Method for preparing bioethanol from animal waste-based saccharified liquid
Through the combined pretreatment technology of animal feces, including drying, crushing, microbial hydrolysis and dilute acid hydrolysis, a saccharification liquid with high content of reduced sugar is generated, which solves the problem of low utilization rate of lignocellulose in animal feces and significantly improves the production efficiency of bioethanol.
Patent Information
- Application Number
- CN202510008702.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-03
- Publication Date
- 2025-03-25
AI Technical Summary
The high content of lignocellulose in animal feces and its complex structure leads to its low utilization rate, limiting the production efficiency of bioethanol.
Using combined pretreatment technology, animal feces are dried, crushed and microbial hydrolyzed, and then hydrolyzed with dilute acids to form a saccharified liquid with high content of reducing sugar, and then bioethanol is prepared by fermentation of Saccharomyces cerevisiae.
Through combined pretreatment technology, the saccharification rate of animal feces has been significantly improved, the production efficiency of bioethanol has been greatly improved, and the high-value utilization of animal feces has been achieved.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of value-added utilization of agricultural waste, and in particular relates to a method for preparing bioethanol by utilizing animal excrement-based saccharification liquid. Background Art
[0002] Bioethanol has become an important alternative to fossil fuels because of its environmental friendliness and renewable nature. In recent years, the raw materials for bioethanol production have shifted from food crops to non-food biomass resources, among which agricultural waste has become an important raw material for sustainable production of bioethanol. The large-scale development of the animal husbandry industry has produced a large amount of animal manure, which is easy to corrupt and produces a pungent odor, which not only pollutes the environment but also poses a threat to human health. Among them, the dry matter of animal manure contains approximately 10.1-24.3% cellulose, 9.9-28.8% hemicellulose and 4.8-15.3% lignin, which has great potential for conversion into bioethanol. However, the high content of lignocellulose in animal manure and its complex structure lead to its low utilization rate, which seriously limits ethanol production. Therefore, the use of effective pretreatment technology to destroy the structure of lignocellulose is of great significance to improve the accessibility of microorganisms or enzymatic hydrolysis and the conversion rate of reducing sugars. In addition, Saccharomyces cerevisiae has excellent multi-environmental adaptability such as acid resistance, salt resistance, and toxicity resistance, as well as efficient ethanol production capacity. Therefore, pretreated lignocellulose can release reducing sugars, which are then converted into metabolites such as ethanol and carbon dioxide by S. cerevisiae.
[0003] At present, a variety of pretreatment technologies have been applied to the conversion of lignocellulosic biomass. For example, the use of microorganisms or biological enzyme preparations to decompose lignocellulose can significantly improve its utilization. Among them, the pretreatment of bamboo stalks with white rot basidiomycetes can effectively improve the effect of enzymatic hydrolysis. In addition, rice straw can dissolve 44% of cellulose after pretreatment with commercial cellulase enzyme preparations. However, disadvantages such as the need for sufficient space, very slow processes, and the need to continuously control microbial growth and activity have hindered the wider application of biological pretreatment in industry. On the other hand, chemical pretreatment increases the degradation rate of cellulose, hemicellulose, and lignin by destroying the covalent cross-linking of lignocellulose. In general, concentrated acid (10-80%) and high temperature (100-250°C) conditions below 100°C are most commonly used for the hydrolysis of lignocellulosic biomass. However, although the concentrated acid hydrolysis method has a high conversion rate, it has problems such as difficult recovery and uneconomical. At present, the combination of multiple pretreatment technologies can further enhance the pretreatment effect. For example, the combined pretreatment of biology and acid can effectively dissolve lignocellulose while limiting the production of inhibitors, thereby increasing the yield of reducing sugars and ethanol. However, there is no report on the technology of combining microorganisms with dilute acid pretreatment of animal manure to increase bioethanol production. Summary of the invention
[0004] In order to overcome the shortcomings and deficiencies in the above-mentioned prior art, the purpose of the present invention is to provide a method for preparing bioethanol using animal manure-based saccharification liquid; this method not only solves the problem of low efficiency in the current direct use of animal manure to produce bioethanol, but also realizes the high-value utilization of animal manure and promotes the sustainable development of the bioethanol industry, and has good industrial application prospects.
[0005] The purpose of the present invention is achieved through the following technical solutions:
[0006] A method for preparing bioethanol using animal excrement-based saccharification liquid comprises the steps of pre-treating the animal excrement to increase its reducing sugar content to obtain saccharification liquid; and then fermenting the saccharification liquid with yeast to obtain bioethanol.
