CN104846033A - Method for preparing bioethanol by recovering and reusing resistant cellulase of coupling surface active agents of recombinant bacteria - Google Patents
Method for preparing bioethanol by recovering and reusing resistant cellulase of coupling surface active agents of recombinant bacteria Download PDFInfo
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Abstract
The invention discloses a method for preparing bioethanol by recovering and reusing resistant cellulase of coupling surface active agents of recombinant bacteria. The method includes the following steps: 1) raw material pretreatment; 2) mixing the pre-treated lignocellulose and nutrient solutions containing Tween 80 to obtain a mixture 1; 3) adding cellulose in the mixture 1; adding recombinant saccharomyces cerevisiae expression fungus for fermentation, conducting solid-liquid separation to obtain fermentation liquor and residues; 4) adding the pre-treated lignocellulose in the fermentation liquor for adsorption, conducting solid-liquid separation to obtain liquid and solid, rectifying the liquid to obtain ethyl alcohol, mixing the solid and the residues, adding the nutrient solutions containing Tween 80 for mixing to obtain the mixture; 5) conducting pre-enzymolysis on the mixture obtained in the step 4), adding the recombinant saccharomyces cerevisiae expression fungus for fermentation, conducting solid-liquid separation to obtain fermentation liquor and residues; 6) repeating the steps 4) and 5). The method avoids replenishing expensive beta-glucosidase in the recovery and reuse processes of cellulose and greatly reduces dosage and cost of cellulose for production of ethyl alcohol.
Description
Technical field
The present invention relates to the cellulase recovery method in a kind of lignocellulose refining bio-ethanol process, belong to industrial biotechnology field.
Background technology
Lignocellulose prepares that bio-ethanol is that development is green, one of the production line of sustainable energy most potentiality, is also solve one of resource, the most effective method of environment and energy problem.Be, in raw material production ethanol process, need use cellulase with lignocellulose, cellulase cost accounts for 50% of total cost of production, has become the major obstacle of cellulosic ethanol commercialization process.Cellulase activity is low, consumption is large and cost height is that its cost continues to occupy high major cause.Therefore, for significantly reducing cellulase cost, cellulase high efficiente callback has become with the exploitation of technique the important channel addressed this problem again.
Cellulase comprises circumscribed β-Isosorbide-5-Nitrae-dextranase (CBH), inscribe β-Isosorbide-5-Nitrae-dextranase (EG) and cellobiase (CB).Heavy absorption method recycled fiber element enzyme, because its technique is simple, equipment requirements is low and with low cost, be widely used in cellulase recycling technique, but this recovery method is mainly applicable to the recovery of resolvase in fermented liquid.In large-scale commercial production, lignocellulose diastatic fermentation carries out usually under high concentration of substrate, meets industrial cost-effectiveness requirement (﹥ 4%) to obtain high concentration ethanol.After high density lignocellulose simultaneous saccharification and fermentation, the cellulase content distributed in solid phase significantly increases, and only the cellulase reclaimed in fermented liquid will greatly reduce enzyme organic efficiency, and on fermentation residue, the recycling of adsorptive enzyme is particularly important.On fermented liquid (heavy absorption method) and fermentation residue, cellulase recovery is mainly for CHB and EG; CB, owing to lacking absorption end, is difficult to realize efficient recovery in above process.Therefore, additionally add with needs usual in process again and act on identical beta-glucosidase to obtain high alcohol yied and high-cellulose transformation efficiency with cellobiase (CB) after enzyme reclaims.But industrially conventional Aspergillus niger strain produces beta-glucosidase, and it is expensive, it is adding again by process the enzyme cost significantly increasing cellulosic ethanol production.
Summary of the invention
The object of the invention is to overcome that prior art exists, utilize reclaim cellulase realize lignocellulose prepare bio-ethanol time, beta-glucosidase and the low deficiency of cellulase organic efficiency need be added, provide a kind of recombinant bacterium coupled surface promoting agent resistant cellulose enzyme to recycle the method preparing bio-ethanol.
