阅读说明：本技术 一种外加电势强化厌氧微生物产中链脂肪酸的方法 (Method for producing medium-chain fatty acid by using external potential to strengthen anaerobic microorganisms ) 是由 党岩 吴胜民 孙德智 刘传奇 陈骞 于 2021-06-28 设计创作，主要内容包括：本发明提供外加电势强化厌氧微生物产中链脂肪酸的方法,为：以厌氧产酸颗粒污泥作为接种菌源,施加电势,进行链延长功能微生物的驯化与富集,在驯化完全的链延长功能微生物的作用下,外加电势,以乙酸、乙醇为底物厌氧发酵生产丁酸和己酸。本发明采用外加电势的方法生产中链脂肪酸,既缩短了反应周期,又提高了丁酸的产量。(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 bacteria source, 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, 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.)

1. A method for strengthening the production of medium-chain fatty acid by anaerobic microorganisms by applying an external potential comprises the following steps: anaerobic acidogenic granular sludge is used as an inoculation bacteria source, 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, potential is applied, and acetic acid and ethanol are used as substrates to produce butyric acid and caproic acid through anaerobic fermentation.

2. The method of claim 1, wherein: the method comprises the following steps:

1) domestication and enrichment of chain-extending functional microorganisms

(1) Collecting anaerobic acid-producing granular sludge, performing heat treatment, and inhibiting the activity of methanogens to obtain pretreated anaerobic granular sludge;

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

(3) inoculating the anaerobic granular sludge prepared in the step (2) into a microbial reactor, applying potential, starting the reactor, performing acclimation and enrichment of chain-extension-function microorganisms under the condition of high organic load, and completing acclimation of the chain-extension-function microorganisms when the concentration of ethanol in fermentation liquid is reduced and the concentration of volatile fatty acid is increased to be stable;

2) under the action of domesticated complete chain-extending functional microorganisms, applying potential, and producing butyric acid and caproic acid by anaerobic fermentation with acetic acid and ethanol as substrates.

3. The method of claim 2, wherein: in the step 1) (1), the anaerobic acid-producing granular sludge is taken from an UASB reactor for treating citric acid wastewater, the average grain diameter of the anaerobic acid-producing granular sludge is 0.6-2.5mm, and the ratio of volatile suspended matters to total suspended matters of the anaerobic sludge is 0.70.

4. A method according to claim 2 or 3, characterized in that: the temperature of the sludge heat treatment in the step (1) is 90-100 ℃, and the treatment time is 1-1.5 h;

aerating anaerobic granular sludge for 20 minutes in step (2), and N₂:CO₂＝80:20。

5. The method according to any one of claims 2-4, wherein: the potential in step (3) is-0.4V (-1) V relative to a standard hydrogen electrode.

6. The method according to any one of claims 2-5, wherein: the high organic load is achieved by:

after the reactor is started, the reactor is fed with water in a continuous flow mode;

the inlet water contains acetic acid, ethanol and 2-BES, wherein the concentrations of the acetic acid, the ethanol and the 2-BES are 7.5g/L, 20ml/L and 5g/L in sequence;

maintaining pH at 5-6.8 during acclimation; the temperature is 28-37 ℃, and the culture time is 30-40 d.

7. The method according to any one of claims 2-6, wherein: in the step 2), the potential is-0.4V (-1) V relative to a standard hydrogen electrode.

8. The application of external potential in domestication and enrichment of chain-extending functional microorganisms.

9. Use according to claim 8, characterized in that: in the application, the potential is-0.4V (-1) V relative to a standard hydrogen electrode.

10. The application of the applied potential to strengthen the production of medium-chain fatty acid by anaerobic microorganisms.

Technical Field

The invention belongs to the field of environmental microorganisms, and particularly relates to a method for producing medium-chain fatty acids by using an external potential reinforced anaerobic microorganism.

Background

The extension of the carbon chain of the microorganism is a novel biotechnology with low energy consumption and low operation cost, the medium-chain fatty acid generated in the process is a resource with higher economic value, and the organic pollutants can be removed while the resource is recycled. The microorganism carbon chain extension is a process of synthesizing medium-chain fatty acid by short-chain fatty acid by adding two carbon atoms in each cycle under the action of functional microorganisms.