[0007] The combined pretreatment specifically follows the following steps: firstly, the animal feces are dried, crushed and sieved to obtain pretreated feces powder, then hydrolyzed by microorganisms, then hydrolyzed by dilute acid, and centrifuged to obtain saccharified liquid.
[0008] The animal manure is one or more of pig manure, chicken manure, cow manure and pigeon manure; the yeast is a conventional brewer's yeast strain with ethanol production ability.
[0009] The microorganism is a conventional Bacillus licheniformis capable of degrading macromolecular substances; and the dilute acid is dilute sulfuric acid.
[0010] The above-mentioned method for preparing bioethanol using animal feces-based saccharification liquid specifically follows the following steps:
[0011] (1) collecting animal feces, drying them in an oven at 55 to 85° C. for 20 to 80 hours, and then crushing and sieving them to obtain feces powder; weighing the obtained feces powder, and adding pure water to obtain a feces mixed liquid with a concentration of 30 to 130 g / L;
[0012] (2) Streaking Bacillus licheniformis on LB solid medium and culturing at 34-38° C. for 40-50 h; picking colonies and placing them in LB liquid medium, culturing at 34-38° C. and 160-200 rpm for 12-20 h to obtain seed solution;
[0013] (3) inoculating the feces mixture of step (1) with the seed solution obtained in step (2) at an inoculum volume of 30 to 70 mL / L, and culturing and fermenting at 34 to 38° C. and 160 to 200 rpm for 30 to 60 hours to obtain a fermentation liquid;
[0014] (4) adding 1-7% by volume dilute sulfuric acid to the fermentation liquid obtained in step (3), stirring and heating in a water bath at 70-130° C. for 1-6 hours; adjusting the pH value of the obtained acid hydrolyzate to 5-7, collecting the supernatant after centrifugation, and obtaining a saccharified liquid;
[0015] (5) Streaking Saccharomyces cerevisiae on YEPD solid medium and culturing at 28-35°C for 30-50 h; picking colonies and placing them on YEPD liquid medium and culturing them at 28-35°C and 180-210 rpm for 18-28 h to obtain seed solution;
[0016] (6) inoculating the saccharified liquid obtained in step (4) with the seed liquid obtained in step (5) at an inoculation rate of 30 to 90 mL / L, and fermenting in an anaerobic bag at a constant temperature of 28 to 35° C. for 24 to 72 hours; and collecting the supernatant after centrifuging the obtained fermentation liquid to obtain bioethanol.
[0017] The sieving in step (1) is to obtain feces powder of 200-400 mesh; the concentration of the feces mixture is 70g / L.
[0018] The pH value of step (4) is adjusted using a 0.1-10M NaOH solution or a 0.1-10M HCl solution.
[0019] The saccharification liquid in step (4) is a solution containing one or more of glucose, fructose, maltose and lactose.
[0020] The centrifugation in step (4) and step (6) is performed using a centrifugal force of 6000 to 10000 g.
[0021] The brewer's yeast in step (5) is commercially available Saccharomyces cerevisiae BNCC 300560.
[0022] Compared with the prior art, the advantages and beneficial effects of the present invention are:
[0023] (1) Compared with the prior art that uses only microorganisms, strong acids, and strong alkalis to pretreat animal manure, the present invention first pretreats the animal manure by drying and crushing, then hydrolyzes it using a microbial method, and finally hydrolyzes it using a dilute acid. This combined pretreatment method can not only avoid the corrosiveness caused by strong acids, but also enable the animal manure to achieve a higher saccharification rate, thereby greatly improving the production efficiency of bioethanol.
[0024] (2) Compared with the sources of raw materials for producing ethanol in the prior art, the raw materials used in the present invention are animal manure, which not only fully utilizes waste resources but also reduces the production cost of the product.
[0025] (3) The raw materials used in the present invention are cheap and easily available, and the preparation process is simple and easy to control, which is suitable for industrial production and has a large market prospect. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 It is a schematic diagram showing the effect of different pretreatment methods on the saccharification of animal manure.
[0027] Figure 2 Schematic diagram of the results of bioethanol production by Saccharomyces cerevisiae using combined pre-treated animal manure. DETAILED DESCRIPTION
[0028] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.
