CN111100882B

CN111100882B - A kind of method for strengthening mixed bacteria group to produce caproic acid

Info

Publication number: CN111100882B
Application number: CN202010052792.2A
Authority: CN
Inventors: 黄振兴; 余江南; 廖家林; 阮文权
Original assignee: Jiangnan University
Current assignee: Jiangsu Daotong Environmental Technology Co ltd
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2021-10-22
Anticipated expiration: 2040-01-17
Also published as: CN111100882A

Abstract

The invention discloses a method for strengthening the production of caproic acid by mixed bacteria group, and belongs to the field of waste recycling. The method is to add chloroform and calcium carbonate to the fermentation mixed solution to carry out anaerobic fermentation; the dosage of chloroform is 0.1-2‰ relative to the mass volume concentration of the fermentation mixed solution; the dosage of calcium carbonate is It is 5-25g/L relative to the mass volume concentration of the fermentation mixture. The combined treatment of chloroform and calcium carbonate in the present invention is superior to the sum of the effects of single chloroform treatment and single calcium carbonate treatment.

Description

Method for producing caproic acid by strengthening mixed flora

Technical Field

The invention relates to a method for producing caproic acid by strengthening mixed flora, belonging to the field of waste recycling.

Background

Various microorganisms existing in the anaerobic mixed flora are double-edged sword for caproic acid fermentation, so that the caproic acid production adaptive to a complex system is improved, and the maximum accumulative concentration of the caproic acid is also improved. The research on the technology also gradually goes into the mechanism exploration and the application development.

However, other functional microorganisms in the mixed flora such as syntrophic oxidation-methanogenesis, homoacetogenesis reduction of carboxylic acid and the like potentially affect caproic acid fermentation. These competing pathways are very active and have a negative impact on hexanoic acid production. The competition diagram for these metabolic pathways is shown in FIG. 1.

The influencing factors and influencing consequences of the by-products such as methane, butanol and butyric acid are not consistent: (1) the mutual-nutrient oxidation-methane production process weakens the caproic acid production, but the process may not seriously threaten the caproic acid fermentation leading microorganism, and even a weak symbiosis phenomenon can occur under certain conditions, namely, the methane bacteria reduce the hydrogen partial pressure or provide a cofactor for the caproic acid bacteria, and indirectly assist the growth of the caproic acid bacteria; (2) butanol accumulation can be considered as reduction after butyric acid is ingested by homoacetogenic bacteria, or the butanol accumulation can be directly obtained by the hydroconversion of self-generated butyryl coenzyme A, the accumulation of a large amount of butanol can cause that the accumulated electronic equivalent can not return to the carbon chain extension cycle, the conversion efficiency is also weakened, and when the concentration of the butanol reaches higher, the production of hexanoic acid is even stopped; (3) butyric acid is a growth-coupled product of caproic acid bacteria as a product of insufficient carbon chain elongation, and is closely related to the proliferation of caproic acid bacteria, and can return to the caproic acid conversion cycle under appropriate conditions despite the slower conversion.

Disclosure of Invention

The method aims at competitive microorganisms in a competitive way, particularly in a butanol way and a methane way, and performs restrictive regulation and control on the competitive microorganisms by adding chloroform so as to strengthen the leading position of the caproic acid bacteria. And the growth promotion of the caproic acid bacteria can be realized by jointly adding calcium carbonate.

The first purpose of the invention is to provide a method for producing hexanoic acid by fermentation, which comprises the steps of adding chloroform and calcium carbonate into fermentation mixed liquor for anaerobic fermentation; the adding amount of the chloroform is 0.1-2 per mill of the mass volume concentration of the fermentation mixed solution; the addition amount of the calcium carbonate is 5-25g/L relative to the mass volume concentration of the fermentation mixed liquor.

In one embodiment of the invention, the fermentation mixed solution is a fermentation solution containing ethanol and carboxylic acid; the mass ratio of the ethanol to the carboxylic acid is (3-5): 1.

in one embodiment of the present invention, the carboxylic acids refer to acetic acid, butyric acid and lactic acid.

In one embodiment of the present invention, the carboxylic acid is acetic acid.

