CN107118988B - High-efficiency composite methane fermentation catalyst - Google Patents
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Abstract
The invention relates to a high-efficiency composite methane fermentation catalyst, which comprises a composite microbial inoculum, and an enzyme activator and a reducing salt which are added into the composite microbial inoculum. The enzymes of the fermentation corresponding stage of the methane generated by the composite microbial inoculum in the fermentation catalyst comprise cellulase and amylase, the enzymes improve the enzyme activity through an enzyme activator, the decomposition and utilization efficiency of each raw material in the methane tank is effectively improved, the yeast and acetic acid bacteria in the composite microbial inoculum increase the accumulation of an important intermediate metabolite, acetic acid, and reducing salts in the fermentation catalyst reduce the oxidation-reduction potential of a methane generating system, reduce dissolved oxygen, provide strict anaerobic environmental conditions for methanogens, are beneficial to the efficient methane generation of the methanogens, and effectively improve the methane yield of the methane tank.
Description
Technical Field
The invention relates to the field of biogas fermentation, in particular to an efficient composite biogas fermentation catalyst.
Background
The biogas fermentation is a process of finally generating biogas by taking various organic matters as raw materials through a series of complex fermentation actions of microorganisms.
The anaerobic fermentation process of the methane is a complex biochemical reaction process with combination and alternate interaction of various microorganisms, and methanogenic bacteria are mutually restricted and dependent.
The biogas fermentation process has the following three stages:
first stage
In the liquefaction stage, microorganisms (cellulolytic bacteria, proteolytic bacteria, etc.) convert solid organic matter into soluble organic matter by the action of extracellular enzymes.
Second stage
In the acid production stage, soluble substances are continuously decomposed and converted into low molecular substances such as methanol, ethanol, formic acid, acetic acid and the like under the action of intracellular enzymes, and simultaneously, hydrogen and carbon dioxide are partially released. In the second stage, the main product is acetic acid, accounting for over 70%.
The third stage
In the methanogenic stage, the strictly anaerobic methanogenic bacteria reduce the small molecular compounds in the acidogenic stage by one or more steps to finally form methane and carbon dioxide to obtain methane.
In order to improve the degradation efficiency of the biogas raw materials and the biogas yield, the purpose of improving the degradation efficiency of the biogas raw materials and the biogas yield is achieved by adding auxiliary agents and improving the enzyme production and decomposition conversion efficiency of microorganisms at each stage. However, these solutions only aim at the first stage and the second stage of biogas fermentation, i.e. improving the utilization efficiency of fermentation raw materials, thus promoting acid production and providing sufficient raw materials for methanogens, but do not improve the conversion utilization efficiency of the methanogens on the small molecular compounds generated in these acid production stages, and do not aim at the overall synergistic promotion measure of the three main stages. Therefore, the current biogas fermentation technology still has a further improved space.
Disclosure of Invention
The invention aims to provide an efficient composite methane fermentation catalyst aiming at the defects of the prior art, enzymes in the corresponding stages of methane fermentation are generated by a composite microbial inoculum in the fermentation catalyst, the enzymes comprise cellulase and amylase, the enzyme activity of the enzymes is improved through an enzyme activator, the decomposition utilization efficiency of each raw material in a methane tank is effectively improved, the accumulation of important intermediate metabolite acetic acid is increased by saccharomycetes and acetic acid bacteria in the composite microbial inoculum, the oxidation reduction potential of a methane production system is reduced by reducing salts in the fermentation catalyst, dissolved oxygen is reduced, strict anaerobic environmental conditions are provided for methanogens, the efficient methane production by the methanogens is facilitated, and the methane yield of the methane tank is effectively improved.
The technical scheme of the invention is as follows: an efficient composite methane fermentation catalyst comprises a composite microbial inoculum, an enzyme activator and a reducing salt which are added in the composite microbial inoculum,
the compound microbial inoculum is prepared from a low-temperature cellulase production bacterial solution, a normal-temperature amylase production bacterial solution,Normal temperature acetic acid bacteria liquid and normal temperature yeast liquid according to the volume ratio of 1-1.5: 1: 1: 1: 1, the bacterial concentration of each bacterial liquid was 1x109cfu/ml；
The enzyme activator added into the complex microbial inoculum comprises a cellulase activator and a methanogenesis-related enzyme activator, wherein the cellulase activator consists of potassium nitrate, zinc chloride and calcium nitrate, and the methanogenesis-related enzyme activator consists of manganese chloride, cobalt nitrate and nickel sulfate;
the reducing salt consists of ferrous sulfate, sodium sulfite, ammonium nitrate and sodium sulfide.
