CN112852888B - Method for improving methanol methane fermentation activity and application thereof - Google Patents
Method for improving methanol methane fermentation activity and application thereof Download PDFInfo
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- CN112852888B CN112852888B CN202110346756.1A CN202110346756A CN112852888B CN 112852888 B CN112852888 B CN 112852888B CN 202110346756 A CN202110346756 A CN 202110346756A CN 112852888 B CN112852888 B CN 112852888B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
- C12P5/023—Methane
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
Abstract
The invention provides a method for improving the fermentation activity of methanol and methane, belonging to the technical field of organic wastewater and methane fermentation. The invention adds quorum sensing signal molecule-N-hexanoyl homoserine lactone (C) into a methanol methane fermentation system 6 HSL) for regulating and controlling a microbial population induction signal system, the metabolic activity of methanogens can be effectively improved, the methanol degradation rate and the methane production rate are further improved, and the economic benefit of methane fermentation of methanol wastewater is improved.
Description
Technical Field
The invention belongs to the technical field of organic wastewater methane fermentation, and particularly relates to a method for improving methanol methane fermentation activity and application thereof.
Background
Methanol (methane, dried, CH) 3 OH) system, is widely applied to organic synthesis, petrochemical industry and forestry chemical production as a supporting product of chemical industry, and the produced wastewater also contains high-concentration methanol. Methanol has high toxicity to human body, the lowest dose of oral poisoning is about 100mg/kg (body weight), and 0.3-1 g/kg (body weight) can kill when orally taken. Meanwhile, the methanol has certain toxicity to microorganisms, the degradation efficiency of organic matters in the biological filter can be reduced by the methanol with the concentration of 790mg/L, and the digestion of sludge in the digestion tank can be inhibited by the methanol with the concentration of 5000 mg/L.
The methanol wastewater refers to methanol-containing wastewater generated in the industrial production or use process of methanol, belongs to easily degradable high-concentration organic wastewater, and is strictly regulated in the country for the discharge of the methanol wastewater, and can be discharged after being treated to reach a certain standard. At present, common methanol wastewater treatment means are mainly divided into physical treatment, chemical treatment, biological treatment and the like. Among them, the anaerobic biological treatment method is widely used because the cost required for treatment is low and renewable energy sources such as hydrogen and methane can be recovered at the same time. The anaerobic degradation of methanol is mainly mediated by methanogens which belong to archaea, have slow growth and metabolism rate and are sensitive to the changes of environmental factors such as pH, temperature, substrate concentration and the like, so the methanogenic step is considered as the rate-limiting step of methane fermentation. Although the biodegradability of methanol is high, high-concentration methanol has certain toxicity to microorganisms, so that the treatment efficiency of high-concentration methanol wastewater is poor. Therefore, how to develop an efficient methanol methane fermentation technology capable of improving the activity of methanogenic flora, increase the methane fermentation efficiency of high-concentration methanol wastewater and reduce the treatment cost of methanol wastewater is a technical problem which needs to be solved urgently in the field.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for improving methanol methane fermentation activity, which can effectively improve the group metabolic activity of methanogens, and further improve the methanol degradation rate and the methane production rate.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for improving the fermentation activity of methanol and methane, which comprises the following steps: n-hexanoyl homoserine lactone is added into a methanol methane fermentation system.
Preferably, the addition amount of the N-hexanoyl homoserine lactone in a methanol methane fermentation system is 4-6 mu g/L.
Preferably, the content of methanol in the methanol methane fermentation system is 1-10 g/L.
Preferably, the methanol methane fermentation system contains anaerobic activated sludge, and the anaerobic activated sludge contains methylotrophic methanogens.
Preferably, the methanol methane fermentation system also comprises a basic anaerobic culture medium.
Preferably, anaerobic activated sludge is inoculated into a basic anaerobic medium containing methanol, and N-hexanoyl homoserine lactone is added for methane anaerobic fermentation.
Preferably, the inoculation amount of the anaerobic activated sludge is 5-20% of the volume of the basic anaerobic culture medium.
Preferably, the anaerobic activated sludge is taken from a methane fermentation reactor.
Preferably, the methanol-methane fermentation temperature is 30-40 ℃.
The invention also provides application of the method in treatment of methanol wastewater.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, N-hexanoyl homoserine lactone is added into a methanol methane fermentation system, so that a microbial quorum sensing signal system can be regulated and controlled, the formic acid degradation rate of anaerobic activated sludge is increased, the group metabolic activity of methanogens is improved, the methanol methane fermentation activity is further improved, and the methane production rate in a culture system is increased.
The invention is beneficial to the methane fermentation system to tolerate high-concentration methanol, improves the methane fermentation efficiency of high-concentration methanol wastewater, reduces the treatment cost of the methanol wastewater, improves the economy and the effectiveness, is easier to be used in engineering practice, and has wide industrial application prospect.