In one embodiment of the invention, the fermentation mixed liquor is prepared by taking inoculated sludge fermentation liquor and naturally acidified fermentation liquor as raw materials.

In one embodiment of the present invention, the method for preparing the naturally acidified fermentation broth comprises: adding water into fruit and vegetable wastes to obtain a fermentation system with the total solid concentration (TS) of 7-10% and the Volatile Solid (VS) content of 80-120 g-VS/L, and then fermenting the fermentation system at the temperature of 34-36 ℃, the pH of 4.0-4.5 and the rotation speed of 12-60 rpm to obtain a naturally acidified fermentation liquid.

In one embodiment of the present invention, the preparation method of the inoculated sludge fermentation broth comprises: adding water into fruit and vegetable wastes to obtain a fermentation system with the total solid concentration (TS) of 7-10% and the Volatile Solid (VS) content of 80-120 g-VS/L, inoculating anaerobic sludge into the fermentation system in an inoculation amount of 50-100 g-VS/L, and fermenting the fermentation system at the temperature of 34-36 ℃, the pH of 4.0-4.5 and the rotation speed of 12-60 rpm to obtain inoculated sludge fermentation liquor.

In one embodiment of the present invention, the fruit and vegetable waste contains sugar.

In one embodiment of the invention, the sugar content of the fruit and vegetable waste is not less than 8% (m/m).

The second purpose of the invention is to provide a method for improving the abundance of caproic acid bacteria, which comprises the steps of adding chloroform and calcium carbonate into fermentation mixed liquor for anaerobic fermentation; the adding amount of the chloroform is 0.1-2 per mill of the mass volume concentration of the fermentation mixed solution; the addition amount of the calcium carbonate is 5-25g/L relative to the mass volume concentration of the fermentation mixed liquor.

The invention has the beneficial effects that:

(1) compared with caproic acid fermentation characteristics under different chloroform adding concentration conditions, methanogen hydrogenotrophus and homoacetogen proliferation, the caproic acid bacteria quantity change dynamically, the caproic acid production strength is not seriously suppressed under a wider low concentration condition, chloroform can actually effectively restrict competition pathway metabolism and activity, and caproic acid bacteria can proliferate but the early growth cycle is delayed; the production of caproic acid is stopped under the condition of overhigh concentration, and the quantity of various functional microorganisms is not increased. And the long-acting inhibitory effect of chloroform further ensures the effectiveness. Therefore, 0.5 per mill (m/m) is selected as the proper chloroform adding amount, and the effective inhibition of competitive microorganisms can be realized.

(2) The experiment offsets part of side effects by jointly adding chloroform-calcium carbonate, and the experimental result shows that the caproic acid yield under the joint adding condition is further improved: remarkably increasing the concentration to 19-21 g/L from about 15g/L under the non-reinforced condition,

(3) the observation of the mixed flora by an electron microscope shows that: the shape of microorganisms in the mixed flora of the calcium carbonate adding group has influence, more bacteria with short and thick shapes exist, and more bacteria with single chloroform adding group have long and thin shapes.

(4) Based on the research of microbial floras, the dominant statuses of the Clostridium kluyveri in the anaerobic mixed floras with the single chloroform addition and the combined chloroform calcium carbonate addition are effectively consolidated and reach more than 30.9 percent, and meanwhile, a great reduction of the intercropping oxidizing bacteria can be found, and the effective restriction on the competitive path is reflected. The calcium carbonate further remarkably improves the abundance of caproic acid bacteria to 49% at most, and the combined treatment of chloroform and calcium carbonate is more superior to the sum of the treatment effects of single chloroform and single calcium carbonate, which shows that the two are mutually supported and supplemented in the aspect of enhancing the production of caproic acid and have synergistic effect.

Drawings

FIG. 1 is a schematic diagram of key metabolic pathways in the process of producing caproic acid by using mixed flora.

FIG. 2 shows the change of the components of the main liquid phase product under different chloroform adding concentration conditions: (A)0.5 per mill is added; (B) adding 1 per mill; (C)5 per mill is added; (D)10 per mill is added.

FIG. 3 is a graph showing the effect of different chloroform dosages on the number of homoacetogenic bacteria.

