CN105177058B - Method for adjusting acid-alcohol ratio in anaerobic gas-feeding microbial fermentation product - Google Patents
Method for adjusting acid-alcohol ratio in anaerobic gas-feeding microbial fermentation product Download PDFInfo
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- CN105177058B CN105177058B CN201510540079.1A CN201510540079A CN105177058B CN 105177058 B CN105177058 B CN 105177058B CN 201510540079 A CN201510540079 A CN 201510540079A CN 105177058 B CN105177058 B CN 105177058B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention aims to provide a culture method for adjusting the proportion of acid and alcohol in a fermentation product of anaerobic gas-feeding microorganisms, namely, the aim of adjusting the proportion of acid and alcohol in the fermentation product is fulfilled by changing culture conditions. In particular to the purpose of adjusting the proportion of alcohol substances of fermentation products in a shaking culture system by changing the conditions of a culture medium. The method for improving the proportion of the alcohol substances in the anaerobic gas-feeding microorganism fermentation product adjusts the initial pH and the mes concentration of a system, and the proportion of the acid substances in the fermentation product can be obviously improved by higher initial pH and lower mes concentration; slightly lower initial pH and mes concentrations can significantly increase the proportion of alcohol in the fermentation product.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for adjusting the proportion of acid and alcohol in an anaerobic gas-feeding microbial fermentation product.
Technical Field
As a world resource consuming nation, China is facing a severe situation that fossil resources are gradually exhausted and carbon emission is increasingly increased, and the excessive use of the fossil resources such as petroleum and natural gas brings many social hot problems such as sustainable development and utilization of energy and chemicals and environmental pollution. The adjustment of energy structure and the development of renewable resources become the inevitable choice for realizing sustainable development in our country. The carbon-containing gas refers to a mixed gas having CO as a main component. The gas composition is mainly CO and CO2And H2It is a cheap raw material with great development value for manufacturing petrochemical chemicals. The anaerobic gas-feeding microorganism can convert carbon-containing components in gas into high-value chemicals, such as ethanol, butanol, PHA, etc., and can be used as petrochemical productThe product is a good substitute. The fermentation product of the microorganism contains not only alcohol substances, but also a plurality of small molecular organic acid substances, and the required product types are different according to different product requirements and downstream process requirements. However, the fermentation characteristics of such microorganisms are complex, and the lack of a purposeful production control means for the fermentation product limits the application of anaerobic gas-feeding microorganisms. Therefore, if the proportion of acid and alcohol in the anaerobic gas-eating bacterium gas fermentation product can be purposefully adjusted, the fermentation application of the anaerobic gas-eating bacterium can be greatly expanded, and the industrial development of the green biological process is realized.
Disclosure of Invention
The invention aims to provide a culture method for adjusting the proportion of acid and alcohol in a fermentation product of anaerobic gas-feeding microorganisms, namely, the aim of adjusting the proportion of acid and alcohol in the fermentation product is fulfilled by changing culture conditions.
The invention relates to a method for adjusting the proportion of acid and alcohol in an anaerobic gas-feeding microorganism fermentation product, which is realized by controlling the initial pH value of a culture system and the dosage of 2-morpholine ethanesulfonic acid MES;
in order to improve the proportion of acid products in anaerobic gas-feeding microorganism fermentation products, the initial pH value of the system is improved, and the using amount of MES is reduced;
wherein the initial pH of the elevated system is preferably adjusted to 6
The reduction of the amount of MES is preferably carried out at a concentration of 0.1g/L
In order to improve the proportion of alcohol products in anaerobic gas-feeding microorganism fermentation products, the initial pH value of the system is reduced, and the using amount of MES is reduced;
wherein the initial pH of the system is increased, preferably by adjusting the pH to 5;
the reduction of the amount of MES is preferably carried out at a concentration of 0.1g/L
The invention also provides a culture medium used for the method, and the culture medium comprises 2-4% mineral element Stock solution (V/V), 0.05% yeast extract (M/V), 0.5% reducing agent Stock solution (V/V) and 0.1% trace element Stock solution (V/V);
wherein the Mineral solution stock (M/V) comprises the following components: 8% sodium chloride, 10% ammonium chloride, 1% potassium chloride, 1% monopotassium phosphate, 2% magnesium sulfate and 0.4% calcium chloride;
the composition of the Reducing Agent Stock (M/V) was 0.9% sodium hydroxide, 4% L-cysteine hydrochloride and 4% sodium sulfide nonahydrate.
