1IU/mL in first group of test, it is to make the complex microbial inoculum that obtains by present embodiment to provide that cellulase activity about 2.0～3.0IU/mL is wherein arranged, and the cellulase activity of second group of test only is 2.1～2.3IU/mL; The present embodiment making obtains complex microbial inoculum and not only has the toxicide effect, also has the activity of cellulase.
The variation comparison diagram of first group of test and the glucose concn of second group of experiment in the fermenting process as shown in Figure 4, the glucose fermentation rate of first group of test has reached 92.9% as can be seen from Figure 4, and second group of glucose fermentation rate of testing only can reach 38.6%; Utilize present embodiment to make the transformation efficiency height of glucose in the cellulose alcoholic fermentation of the complex microbial inoculum that obtains, help improving ethanol yield.
The variation comparison diagram that first group of test and second group are tested the alcohol concn in the fermenting process as shown in Figure 5, as can be seen from Figure 5, under the identical situation of fermentation time, the alcoholic acid maximum concentration of first group of test can reach 33g/L, and the ethanol maximum concentration of second group of experiment only can reach 16g/L, hence one can see that utilizes present embodiment to make the whole productive rate height of the resulting ethanol of cellulose ethanol of the complex microbial inoculum that obtains, and the complex microbial inoculum that utilizes present embodiment to make to obtain does not need to make and washes detoxifcation with water, also can carry out cellulose alcoholic fermentation, greatly reduce production costs of cellulosic ethanol.

Claims (8)

1. a complex microbial inoculum that is used for cellulosic ethanol production is characterized in that complex microbial inoculum is 10 by concentration 8The mould spore suspension of healthy and free from worry wood of individual/mL, concentration are 10 8The aspergillus oryzae spore suspension of individual/mL and concentration are 10 8The angle chaetomium spore suspension of individual/mL is formed according to 1: 1: 1 volume ratio.
2. a kind of complex microbial inoculum that is used for cellulosic ethanol production according to claim 1, it is characterized in that complex microbial inoculum also comprises substratum, substratum is made up of 7～9 parts wheat bran, 11～13 parts Microcrystalline Cellulose and 14～16 parts inorganic salt solution according to parts by weight; Wherein inorganic salt solution is KH 2PO 4Mass concentration be 0.7%, (NH 4) 2SO 4Mass concentration be 3.3%, K 2HPO 4Mass concentration be 0.7% and MgSO 4Mass concentration be 0.1% the aqueous solution, the pH value of inorganic salt solution is 5～7.
3. a making method that is used for the complex microbial inoculum of cellulosic ethanol production is characterized in that the making method of complex microbial inoculum is carried out according to following steps: be 10 with concentration one, 8The mould spore suspension of healthy and free from worry wood of individual/mL, concentration are 10 8The aspergillus oryzae spore suspension of individual/mL and concentration are 10 8The angle chaetomium spore suspension of individual/mL obtains mixing spore liquid according to 1: 1: 1 volume ratio mixing; Two, under aseptic condition, in the mixing spore liquid access substratum with step 1, static cultivation 48～72h under 30～40 ℃ of conditions promptly makes and obtains complex microbial inoculum, and the volume ratio of wherein mixing spore liquid and substratum is 2～3: 25.
4. a kind of making method that is used for the complex microbial inoculum of cellulosic ethanol production according to claim 3, the making method that it is characterized in that the mould spore suspension of healthy and free from worry wood in the step 1 is: be inoculated on the potato dextrose agar inclined-plane healthy and free from worry wood is mould, under 28～32 ℃ of conditions, cultivate 48～72h and obtain the mould spore of healthy and free from worry wood, wash cultivating the mould spore of healthy and free from worry wood that obtains on the potato dextrose agar inclined-plane with sterilized water then, and sterilized water and the mould spore uniform mixing of healthy and free from worry wood are promptly obtained the mould spore suspension of healthy and free from worry wood.
5. according to claim 3 or 4 described a kind of making methods that are used for the complex microbial inoculum of cellulosic ethanol production, the making method that it is characterized in that aspergillus oryzae spore suspension in the step 1 is: aspergillus oryzae is inoculated on the potato dextrose agar inclined-plane, under 28～32 ℃ of conditions, cultivate 48～72h and obtain the aspergillus oryzae spore, wash cultivating the aspergillus oryzae spore that obtains on the potato dextrose agar inclined-plane with sterilized water then, and sterilized water and aspergillus oryzae spore uniform mixing are promptly obtained the aspergillus oryzae spore suspension.
6. a kind of making method that is used for the complex microbial inoculum of cellulosic ethanol production according to claim 5, the making method that it is characterized in that chaetomium spore suspension in angle in the step 1 is: the angle chaetomium is inoculated on the potato dextrose agar inclined-plane, under 28～32 ℃ of conditions, cultivate 48～72h and obtain angle chaetomium spore, wash cultivating the angle chaetomium spore that obtains on the potato dextrose agar inclined-plane with sterilized water then, and sterilized water and aspergillus oryzae spore uniform mixing are promptly obtained angle chaetomium spore suspension.
7. according to claim 3,4 or 6 described a kind of making methods that are used for the complex microbial inoculum of cellulosic ethanol production, it is characterized in that the substratum in the step 2 is made up of 7～9 parts wheat bran, 11～13 parts Microcrystalline Cellulose and 14～16 parts inorganic salt solution according to parts by weight; KH in the inorganic salt solution wherein 2PO 4Mass concentration be 0.7%, (NH 4) 2SO 4Mass concentration be 3.3%, K 2HPO 4Mass concentration be 0.7% and MgSO 4Mass concentration be 0.1% the aqueous solution, the pH value of inorganic salt solution is 5～7.
8. a kind of making method that is used for the complex microbial inoculum of cellulosic ethanol production according to claim 7 is characterized in that in the step 2 static cultivation 62h under 40 ℃ of conditions.
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CN102424808A (en) * 2011-12-26 2012-04-25 南开大学 Straw-degrading composite microbial inoculum and application thereof in pretreatment of ethanol production

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CN102424808A (en) * 2011-12-26 2012-04-25 南开大学 Straw-degrading composite microbial inoculum and application thereof in pretreatment of ethanol production
CN102424808B (en) * 2011-12-26 2013-04-24 南开大学 Preparation method of straw-degrading composite microbial inoculum and application thereof

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