FIG. 4 is a schematic diagram of acetyl-CoA clearance pathway of homoacetogenic bacteria.

FIG. 5 shows the inhibition of methanogens hydrogenotrophus by chloroform at different concentrations.

FIG. 6 shows the effect of different chloroform dosages on caproic acid bacteria proliferation: (A)0.5 per mill; (B)1 per mill; (C)5 per mill; (D)10 per mill.

FIG. 7 shows the caproic acid fermentation characteristics under the condition of combined addition of chloroform-calcium carbonate, and A, B, C shows the caproic acid fermentation characteristics under the condition of 0.5 per mill of added chloroform, wherein the calcium carbonate dosage is respectively 5,10 and 15 g/L.

FIG. 8 shows caproic acid fermentation mixed flora under the field of scanning electron microscope: the concentration of chloroform added into each group A, B and C is 0.5 per mill, 1 per mill and 5 per mill respectively; and C, jointly adding 0.5 per mill of chloroform-calcium carbonate into the groups D, E and F respectively, wherein the adding amount of the calcium carbonate is 5,10 and 15 g/L.

FIG. 9 shows the species level differences of mixed bacteria for caproic acid fermentation in each group: LF1,2 and 3 are chloroform groups which are independently added, and the concentration gradients are respectively 0.5 per mill, 1 per mill and 5 per mill; LH1, LH 892 and LH 3 are combined addition of chloroform-calcium carbonate groups, and the addition amount of calcium carbonate is 5g/L, 10g/L and 15g/L respectively; the selected microorganisms in the graph have a relative abundance of greater than 1% in at least one set of experiments, and the remaining microorganism abundances are classified as others.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.

The fruit and vegetable wastes related in the following embodiments are collected from the farm product markets around the south of the Yangtze river university and consist of rotten apples, bananas, pineapples and pears; anaerobic sludge referred to in the following examples was taken from an anaerobic wastewater treatment unit of a certain syrup plant in the tin-free market; dry Angel yeast referred to in the examples below was purchased from Angel Yeast Inc. (SY, Saccharomyces cerevereviiae).

The high throughput sequencing method for bacteria involved in the following examples was as follows:

first use

(MoBio, 12888-50) extracting genome DNA of a fermentation system by using a soil DNA extraction kit, amplifying a V3-V4 region of a 16S rRNA gene of bacteria by using a universal primer, and finally performing high-throughput sequencing on an amplification product by using an Illumina Miseq sequencing platform (Kajie transform medicine research Co., Ltd., Suzhou);

wherein, the universal primers are as follows:

338F:5'-ACTCCTACGGGAGGCAGCAG-3'(SEQ ID NO.1)；

806R:5'-GGACTACHVGGGTWTCTAAT-3'(SEQ ID NO.2)。

the fungal high throughput sequencing methods referred to in the following examples are as follows:

first use

(MoBio, 12888-50) extracting genome DNA of a fermentation system by using a soil DNA extraction kit, amplifying an ITS1F-ITS2 region of a fungal genome by using a universal primer, and finally performing high-throughput sequencing on an amplification product by using an Illumina Miseq sequencing platform (Kajie transform medicine research Co., Ltd., Suzhou);

wherein, the universal primers are as follows:

ITS1F:5'-CTTGGTCATTTAGAGGAAGTAA-3'(SEQ ID NO.3)；

ITS2R:5'-GCTGCGTTCTTCATCGATGC-3(SEQ ID NO.4)'。

the fluorescent quantitative PCR test method referred to in the following examples is as follows:

sludge sample DNA extraction was as described in 2.23. qPCR assays for Clostridium kluyveri were as 2.2.5; the fluorescence quantitative PCR aiming at homoacetogenic bacteria adopts a primer fhs-F (5 '-GTWTGGGCWAARGGYGGMGAAGG-3'); an fhs-R (5 '-GTATTGDGTYTTRGCCATACA-3') qPCR reaction system and an amplification calculation reference are used for researching experimental conditions in a microbial transformation mechanism of synthesis gas mixed fermentation under a high-temperature condition [ D ]. Jiangnan university, 2018 ].