The low-temperature cellulase producing bacteria are pseudomonas.
The normal-temperature cellulase producing bacteria are bacillus subtilis subspecies subtilis.
The normal-temperature amylase producing bacteria are bacillus subtilis.
The normal-temperature acetic acid bacteria is acetobacter pasteurianus.
The normal temperature yeast is saccharomyces cerevisiae.
The concentrations of the potassium nitrate, the zinc chloride and the calcium nitrate are all 30-60 mg/ml.
The concentrations of the manganese chloride, the cobalt nitrate and the nickel sulfate are all 30-60 mg/ml.
The concentrations of the ferrous sulfate, the sodium sulfite, the ammonium nitrate and the sodium sulfide are all 30-60 mg/ml.
0.12-0.15wt% of tween is added into the composite microbial inoculum.
The methanogen is obligate strict anaerobe, has strict requirements on oxidation-reduction potential, the most suitable oxidation-reduction potential is-400 to-500 mv, and particularly, the oxidation-reduction potential can not be even higher than-330 mv at the initial culture stage. The methane tank can not avoid bringing air in the feeding process, so that oxygen is dissolved in the liquid raw material, and methane is not produced by methanogens.
The low-temperature cellulase producing bacteria of the composite microbial inoculum in the high-efficiency composite methane fermentation catalyst produce cellulase in a low-temperature environment, cellulose in decomposed raw materials is soluble organic substances (saccharides), the normal-temperature cellulase producing bacteria and the normal-temperature amylase producing bacteria produce cellulase and amylase respectively in a normal-temperature environment, and the cellulose and the starch in the decomposed raw materials are corresponding soluble organic substances (saccharides), so that the methane fermentation raw materials can be quickly decomposed into the soluble organic substances under the conditions of low temperature and normal temperature to form substrates for producing acetic acid and ethanol. The normal temperature yeast and the normal temperature acetic acid bacteria in the composite microbial inoculum further convert soluble organic substances (saccharides) into acetic acid to generate important intermediate metabolites for producing methane, the enzyme activity of cellulose is improved by adding a cellulase activator, the decomposition rate of the cellulose is promoted, the enzyme activity of related enzymes is improved by adding a methanogenic related enzyme activator, the utilization efficiency of the methanogenic bacteria on the acetic acid is improved, and the gas production rate of the methane are improved. The efficient composite methane fermentation catalyst disclosed by the invention has the advantages that the reductive salt ferrous sulfate, sodium sulfite, ammonium nitrate and sodium sulfide are added, so that the oxidation-reduction potential of a methane production system is reduced, the dissolved oxygen is reduced, a strict anaerobic environment condition is provided for the fermentation system, a proper oxidation-reduction potential condition is created for the growth of methanogens, and the growth and the propagation of methanogenic bacteria are facilitated. Along with the reduction of the oxidation-reduction potential, the activity of various microorganisms is changed, and the activities are firstly expressed as nitrogen respiration to generate ammonia nitrogen and nitrite; the oxidation-reduction potential is continuously reduced, which is shown as the breathing of ferro-manganese, ferric iron is gradually reduced into ferrous iron, oxygen is consumed to produce acid in the process, and the pH value of the fermentation environment is reduced; the oxidation-reduction potential continues to be reduced, which is expressed by sulfur respiration, the sulfate radical originally existing is reduced into hydrogen sulfide, and the hydrogen sulfide reacts with ferrous iron (ferrous ion) to generate black and odorous phenomena; finally, under the condition of extreme oxygen deficiency in the fermentation environment, the methanogen decomposes organic matters to generate methane, thereby realizing the purposes of efficiently generating methane and effectively improving the methane yield of the methane tank.
0.12-0.15wt% of tween is added into the composite microbial inoculum for improving the uniform distribution degree of an enzyme activator, reducing salt and bacteria.