Technical scheme of the present invention is summarized as follows:
Recombinant bacterium coupled surface promoting agent resistant cellulose enzyme recycling prepares a method for bio-ethanol, comprises the following steps:
1) raw materials pretreatment: by the lignocellulose pre-treatment after pulverizing, oven drying at low temperature;
2) preparating mixture: in proportion by 2.5-10g step 1) lignocellulose that obtains and the 50mL nutritive medium that contains Tween80 is mixed to get mixture 1;
3) simultaneous saccharification and fermentation: mixture 1 is placed in reactor, at 45-50 DEG C, calculate by gauge cellulosic in lignocellulose, every g Mierocrystalline cellulose adds 10-50FPU cellulase, pre-enzymolysis 12-24 hour; Be cooled to 30-37 DEG C, add the recombinant Saccharomyces cerevisiae expression bacterium that can secrete beta-glucosidase and make concentration be 1g/L-5g/L, at 30-37 DEG C of fermentation 72-168 hour, solid-liquid separation, obtains fermented liquid and fermentation residue;
4) cellulase recycling: add and step 2 in fermented liquid) the pretreated lignocellulose of equal in quality, 60-120min is adsorbed at 4-25 DEG C, solid-liquid separation, obtain liquid and solid, described liquid obtains ethanol through rectifying separation, solid and fermentation residue mixing, add and step 2) nutritive medium containing Tween80 of nutritive medium same volume containing Tween80 mixes, and obtains mixture;
5) simultaneous saccharification and fermentation again: by step 4) mixture that obtains is placed in reactor, at 45-50 DEG C, pre-enzymolysis 12-24 hour; Be cooled to 30-37 DEG C, add the recombinant Saccharomyces cerevisiae expression bacterium that can secrete beta-glucosidase and make concentration be 1g/L-5g/L, at 30-37 DEG C of fermentation 72-168 hour, solid-liquid separation, obtains fermented liquid and fermentation residue;
6) repeating step 4)-5); Be 0.5g/L-20g/L containing the concentration of Tween80 in the nutritive medium of Tween80.
Lignocellulose is preferably the corn cob after pulverizing, bagasse, maize straw, straw, straw, wood chip, waste paper or switchgrass.
Step (1) pretreated method is preferably dilute acid pretreatment, alkaline process pre-treatment, mechanical pretreatment, Biological Pretreatment, ammonia leaching pre-treatment, sulfuric acid-sodium hydroxide coupling pre-treatment or the pre-treatment of Sulfuric Acid-Ammonia water coincidence, the preferably pre-treatment of Sulfuric Acid-Ammonia water coincidence.
Advantage of the present invention:
(1) method of the present invention expresses bacterium owing to have employed the recombinant Saccharomyces cerevisiae secreting beta-glucosidase, avoid adding of beta-glucosidase expensive in cellulase recycling process, significantly reduce cellulase consumption and the cost of alcohol production;
(2) fermented liquid and fermentation residue cellulase reclaim the enzyme organic efficiency significantly improved under high concentration of substrate after simultaneous saccharification and fermentation altogether;
(3) organic efficiency of the further resistant cellulose enzyme of tensio-active agent Tween80 is added;
(4) the method technique is simple, easy to operate, with low cost, is easy to set up economically viable cellulase recovery process, in ethanol industryization is produced, have very important application prospect.
Accompanying drawing explanation
Fig. 1 is embodiment 1 cellulase recycling design sketch.
Fig. 2 is embodiment 2 cellulase recycling design sketch.
Specific implementation method
Below by specific embodiment, the present invention is further illustrated.
The following examples are to enable those skilled in the art understand the present invention better, but are not limited in any way claim.