[0029] The LB liquid culture medium used in the following examples has the following formula: by mass fraction, it includes 1% tryptone, 1% NaCl, 0.5% yeast powder, and the balance is water; the LB solid culture medium needs to add 1.5% agar to the above LB liquid culture medium formula, and the preparation method is to mix the ingredients evenly and sterilize at 121°C for 15 minutes.
[0030] The formula of the YEPD liquid culture medium used in the following examples is as follows: by mass fraction, it includes 2% peptone, 2% glucose, 1% yeast powder, and the remainder is water; the YEPD solid culture medium is to add 1.5% agar to the above YEPD liquid culture medium formula, and the preparation method is to mix the ingredients evenly and sterilize at 121°C for 20 minutes.
[0031] Example 1
[0032] This example adopts a combined pretreatment method to increase the reducing sugar content of animal feces, and measures the effects of different pretreatment methods on the saccharification effect of animal feces.
[0033] 1) The animal feces collected from the farm were dried in an oven at 85°C for 50 hours, and then crushed by a grinder and passed through a 400-mesh sieve to obtain pretreated feces powder; the feces powder was weighed, and pure water was added to obtain a feces mixed solution with a concentration of 90.5 g / L.
[0034] 2) Streak the commercially available Bacillus licheniformis CICC 10092 on LB solid medium and culture at 37°C for 48h; pick the colony of Bacillus licheniformis CICC 10092 on the solid medium and place it in LB liquid medium, culture it at 37°C and 180rpm for 13h to obtain seed liquid. Then, inoculate the seed liquid into the feces mixture obtained in step 1) with an inoculum amount of 50mL / L, culture and ferment it at 37°C and 180rpm for 40h to obtain fermentation liquid. Centrifuge the obtained fermentation liquid at 8000g for 20min, and collect the supernatant. The reducing sugar concentration of the supernatant was detected using a reducing sugar content determination kit (Beijing Solebold Technology Co., Ltd.).
[0035] 3) Add 500 mL of pure water to a 1 L beaker, and slowly add 10.44 mL of H2SO4 with a mass concentration of 96% to the pure water while stirring continuously to obtain a dilute sulfuric acid solution with a volume fraction of about 2%. Add the obtained dilute sulfuric acid solution to the feces mixture obtained in step 1), place it in an 80°C water bath, stir and heat for 2 hours, and obtain an acid hydrolysis mixture. Cool the obtained acid hydrolysis mixture to room temperature, adjust the pH value to 6.5 with 1M NaOH solution or 1M HCl solution, and then centrifuge at 8000g for 20 minutes to collect the supernatant. The reducing sugar content determination kit (Beijing Solebow Technology Co., Ltd.) was used to detect the reducing sugar concentration of the supernatant.
[0036] 4) Add the dilute sulfuric acid solution obtained in step 3) to the fermentation broth obtained in step 2), place in a water bath at 80°C and stir for 2h to obtain an acid hydrolysis mixture; cool the obtained acid hydrolysis mixture to room temperature, adjust the pH value to 6.5 with 1M NaOH solution or 1M HCl solution, and then centrifuge at 8000g for 20min to collect the supernatant. Use a reducing sugar content determination kit (Beijing Solebow Technology Co., Ltd.) to detect the reducing sugar concentration of the supernatant.
[0037] 5) The control group is the feces mixture with a concentration of 90.5 g / L obtained in step 1), which is cultured and fermented at 37°C and 180 rpm for 40 hours to obtain a control fermentation liquid. The obtained control fermentation liquid is placed in an 80°C water bath and stirred and heated for 2 hours to obtain a thermal reaction mixture. The obtained thermal reaction mixture is cooled to room temperature, the pH value is adjusted to 6.5 with 0.1M NaOH solution or 0.1M HCl solution, and then centrifuged at 8000g for 20 minutes to collect the supernatant. The reducing sugar concentration of the supernatant is detected using a reducing sugar content determination kit (Beijing Solebold Technology Co., Ltd.).
[0038] like Figure 1As shown in the figure, animal feces pretreated with Bacillus licheniformis CICC 10092, pretreated with dilute sulfuric acid, and pretreated with Bacillus licheniformis CICC 10092 and dilute sulfuric acid can effectively increase the reducing sugar content. Among them, the most effective pretreatment method is the combined pretreatment. Compared with the control group, the reducing sugar content (19.4 g / L) of animal feces after combined pretreatment increased by 1469.5%. This may be because Bacillus licheniformis efficiently produces cellulase to decompose cellulose and lignin in animal feces, thereby releasing sugars; while dilute sulfuric acid pretreatment quickly destroys the covalent cross-linking (such as hydrogen bonds and covalent bonds) of lignocellulose in animal feces in a short time, enhancing the solubility of cellulose and hemicellulose. Therefore, combined pretreatment with Bacillus licheniformis and pretreatment with dilute sulfuric acid can further promote the efficient conversion of polysaccharides into small molecule sugars, thereby increasing the yield of reducing sugars. It shows that combined pretreatment of animal feces is significantly better than single pretreatment and can effectively increase the yield of reducing sugars.