Absolute quantitative reaction systems and methods of methanogens hydrogenotrophus (Methanobacteria) (primers F: CGWAGGGAAGCTGTTAAGT; R: TACCGTCGTCCACTCCTT) and Methanococcus acetovorans (Methanosarcinales) (primers F: GTAAACGATRYTCGCTAGGT; R: GGTCCCCACAGWGTACC) the experimental conditions in the reference "dungyu. co-culture flora enhanced straw anaerobic digestion and microbiological mechanism research [ D ]. university of south of the Yangtze river, 2017".

The detection methods referred to in the following examples are as follows:

the detection method of total solid concentration (TS), Total Chemical Oxygen Demand (TCOD) and Volatile Solid (VS) content comprises the following steps: reference may be made in particular to the Standard Methods described in "APHA, 2012, Standard Methods for the interpretation of 410Water and Water, American Public Health Association/American Water Works Association/Water environmental Federation, Washington, DC, USA".

The detection method of the content of ethanol and carboxylic acid comprises the following steps: taking the fermentation liquor, centrifuging at 12000rpm, filtering by a 0.45 mu m water system filter membrane and pre-acidifying by 3mol/L phosphoric acid, and simultaneously determining the concentration of carboxylic acid or various carboxylic acids by using a gas chromatograph (GC-2010plus, Shimadzu) provided with a Flame Ionization Detector (FID) and a 30m x 0.25mm x 0.25 mu m capillary column (Intercap FFAP); the instrument is set as follows: the temperature settings of the injection port (INJ) and the detector (FID) are both 250 ℃; the column box temperature control program is set as follows: firstly, maintaining the initial temperature of 70 ℃ for 2min, raising the temperature to 210 ℃ at the ramp heating rate of 15 ℃/min, maintaining the temperature at 210 ℃ for 2min, then cooling, and entering the next detection period; carrier gas (99.999% N)₂Xin xi apparatus science and technology Co., Ltd., Wuxi city) at a flow rate of 60 ml/min; the flow rates of hydrogen and high-pressure air are respectively set to be 40 and 400 mL/min; comparing the peak time of each component obtained by detection with the peak time of a standard product for qualitative determination, and quantifying the peak concentration according to the peak area of each component and the external standard concentration (external standard method).

Calculation formula of conversion: conversion (%) ═ COD_Ethanol+COD_{Carboxylic acids})/COD_{Raw materials}。

Formula for calculating maximum ethanol production rate: the maximum ethanol production rate (g/L · d) ═ max (δ ethanol concentration net yield/δ time).

Calculation formula of ethanol yield: ethanol yield (%) ═ COD_Ethanol/COD_{Raw materials}。

And (4) judging the end of fermentation: the end of fermentation is determined when the main product reaches 95% of the maximum yield.

Calculation formula of alcohol-acid ratio (COD-ethanol/COD-carboxylic acid): alcohol acid ratio (COD)_Ethanol/(COD_{Acetic acid}+COD_{Butyric acid})。

Example 1: production of caproic acid fermentation broth

The experimental raw materials adopt natural acidification fermentation liquor and inoculated sludge fermentation mixed liquor obtained in patent with application number of 201911354619.1, wherein:

and (3) natural acidification group: adding water into fruit and vegetable wastes to obtain a fermentation system with the total solid concentration (TS) of 8% and the Volatile Solid (VS) content of 78g-VS/L, and then fermenting the fermentation system for 17d under the conditions that the temperature is 34-36 ℃, the pH value is 4.0-4.5 and the rotating speed is 60rpm to obtain a fermentation liquid (the fermentation liquid is the ethanol-type acidified fermentation liquid).

Inoculating a sludge group: adding water into fruit and vegetable wastes to obtain a fermentation system with the total solid concentration (TS) of 8% and the Volatile Solid (VS) content of 78g-VS/L, inoculating anaerobic sludge into the fermentation system with the inoculation amount of 70g-VS/L, and fermenting the fermentation system for 17d under the conditions that the temperature is 34-36 ℃, the pH is 4.0-4.5 and the rotating speed is 60rpm to obtain fermentation liquor. Firstly, the fermentation mixed liquor passes through a 200-mesh screen to respectively obtain inoculated sludge fermentation liquor and naturally acidified fermentation liquor, and in order to obtain a fermentation substrate under a better condition, the fermentation mixed liquor with the ratio of alcohol to acid being approximately 4:1 is prepared to be used as a raw material for fermenting the caproic acid.