It is with China Patent No. 201110034926.9 that the recombinant Saccharomyces cerevisiae secreting beta-glucosidase of various embodiments of the present invention employing expresses bacterium, denomination of invention is: the gene of encoding beta-glucosidase and recombinant expression vector and recombinant Saccharomyces cerevisiae expression strain and application disclosed " recombinant Saccharomyces cerevisiae " are example, but not as restriction, every recombinant Saccharomyces cerevisiae secreting beta-glucosidase is expressed bacterium and all be can be used for the present invention.
Be YPD substratum for the substratum of the enlarged culturing of the recombination microzyme of encoding beta-glucosidase can be secreted in each embodiment:
Nutritive medium containing Tween80 the: with the addition of (NH that final concentration is 0.5g/L in pH=4.9,50mM sodium citrate buffer solution
4)
2hPO
4, the MgSO of 0.025g/L
4.7H
2the yeast extract paste of O, 1.0g/L, the tsiklomitsin of 40mg/L and Tween80.
Embodiment 1
Recombinant bacterium coupled surface promoting agent resistant cellulose enzyme recycling prepares a method for bio-ethanol, comprises the following steps:
1) raw materials pretreatment: by the method pre-treatment of the corn cob (lignocellulose) after pulverizing by Sulfuric Acid-Ammonia water coincidence, oven drying at low temperature;
2) preparating mixture: by 5g step 1) obtain the nutritive medium that lignocellulose and 50mL contain Tween80 and be mixed to get mixture 1;
3) simultaneous saccharification and fermentation: mixture 1 is placed in shaking flask, at 50 DEG C, cellulase is added in shaking flask, calculate by gauge cellulosic in lignocellulose, every g Mierocrystalline cellulose adds cellulase 30FPU (mass ratio that Mierocrystalline cellulose accounts for lignocellulose is 70%), pre-enzymolysis 12 hours; Be cooled to 30 DEG C, add the recombinant Saccharomyces cerevisiae expression bacterium that can secrete beta-glucosidase after enlarged culturing and make concentration be 2g/L, 30 DEG C of fermentations 96 hours, solid-liquid separation, obtained fermented liquid and fermentation residue;
4) cellulase recycling: add the pretreated lignocellulose of 5g in fermented liquid, 120min is adsorbed at 20 DEG C, solid-liquid separation, obtain liquid and solid, described liquid obtains ethanol through rectifying separation, solid and fermentation residue mixing, add the nutritive medium mixing that 50mL contains Tween80, obtain mixture;
5) simultaneous saccharification and fermentation again: by step 4) mixture that obtains is placed in shaking flask, at 50 DEG C, pre-enzymolysis 12 hours; Be cooled to 30 DEG C, add the recombinant Saccharomyces cerevisiae expression bacterium that can secrete beta-glucosidase after enlarged culturing and make concentration be 2g/L, 30 DEG C of fermentations 96 hours, solid-liquid separation, obtained fermented liquid and fermentation residue;
6) 1 step 4)-5 is repeated); Be 5g/L containing the concentration of Tween80 in the nutritive medium of Tween80.
Time (at repeating step 4), step 4) fermented liquid be last reactions steps 5) fermented liquid that obtains)
With alcohol concn and cellulase production ability for inspection target, evaluate cellulase recycle efficiency, as shown in Figure 1.
First time simultaneous saccharification and fermentation, i.e. step 1)-3), from step 3) fermented liquid that obtains gets trace detection alcohol concn;
Second time simultaneous saccharification and fermentation, i.e. step 5), from step 5) fermented liquid that obtains gets trace detection alcohol concn;
For the third time, namely repeat 1 step 4)-5), the step 5 from repetition 1 time) fermented liquid that obtains gets trace detection alcohol concn;
Survey the concentration of ethanol in fermented liquid: three times gained alcohol concn is followed successively by 33.05g/L, 29.98g/L and 28.36g/L, producing the alcohol ability rate of recovery is 90.7% and 85.8%.
