CN113430234B - Method for producing medium-chain fatty acid by using external potential to strengthen anaerobic microorganisms - Google Patents

Abstract

The invention provides a method for producing medium-chain fatty acid by strengthening anaerobic microorganisms with external potential, which comprises the following steps: anaerobic acidogenic granular sludge is used as an inoculation bacterial source, electric potential is applied to domesticate and enrich microorganisms with chain extension function, and under the action of the microorganisms with the chain extension function completely domesticated, the electric potential is applied, and acetic acid and ethanol are used as substrates to produce butyric acid and caproic acid through anaerobic fermentation. The invention adopts the method of external potential to produce medium-chain fatty acid, which not only shortens the reaction period, but also improves the yield of butyric acid.

Description

A method for enhancing the production of medium-chain fatty acids by anaerobic microorganisms by applying an electric potential

technical field

The invention belongs to the field of environmental microorganisms, and in particular relates to a method for enhancing the production of medium-chain fatty acids by anaerobic microorganisms by applying an electric potential.

Background technique

Microbial carbon chain extension is a new type of biotechnology with low energy consumption and low operating cost. The medium-chain fatty acid produced in this process is a resource with high economic value, which can realize the recycling of resources and the elimination of organic pollutants at the same time. remove. Microbial carbon chain elongation is a process in which short-chain fatty acids are synthesized into medium-chain fatty acids by increasing two carbon atoms per cycle under the action of functional microorganisms.

At present, the production methods of butyric acid that have been practically used have the disadvantages of strict technical conditions, high cost, and the generation of secondary pollutants, and are often accompanied by certain product losses. Microbial anaerobic fermentation technology has the advantages of mild reaction conditions, low processing cost, and environmental friendliness. Compared with other upgrading technologies, the microbial method has better economic benefits, but is limited by the participation of a variety of functional microorganisms (mainly Clostridium genus Clostridium), and the carbon chain elongation reaction is susceptible to environmental factors and functional microbial activity. Influence (such as pH, substrate concentration, etc.).

Patent CN112391292A reported a method and application of enrichment and application of medium-chain fatty acid carbon chain extension functional microorganisms, and finally obtained 3.4g/L butyric acid and 2.5g/L hexanoic acid; patent CN111909970A reported that adding exogenous medium through Anaerobic microorganisms strengthen syngas to produce medium-chain fatty acids. Adding iron-loaded platinum solid medium to the reaction system can achieve the maximum hexanoic acid production of 4.97g/L, and adding brown manganese to the reaction system can achieve the maximum butyric acid production of 3.43g/L; Patents CN110643644A and CN110643644B reported the method for producing medium-chain fatty acids from livestock and poultry manure fermentation liquid; patent CN110656133A reported pretreatment of anaerobic sludge by ammonium chloride solution, and carried out anaerobic fermentation to produce medium-chain fatty acids; patent CN110734934A reported the use of nitrous acid Sodium solution pretreats anaerobic sludge to produce medium-chain fatty acids; patent CN110734933A reports the addition of zero-valent iron powder for anaerobic fermentation to produce medium-chain fatty acids.

As described in the patents published above, the methods for producing medium-chain fatty acids by anaerobic microorganisms are mostly methods of adding exogenous media. Although the production of butyric acid is promoted, the output of butyric acid is still low, and the reaction cycle is relatively short. long.

Contents of the invention

In order to overcome the shortcomings of traditional anaerobic microbial chain extension substrate utilization rate is low, butyric acid, hexanoic acid yield is not high, the purpose of the present invention is to provide a kind of method that external electric potential strengthens anaerobic microorganism to produce medium-chain fatty acid, and this method can Enrich chain-extended acid-producing microorganisms and increase butyric acid production in a short period of time.

The invention realizes the acclimatization and application of chain-extending functional microorganisms through external potential. Provide electron donors and electron acceptors (such as ethanol and acetic acid), and realize the extension of carbon chains under the action of chain extension functional microorganisms. The present invention aims at increasing the concentration of butyric acid and hexanoic acid, endowing higher commercial value and economic value.

The device for carbon chain extension provided by the present invention, as shown in Figure 1, takes anaerobic granular sludge as the environmental source of chain extension functional microorganisms, applies a working potential to the system, and successfully realizes domestication, Enrich chain extension functional microorganisms, and achieve the purpose of rapid and efficient production of butyric acid and hexanoic acid. Therefore, the invention helps our country get rid of the plight of low production of medium-chain fatty acids, and has important economic value and research significance.