Drawings
FIG. 1 is a graph of gas production during fermentation in example 1;
FIG. 2 is a graph of gas production during fermentation in example 2.
Detailed Description
In the invention, potassium nitrate, zinc chloride, calcium nitrate, manganese chloride, cobalt nitrate, nickel sulfate, ferrous sulfate, sodium sulfite, ammonium nitrate, sodium sulfide and tween are all purchased from Chongqing YunyiboPakou technology ltd and are all in chemical purity.
In the invention, the sources of all strains are as follows:
pseudomonas bacteria (Pseudomonadaceae) Purchased from China center for culture collection and management of industrial microorganisms, and the strain collection number is as follows: CICC 10441.
Bacillus subtilis subspecies (B.subtilis)Bacillus subtilis subspecies) Purchased from China center for culture collection and management of industrial microorganisms, and the strain collection number is as follows: CICC 10832.
Acetobacter pasteurianus (A), (B), (C)Acetobacter Pasteurianus) Purchased from Guangdong province microbial strain preservation center, and the strain numbers are as follows: GIM 1.67.
Saccharomyces cerevisiae (Saccharomyces cerevisiae) Purchased from China center for culture collection and management of industrial microorganisms, and the strain number is as follows: CICC 1049.
Bacillus subtilis (A. subtilis)Bacillus subtilis) Purchased from China center for culture collection and management of industrial microorganisms, and the strain collection number is as follows: cic 10066.
Example 1:
test groups:
culturing purchased Pseudomonas strain in beef extract peptone medium (1000 ml distilled water added with 5.0g peptone, 3.0g beef extract, 5.0g NaCl, 15.0g agar) at 28 deg.C for 60-72 hr to concentration of 1 × 109cfu/ml；
Culturing purchased Bacillus subtilis subspecies in beef extract peptone medium (1000 ml distilled water added with 5.0g peptone, 3.0g beef extract, 5.0g NaCl, 15.0g agar) for 60-72h at 28 deg.C to concentration of 1x109cfu/ml；
Taking Acetobacter pasteurianus purchased, adding 100g glucose, 10g yeast extract, 20g CaCO into YPD culture medium (1000 ml distilled water)315g agar, pH adjusted to 6.8) for 80h at 28 ℃ to a concentration of 1X109cfu/ml；
The purchased Saccharomyces cerevisiae was cultured for 75h in 5 Bee wort agar medium (1.0L of 5B Bee wort supplemented with 15.0g of agar, natural pH) at 28-30 deg.C to a concentration of 1X109cfu/ml；
Culturing purchased Bacillus subtilis in beef extract peptone medium (1000 ml distilled water added with 5.0g peptone, 3.0g beef extract, 5.0g NaCl, 15.0g agar) at 37 deg.C to concentration of 1 × 10 for 60 hr9cfu/ml。
Taking prepared bacterial liquids, and mixing the prepared bacterial liquids according to the volume ratio of 1.5: 1: 1: 1: 1, mixing to obtain the compound microbial inoculum.
Taking 3ml of the prepared composite microbial inoculum, adding 150mg of potassium nitrate, 150mg of zinc chloride, 150mg of calcium nitrate, 150mg of manganese chloride, 150mg of cobalt nitrate, 150mg of nickel sulfate, 150mg of ferrous sulfate, 150mg of sodium sulfite, 150mg of ammonium nitrate, 150mg of sodium sulfide and 3.9mg of tween, and uniformly mixing to prepare the high-efficiency composite methane fermentation catalyst.
Taking 260ml of waste water of a fresh livestock slaughter house, adjusting the solid matter content (TS) in the waste water to 10 percent, and taking the waste water as a fermentation raw material.
Adding 260ml and 3ml of high-efficiency composite methane fermentation catalyst into a constant-temperature anaerobic methane fermentation device, and fermenting at the medium temperature of 30 ℃.
Setting 3 parallel, regularly recording the methane yield every 2 days, sampling and storing, mixing all samples, and measuring methane (CH) in methane4) And (4) content.
Control group:
taking 260ml of waste water of a fresh livestock slaughter house, adjusting the solid matter content (TS) in the waste water to 10 percent, and taking the waste water as a fermentation raw material.