Example 2: selection of chloroform dosage

The experiment adopts a batch fermentation mode, fed-batch fermentation is carried out in a 1L anaerobic device, anaerobic sludge is inoculated into the fermentation mixed liquid finally obtained in the embodiment 1 for anaerobic fermentation, chloroform is added into the fermentation device, the inoculation amount of the sludge is 20g-TS/L, different chloroform adding concentrations (0.5 thousandth, 1 thousandth, 5 thousandth, 10 thousandth, m/m, g-chloroform/g-sludge) are set, and the change of liquid phase components in the fermentation process is detected. And performing fluorescent quantitative PCR detection on the quantity of caproic acid bacteria, methanogen hydrogenotrophus and homoacetogenic bacteria in the biological phase to obtain the optimized chloroform adding concentration.

The main liquid phase product dynamics under different chloroform adding concentration conditions are shown in figure 2.

Compared with the fermentation performance of caproic acid with different chloroform adding concentrations, the ethanol and acetic acid are rapidly taken in at the initial stage of fermentation (0-10 days) under the condition of lower chloroform adding amount (0.5 per thousand and 1 per thousand) to generate butyric acid and caproic acid, the accumulated butyric acid is further converted into caproic acid along with the reaction, and the final production of the caproic acid reaches 15.8-16.1 g/L. The cumulative butanol concentration at this time was only 0.23-0.25g/L (see Table 1), while the detected methane concentration in the gas phase was less than 1%, and the production of by-products was significantly reduced (p < 0.05). The caproic acid production intensity was almost identical to the initial conditions. However, the whole fermentation period is delayed from 30-40 days under the initial condition to about 40-53 days, and the delay of the fermentation period is more obvious compared with that of the fermentation period reinforced by adding calcium carbonate (about 20 days).

With the addition of chloroform, the dosage is increased to 5 per thousand, the metabolic products of methane and butanol in the competitive path are further reduced, but the fermentation performance is obviously weakened. Not only the consumption of ethanol and acetic acid is slowed down in the early fermentation stage, but also the production of the caproic acid is stopped under the condition that relatively sufficient ethanol and carboxylic acid still exist in the liquid phase in the middle fermentation stage, and the maximum caproic acid yield is reduced to 10.0 g/L. Further increasing the chloroform concentration to 10 per mill, the whole fermentation production is seriously inhibited, the intake of ethanol and acetic acid is almost completely stopped, and the production of caproic acid is weak.

TABLE 1 fermentation Performance under different chloroform addition conditions

The effect of chloroform at each dose on homoacetogens is shown in FIG. 3. The number of homoacetogenic bacteria under all conditions can be greatly reduced, finally, the number of homoacetogenic bacteria under the adding conditions of 0.5,1 and 5 per thousand is reduced to 103 copy numbers/g-VS, homoacetogenic bacteria under the condition of 10 per thousand chloroform are added, no detection is carried out after 25 days, and the adding amount of chloroform and the number of homoacetogenic bacteria show obvious negative correlation (r is less than-0.8). From the previous monitoring data (days 5 to 15), it can be seen that the number of homoacetogens decreased more rapidly as the amount of chloroform was increased. In addition, chloroform has strong persistence on the inhibition of homoacetogenic bacteria, and no obvious proliferation process of homoacetogenic bacteria under various concentration conditions occurs in the whole detection time of 100 days. The butanol yield in the combination process can show that the number of homoacetogens and the butanol yield present obvious positive correlation (r is more than 0.8), and further verifies that the butanol production and the homoacetogens are closely related.

The main mechanism of chloroform inhibition of homoacetogens is widely reported: chloroform produces toxic intermediates during the conversion of homoacetogens of CO2 to acetyl-coa. A schematic diagram of the acetyl-CoA pathway of homoacetogens is shown in FIG. 4.

