CN104745641B - Method for producing ethanol by fermenting lignocellulose through natural pH - Google Patents
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Abstract
The invention discloses a method for producing ethanol by fermenting lignocellulose with natural pH, which adopts an amplification culture medium containing C5 and C6 as carbon sources to carry out specific induction culture on specific strains, so that the strains keep better fermentation activity, the fermentation with natural pH is realized, and the fermentation performance of the strains is better. Meanwhile, the meta-acid of the fermentation system can effectively inhibit the growth of mixed bacteria such as lactic acid bacteria and the like, and the concentration of lactic acid, acetic acid and the like generated by the mixed bacteria in the fermentation liquor is obviously reduced.
Description
Technical Field
The invention belongs to the field of microbial fermentation, and particularly relates to a method for producing ethanol by fermenting lignocellulose through natural pH.
Background
The increasing exhaustion of many non-renewable fossil energy sources such as petroleum leads to the growing concern of renewable energy sources, especially biofuel, and brings great commercial and social significance.
Ethanol is a clean renewable liquid fuel and many countries have begun to use gasoline-gasoline alcohol with a proportion of ethanol added to replace gasoline consumption. The novel fuel can relieve the consumption rate of petroleum, can reduce the pollution of automobile exhaust, and has great application and development potential. China starts to popularize and use gasoline alcohol from 2001, and the gasoline alcohol accounts for about 20% of the total consumption of gasoline fuels at present and is at the trend of increasing year by year.
At present, grain crops such as corn and the like are used as main raw materials for producing ethanol at home and abroad. With the increasing world population, food is in increasing shortage, so food crops are not ideal raw materials for producing ethanol in the long run.
Biomass energy is an important renewable energy source in the future energy field. In recent years, all countries in the world have vigorously developed related technologies for comprehensively utilizing biomass, and certain achievements are achieved in the aspects of producing fuel ethanol, biodiesel, biohydrogen, biobiogas and the like by utilizing biomass, so that the related technologies of biomass energy sources are hot spots for research and development of all countries in the future. China has abundant biomass resources, and according to statistics, the yield of only straws in China can reach 7 hundred million tons every year, so that considerable biomass energy is stored in China and further development and utilization are still needed.
At present, the related technology for producing ethanol by adopting a biomass fermentation mode, namely the related technology for producing ethanol by fermenting lignocellulose in biomass, has already formed a certain industrialization scale. However, the following problems are common: 1) in order to ensure that the fermentation strain is in the most suitable pH environment and has stronger activity so as to improve the yield of ethanol, a proper amount of alkali has to be added in the fermentation process to adjust the pH of the fermentation liquor in real time, so that the operation in the fermentation process is complicated, the fermentation yield is easily influenced by the adjustment and control error, the growth of mixed bacteria such as lactic acid bacteria and the like is facilitated in the environment of adding the alkali, and the content of the mixed bacteria in a fermentation system is easily increased.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a method for producing ethanol by fermenting lignocellulose without adjusting pH in the fermentation process.
The method for producing ethanol by fermenting lignocellulose through natural pH comprises the following steps:
1) taking a Ho-Purdue yeast 424A (LNH-ST) strain for amplification culture;
2) carrying out enzymolysis on a lignocellulose-containing raw material to obtain a fermentation culture medium containing lignocellulose-containing raw material enzymolysis liquid;
3) inoculating the strain subjected to the amplification culture in the step 1) into the fermentation medium obtained in the step 2), controlling the temperature to be 28-35 ℃, the rotating speed to be 50-100rpm/min and the natural pH value, and fermenting for 24-72 hours to obtain a fermentation liquid;
4) separating and extracting ethanol in the fermentation liquor obtained in the step 3).
Wherein the Ho-Purdue yeast 424A (LNH-ST) is commercially available from Green technology, Inc., USA.
In the step 1), the culture conditions in the step of expanding culture are specifically as follows: the pH value of the amplification culture medium is controlled to be 4-6, the amplification temperature is controlled to be 28-35 ℃, and the rotation speed is controlled to be 100-200 rpm/min.