The microelement liquid storage solution (M/V) comprises 1% of nitrilotriacetic acid, 0.5% of magnesium sulfate, 0.4% of ammonium ferrous sulfate, 0.1% of cobalt chloride, 0.1% of zinc sulfate, 0.01% of nickel chloride, 0.01% of sodium selenate and 0.01% of sodium tungstate.
The method can effectively adjust the proportion of alcohol substances in the anaerobic gas-feeding microorganism gas fermentation product, and effectively improve the technical value.
Detailed Description
The gas metabolism process of anaerobic gas-feeding bacteria is very complex, and relates to the production and the reuse of various fermentation products and the balance of the production and the consumption of energy ATP. The regulation and control of the fermentation system conditions have great influence on the final fermentation result. Applicants found that pH and pH buffer MES can significantly affect the ratio of organic acids to alcohols in the fermentation product. Higher pH and MES concentrations favor the accumulation of organic acids and ATP production, whereas lower pH and MES concentrations favor the accumulation of alcohols, but result in lower overall yields due to lack of accumulation of ATP during the acidogenic phase. Therefore, how to balance the process of bacterial fermentation and maximize the yield of the target product is very critical.
Wherein the acid products in the anaerobic gas-eating microorganism fermentation product are acetic acid, n-butyric acid and n-hexanoic acid, and the alcohol products are ethanol, n-butanol and n-hexanol
The microorganisms used in the examples of the present invention are anaerobic gas-feeding microorganisms, such as the strain Clostridium carboxidigorans P7(DSM15243) and the strain Clostridium autoethanogenum DSM 10060, purchased from the German Collection of microorganisms and cell cultures, the institute of Lapanicz, Brenneck, Germany.
The culture medium used includes, but is not limited to, the culture medium of Clostridium carboxidivorans strain and the strain Clostridium autoethanogenum.
The present invention will be described in detail with reference to specific examples.
The operation method in the examples is as follows:
constant temperature shaking culture: adding 2ml of mineral solution Stock and 0.05g of yeast extract into a 100ml system, adjusting the initial pH range of the system to 5-6, filling 99.999% nitrogen at the MES concentration of 0.1-5g/L and 5ml of reducing agent Stock for 5min at the speed of 2L/min, sealing, and sterilizing at the high temperature of 121 ℃ for 20 min. Cooling to about 40 ℃ after sterilization, and then adding 0.1ml of microelement stock solution to complete the construction of the culture system. Inoculating 10ml culture solution of Clostridium carboxidigorans strain P7 or Clostridium autoethanogenum DSM 10060, culturing to logarithmic growth phase, and continuously introducing simulated syngas (gas composition: 50% CO, 35% CO) at flow rate of 2L/min and pressure of 0.4bar2、15％H2) Equilibrate for 5min, pressurize to 0.6bar and seal. Shaking and culturing at 37 deg.C and 180rpm, and sampling at regular intervals for fermentation product detection.
Example 1
Adjusting the initial pH of the system to 6.0, adjusting the MES concentration to 5g/L, and performing shaking culture in a constant-temperature shaking table under other conditions, taking out samples at regular intervals, and detecting the concentrations of acetic acid, ethanol and butanol in the fermentation broth by an HPLC method. After fermenting for 6 days, the concentration of ethanol in the system is 0.34g/L, butanol is 0g/L, acetic acid is 1.47g/L, and the ratio of acid-alcohol substances is 4.3: 1, under the conditions of higher pH and MES concentration, the yield of the organic acid substances is higher.