[0039] Example 2
[0040] Effects of combined pretreatment of animal manure with different concentrations on bioethanol production by Saccharomyces cerevisiae
[0041] 1) The animal feces collected from the farm were dried in an oven at 85°C for 50 h, and then crushed by a grinder and passed through a 400-mesh sieve to obtain pretreated feces powder; the feces powder was weighed and added with pure water to prepare feces mixed solutions with concentrations of 30, 50, 70, 90, 110, and 130 g / L, respectively.
[0042] 2) The commercially available Bacillus licheniformis CICC 10092 was streaked on LB solid medium and cultured at 37°C for 49h. The colonies of Bacillus licheniformis CICC 10092 on the solid medium were picked and placed in LB liquid medium, and cultured at 37°C and 180rpm for 14h to obtain seed liquid. Then, the seed liquid was inoculated into the feces mixture of different concentrations obtained in step 1) at an inoculum amount of 50mL / L, and cultured and fermented at 37°C and 180rpm for 42h to obtain fermentation liquid. The dilute sulfuric acid solution obtained in step 3) of Example 1 was added to the above-mentioned fermentation liquid, placed in an 80°C water bath, stirred and heated for 2h to obtain an acid hydrolysis mixture. The obtained acid hydrolysis mixture was cooled to room temperature, the pH value was adjusted to 6.5 with 1M NaOH solution or 1M HCl solution, and then centrifuged at 8000g for 20min to collect the supernatant.
[0043] 3) The commercially available Saccharomyces cerevisiae BNCC 300560 was streaked on a YEPD solid medium and incubated at 30°C for 48 hours. The colony of Saccharomyces cerevisiae BNCC 300560 on the solid medium was picked and placed in a YEPD liquid medium, and incubated at 30°C and 180rpm for 20 hours to obtain a Saccharomyces cerevisiae seed solution. Then, the Saccharomyces cerevisiae seed solution was inoculated into the supernatant obtained in step 2) at an inoculum of 50mL / L, and the fermentation was continued at a constant temperature of 30°C in an anaerobic bag for 28 hours to obtain a fermentation liquid. The obtained fermentation liquid was centrifuged, the supernatant was collected, and ultrasonic treatment was performed, diluted to an appropriate concentration, and then filtered through a membrane (0.22μm), and the ethanol concentration was determined by gas chromatography.
[0044] 4) The control group is the feces mixture of different concentrations described in step 1), which is cultured and fermented at 37°C and 180rpm for 40h to obtain control fermentation liquid of different concentrations. The control fermentation liquid of different concentrations is placed in an 80°C water bath and stirred and heated for 2h to obtain a thermal reaction mixture of different concentrations. The obtained thermal reaction mixture of different concentrations is cooled to room temperature, the pH value is adjusted to 6.5 with 0.1M NaOH solution or 0.1M HCl solution, and then centrifuged at 8000g for 20min to collect supernatants of different concentrations. Then, the brewer's yeast seed liquid obtained in step 3) is inoculated into the supernatants of different concentrations obtained above at an inoculation amount of 50mL / L, and fermented at a constant temperature of 30°C anaerobic bag for 28h to obtain a fermentation liquid. After the obtained fermentation liquid is centrifuged, the supernatant is collected, ultrasonically treated, diluted to an appropriate concentration, and then filtered through a membrane (0.22μm), and the ethanol concentration is determined by gas chromatography.