The method for enhancing the production of medium-chain fatty acids by anaerobic microorganisms with an external potential provided by the present invention is as follows: using anaerobic acid-producing granular sludge as an inoculation source, applying an electric potential to carry out domestication and enrichment of microorganisms with chain extension functions, Under the action of microorganisms with the chain extension function, an external potential is applied to produce butyric acid and caproic acid by anaerobic fermentation with acetic acid and ethanol as substrates.

Described method comprises the steps:

1) Domestication and enrichment of microorganisms with chain elongation function

(1) collecting anaerobic acid-producing granular sludge, heat treatment, inhibiting the activity of methanogenic bacteria, and obtaining pretreated anaerobic granular sludge;

(2) aerating the anaerobic granular sludge prepared in step (1) to maintain an anaerobic environment;

(3) Inoculate the anaerobic granular sludge prepared in step (2) into the microbial reactor, apply a potential, start the reactor, and carry out the domestication and enrichment of the microorganisms with chain extension function under the condition of high organic load, and wait for fermentation When the concentration of ethanol in the liquid drops and the concentration of volatile fatty acids rises to a stable level, the acclimatization of the microorganisms with chain extension function is completed;

2) Under the action of fully domesticated microorganisms with chain extension function, an external potential is applied to produce butyric acid and hexanoic acid by anaerobic fermentation with acetic acid and ethanol as substrates.

In step 1)(1) of the above method, the anaerobic acid-producing granular sludge is taken from the residual sludge anaerobic digestion device of Beijing Xiaohongmen Sewage Treatment Plant, and its average particle size is 0.6-2.5mm. The ratio of volatile suspended matter to total suspended matter (VSS/TSS) of mud is 0.4-0.8, specifically 0.7;

The temperature of the sludge heat treatment in step (1) can be 90-100°C, and the treatment time can be 1-1.5h;

In step (2), the anaerobic granular sludge is aerated for 20 minutes (N ₂ :CO ₂ =80:20);

The potential in step (3) can be -0.4V-(-1)V (relative to the standard hydrogen electrode), specifically -0.7V;

In step (3), described high organic load realizes by following operations:

After starting the reactor, the reactor is fed with water in a continuous flow;

Influent water contains acetic acid, ethanol and 2-BES (sodium 2-bromoethanesulfonate: inhibitor of methanogens), among which, the concentration of acetic acid, ethanol and 2-BES can be 6.8-30g/L, 4.8-25ml/ L, 1-10g/L;

During the acclimatization period, the pH is maintained at 5-6.8, specifically 5.5-6.5; the temperature can be 28°C-37°C, and the culture time can be 30-40d.

In step 2), the potential may be -0.4V-(-1)V (relative to the standard hydrogen electrode), specifically -0.8V.

The application of external potential in the domestication and enrichment of microorganisms with chain extension function also belongs to the protection scope of the present invention.

In the application, the potential may be -0.4V-(-1)V (relative to the standard hydrogen electrode), specifically -0.7V.

The application of an external potential to strengthen the production of medium-chain fatty acids by anaerobic microorganisms also belongs to the protection scope of the present invention.

Application mechanism and effect of the present invention are:

(1) The addition of the carbon brush electrode (12) strengthens the stability of the anaerobic system and the enrichment ability of microorganisms;

(2) The hydrogen ions delivered to the cathode region play a positive role in the reduction of acetic acid;

(3) Lower pH can inhibit the growth of methanogens and promote the accumulation of butyrate;

(4) The molar ratio of acetic acid and ethanol is low, and the larger organic load improves the butyric acid productivity;

(5) 2-BES inhibits the growth of methanogens and promotes the chain elongation reaction;

(6) The present invention enriches chain-extending functional microorganisms from anaerobic granular sludge, and solves the problem of limited sources of chain-extending microorganisms.

The invention adopts the method of applying electric potential to produce medium-chain fatty acid, which not only shortens the reaction cycle, but also improves the output of butyric acid.

Description of drawings

Figure 1 is a schematic diagram of the reaction device used in the present invention. Among them, 1: water inlet peristaltic pump; 2: water inlet; 3: sampling port; 4: water outlet; 5: gas collection port; 6: electrochemical workstation; 7: computer; 8: gas collection bag; 9: reference Electrode; 10: anode (graphite rod electrode); 11: three-phase separator; 12: cathode (carbon brush electrode); 13: heating cable; 14: temperature control probe; 15: water inlet bucket; 16: water outlet bucket.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The invention provides a method for strengthening the production of medium-chain fatty acids by anaerobic microorganisms with an external potential, comprising the following steps:

1) Domestication and enrichment of microorganisms with chain elongation function

(1) collecting anaerobic acid-producing granular sludge, heat treatment, inhibiting the activity of methanogenic bacteria, and obtaining pretreated anaerobic granular sludge;

(2) aerating the anaerobic granular sludge prepared in step (1) to maintain an anaerobic environment;

(3) Inoculate the anaerobic granular sludge prepared in step (2) into the microbial reactor, apply a potential, start the reactor, and carry out the domestication and enrichment of the microorganisms with chain extension function under the condition of high organic load, and wait for fermentation When the concentration of ethanol in the liquid drops and the concentration of volatile fatty acids rises to a stable level, the acclimatization of the microorganisms with chain extension function is completed;

2) Under the action of fully domesticated microorganisms with chain extension function, an external potential is applied to produce butyric acid and hexanoic acid by anaerobic fermentation with acetic acid and ethanol as substrates.