Adding 260ml of fermentation raw materials into a constant-temperature anaerobic biogas fermentation device, and fermenting at the medium temperature of 30 ℃.
Setting 3 parallel, regularly recording the methane yield every 2 days, sampling and storing, mixing all samples, and measuring methane (CH) in methane4) And (4) content.
As a result:
the gas production of the test group and the control group are shown in FIG. 1, and the test groupAverage methane (CH) in gas production4) The content of the product gas is 53.8 percent, the total product gas during the fermentation period is 39.2 ml, and the average methane (CH) in the product gas of a control group4) The content is 50.15%, and the gas is generated in 27.64 ml during the fermentation.
Example 2:
test groups:
culturing purchased Pseudomonas strain in beef extract peptone medium (1000 ml distilled water added with 5.0g peptone, 3.0g beef extract, 5.0g NaCl, 15.0g agar) at 28 deg.C for 60-72 hr to concentration of 1 × 109cfu/ml；
Culturing purchased Bacillus subtilis subspecies in beef extract peptone medium (1000 ml distilled water added with 5.0g peptone, 3.0g beef extract, 5.0g NaCl, 15.0g agar) for 60-72h at 28 deg.C to concentration of 1x109cfu/ml；
Taking Acetobacter pasteurianus purchased, adding 100g glucose, 10g yeast extract, 20g CaCO into YPD culture medium (1000 ml distilled water)315g agar, adjusting pH to 6.8) for 80h, at 28 ℃ to a concentration of 1 × 109cfu/ml；
The purchased Saccharomyces cerevisiae was cultured for 75h in 5 Bee wort agar medium (1.0L of 5B Bee wort supplemented with 15.0g of agar, natural pH) at 28-30 deg.C to a concentration of 1X109cfu/ml；
Culturing purchased Bacillus subtilis in beef extract peptone medium (1000 ml distilled water added with 5.0g peptone, 3.0g beef extract, 5.0g NaCl, 15.0g agar) at 37 deg.C to concentration of 1 × 10 for 60 hr9cfu/ml。
Taking prepared bacterial liquids, and mixing the prepared bacterial liquids according to the volume ratio of 1.5: 1: 1: 1: 1, mixing to obtain the compound microbial inoculum.
Taking 15L of the composite microbial agent, adding 750g of potassium nitrate, 750g of zinc chloride, 750g of calcium nitrate, 750g of manganese chloride, 750g of cobalt nitrate, 750g of nickel sulfate, 750g of ferrous sulfate, 750g of sodium sulfite, 750g of ammonium nitrate, 750g of sodium sulfide and 19.5g of tween, and uniformly mixing to obtain the high-efficiency composite methane fermentation catalyst.
Taking 1000L of waste water of a fresh livestock slaughter house, adjusting the solid matter content (TS) in the waste water to 8 percent, and taking the waste water as a fermentation raw material.
Adding 1000L and 15L of fermentation raw materials into a large-scale fermentation tank, and fermenting at normal temperature.
Regularly recording the gas production amount every 2 days, sampling and storing, mixing all samples, and measuring methane (CH) in the methane4) And (4) content.
Control group:
taking 1000L of waste water of a fresh livestock slaughter house, adjusting the solid matter content (TS) in the waste water to 8 percent, and taking the waste water as a fermentation raw material.
1000L of fermentation raw materials are added into a large-scale fermentation tank for fermentation at normal temperature.
Regularly recording the gas production amount every 2 days, sampling and storing, mixing all samples, and measuring methane (CH) in the methane4) And (4) content.
Results
The gas production rates of the test group and the control group are shown in FIG. 2, and the average methane (CH) in the gas production rate of the test group4) The content of the product is 54.3 percent, the co-produced gas during the fermentation period is 197.55L, and the average methane (CH) in the produced gas of a control group4) The content is 50.35 percent, and 140.6L of gas is generated in the fermentation period.