Experiment proves, substitute the corn cob of the present embodiment with straw, wood chip, waste paper or switchgrass, other same the present embodiment, its effect is similar to the effect of the present embodiment.
Experiment proves, dilute acid pretreatment, mechanical pretreatment, Biological Pretreatment substitute other same the present embodiment of Sulfuric Acid-Ammonia water coincidence pre-treatment of the present embodiment, and its effect is similar to the effect of the present embodiment.
Embodiment 2
Recombinant bacterium coupled surface promoting agent resistant cellulose enzyme recycling prepares the method for bio-ethanol, comprises the following steps:
1) raw materials pretreatment: the method pre-treatment that the maize straw (lignocellulose) after pulverizing is soaked by ammonia, oven drying at low temperature;
2) preparating mixture: by 5g step 1) obtain the nutritive medium that lignocellulose and 50mL contain Tween80 and be mixed to get mixture 1;
3) simultaneous saccharification and fermentation: mixture 1 is placed in shaking flask, at 45 DEG C, adds cellulase in shaking flask, and calculate by gauge cellulosic in lignocellulose, every g Mierocrystalline cellulose adds cellulase 30FPU, pre-enzymolysis 24 hours; Be cooled to 35 DEG C, add the recombinant Saccharomyces cerevisiae expression bacterium that can secrete beta-glucosidase after enlarged culturing and make concentration be 1g/L, 35 DEG C of fermentations 168 hours, solid-liquid separation, obtained fermented liquid and fermentation residue;
4) cellulase recycling: add the pretreated lignocellulose of 5g in fermented liquid, 120min is adsorbed at 4 DEG C, solid-liquid separation, obtain liquid and solid, described liquid obtains ethanol through rectifying separation, described solid and fermentation residue mixing, add the nutritive medium mixing that 50mL contains Tween80, obtain mixture;
5) simultaneous saccharification and fermentation again: by step 4) mixture that obtains is placed in shaking flask, at 45 DEG C, pre-enzymolysis 24 hours; Be cooled to 35 DEG C, add the recombinant Saccharomyces cerevisiae expression bacterium that can secrete beta-glucosidase after enlarged culturing and make concentration be 1g/L, 35 DEG C of fermentations 168 hours, solid-liquid separation, obtained fermented liquid and fermentation residue;
6) 1 step 4)-5 is repeated); Be 0.5g/L containing the concentration of Tween80 in the nutritive medium of Tween80.
With alcohol concn and cellulase production ability for inspection target, evaluate cellulase recycle efficiency, as shown in Figure 2.
First time simultaneous saccharification and fermentation, i.e. step 1)-3), from step 3) fermented liquid that obtains gets trace detection alcohol concn;
Second time simultaneous saccharification and fermentation, i.e. step 5), from step 5) fermented liquid that obtains gets trace detection alcohol concn;
For the third time, namely repeat 1 step 4)-5), the step 5 from repetition 1 time) fermented liquid that obtains gets trace detection alcohol concn;
Survey the concentration of ethanol in fermented liquid: three times gained alcohol concn is followed successively by 32.23g/L, 27.65g/L and 24.53g/L, producing the alcohol ability rate of recovery is 85.8% and 76.1%.