In step 1)(1) of the above method, the anaerobic acid-producing granular sludge is taken from the residual sludge anaerobic digestion device of Beijing Xiaohongmen Sewage Treatment Plant, and its average particle size is 0.6-2.5mm. The ratio of volatile suspended matter to total suspended matter (VSS/TSS) of mud is 0.70;

The temperature of the sludge heat treatment in step (1) can be 90-100°C, and the treatment time can be 1-1.5h;

In step (2), the anaerobic granular sludge is aerated for 20 minutes (N ₂ :CO ₂ =80:20);

The potential in step (3) can be -0.4V-(-1)V (relative to the standard hydrogen electrode), specifically -0.7V;

In step (3), described high organic load realizes by following operations:

After starting the reactor, the reactor is fed with water in a continuous flow;

Influent water contains acetic acid, ethanol and 2-BES (sodium 2-bromoethanesulfonate: inhibitor of methanogens), among which the concentration of acetic acid, ethanol and 2-BES can be 7.5g/L, 20ml/L, 5g /L;

During the acclimatization period, the pH is maintained at 5-6.8; the temperature can be 28°C-37°C, and the culture time can be 30-40d.

In step 2), the potential may be -0.4V-(-1)V (relative to the standard hydrogen electrode), specifically -0.8V.

The application of external potential in the domestication and enrichment of microorganisms with chain extension function also belongs to the protection scope of the present invention.

In the application, the potential may be -0.4V-(-1)V (relative to the standard hydrogen electrode), specifically -0.7V.

The application of an external potential to strengthen the production of medium-chain fatty acids by anaerobic microorganisms also belongs to the protection scope of the present invention.

The invention adopts the method of applying electric potential to produce medium-chain fatty acid, which not only shortens the reaction cycle, but also improves the output of butyric acid.

Example 1

Microorganisms with chain elongation function are domesticated and enriched by the following methods:

(1) Collect fresh and come from Beijing Xiaohongmen Sewage Treatment Plant excess sludge anaerobic digestion unit, its average particle size is 0.6-2.5mm, the ratio of volatile suspended matter and total suspended matter of anaerobic sludge ( VSS/TSS) is 0.70, after shaking evenly, place it in a beaker for heat treatment at 100°C in a water bath for 1 hour to inhibit the activity of methanogens and obtain pretreated anaerobic granular sludge;

(2) aerating the anaerobic granular sludge prepared in step (1) with N ₂ : CO ₂ =80:20 mixed gas for 20 minutes to maintain an anaerobic environment;

(3) the activated sludge after the treatment is inoculated into a volume of 1L microbial reactor (shown in Figure 1); open the electrochemical workstation 6, and apply a Specific potential -0.7V (relative to the standard hydrogen electrode), through the laboratory peristaltic pump (Baoding Lange BT100-2J), inflow of water in a continuous flow; the inflow water contains acetic acid, ethanol and 2-BES (2- Sodium bromoethanesulfonate: methanogen inhibitor), the specific concentrations of acetic acid, ethanol, and 2-BES are 7.5g/L, 20ml/L, and 5g/L respectively; the pH is maintained at 5.8 during acclimation; the temperature is 35°C; Acclimatization and enrichment of microorganisms with chain elongation function, the culture time is 40 days.

(4) When the concentration of ethanol in the fermentation liquid drops and the concentration of volatile fatty acids rises to a stable level, the acclimatization of the microorganisms with chain extension function is completed.

Example 2

The device provided by the present invention for the production of medium-chain fatty acids by microbial carbon chain extension is shown in FIG. 1 .