At present, the method for producing butyric acid which is actually applied has the defects of strict technical conditions, high cost, secondary pollutant generation and the like, and is often accompanied with certain product loss. The microbial anaerobic fermentation technology has the advantages of mild reaction conditions, low treatment cost, environmental friendliness and the like. Compared with other upgrading technologies, the microbiological method has better economic benefits, but is limited by the participation of various functional microorganisms (mainly Clostridium clarkii), and the carbon chain elongation reaction is easily influenced by environmental factors and the activity of the functional microorganisms (such as pH, substrate concentration and the like).

Patent CN112391292A reports an enrichment method and application of microorganisms with medium-chain fatty acid carbon chain extension function, and 3.4g/L of butyric acid and 2.5g/L of caproic acid are finally obtained; patent CN111909970A reports that medium-chain fatty acids are produced by adding exogenous media and strengthening synthesis gas through anaerobic microorganisms, the maximum production of caproic acid can be 4.97g/L by adding iron-loaded platinum solid media in a reaction system, and the maximum production of butyric acid can be 3.43g/L by adding pyrolusite in the reaction system; patents CN110643644A and CN110643644B report methods for producing medium-chain fatty acids from fermentation broth of livestock and poultry manure; patent CN110656133A reports the production of medium-chain fatty acids by anaerobic fermentation of anaerobic sludge pretreated with ammonium chloride solution; patent CN110734934A reports the production of medium-chain fatty acids by pretreating anaerobic sludge with sodium nitrite solution; patent CN110734933A reports the production of medium-chain fatty acids by anaerobic fermentation with addition of zero-valent iron powder.

The methods for producing medium-chain fatty acids using anaerobic microorganisms described in the above-mentioned published patents are mostly methods in which an exogenous medium is added, and although production of butyric acid is promoted, the yield of butyric acid is low and the reaction period is long.

Disclosure of Invention

In order to overcome the defects of low utilization rate of a traditional anaerobic microorganism chain extension substrate, low yield of butyric acid and caproic acid and the like, the invention aims to provide a method for producing medium-chain fatty acid by using an external potential reinforced anaerobic microorganism.

The invention realizes domestication and application of chain extension functional microorganisms through external potential. Providing an electron donor and an electron acceptor (such as ethanol and acetic acid), and realizing the extension of the carbon chain under the action of the chain extension functional microorganism. The invention aims to improve the concentration of butyric acid and caproic acid and endow higher commercial value and economic value.

As shown in figure 1, the device for carbon chain extension provided by the invention successfully realizes domestication and enrichment of chain extension functional microorganisms from anaerobic sludge by taking anaerobic granular sludge as an environmental source of the chain extension functional microorganisms and applying a working potential to a system, and achieves the aim of quickly and efficiently producing butyric acid and caproic acid. Therefore, the method is beneficial to getting rid of the dilemma of low yield of the medium-chain fatty acid in China, and has important economic value and research significance.

The invention provides a method for producing medium-chain fatty acid by using an external potential to strengthen anaerobic microorganisms, which comprises the following steps: anaerobic acidogenic granular sludge is used as an inoculation bacteria source, 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, potential is applied, and acetic acid and ethanol are used as substrates to produce butyric acid and caproic acid through anaerobic fermentation.

The method comprises the following steps:

1) domestication and enrichment of chain-extending functional microorganisms

(1) Collecting anaerobic acid-producing granular sludge, performing heat treatment, and inhibiting the activity of methanogens to obtain pretreated anaerobic granular sludge;

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

(3) inoculating the anaerobic granular sludge prepared in the step (2) into a microbial reactor, applying potential, starting the reactor, performing acclimation and enrichment of chain-extension-function microorganisms under the condition of high organic load, and completing acclimation of the chain-extension-function microorganisms when the concentration of ethanol in fermentation liquid is reduced and the concentration of volatile fatty acid is increased to be stable;

2) under the action of domesticated complete chain-extending functional microorganisms, applying potential, and producing butyric acid and caproic acid by anaerobic fermentation with acetic acid and ethanol as substrates.

In the step 1) (1) of the method, the anaerobic acid-producing granular sludge is obtained from an anaerobic digestion device of excess sludge of a sewage treatment plant of Minican in Beijing, the average grain diameter of the anaerobic digestion device is 0.6-2.5mm, and the ratio (VSS/TSS) of volatile suspended matters and total suspended matters of the anaerobic sludge is 0.4-0.8, and particularly 0.7;

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

anaerobic granular sludge aeration for 20 minutes in step (2) (N)₂:CO₂＝80:20)；

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

in the step (3), the high organic load is realized by the following operations:

after the reactor is started, the reactor is fed with water in a continuous flow mode;

the inlet water contains acetic acid, ethanol and 2-BES (2-bromoethyl sodium sulfonate: methanogen inhibitor), wherein the concentrations of the acetic acid, the ethanol and the 2-BES can be 6.8-30g/L, 4.8-25ml/L and 1-10g/L in sequence;

maintaining pH at 5-6.8, specifically 5.5-6.5 during acclimation period; the temperature can be 28-37 ℃, and the culture time can be 30-40 d.