Embodiment 3
Recombinant bacterium coupled surface promoting agent resistant cellulose enzyme recycling prepares the method for bio-ethanol, comprises the following steps:
1) raw materials pretreatment: by the straw (lignocellulose) after pulverizing by alkaline process (20g/L aqueous sodium hydroxide solution) pre-treatment, oven drying at low temperature;
2) preparating mixture: by 10g step 1) obtain the nutritive medium that lignocellulose and 50mL contain Tween80 and be mixed to get mixture 1;
3) simultaneous saccharification and fermentation: mixture 1 is placed in shaking flask, at 50 DEG C, adds cellulase in shaking flask, and calculate by gauge cellulosic in lignocellulose, every g Mierocrystalline cellulose adds cellulase 50FPU, pre-enzymolysis 12 hours; Be cooled to 37 DEG C, add the recombinant Saccharomyces cerevisiae expression bacterium that can secrete beta-glucosidase after enlarged culturing and make concentration be 5g/L, 37 DEG C of fermentations 72 hours, solid-liquid separation, obtained fermented liquid and fermentation residue;
4) cellulase recycling: add the pretreated lignocellulose of 10g in fermented liquid, 60min is adsorbed at 25 DEG C, solid-liquid separation, obtain liquid and solid, described liquid obtains ethanol through rectifying separation, described solid and fermentation residue mixing, add the nutritive medium mixing that 50mL contains Tween80, obtain mixture;
5) simultaneous saccharification and fermentation again: by step 4) mixture that obtains is placed in shaking flask, at 50 DEG C, pre-enzymolysis 12 hours; Be cooled to 37 DEG C, add the recombinant Saccharomyces cerevisiae expression bacterium that can secrete beta-glucosidase after enlarged culturing and make concentration be 5g/L, 37 DEG C of fermentations 72 hours, solid-liquid separation, obtained fermented liquid and fermentation residue;
6) 2 step 4)-5 are repeated); Be 20g/L containing the concentration of Tween80 in the nutritive medium of Tween80.
With alcohol concn and cellulase production ability for inspection target, evaluate cellulase recycle efficiency.
First time simultaneous saccharification and fermentation, i.e. step 1)-3), from step 3) fermented liquid that obtains gets trace detection alcohol concn;
Second time simultaneous saccharification and fermentation, i.e. step 5), from step 5) fermented liquid that obtains gets trace detection alcohol concn;
For the third time, namely repeat 1 step 4)-5), the step 5 from repetition 1 time) fermented liquid that obtains gets trace detection alcohol concn;
4th time, namely repeat 1 step 4)-5), the step 5 from repetition 1 time) fermented liquid that obtains gets trace detection alcohol concn;
Survey the concentration of ethanol in fermented liquid: four times gained alcohol concn is followed successively by 51.62g/L, 47.41g/L, 43.85g/L and 35.52g/L, producing the alcohol ability rate of recovery is 91.8%, 84.9% and 68.8%.
Embodiment 4
Recombinant bacterium coupled surface promoting agent resistant cellulose enzyme recycling prepares the method for bio-ethanol, comprises the following steps:
1) raw materials pretreatment: by the bagasse (lignocellulose) after pulverizing by sulfuric acid-sodium hydroxide coupling pre-treatment, oven drying at low temperature;
2) preparating mixture: by 2.5g step 1) obtain the nutritive medium that lignocellulose and 50mL contain Tween80 and be mixed to get mixture 1;
3) simultaneous saccharification and fermentation: mixture 1 is placed in shaking flask, at 50 DEG C, adds cellulase in shaking flask, and calculate by gauge cellulosic in lignocellulose, every g Mierocrystalline cellulose adds cellulase 10FPU, pre-enzymolysis 12 hours; Be cooled to 37 DEG C, add after enlarged culturing can secrete β ?the recombinant Saccharomyces cerevisiae of glucuroide express bacterium and make concentration be 5g/L, 37 DEG C of fermentations 168 hours, solid-liquid separation, obtained fermented liquid and fermentation residue;
4) cellulase recycling: add the pretreated lignocellulose of 2.5g in fermented liquid, 60min is adsorbed at 25 DEG C, solid-liquid separation, obtain liquid and solid, described liquid obtains ethanol through rectifying separation, described solid and fermentation residue mixing, add the nutritive medium mixing that 50mL contains Tween80, obtain mixture;
5) simultaneous saccharification and fermentation again: by step 4) mixture that obtains is placed in shaking flask, at 50 DEG C, pre-enzymolysis 12 hours; Be cooled to 37 DEG C, add the recombinant Saccharomyces cerevisiae expression bacterium that can secrete beta-glucosidase after enlarged culturing and make concentration be 5g/L, 37 DEG C of fermentations 168 hours, solid-liquid separation, obtained fermented liquid and fermentation residue;
6) 1 step 4)-5 is repeated); Be 20g/L containing the concentration of Tween80 in the nutritive medium of Tween80.