Drawings
FIG. 1 shows addition of C 6 -HSL、C 4 -HSL、C 12 -HSL and C 14 -methanol content after HSL over time curve;
FIG. 2 shows addition of C 6 -HSL、C 4 -HSL、C 12 -HSL and C 14 Cumulative methane production after HSL over time curve.
Detailed Description
The invention provides a method for improving the fermentation activity of methanol methane, which comprises the step of adding N-hexanoyl homoserine lactone (C) into a methanol methane fermentation system 6 -HSL)。
Quorum Sensing (QS) system can control activities of microorganisms and synthesis of extracellular polymers, wherein the QS system mediated by AHLs is a microbial communication system depending on cell density, and secretion of AHLs is synchronously increased along with increase of microbial density, howeverMost of the functional flora involved in methane fermentation will secrete AHLs in large amounts only when subjected to starvation stress. C 6 HSL belongs to one of microorganism QS signal molecules AHLs, and is closely related to the growth and metabolism of propionic acid nutritional hydrogen-producing acetogenic flora and methanotrophic acetogenic flora. Invention pair C 6 The specific source of HSL is not limited.
The anaerobic degradation of the methanol can be finished under the action of methanogens, but because the methanol has certain toxicity to microorganisms, the decomposition effect of organic matters in the biological filter can be weakened by the methanol with the mass concentration of 790mg/L, and the digestion reaction of sludge in the digestion tank can be inhibited by the methanol with the mass concentration of 5000mg/L, so that a methane fermentation system of high-concentration methanol is easy to acidify, and the methane fermentation reaction fails. The present invention found that a signal molecule-C is induced by addition of a population 6 HSL, which can regulate the microbial quorum sensing signal system, can increase the concentration of extracellular polymers of microbes, form floccules from anaerobic activated sludge, and increase the resistance of microbes to adverse environments. At the same time, C is added 6 After HSL, the quorum sensing system can improve the quorum metabolic activity of methanogens by controlling the quorum activity of microorganisms, so that the methanol methane fermentation activity is improved, and the utilization efficiency of methanol is improved.
In the present invention, C 6 The addition amount of the HSL in the methanol methane fermentation system is 4-6 mu g/L, and more preferably 5 mu g/L.
The content of methanol in the methanol methane fermentation system is 1-10 g/L, preferably 3-9 g/L, and more preferably 5-7 g/L. The invention discovers that C is added 6 After HSL, the resistance of methanogens can be improved, and when the mass concentration of methanol in a fermentation system is 1-10 g/L, the phenomena that the activity of the methanogens is reduced and the methane fermentation reaction is inhibited do not occur.
The methanol methane fermentation system can also contain a basic anaerobic culture medium, and the basic anaerobic culture medium preferably contains 2-10 g of methanol per liter of culture medium. The invention does not limit the concrete formula and the preparation method of the basic anaerobic culture medium. As an alternative embodiment, the formulation of the basic anaerobic culture mediumThe method comprises the following steps: 1L of deionized water was added with 10g of methanol and NaHCO 3 4g，Na 2 HPO 4 ·2H 2 O530mg，KH 2 PO 4 410mg，NH 4 Cl 300mg，CaCl 2 ·2H 2 O 110mg，MgCl 2 ·6H 2 O100mg，NaCl 300mg，FeCl 2 ·4H 2 O 4.5mg，EDTA·Na 2 1.65mg, 1mL of trace element liquid and 1mL of vitamin liquid. The formula (mg/L) of the trace element liquid is as follows: h 3 BO 4 50，ZnCl 2 50，CuCl 2 ·H 2 O 38，MnCl 2 ·4H 2 O 50，CoCl 2 ·6H 2 O 50，NiCl 2 ·6H 2 O 92，Na 2 SeO 3 ·5H 2 O 26，Na 2 WO 4 ·2H 2 O 33，Na 2 MoO 4 ·2H 2 And O24. Vitamin liquid formula (mg/L): biotin 4, nicotinic acid 40, vitamin B610, vitamin B220, vitamin B140, vitamin B1220, folic acid 20, lipoic acid 40 and p-aminobenzoic acid 20. Adding deionized water into an electric heating pot, continuing heating after heating and boiling, introducing high-purity nitrogen into the pot for blowing off dissolved oxygen in water, mixing boiling water with culture medium components after the content of the dissolved oxygen in the water is reduced, continuing introducing the high-purity nitrogen, and adjusting the pH to be 6.5-7.5 by using 1-2 mol/L NaOH and HCl when the temperature of the culture medium is reduced to 35-40 ℃.