In the conversion of homoacetogenic acid, CO₂Participate in two ways, (1) the methyl pathway: CO2₂Is converted into formic acid (formyl), then is reduced and converted into methyl-tetrahydrofolate (methyl-tetrahydrofolate) by combining with tetrahydrofolate, and the carried methyl passes through iron-sulfur protein to form the methyl part of acetyl coenzyme A (acetyl-CoA); (2) carbonyl route: CO2₂Under the driving action of an electron donor such as hydrogen, the carbonyl moiety of acetyl-CoA is further reduced by the action of CO dehydrogenase. The whole process is called acetyl-CoA clearance pathway to generate acetyl-CoA which can be further converted into a cell constituent substance or participate in the generation of substances such as acetic acid, ethanol or butanol. After chloroform enters cells, dichlorocarbene (dichlorocarbene) reaction activity generated by dechlorination is extremely active by combining carbon monoxide dehydrogenase (CODH) of homoacetogenic bacteria, and the substance can damage intracellular metabolic balance and key enzyme activity, thereby causing homoacetogenic bacteria poisoning. Thereby playing the role of effectively inhibiting the homoacetogenesis.

The inhibition effect of chloroform on methanogens hydrogenotropes (figure 5) and the chloroform addition concentration showed a positive correlation (r > 0.8). Meanwhile, compared with homoacetogenic bacteria, the inhibition effect of chloroform under various concentration conditions is more thorough, and the initial 10 days of the whole 5 days⁴The copy number/g-VS is reduced to 10³Even lower. Then, as the reaction proceeds, the detection limit of the fluorescent quantitative PCR is gradually lowered (1X 10)³) Copy number/g-VS.

The chloroform inhibitory mechanism of methanobacteria is widely reported and applied, mainly based on that chloroform can affect the methane production process. Methanogens such as hydrogenotrophs are converted to methyl groups by a series of enzymatic systems using carbon dioxide molecules: carbon dioxide is first converted to formyl-methylfuran and the formyl groups undergo a stepwise conversion of tetrahydromethylpterin to produce methyl-tetrahydromethylpterin, the methyl groups of which are finally bound via coenzyme M and finally reduced to methane. Wherein the tetrahydromethyl pterin and the tetrahydrofolic acid have similar structures and more consistent functions, and both relate to the process of reducing formyl to methyl. The methanogen acetophaga phosphorylates the level of an acetic acid substrate, converts the acetic acid substrate into acetyl coenzyme A, and converts the acetyl coenzyme A into methyl-tetrahydromethyl pterin and carbon monoxide through catalysis. The methyl group therein is finally reduced to methane via coenzyme M. Therefore, the two methane bacteria can realize regeneration by effectively converting methyl coenzyme M, and can finally obtain the energy released in the methane conversion process to maintain the cell activity. Chloroform can effectively inhibit functions of corrinoid enzyme and coenzyme M through competitive inhibition, so that key methanogenic enzyme cannot be effectively regenerated, and further energy released in the process is difficult to obtain.

The effect of adding different doses of chloroform on the proliferation of caproic acid bacteria is shown in FIG. 6. It can be found that except for a short stagnation adaptation stage in the initial fermentation stage, the proliferation rule of the caproic acid bacteria under the condition of adding chloroform with a lower concentration (0.5-1 per thousand) is closer to that of the caproic acid bacteria which are not added before for fermentation (as shown in figures 6A and B), the caproic acid bacteria are firstly rapidly increased, and then the number of the caproic acid bacteria is gradually reduced. In addition, the maximum caproic acid bacteria number is increased from 0.5 per thousand to 1 per thousand along with the chloroform addition, a certain reduction is generated, but the difference is not obvious. (1.52. + -. 0.17). times.108, (1.32. + -. 0.18). times.108 copies/g-VS, respectively.