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

The application of the external potential in the domestication and enrichment of the chain-extending functional microorganisms also belongs to the protection scope of the invention.

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

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

The application mechanism and the effect of the invention are as follows:

(1) the stability of an anaerobic system and the enrichment capacity of microorganisms are enhanced by adding the carbon brush electrode (12);

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

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

(4) the mole ratio of acetic acid and ethanol is lower, and the larger organic load improves the production rate of butyric acid;

(5)2-BES inhibits the growth of methanogens and promotes chain extension reactions;

(6) the invention enriches chain-extension functional microorganisms from anaerobic granular sludge, and solves the problem of limited source of chain-extension microorganisms.

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.

Drawings

FIG. 1 is a schematic view of a reaction apparatus used in the present invention. Wherein, 1: a water inlet peristaltic pump; 2: a water inlet; 3: a sampling port; 4: a water outlet; 5: a gas collection port; 6: an electrochemical workstation; 7: a computer; 8: a gas collection bag; 9: a reference electrode; 10: anode (graphite rod electrode); 11: a three-phase separator; 12: a cathode (carbon brush electrode); 13: a heat tracing band; 14: a temperature control probe; 15: a water inlet barrel; 16: and (6) discharging the water bucket.

Detailed Description

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

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The invention provides a method for producing medium-chain fatty acid by using an external potential to strengthen anaerobic microorganisms, which comprises the following steps:

1) domestication and enrichment of chain-extending functional microorganisms

(1) Collecting anaerobic acid-producing granular sludge, performing heat treatment, and inhibiting the activity of methanogens to obtain pretreated anaerobic granular sludge;

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

(3) inoculating the anaerobic granular sludge prepared in the step (2) into a microbial reactor, applying potential, starting the reactor, performing acclimation and enrichment of chain-extension-function microorganisms under the condition of high organic load, and completing acclimation of the chain-extension-function microorganisms when the concentration of ethanol in fermentation liquid is reduced and the concentration of volatile fatty acid is increased to be stable;

2) under the action of domesticated complete chain-extending functional microorganisms, applying potential, and producing butyric acid and caproic acid by anaerobic fermentation with acetic acid and ethanol as substrates.

In the step 1) (1) of the method, the anaerobic acid-producing granular sludge is obtained from an anaerobic digestion device of excess sludge of a small Hongmen sewage treatment plant in Beijing, the average grain diameter of the anaerobic digestion device is 0.6-2.5mm, and the ratio (VSS/TSS) of volatile suspended matters and total suspended matters of the anaerobic sludge is 0.70;

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

anaerobic granular sludge aeration for 20 minutes in step (2) (N)₂:CO₂＝80:20)；

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

in the step (3), the high organic load is realized by the following operations:

after the reactor is started, the reactor is fed with water in a continuous flow mode;

the inlet water contains acetic acid, ethanol and 2-BES (2-bromoethanesulfonic acid sodium salt: methanogen inhibitor), wherein the concentrations of acetic acid, ethanol and 2-BES can be 7.5g/L, 20ml/L and 5g/L in sequence;

maintaining pH at 5-6.8 during acclimation; the temperature can be 28-37 ℃, and the culture time can be 30-40 d.

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

The application of the external potential in the domestication and enrichment of the chain-extending functional microorganisms also belongs to the protection scope of the invention.

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

The application of the applied potential to strengthen the production of the medium-chain fatty acid by the anaerobic microorganisms also belongs to the protection scope of the invention.

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.