In the step 1), the amplification culture medium selected in the amplification culture step comprises 2-10 wt% of glucose and 1-6 wt% of xylose. Preferably, the content of glucose in the culture medium of the expanded culture is 4-6% by weight, and the content of xylose is 1-4% by weight.
The expanding culture medium also comprises one or more of corn mash, molasses and sweet sorghum stalk juice as carbon sources, and one or more of soybean cake powder, corn steep liquor, fish meal, peptone and yeast extract as nitrogen sources.
The expanding culture medium also comprises urea with the content of 2-5g/L and/or inorganic salt with the content of 0.5-4 g/L. Wherein the inorganic salt is one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and diammonium hydrogen phosphate. Preferably, the culture medium for the expanded culture contains 1.5g/L of the inorganic salt potassium dihydrogen phosphate and 1.5g/L of diammonium hydrogen phosphate.
Further, the expanding culture is to inoculate and expand the culture to (0.2-0.3) multiplied by 10 by 5% -20% of the inoculation amount9/ml。
In the step 2), the step of enzymolysis of the lignocellulose-containing raw material specifically comprises: adding 100-200fpu filter paper enzyme activity unit enzyme into the lignocellulose-containing raw material, mixing at 40-60 deg.C for 48-96h to obtain lignocellulose raw material enzymolysis liquid with sugar concentration of 8-16%, and adjusting pH of the enzymolysis liquid to 4-6.
The lignocellulose-containing material is selected from corn stover, corn cobs, hardwoods, softwoods, nut shells, grasses, paper, barley straw, wheat straw, leaves, cottonseed batting, newspapers, willows, or oat hulls.
Structurally, lignocellulose mainly includes cellulose, hemicellulose and lignin. In a preferred embodiment, the lignocellulose-containing feedstock comprises at least 30 wt.%, preferably at least 50 wt.%, more preferably at least 70 wt.%, even more preferably at least 90 wt.% lignocellulose. It is to be understood that the lignocellulose-containing material may also comprise other components, such as proteinaceous material, starch, sugars, such as fermentable sugars and/or non-fermentable sugars. Preferably, the lignocellulose-containing material is corn stover.
Wherein, the cellulase includes but is not limited to cellobiohydrolases (cellobiohydrolase I and cellobiohydrolase II) as well as endoglucanases and β -glucosidase.
The lignocellulose-containing raw material enzymolysis liquid also comprises an inhibitor with the working concentration of 2-20 units/mL; the inhibitor is one or more of penicillin, ampicillin, streptomycin, chloramphenicol, oxytetracycline, and tetracycline.
The enzymolysis solution of the lignocellulose-containing raw material also contains 2-5g/L of urea and 0.5-4g/L of inorganic salt.
Preferably, before the enzymolysis of the lignocellulosic raw material in step 2), the lignocellulosic raw material is pretreated by one or more of a cooking method, an acid leaching method, an alkali treatment, a steam explosion and a colloid mill crushing method, which are commonly used in the art.
In the step 3), the strain subjected to the amplification culture in the step 1) is inoculated into the fermentation medium obtained in the step 2), and the inoculation amount is 5-20%.
In the step 1), the method further comprises a step of performing seed liquid culture on the strain before the step of expanding culture, wherein the conditions of the seed liquid culture step are as follows: the pH value is controlled to be 4-6, the culture temperature is controlled to be 28-35 ℃, and the rotation speed is 100-.
The seed liquid culture medium in the seed liquid culture step comprises 10g/L of yeast powder, 20g/L of peptone and 20g/L of glucose. The seed liquid culture medium is prepared by mixing yeast powder, peptone and glucose after subpackaging and sterilizing.