And (4) conclusion:
from the results of the embodiment 1 and the embodiment 2, it can be known that the gas production rate of the test group during the whole fermentation period is much larger than that of the control group by adding the high-efficiency composite methane fermentation catalyst into the fermentation raw materials, compared with the control group, and the high-efficiency composite methane fermentation catalyst can be proved to be capable of effectively improving the degradation efficiency of macromolecular substances and the utilization efficiency of methanogens on acetic acid, and improving the gas production rate and the gas production rate of methane. And methane (CH) in the biogas of the test group relative to the control group4) The content is higher, and the high-efficiency composite methane fermentation catalyst can generate more appropriate oxidation-reduction potential conditions and is beneficial to the growth and metabolism of methanogens.
Claims (5)
1. An efficient composite methane fermentation catalyst is characterized in that: comprises a complex microbial inoculum, an enzyme activator and a reducing salt which are added into the complex microbial inoculum,
the composite microbial inoculum is prepared from a low-temperature cellulase production bacterial liquid, a normal-temperature amylase production bacterial liquid, a normal-temperature acetic acid bacterial liquid and a normal-temperature yeast bacterial liquid according to the volume ratio of 1-1.5: 1: 1: 1: 1, the bacterial concentration of each bacterial liquid was 1x109cfu/ml；
The enzyme activator added into the complex microbial inoculum comprises a cellulase activator and a methanogenesis-related enzyme activator, wherein the cellulase activator consists of potassium nitrate, zinc chloride and calcium nitrate, and the methanogenesis-related enzyme activator consists of manganese chloride, cobalt nitrate and nickel sulfate;
the reducing salt consists of ferrous sulfate, sodium sulfite, ammonium nitrate and sodium sulfide;
the low-temperature cellulase producing bacteria are pseudomonas; the normal-temperature cellulase producing bacteria are bacillus subtilis subspecies; the normal-temperature amylase producing bacteria are bacillus subtilis; the normal-temperature acetic acid bacteria are acetobacter pasteurianus; the normal temperature yeast is saccharomyces cerevisiae.
2. The high-efficiency composite methane fermentation catalyst according to claim 1, wherein: the concentrations of the potassium nitrate, the zinc chloride and the calcium nitrate are all 30-60 mg/ml.
3. The high-efficiency composite methane fermentation catalyst according to claim 1, wherein: the concentrations of the manganese chloride, the cobalt nitrate and the nickel sulfate are all 30-60 mg/ml.
4. The high-efficiency composite methane fermentation catalyst according to claim 1, wherein: the concentrations of the ferrous sulfate, the sodium sulfite, the ammonium nitrate and the sodium sulfide are all 30-60 mg/ml.
5. The high-efficiency composite methane fermentation catalyst according to claim 1, wherein: 0.12-0.15wt% of tween is added into the composite microbial inoculum.
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| CN101381761A (en) * | 2008-10-17 | 2009-03-11 | 山东省农业科学院土壤肥料研究所 | Application of duel functional composite bacteria agent capable of decomposition of stalk and low-temperature production of biogas |
| CN102433262A (en) * | 2011-12-13 | 2012-05-02 | 新疆农业科学院微生物应用研究所 | Complex microbial agent for low-temperature methane fermentation and preparation method thereof |
| WO2013020118A1 (en) * | 2011-08-04 | 2013-02-07 | Danisco Us Inc. | Production of isoprene, isoprenoid precursors, and isoprenoids using acetoacetyl-coa synthase |
| CN105368745A (en) * | 2015-12-01 | 2016-03-02 | 深圳市大治生光环保科技有限公司 | Composite microbial preparation for treating black and odorous river and preparation method thereof |
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| CN101381761A (en) * | 2008-10-17 | 2009-03-11 | 山东省农业科学院土壤肥料研究所 | Application of duel functional composite bacteria agent capable of decomposition of stalk and low-temperature production of biogas |
| WO2013020118A1 (en) * | 2011-08-04 | 2013-02-07 | Danisco Us Inc. | Production of isoprene, isoprenoid precursors, and isoprenoids using acetoacetyl-coa synthase |
| CN102433262A (en) * | 2011-12-13 | 2012-05-02 | 新疆农业科学院微生物应用研究所 | Complex microbial agent for low-temperature methane fermentation and preparation method thereof |
| CN105368745A (en) * | 2015-12-01 | 2016-03-02 | 深圳市大治生光环保科技有限公司 | Composite microbial preparation for treating black and odorous river and preparation method thereof |
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