[0045] Figure 2 The results show that with the increase of the amount of combined pre-treated animal feces, the production of bioethanol by Saccharomyces cerevisiae BNCC 300560 after fermentation for 28h first increases and then decreases, and the ethanol production reaches the maximum (1013.7mg / L) at 70g / L animal feces. However, since the control group was not pre-treated, its final ethanol production did not change significantly with the increase of the amount of animal feces added, and was lower than 60mg / L. Moreover, when the amount of animal feces added was 70g / L, the ethanol production of the combined pre-treatment method increased by 1682.8% compared with the control group. This may be because the combined pre-treatment of animal feces significantly increases its reducing sugar content, providing a good fermentation environment for Saccharomyces cerevisiae, thereby promoting Saccharomyces cerevisiae to metabolize fermentable sugars to produce ethanol. It shows that the combined pre-treatment of animal feces of the present invention is significantly better than single pre-treatment, not only can it efficiently saccharify animal feces, but also can further increase the production of bioethanol by Saccharomyces cerevisiae using animal feces.
[0046] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Anyone can derive other various forms of products under the inspiration of the present invention, but all embodiments without innovative changes and research belong to the protection scope of the present invention.
Claims
1. A method for preparing bioethanol using animal feces-based saccharification liquid, characterized in that The method comprises the following steps: pre-treating animal feces to increase the reducing sugar content thereof to obtain saccharification liquid; and then fermenting the saccharification liquid with yeast to obtain bioethanol.
2. The method for preparing bioethanol using animal feces-based saccharification liquid according to claim 1, characterized in that: The combined pretreatment specifically follows the following steps: firstly, the animal feces are dried, crushed and sieved to obtain pretreated feces powder, then hydrolyzed by microorganisms, then hydrolyzed by dilute acid, and centrifuged to obtain saccharified liquid.
3. The method for preparing bioethanol using animal feces-based saccharification liquid according to claim 1, characterized in that: The animal manure is one or more of pig manure, chicken manure, cow manure and pigeon manure; the yeast is a conventional brewer's yeast strain with ethanol production ability.
4. The method for preparing bioethanol using animal feces-based saccharification liquid according to claim 2, characterized in that: The microorganism is a conventional Bacillus licheniformis capable of degrading macromolecular substances; and the dilute acid is dilute sulfuric acid.
5. The method for preparing bioethanol using animal feces-based saccharification liquid according to claim 1, characterized in that Follow these steps: (1) collecting animal feces, drying them in an oven at 55 to 85° C. for 20 to 80 hours, and then crushing and sieving them to obtain feces powder; weighing the obtained feces powder, and adding pure water to obtain a feces mixed liquid with a concentration of 30 to 130 g / L; (2) Streaking Bacillus licheniformis on LB solid medium and culturing at 34-38° C. for 40-50 h; picking colonies and placing them in LB liquid medium, culturing at 34-38° C. and 160-200 rpm for 12-20 h to obtain seed solution; (3) inoculating the feces mixture of step (1) with the seed solution obtained in step (2) at an inoculum volume of 30 to 70 mL / L, and culturing and fermenting at 34 to 38° C. and 160 to 200 rpm for 30 to 60 hours to obtain a fermentation liquid; (4) adding 1-7% by volume dilute sulfuric acid to the fermentation liquid obtained in step (3), stirring and heating in a water bath at 70-130° C. for 1-6 hours; adjusting the pH value of the obtained acid hydrolyzate to 5-7, collecting the supernatant after centrifugation, and obtaining a saccharified liquid; (5) Streaking Saccharomyces cerevisiae on YEPD solid medium and culturing at 28-35°C for 30-50 h; picking colonies and placing them on YEPD liquid medium and culturing them at 28-35°C and 180-210 rpm for 18-28 h to obtain seed solution; (6) inoculating the saccharified liquid obtained in step (4) with the seed liquid obtained in step (5) at an inoculation rate of 30 to 90 mL / L, and fermenting in an anaerobic bag at a constant temperature of 28 to 35° C. for 24 to 72 hours; and collecting the supernatant after centrifuging the obtained fermentation liquid to obtain bioethanol.
6. The method for preparing bioethanol using animal feces-based saccharification liquid according to claim 5, characterized in that: The sieving in step (1) is to obtain feces powder of 200-400 mesh; the concentration of the feces mixture is 70g / L.
7. The method for preparing bioethanol using animal feces-based saccharification liquid according to claim 5, characterized in that: The pH value of step (4) is adjusted using a 0.1-10M NaOH solution or a 0.1-10M HCl solution.
8. The method for preparing bioethanol using animal feces-based saccharification liquid according to claim 5, characterized in that: The saccharification liquid in step (4) is a solution containing one or more of glucose, fructose, maltose and lactose.
9. The method for preparing bioethanol using animal feces-based saccharification liquid according to claim 5, characterized in that: The centrifugation in step (4) and step (6) is performed using a centrifugal force of 6000 to 10000 g.