The methanol methane fermentation system contains anaerobic activated sludge, and preferably, the anaerobic activated sludge contains methylotrophic methanogens. As an optional implementation mode, the anaerobic activated sludge can be directly taken from the upflow anaerobic sludge bed reactor to be used as an inoculum, and can also be used as the inoculum after 2-3 times of enrichment culture through a methane fermentation system. The specific source of the anaerobic activated sludge is not limited in the present invention. The present inventors have found that 6 HSL is mainly regulated and controlled against methylotrophic methanogens in anaerobic activated sludge, and the methane yield is increased by improving the resistance of thalli to adverse environments and the colony activity.
The invention explains the enrichment culture method of anaerobic activated sludge in detail: inoculating anaerobic activated sludge in the basic anaerobic culture medium, culturing the anaerobic activated sludge in a constant-temperature shaking table, transferring the anaerobic activated sludge into a new fermentation system after methanol in a culture system is completely degraded, and repeating the steps for 2-3 times to obtain inoculated sludge after enrichment culture; preferably, the inoculation amount of the anaerobic activated sludge is 15-25% of the volume of the basic anaerobic culture medium, and more preferably 20%; the culture temperature of the constant temperature shaking table is 30-40 ℃, and the rotating speed is 120-140 rpm. The invention discovers that the abundance of methylotrophic methanogens in the anaerobic activated sludge can be improved and the subsequent experimental time can be reduced by repeated enrichment culture. The enrichment culture in the present invention is not an essential step.
The invention inoculates anaerobic activated sludge into a basic anaerobic culture medium containing methanol, and adds C 6 HSL for methane anaerobic fermentation. The inoculation amount of the anaerobic activated sludge is 5-20% of the volume of the basic anaerobic culture medium, and is preferably 6-10%. The invention is used for anaerobic activated sludge and C 6 The order and manner of addition of HSL is not limited. As an alternative embodiment, the prepared basic anaerobic culture medium is transferred into a serum bottle by using a syringe, the serum bottle is blown off by high-purity nitrogen to remove air in advance, the culture medium is filled into the serum bottle and then is blown off by the high-purity nitrogen for 5min, anaerobic activated sludge with 10 percent of the volume of the culture medium is inoculated, the culture medium is sealed by using a butyl rubber plug, and 5 mu g/L of C is added into a fermentation system by using the syringe 6 -HSL。
The reaction temperature of the methanol methane fermentation system is 30-40 ℃, and the preferable temperature is 35-37 ℃. The invention discovers that the reaction temperature is the optimum temperature for growth and metabolism of methanogens, and anaerobic fermentation at the temperature is more favorable for improving the activity of strains so as to improve the methanogenesis rate.
The invention also provides the industrial application of the method in treating the methanol wastewater. As an alternative embodiment, in the actual industrial wastewater treatment process, the C is directly added into the methanol methane fermentation system without adding a basic anaerobic culture medium 6 HSL, can achieve the effect of improving the resistance of methanogens to adverse environment and the colony activity, thereby improving the yield and the rate of methane production. Industrial waste waterWhich is identical to the basic anaerobic medium of the invention described above, both function identically.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The inoculum is obtained from anaerobic activated sludge of an upflow anaerobic sludge blanket reactor with an organic load of 5 kg/(m) 3 D) daily volumetric gas production rate of 2.1L/(L d).
The basic anaerobic culture medium formula comprises: 1L of deionized water was added with 10g of methanol and NaHCO 3 4g，Na 2 HPO 4 ·2H 2 O 530mg，KH 2 PO 4 410mg，NH 4 Cl 300mg，CaCl 2 ·2H 2 O 110mg，MgCl 2 ·6H 2 O 100mg，NaCl 300mg，FeCl 2 ·4H 2 O 4.5mg，EDTA·Na 2 1.65mg, 1mL of trace element liquid and 1mL of vitamin liquid.
The formula (mg/L) of the trace element liquid is as follows: h 3 BO 4 50，ZnCl 2 50，CuCl 2 ·H 2 O 38，MnCl 2 ·4H 2 O 50，CoCl 2 ·6H 2 O 50，NiCl 2 ·6H 2 O 92，Na 2 SeO 3 ·5H 2 O 26，Na 2 WO 4 ·2H 2 O 33，Na 2 MoO 4 ·2H 2 O 24。
Vitamin liquid formula (mg/L): biotin 4, nicotinic acid 40, vitamin B610, vitamin B220, vitamin B140, vitamin B1220, folic acid 20, lipoic acid 40 and p-aminobenzoic acid 20.
The preparation method of the basic anaerobic culture medium is as follows (1L): the medicines are weighed according to the components of the culture medium and then added into the conical flask. Adding deionized water into an electric heating pot, continuing heating after the deionized water is heated and boiled, and introducing high-purity nitrogen into the pot for blowing off dissolved oxygen in the water. After the content of dissolved oxygen in water is reduced, injecting boiling water into the conical flask, continuously introducing high-purity nitrogen, and adjusting the pH value to 7 by using 1mol/L NaOH and HCl when the temperature of the culture medium is reduced to 40 ℃.