Example 2
Adjusting the initial pH of the system to 6.0, adjusting the MES concentration to 0.1g/L, and performing shaking culture in a constant-temperature shaking table under other conditions, taking out samples at regular intervals, and detecting the concentrations of acetic acid, ethanol and butanol in the fermentation broth by an HPLC method. After fermenting for 6 days, the concentration of ethanol in the system is 0.11g/L, butanol is 0g/L, acetic acid is 1.79g/L, and the ratio of acid to alcohol is 16.3: 1.
the results of examples 1 and 2 show that increasing the initial pH of the system, while reducing the amount of MES, can significantly increase the proportion of organic acids in the fermentation product.
Example 3
Adjusting the initial pH of the system to 5.0, adjusting the MES concentration to 0.1g/L, and performing shaking culture in a constant-temperature shaking table under other conditions, taking out samples at regular intervals, and detecting the concentrations of acetic acid, ethanol and butanol in the fermentation broth by an HPLC method. After fermenting for 6 days, the concentration of ethanol in the system is 0.51g/L, the concentration of butanol is 0.065g/L, the concentration of acetic acid is 0.12g/L, and the ratio of acid to alcohol is 0.21: 1, the yield of alcohol is higher in the MES system with slightly lower pH and low concentration.
Example 4
Adjusting the initial pH of the system to 5.0, adjusting the MES concentration to 5g/L, and performing shaking culture in a constant-temperature shaking table under other conditions, taking out samples at regular intervals, and detecting the concentrations of acetic acid, ethanol and butanol in the fermentation broth by an HPLC method. After fermenting for 6 days, the concentration of ethanol in the system is 0.44g/L, butanol is 0.16g/L, acetic acid is 0.59g/L, and the ratio of acid-alcohol substances is 1: 1.
the results of examples 3 and 4 show that reducing the initial pH of the system, while reducing the amount of MES, can significantly increase the proportion of alcohol in the fermentation product.
The culture medium used by the method comprises 2-4% of mineral element Stock solution (V/V), 0.05% of yeast extract (M/V), 0.5% of reducing agent Stock solution (V/V) and 0.1% of trace element Stock solution (V/V);
wherein the Mineral solution stock (M/V) comprises the following components: 8% sodium chloride, 10% ammonium chloride, 1% potassium chloride, 1% monopotassium phosphate, 2% magnesium sulfate and 0.4% calcium chloride; the composition of the Reducing Agent Stock (M/V) was 0.9% sodium hydroxide, 4% L-cysteine hydrochloride and 4% sodium sulfide nonahydrate.
The microelement liquid storage solution (M/V) comprises 1% of nitrilotriacetic acid, 0.5% of magnesium sulfate, 0.4% of ammonium ferrous sulfate, 0.1% of cobalt chloride, 0.1% of zinc sulfate, 0.01% of nickel chloride, 0.01% of sodium selenate and 0.01% of sodium tungstate.
The above-mentioned medium is formulated based on the physicochemical properties of the strain Clostridium carboxidivorans and the strain Clostridium autoethanogenum, and the medium used in the examples is more effective for producing acid alcohols than other medium containing inorganic salts.