Further, the seed liquid culture comprises: culturing the Ho-Purdue yeast 424A (LNH-ST) strain to (0.1-0.5) x 10 with the seed liquid culture medium9/ml。
Compared with the prior art, the technical scheme of the invention has the following advantages:
(1) according to the method for producing ethanol by fermentation, the specific strain is selected to ferment the enzymolysis liquid of the lignocellulose-containing raw material to produce ethanol, the change of the pH of the fermentation liquid in the whole fermentation process has little influence on the activity of the strain, the fermentation of the ethanol can be realized under the condition of not adjusting the natural pH of the fermentation liquid, the higher ethanol yield is realized, and the complex operation and the product inhibition caused by continuously adding alkali in the fermentation process are avoided; meanwhile, the pH value is relatively low in the fermentation process, so that the growth of mixed bacteria such as lactic acid bacteria and the like is inhibited, and the concentration of lactic acid, acetic acid and the like generated by the mixed bacteria in the fermentation liquid is obviously low through experimental determination;
(2) the method for producing the ethanol by fermentation adopts the selected enlarged culture medium containing C5 and C6 as carbon sources to carry out specific induction culture on specific strains, so that the strains keep better fermentation activity, the fermentation of natural pH is realized, and the fermentation performance of the strains is better.
Detailed Description
Example 1
The method for producing ethanol by fermenting lignocellulose through natural pH comprises the following steps:
1) taking the Ho-Purdue yeast 424A (LNH-ST) strain to carry out seed culture and amplification culture; the cultivation process specifically comprises the following steps:
seed culture: subpackaging and sterilizing yeast powder 10g/L, peptone 20g/L and glucose 20g/L, mixing to obtain a seed liquid culture medium, inoculating the Ho-Purdue yeast 424A (LNH-ST) strain to the culture medium, and culturing for 20h until the strain concentration reaches (0.2-0.3) multiplied by 109/ml。
And (3) amplification culture: inoculating the thallus obtained from the seed culture into a shake flask containing an expanding culture medium according to the inoculation amount of 5%, wherein the expanding culture temperature is 30 ℃, the rotation speed is 200rpm, the culture time is 16h, inoculating the first-stage thallus 5% into a second-stage expanding culture medium for culture, adjusting the pH value to 5, the expanding culture temperature is 30 ℃, the rotation speed is 200rpm, expanding culture is 16h, and expanding culture is carried out until the temperature is 0.2-0.3 multiplied by 109/ml。
The culture medium for the enlarged culture is 2% glucose and 6% xylose, and contains glucose 2% (by mass), xylose 6% (by mass), and corn steep liquor (dry matter content 40%) 15g/L, KH2PO41.5g/L、(NH4)2HPO41.5 g/L; preparing solution with deionized water or softened water, adjusting pH to 6.0, and separatingSterilizing, cooling, mixing, and adding 3g/L urea.
2) Adding cellulase with the activity unit of 200fpu filter paper enzyme into the lignocellulose raw material, preserving heat and mixing for 72 hours at 50 ℃ to obtain lignocellulose raw material enzymatic hydrolysate with the sugar concentration of 8%, and adjusting the pH value to 5.0; wherein the lignocellulose raw material is corn straw.
3) Inoculating the strain obtained in the enlarged culture into the lignocellulose raw material enzymolysis liquid obtained in the step 2) by 10 percent of inoculation amount, keeping the temperature at 30 ℃, shaking the bottle at the rotating speed of 75rpm/min, and fermenting for 72 hours at natural pH value.
4) Separating the ethanol in the fermentation liquor obtained in the step 3).
Example 2
The method for producing ethanol by fermenting lignocellulose through natural pH comprises the following steps:
1) taking the Ho-Purdue yeast 424A (LNH-ST) strain to carry out seed culture and amplification culture; the cultivation process specifically comprises the following steps:
seed culture: subpackaging and sterilizing yeast powder 10g/L, peptone 20g/L and glucose 20g/L, mixing to obtain a seed liquid culture medium, inoculating the Ho-Purdue yeast 424A (LNH-ST) strain to the culture medium, culturing at 30 ℃ and pH6.0 until the strain concentration reaches (0.1-0.2) multiplied by 109/ml。
And (3) amplification culture: inoculating the thallus obtained from the seed culture into a shake flask containing an expanding culture medium according to the inoculum size of 20%, adjusting pH to 4, expanding culture temperature to 35 deg.C, rotating speed of 100rpm, and culturing for 14h, and expanding culture to (0.2-0.3) x 109/ml。
The culture medium for the enlarged culture comprises sweet sorghum stalk juice containing 10% of glucose and 1% of xylose in percentage by weight, 5g/L of corn cake powder and 4g/L of monopotassium phosphate; preparing solution with deionized water or softened water, subpackaging, sterilizing, cooling, mixing, and adding 2g/L urea.