5 parts of the prepared culture medium are taken for carrying out a single-factor control experiment:
experimental groups: adding C 6 -HSL；
Control group 1: addition of N-butyrylhomoserine lactone (C) 4 -HSL)；
Control group 2: addition of N-lauroyl homoserine lactone (C) 12 -HSL)；
Control group 3: addition of N-tetradecanoylhomoserine lactone (C) 14 -HSL)；
Blank control group: deionized water was added after boiling to the same volume as the experimental group.
Transferring 100mL of culture medium into 250mL of serum bottle by using syringe with long needle for animals, removing air by blowing high-purity nitrogen gas, filling the culture medium, continuously blowing high-purity nitrogen gas for 4min, inoculating 9mL of anaerobic activated sludge, sealing by using butyl rubber plug, and adding C into each group by using syringe 4 -HSL、C 6 -HSL、C 12 -HSL、C 14 HSL and deionized water to a final concentration of 5. mu.g/L. After the fermentation system is constructed, the mixture is placed in an air bath constant temperature shaking table at 36 ℃ for culture. The gas production, gas composition and methanol content were measured every two days.
The above 5 experiments were conducted in 3 replicates, and the methanol content duration curve (see fig. 1 for details) and the cumulative methane production duration curve (see fig. 2 for details) were plotted as the average of the three replicates.
As can be seen from FIG. 1, the methanol content in the fermentation system gradually decreases with time, and compared with the blank control group, the anaerobic degradation rate of methanol in the experimental group is increased by 9.4%, and the anaerobic degradation rates of methanol in the control groups 1 to 3 are respectively decreased by 2.6%, 2.3% and 9.0%. Shows that C 6 HSL increases the methanol degradation rate of anaerobic activated sludge, being C of the QS signal molecule of the microorganism 4 -HSL、C 12 -HSL and C 14 HSL inhibits the methanol degradation rate of anaerobic activated sludge.
As can be seen from FIG. 2, as time increases, the methane content in the fermentation system gradually increases, and compared with a blank control group, the methane fermentation rate of the experimental group is increased by 9.4%, and the methane fermentation rates of the control groups 1-3 are respectively reduced by 1.4%, 1.1% and 7.9%. Shows that C 6 HSL is capable of increasing the methanogenesis rate of anaerobic activated sludge, together with C of the QS signal molecule of the microorganism 4 -HSL、C 12 -HSL、C 14 HSL then reduces the methane production rate of anaerobic activated sludge.
Example 2
This example will C 6 HSL was added directly to the feed water of the methane fermentation reactor for a single factor control experiment.
Two upflow anaerobic sludge blanket reactors with effective volume of 4L are selected, the water inlet is formed by diluting methanol to COD of 6000mg/L, and sodium bicarbonate is added to adjust the alkalinity to 3000mg/L (CaCO) 3 Metering), the inoculated sludge is mature anaerobic granular sludge, and the inoculum size is 1L.
C is added to the feed water of the reactor R1 to a final concentration of 5. mu.g/L 6 HSL, reactor R2 as control, without addition of C to the feed water 6 -HSL. The two upflow anaerobic sludge bed reactors are operated under the conditions that the hydraulic retention time is 24 hours and the temperature is 35 ℃, and the COD values of the effluent of the two reactors are respectively measured.
As a result: the average COD value of the effluent of R1 is 259.61mg/L, and the average COD value of the effluent of R2 is 826.37 mg/L.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A method for improving the fermentation activity of methanol methane is characterized in that: adding N-hexanoyl homoserine lactone into a methanol methane fermentation system;
the addition amount of the N-caproyl homoserine lactone in a methanol methane fermentation system is 4-6 mu g/L;
the content of methanol in the methanol methane fermentation system is 5-10 g/L.
2. The method of claim 1, wherein: the methanol methane fermentation system contains anaerobic activated sludge, and the anaerobic activated sludge contains methylotrophic methanogens.
3. The method according to claim 1 or 2, characterized in that: the methanol methane fermentation system also contains a basic anaerobic culture medium.
4. The method of claim 3, wherein: inoculating anaerobic activated sludge into a basic anaerobic culture medium containing methanol, and adding N-caproyl homoserine lactone to perform methane anaerobic fermentation.
5. The method of claim 4, wherein: the inoculation amount of the anaerobic activated sludge is 5-20% of the volume of the basic anaerobic culture medium.
6. The method of claim 4, wherein: the anaerobic activated sludge is taken from a methane fermentation reactor.
7. The method of claim 4, wherein: the fermentation temperature of the methanol methane is 30-40 ℃.
8. Use of the method according to any one of claims 1 to 7 for the treatment of methanol wastewater.
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