Example 1

The chain extension functional microorganism is domesticated and enriched by the following method:

(1) collecting a fresh residual sludge anaerobic digestion device from a small Hongmen sewage treatment plant in Beijing, wherein the average particle size of the residual sludge anaerobic digestion device is 0.6-2.5mm, the ratio (VSS/TSS) of volatile suspended matters and total suspended matters of anaerobic sludge is 0.70, uniformly shaking the residual sludge anaerobic digestion device, placing the residual sludge anaerobic digestion device in a beaker for heating treatment in a water bath at 100 ℃, wherein the treatment time is 1h, and inhibiting the activity of methanogens to obtain pretreated anaerobic granular sludge;

(2) n is used for anaerobic granular sludge prepared in the step (1)₂：CO₂Aerating mixed gas at the ratio of 80:20 for 20min, and keeping an anaerobic environment;

(3) inoculating the treated activated sludge into a 1L microbial reactor (shown in figure 1); opening the electrochemical workstation 6, applying a specific potential of-0.7V (relative to a standard hydrogen electrode) to the working electrode 12 (cathode: carbon brush electrode) of the anaerobic reactor, feeding water in a continuous flow by means of a laboratory peristaltic pump (Badin Lange BT 100-2J); the inlet water contains acetic acid, ethanol and 2-BES (2-bromoethyl sodium sulfonate: methanogen inhibitor), and the concentrations of the acetic acid, the ethanol and the 2-BES are respectively 7.5g/L, 20ml/L and 5 g/L; during acclimation the pH was maintained at 5.8; the temperature is 35 ℃; domestication and enrichment of chain extension functional microorganisms are carried out, and the culture time is 40 days.

(4) And (3) completing the domestication of the chain-extending functional microorganisms when the concentration of the ethanol in the fermentation solution is reduced and the concentration of the volatile fatty acid is increased to be stable.

Example 2

The device for producing medium-chain fatty acid by microorganism carbon chain extension is shown in figure 1.

Inoculating the fully domesticated functional microorganisms of example 1 into 2 anaerobic reactors as shown in figure 1;

the continuous flow mode water inlet comprises water inlet containing acetic acid, ethanol and 2-BES (2-bromoethanesulfonic acid sodium salt: methanogen inhibitor), wherein the concentrations of the acetic acid, the ethanol and the 2-BES are respectively 6.8g/L, 22ml/L and 2.5 g/L; the temperature is 35 ℃;

opening the electrochemical workstation 6, applying a specific potential of-0.8V (relative to a standard hydrogen electrode) to the working electrode 12 (cathode: carbon brush electrode) of one of the anaerobic reactors; the other is not applied with electric potential;

by adopting the method, the invention can achieve the following effects:

the reaction is applied with a cathode potential of-0.8V, butyric acid is rapidly produced in the system, the yield of butyric acid reaches 137.5mM in the fifth day, and the yield of caproic acid is 3.3 mM; in the next 15 days, the yield of butyric acid is reduced and is maintained at about 100mM, and the yield of caproic acid is increased to 24.5 mM; the acetic acid conversion rate reaches 80-90%; the conversion rate of the ethanol reaches 85 to 90 percent; the yield of butyric acid is reduced from 60% to about 45% along with the generation of caproic acid; the total carbon efficiency is basically kept at about 70-80%; in the reactor without the working potential, butyric acid reached a maximum concentration of 125.1mM around the twentieth day and caproic acid reached 15.2mM at day 60; the acetic acid conversion rate is basically maintained at 80-85%, and the ethanol conversion rate is about 70-80%. Compared with the potential without adding, the butyric acid and the caproic acid generated by the potential with-0.8V are respectively increased by 12.4mM and 9.3mM, the acetic acid conversion rate is increased by about 5 percent, and the ethanol conversion rate is increased by 10 to 15 percent.

The reaction system can generate 137.5mM butyric acid and 24.5mM caproic acid in a short time by applying-0.8V potential, high utilization and conversion of acetic acid and ethanol are realized, meanwhile, the carbon brush electrode promotes the enrichment of functional microorganisms, and electrons in the cathode region play a positive reduction role on the acetic acid, thereby promoting the generation of the butyric acid and the caproic acid.

The reaction system has certain influence on the production of butyric acid and caproic acid by applying different working potentials. The reaction is applied with a cathode potential of-0.7V, the yield of butyric acid is basically maintained at about 97mM, and the highest yield of caproic acid is 19.5 mM; the reaction is applied with a cathode potential of-0.6V, the yield of butyric acid is basically maintained at about 116mM, and meanwhile, the yield of caproic acid is reduced to about 13 mM; the reaction is applied with a cathode potential of-0.5V, the yield of butyric acid is maintained at about 100mM, and the caproic acid is increased to 19 mM; the yield of butyric acid and caproic acid of the reaction at-0.4V applied with a cathodic potential was maintained at about 120mM and at about 5 mM.

The production of butyric acid and caproic acid can be realized in a short time by applying the working potential, but the production of butyric acid and caproic acid is different by different potentials.