2) Adding amylase with the activity unit of 100fpu filter paper enzyme and tetracycline with the working concentration of 2 units/mL into the lignocellulose raw material, preserving the heat at 40 ℃ and mixing for 48 hours to obtain lignocellulose raw material enzymatic hydrolysate with the sugar concentration of 16%, and adjusting the pH to 4.0; wherein the lignocellulose raw material is leaf and cotton seed wadding.
3) Inoculating the strain obtained in the enlarged culture into the lignocellulose raw material enzymolysis liquid obtained in the step 2) by using the inoculation amount of 20%, keeping the temperature at 28 ℃, keeping the shaking bottle rotating speed at 50rpm/min, keeping the pH value natural, and fermenting for 24 hours;
4) separating ethanol in the fermentation liquor obtained in the step 3) to obtain the product;
example 3
The method for producing ethanol by fermenting lignocellulose through natural pH comprises the following steps:
1) taking the Ho-Purdue yeast 424A (LNH-ST) strain to carry out seed culture and amplification culture; the cultivation process specifically comprises the following steps:
seed culture: subpackaging and sterilizing yeast powder 10g/L, peptone 20g/L and glucose 20g/L, mixing to obtain a seed liquid culture medium, inoculating the Ho-Purdue yeast 424A (LNH-ST) strain to the culture medium, and culturing until the strain concentration reaches (0.4-0.5) multiplied by 109/ml。
And (3) amplification culture: inoculating the thallus obtained from the seed culture into a shake flask containing an expanding culture medium according to the inoculum size of 10%, adjusting pH to 6, expanding culture temperature to 28 deg.C, rotating speed of 150rpm, and culturing for 14h, and expanding culture to (0.2-0.3) x 109/ml。
The culture medium for the enlarged culture is a molasses culture medium containing 6 percent of glucose and 4 percent of xylose, and contains 6 percent of glucose (mass percent), 4 percent of xylose (mass percent), 10g/L of fish meal and 0.5g/L of sodium dihydrogen phosphate; preparing solution with deionized water or softened water, adjusting pH to 7.0, packaging, sterilizing, cooling, mixing, and adding 5g/L urea.
2) Soaking oat hulls serving as a lignocellulose raw material in alkali, adding protease with 150fpu filter paper enzyme activity units, streptomycin with the working concentration of 20 units/mL, urea with the concentration of 2g/L and inorganic salt sodium dihydrogen phosphate with the concentration of 4g/L, preserving heat and mixing for 96 hours at the temperature of 60 ℃ to obtain lignocellulose raw material enzymolysis liquid with the sugar concentration of 12%, and adjusting the pH value to 6;
3) inoculating the strain obtained in the enlarged culture into the lignocellulose raw material enzymolysis liquid obtained in the step 2) by an inoculation amount of 5%, keeping the temperature at 32 ℃, shaking the flask at a rotating speed of 100rpm/min, and fermenting for 48 hours, wherein the pH value is natural;
4) separating ethanol in the fermentation liquor obtained in the step 3) to obtain the product;
example 4
The method for producing ethanol by fermenting lignocellulose through natural pH comprises the following steps:
1) taking the Ho-Purdue yeast 424A (LNH-ST) strain to carry out seed culture and amplification culture; the cultivation process specifically comprises the following steps:
seed culture: subpackaging and sterilizing yeast powder 10g/L, peptone 20g/L and glucose 20g/L, mixing to obtain a seed liquid culture medium, inoculating the Ho-Purdue yeast 424A (LNH-ST) strain to the culture medium, and culturing until the strain concentration reaches (0.4-0.5) multiplied by 109/ml。
And (3) amplification culture: inoculating the thallus obtained from the seed culture into a shake flask containing an expanding culture medium according to the inoculum size of 5%, adjusting pH to 4, expanding culture temperature of 37 deg.C, rotating speed of 200rpm, and culturing for 14h, and expanding culture to (0.2-0.3) x 109/ml。
The culture medium for the enlarged culture is corn mash with 4% of glucose and 2% of xylose, and contains 4% (mass percent) of glucose, 2% (mass percent) of xylose, 7g/L of soybean cake powder and 2.3g/L of dipotassium phosphate; preparing solution with deionized water or softened water, subpackaging, sterilizing, cooling, mixing, and adding 3g/L urea.