Claims (1)
1. A method for adjusting the proportion of acid and alcohol in an anaerobic gas-feeding microorganism fermentation product is characterized in that the method is realized by controlling the initial pH value of a culture system and the using amount of 2-morpholine ethanesulfonic acid MES;
wherein the proportion of acid products in the anaerobic gas-feeding microorganism fermentation product is increased by increasing the initial pH value of the culture system and simultaneously reducing the dosage of MES; the initial pH value of the system is increased by adjusting the pH value to 6; the MES dosage is reduced by adjusting the addition concentration of MES to 0.1g/L in a culture system;
wherein, the proportion of alcohol products in the anaerobic gas-feeding microorganism fermentation products is increased by reducing the initial pH value of the system and simultaneously reducing the dosage of MES; the initial pH value of the reducing system is adjusted to 5; the MES dosage is reduced by adjusting the addition concentration of MES to 0.1g/L in a culture system;
the culture medium used in the culture system comprises 2-4% of mineral element stock solution, 0.05% of yeast extract, 0.5% of reducing agent stock solution and 0.1% of trace element stock solution;
the mineral element stock solution comprises the following components: 8% sodium chloride, 10% ammonium chloride, 1% potassium chloride, 1% monopotassium phosphate, 2% magnesium sulfate and 0.4% calcium chloride;
the reducing agent stock solution comprises 0.9% of sodium hydroxide, 4% of L-cysteine hydrochloride and 4% of sodium sulfide nonahydrate;
the storage liquid of the microelement liquid comprises 1% of nitrilotriacetic acid, 0.5% of magnesium sulfate, 0.4% of ammonium ferrous sulfate, 0.1% of cobalt chloride, 0.1% of zinc sulfate, 0.01% of nickel chloride, 0.01% of sodium selenate and 0.01% of sodium tungstate.
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| CN107779477B (en) * | 2016-08-25 | 2021-10-12 | 中国科学院天津工业生物技术研究所 | Culture system and culture method for producing alcohol by fermenting synthesis gas by using gas-feeding anaerobic bacteria |
| CN107779478B (en) * | 2016-08-25 | 2021-08-24 | 中国科学院天津工业生物技术研究所 | Culture system and culture method for producing alcohol by fermenting synthesis gas by using gas-feeding anaerobic bacteria |
| CN108486172B (en) * | 2018-04-04 | 2020-12-29 | 江南大学 | Method for producing caproic acid by two-phase anaerobic fermentation |
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| CN1444658A (en) * | 2000-07-25 | 2003-09-24 | 生物工程资源股份有限公司 | Methods for increasing production of ethanol from microbial fermentation |
| CN101998997A (en) * | 2008-03-12 | 2011-03-30 | 蓝瑟科技纽西兰有限公司 | Microbial alcohol production process |
| CN102016052A (en) * | 2007-08-15 | 2011-04-13 | 兰扎泰克新西兰有限公司 | Processes of producing alcohols |
| CN102361989A (en) * | 2009-01-29 | 2012-02-22 | 兰扎泰克新西兰有限公司 | Alcohol production process |
| CN104531780A (en) * | 2015-01-07 | 2015-04-22 | 中国科学院天津工业生物技术研究所 | Method for improving fermentation efficiency of anaerobic gas feeding microorganisms |
| CN104619849A (en) * | 2012-07-11 | 2015-05-13 | 科斯卡塔公司 | Method for producing c2 oxygenates by fermentation using high oxidation state sulfur |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1444658A (en) * | 2000-07-25 | 2003-09-24 | 生物工程资源股份有限公司 | Methods for increasing production of ethanol from microbial fermentation |
| CN102016052A (en) * | 2007-08-15 | 2011-04-13 | 兰扎泰克新西兰有限公司 | Processes of producing alcohols |
| CN101998997A (en) * | 2008-03-12 | 2011-03-30 | 蓝瑟科技纽西兰有限公司 | Microbial alcohol production process |
| CN102361989A (en) * | 2009-01-29 | 2012-02-22 | 兰扎泰克新西兰有限公司 | Alcohol production process |
| CN104619849A (en) * | 2012-07-11 | 2015-05-13 | 科斯卡塔公司 | Method for producing c2 oxygenates by fermentation using high oxidation state sulfur |
| CN104531780A (en) * | 2015-01-07 | 2015-04-22 | 中国科学院天津工业生物技术研究所 | Method for improving fermentation efficiency of anaerobic gas feeding microorganisms |
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