The fully domesticated functional microorganisms in Example 1 were inoculated into 2 anaerobic reactors as shown in Figure 1;

Continuous flow mode feed water, containing acetic acid, ethanol and 2-BES (sodium 2-bromoethanesulfonate: inhibitor of methanogens) in the feed water, the specific concentrations of acetic acid, ethanol and 2-BES are 6.8g/L respectively , 22ml/L, 2.5g/L; the temperature is 35°C;

Open the electrochemical workstation 6, and apply a specific potential-0.8V (relative to the standard hydrogen electrode) to the working electrode 12 (cathode: carbon brush electrode) of one of the anaerobic reactors; the other does not apply potential;

By adopting the above method, the present invention can achieve the following effects:

A cathodic potential of -0.8V was applied to the reaction, and butyric acid was rapidly produced in the system, and the butyric acid production reached 137.5 mM in the fifth day, and the hexanoic acid production was 3.3 mM; in the next 15 days, the butyric acid production was Reduced, maintained at about 100mM, the output of hexanoic acid increased to 24.5mM; the conversion rate of acetic acid reached 80%-90%; the conversion rate of ethanol reached 85%-90%; 60% dropped to about 45%; the total carbon efficiency basically remained at about 70%-80%; in the reactor without applied working potential, butyric acid reached the maximum concentration of 125.1mM on the 20th day, and hexanoic acid reached the maximum concentration on the 60th day It reaches 15.2mM; the conversion rate of acetic acid is basically maintained at 80%-85%, and the conversion rate of ethanol is about 70%-80%. Compared with that without potential, the butyric acid and hexanoic acid produced by applying -0.8V potential increased by 12.4mM and 9.3mM respectively, the conversion rate of acetic acid increased by about 5%, and the conversion rate of ethanol increased by 10%-15%.

Applying a -0.8V potential to the reaction system can generate 137.5mM butyric acid and 24.5mM hexanoic acid in a short time, realizing the high utilization and conversion of acetic acid and ethanol. At the same time, the carbon brush electrode promotes the enrichment of functional microorganisms, and the cathode area The electrons play an active reducing role on acetic acid, promoting the formation of butyric acid and hexanoic acid.

The production of butyric acid and hexanoic acid was affected by applying different working potentials to the reaction system. The reaction applied a cathodic potential of -0.7V, the butyric acid production was basically maintained at about 97mM, and the hexanoic acid was up to 19.5mM; About 13mM; the reaction applied a cathodic potential of -0.5V, the output of butyric acid was maintained at about 100mM, and the hexanoic acid increased to 19mM; the reaction was applied with a cathodic potential of -0.4V, and the output of butyric acid was maintained at about 120mM, and the output of hexanoic acid was about 5mM.

Applying the above working potentials can realize the production of butyric acid and hexanoic acid in a short time, but the potentials are different, and there are differences in the production of butyric acid and hexanoic acid.

Claims (5)

1. a kind of method that external electric potential strengthens anaerobic microorganism to produce medium-chain fatty acid, comprises the steps: 1) Domestication and enrichment of microorganisms with chain elongation function (1) collecting anaerobic acid-producing granular sludge, heat treatment, inhibiting the activity of methanogenic bacteria, and obtaining pretreated anaerobic granular sludge; (2) aerating the anaerobic granular sludge prepared in step (1) to maintain an anaerobic environment; (3) Inoculate the anaerobic granular sludge prepared in step (2) into the microbial reactor, apply a potential, start the reactor, and carry out the domestication and enrichment of the microorganisms with chain extension function under the condition of high organic load, and wait for fermentation When the concentration of ethanol in the liquid drops and the concentration of volatile fatty acids rises to a stable level, the acclimatization of the microorganisms with chain extension function is completed; 2) Under the action of fully domesticated microorganisms with chain extension functions, an external potential is applied to produce butyric acid and hexanoic acid by anaerobic fermentation with acetic acid and ethanol as substrates; In step 2), the potential is -0.4V, relative to the standard hydrogen electrode. 2. method according to claim 1, is characterized in that: in step 1) (1), described anaerobic acid production granular sludge is taken from the UASB reactor of processing citric acid waste water, and its average particle diameter is 0.6- 2.5mm, the ratio of volatile suspended matter to total suspended matter of anaerobic sludge is 0.70. 3. The method according to claim 1 or 2, characterized in that: the temperature of the sludge heat treatment in step (1) is 90-100°C, and the treatment time is 1-1.5h; In step (2), the anaerobic granular sludge is aerated for 20 minutes, and N ₂ :CO ₂ =80:20. 4. The method according to claim 1 or 2, characterized in that: the potential in step (3) is -0.4V-(-1)V, relative to the standard hydrogen electrode. 5. The method according to claim 1 or 2, characterized in that: the high organic load is achieved through the following operations: After starting the reactor, the reactor is fed with water in a continuous flow; Influent water contains acetic acid, ethanol and 2-BES, among which, the concentrations of acetic acid, ethanol and 2-BES are 7.5g/L, 20ml/L and 5g/L in sequence; During the acclimatization period, the pH is maintained at 5-6.8; the temperature is 28°C-37°C, and the culture time is 30-40d.

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