2) Taking corncobs and hardwoods as lignocellulose raw materials, cooking for 1h, adding cellulose with the activity unit of 120fpu filter paper enzyme, ampicillin with the working concentration of 10 units/mL, 5g/L urea and 0.5g/L ammonium dihydrogen phosphate, and carrying out heat preservation and mixing for 72h at 50 ℃ to obtain lignocellulose raw material enzymatic hydrolysate;
3) inoculating the strain obtained in the enlarged culture into the lignocellulose raw material enzymolysis liquid obtained in the step 2) by 10 percent of inoculation amount, keeping the temperature at 35 ℃, shaking the flask at the rotating speed of 100rpm/min, and fermenting for 72 hours, wherein the pH value is natural;
4) separating the ethanol in the fermentation liquor obtained in the step 3).
Example 5
The method for producing ethanol by fermenting lignocellulose through natural pH comprises the following steps:
1) taking the Ho-Purdue yeast 424A (LNH-ST) strain to carry out seed culture and amplification culture; the cultivation process specifically comprises the following steps:
seed culture: subpackaging and sterilizing yeast powder 10g/L, peptone 20g/L and glucose 20g/L, mixing to prepare a seed liquid culture medium, inoculating the Ho-Purdue yeast 424A (LNH-ST) strain to the culture medium, culturing for 14h, expanding and culturing to (0.4-0.5) multiplied by 109/ml。
And (3) amplification culture: inoculating the thallus obtained from the seed culture into a shake flask containing an expanding culture medium according to the inoculum size of 10%, expanding culture at 30 deg.C, rotating speed of 200rpm, culturing for 14h, and expanding culture to (0.2-0.3) × 109/ml。
The culture medium for the enlarged culture is a mixture of molasses and sweet sorghum stalk juice, and contains 4 percent (mass percent) of glucose, 5 percent (mass percent) of xylose, 5g/L of a mixture of peptone and yeast extract, 1.5g/L of disodium hydrogen phosphate and 1.5g/L of diammonium hydrogen phosphate; preparing solution with deionized water or softened water, subpackaging, sterilizing, cooling, mixing, and adding 3g/L urea.
2) Adding glucoamylase, 3g/L urea and 2.3g/L ammonium dihydrogen phosphate into the lignocellulose raw material, and carrying out heat preservation and mixing at 50 ℃ for 72h to obtain lignocellulose raw material enzymatic hydrolysate;
3) inoculating the strain obtained in the enlarged culture into the lignocellulose raw material enzymolysis liquid obtained in the step 2) by 10 percent of inoculation amount, keeping the temperature at 30 ℃, keeping the shaking bottle rotating speed at 80rpm/min, and fermenting for 72 hours, wherein the pH value is natural;
4) separating the ethanol in the fermentation liquor obtained in the step 3).
Comparative example
In the method for producing ethanol of the present comparative example, commercially available candida tropicalis candida is used, the seed culture and the expanded culture are performed by the same method as in example 1, and the fermentation is performed by the same fermentation broth and the same fermentation conditions, and the difference is that: and adding ammonia water to adjust the pH value in the fermentation process, and supplementing 20% ammonia water according to the change of the pH value in real time by using an online pH value automatic adjustment system to keep the pH value between 4.6 and 5.5.
Experimental examples of Effect
Ethanol was obtained according to the method of examples 1 to 5 and comparative example.
And measuring the initial sugar concentration, the final sugar concentration, the xylose consumption rate, the theoretical total sugar-alcohol conversion rate, the lactic acid concentration and the acetic acid concentration in the fermented liquid after fermentation.