With alcohol concn and cellulase production ability for inspection target, evaluate cellulase recycle efficiency.
First time simultaneous saccharification and fermentation, i.e. step 1)-3), from step 3) fermented liquid that obtains gets trace detection alcohol concn;
Second time simultaneous saccharification and fermentation, i.e. step 5), from step 5) fermented liquid that obtains gets trace detection alcohol concn;
For the third time, namely repeat 1 step 4)-5), the step 5 from repetition 1 time) fermented liquid that obtains gets trace detection alcohol concn;
Survey the concentration of ethanol in fermented liquid: three times gained alcohol concn is followed successively by 18.23g/L, 16.22g/L and 13.68g/L, producing the alcohol ability rate of recovery is 88.9% and 75.0%.
Claims (4)
1. recombinant bacterium coupled surface promoting agent resistant cellulose enzyme recycling prepares a method for bio-ethanol, it is characterized in that comprising the following steps:
1) raw materials pretreatment: by the lignocellulose pre-treatment after pulverizing, oven drying at low temperature;
2) preparating mixture: in proportion by 2.5-10g step 1) lignocellulose that obtains and the 50mL nutritive medium that contains Tween80 is mixed to get mixture 1;
3) simultaneous saccharification and fermentation: mixture 1 is placed in reactor, at 45-50 DEG C, calculate by gauge cellulosic in lignocellulose, every g Mierocrystalline cellulose adds 10-50FPU cellulase, pre-enzymolysis 12-24 hour; Be cooled to 30-37 DEG C, add the recombinant Saccharomyces cerevisiae expression bacterium that can secrete beta-glucosidase and make concentration be 1g/L-5g/L, at 30-37 DEG C of fermentation 72-168 hour, solid-liquid separation, obtains fermented liquid and fermentation residue;
4) cellulase recycling: add and step 2 in fermented liquid) the pretreated lignocellulose of equal in quality, 60-120min is adsorbed at 4-25 DEG C, solid-liquid separation, obtain liquid and solid, described liquid obtains ethanol through rectifying separation, solid and fermentation residue mixing, add and step 2) nutritive medium containing Tween80 of nutritive medium same volume containing Tween80 mixes, and obtains mixture;
5) simultaneous saccharification and fermentation again: by step 4) mixture that obtains is placed in reactor, at 45-50 DEG C, pre-enzymolysis 12-24 hour; Be cooled to 30-37 DEG C, add the recombinant Saccharomyces cerevisiae expression bacterium that can secrete beta-glucosidase and make concentration be 1g/L-5g/L, at 30-37 DEG C of fermentation 72-168 hour, solid-liquid separation, obtains fermented liquid and fermentation residue;
6) repeating step 4)-5); Be 0.5g/L-20g/L containing the concentration of Tween80 in the nutritive medium of Tween80.
2. method according to claim 1, is characterized in that described lignocellulose is corn cob after pulverizing, bagasse, maize straw, straw, straw, wood chip, waste paper or switchgrass.
3. method according to claim 1, is characterized in that step (1) pretreated method is dilute acid pretreatment, alkaline process pre-treatment, mechanical pretreatment, Biological Pretreatment, ammonia leaching pre-treatment, sulfuric acid-sodium hydroxide coupling pre-treatment or the pre-treatment of Sulfuric Acid-Ammonia water coincidence.
4. method according to claim 3, is characterized in that step (1) pretreated method is the pre-treatment of Sulfuric Acid-Ammonia water coincidence.
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