The test results are as follows:
therefore, the ethanol produced by the method can achieve higher ethanol yield, and meanwhile, pH adjustment is not needed in the ethanol fermentation process, so that the cost is reduced, and the operation in the fermentation process is simplified. In addition, when the method is used for producing the ethanol, the concentrations of the lactic acid and the acetic acid are both obviously reduced, which shows that the content of mixed bacteria such as lactic acid bacteria and the like in a fermentation system is reduced.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A method for producing ethanol by fermenting lignocellulose through natural pH is characterized by comprising the following steps:
1) taking a Ho-Purdue yeast 424A (LNH-ST) strain to perform seed liquid culture and amplification culture; the expanding culture medium selected in the expanding culture step comprises 2-10% of glucose and 1-6% of xylose by weight;
culturing the strain to (0.1-0.5) x 10 with the seed solution9/ml；
The strain is cultured in an enlarged manner to (0.2-0.3). times.109/ml；
The inoculum size of the strain inoculated into the expanded culture medium after the seed liquid culture is 5-20%;
2) carrying out enzymolysis on a lignocellulose-containing raw material to obtain a fermentation culture medium containing lignocellulose-containing raw material enzymolysis liquid;
3) inoculating the strain subjected to the amplification culture in the step 1) into the fermentation medium obtained in the step 2), controlling the temperature to be 28-35 ℃, the rotating speed to be 50-100rpm/min and the natural pH value, and fermenting for 24-72 hours to obtain a fermentation liquid;
the inoculation amount of the strain after the expanded culture inoculated into the fermentation medium is 5-20%;
4) separating and extracting ethanol in the fermentation liquor obtained in the step 3).
2. The method of producing ethanol by natural pH fermentation of lignocellulose as recited in claim 1, wherein:
in the step 1), the culture conditions in the step of expanding culture are specifically as follows: the pH value of the amplification culture medium is controlled to be 4-6, the amplification temperature is controlled to be 28-35 ℃, and the rotation speed is controlled to be 100-200 rpm/min.
3. The method of producing ethanol by natural pH fermentation of lignocellulose as recited in claim 1, wherein:
the expanding culture medium also comprises one or more of corn mash, molasses and sweet sorghum stalk juice as carbon sources, and one or more of soybean cake powder, corn steep liquor, fish meal, peptone and yeast extract as nitrogen sources.
4. The method of producing ethanol by natural pH fermentation of lignocellulose as recited in claim 1 or 3, wherein: the expanding culture medium also comprises urea with the content of 2-5g/L and/or inorganic salt with the content of 0.5-4 g/L.
5. The method of producing ethanol by natural pH fermentation of lignocellulose as recited in claim 1 or 3, wherein: in the step 2), the step of enzymolysis of the lignocellulose-containing raw material specifically comprises: adding 100-200fpu filter paper enzyme activity unit enzyme into the lignocellulose-containing raw material, keeping the temperature at 40-60 ℃, mixing for 48-96h to obtain lignocellulose raw material enzymolysis liquid with the sugar concentration of 8-16%, and adjusting the pH of the enzymolysis liquid to 4-6.
6. The method of producing ethanol by natural pH fermentation of lignocellulose as recited in claim 5, wherein:
the lignocellulose-containing material is selected from corn stover, corn cobs, hardwoods, softwoods, nut shells, grasses, paper, barley straw, wheat straw, leaves, cottonseed batting, newspapers, willows, or oat hulls.
7. The method of producing ethanol by natural pH fermentation of lignocellulose as recited in claim 5, wherein:
the enzyme is selected from cellulase, hemicellulase, amylase, protease, glucoamylase or lipase.
8. The method of producing ethanol by natural pH fermentation of lignocellulose as recited in claim 5, wherein:
the lignocellulose-containing raw material enzymolysis liquid also comprises an inhibitor with the working concentration of 2-20 units/mL; the inhibitor is one or more of penicillin, ampicillin, streptomycin, chloramphenicol, oxytetracycline, and tetracycline.
9. The method of producing ethanol by natural pH fermentation of lignocellulose as recited in claim 1, wherein:
the seed liquid culture conditions are as follows: the pH value is controlled to be 4-6, the culture temperature is controlled to be 28-35 ℃, and the rotation speed is 100-.
10. The method of producing ethanol by natural pH fermentation of lignocellulose as recited in claim 9, wherein: the seed liquid culture medium in the seed liquid culture step comprises 10g/L of yeast powder, 20g/L of peptone and 20g/L of glucose.
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