Further introducing higher concentration chloroform to 5 ‰ brings certain difference (as shown in fig. 6C), at first, even slight decline of caproic acid bacteria number occurs in the initial stage of fermentation, and the highest caproic acid bacteria number in the middle stage of fermentation is reduced to (1.06 ± 0.10) × 108 copy number/g-VS, and under the condition that the toxicity of caproic acid is weaker than that in the former group, the decline of caproic acid bacteria in the later stage is more obvious due to higher concentration chloroform, and by 113 days, the caproic acid bacteria number is (3.32 ± 0.78) × 107 copy number/g-VS. The addition of excessive chloroform (10 per thousand) can completely inhibit the production of caproic acid, the caproic acid bacteria can not effectively proliferate under the condition (as shown in figure 6D), the excessive chloroform causes the number of the caproic acid bacteria to rapidly decrease, and the number is only (4.21 +/-0.21) multiplied by 106 copy numbers/g-VS at the end of fermentation. The overall caproic acid bacteria count was reduced by an order of magnitude compared to the initial inoculation period.

In combination with the relative 'normal' proliferation process of the caproic acid bacteria under the condition and the dynamic quantity of the massive decline of the methanogens hydrogenotrophicus and homoacetogens, the microorganisms do not present an obvious 'mutualistic symbiosis' relationship, and the judgment of the method is further verified, namely the methanogens, homoacetogens and caproic acid bacteria have weak symbiotic relationship.

In addition, in the later stage of fermentation, the toxicity of chloroform is superposed with the feedback inhibition of caproic acid, and the reduction of the number of caproic acid bacteria can still be caused. The overall proliferation rule of the caproic acid bacteria is similar to that of the caproic acid bacteria without adding chloroform in the early stage. This further suggests that although the chloroform inhibitor has low and controllable side effects and can effectively restrict competitive metabolic pathways, the chloroform inhibitor still potentially threatens the high-intensity production of caproic acid, and how to alleviate the side effects of the chloroform inhibitor on the premise of fully exerting the effective restriction effect of the chloroform is worthy of further research.

Example 3: combined treatment of chloroform and calcium carbonate

Based on the side effect of chloroform on the caproic acid fermentation process to a certain extent in example 2, the side effect is relieved by adding calcium carbonate. Different calcium carbonate adding concentrations are set to be combined with a chloroform experimental group, so that a better caproic acid fermentation efficiency is obtained, under the condition that the adding concentration of chloroform is 0.5 per thousand, the calcium carbonate gradient is set to be 5,10 and 15g/L, and the caproic acid fermentation characteristics of different calcium carbonate doses (5,10 and 15g/L) are shown in figure 7. It can be found that in the early stage (0-8 days) of caproic acid fermentation, all groups of differences are not obvious, and obvious retardation/adaptation stages appear, and compared with fig. 2, calcium carbonate can be found to not obviously change the caproic acid fermentation characteristics in the stage.

And entering a rapid synthesis period (8-20 days) of butyric acid and caproic acid along with the passing of the adaptation period of the mixed flora. At this stage, each group shows the characteristic of rapid consumption of ethanol and acetic acid, and butyric acid and caproic acid are rapidly synthesized. In this stage, the difference of caproic acid synthesis appears under different calcium carbonate adding conditions, namely, the obvious phenomenon (5g/L) that butyric acid is accumulated earlier and caproic acid synthesis is slightly delayed is shown when the calcium carbonate adding amount is relatively less; in the case of more calcium carbonate addition, the synthesis of caproic acid is more advanced, and the synthesis of butyric acid even presents a phenomenon of 'synchronization' (15 g/L). However, the accumulated amount of caproic acid at this stage did not show a significant difference. Compared with the fermentation period, the synthesis rate of the caproic acid at the stage is obviously improved compared with the condition of not adding calcium carbonate, and the problem of the retardation of the synthesis rate of the caproic acid caused by adding chloroform is well relieved. In the late stage of fermentation, with complete exhaustion of ethanol and acetic acid, ethanol and acetic acid were added in the experiment in order to further verify the synthesis efficiency and production intensity of caproic acid.

When the reaction enters a continuous accumulation period of the caproic acid (21-90 days), the most obvious characteristic of each group in the period is accumulation of the caproic acid, but the production/consumption of the butyric acid is not obvious, and the consumption rate of the whole ethanol and the acetic acid is slowed down. However, the final accumulated concentration of the caproic acid reaches 19.01, 21.02 and 20.12/L, which is obviously higher (p is less than 0.05) than that of the caproic acid which is not added with chloroform (14.9g/L) or treated with chloroform alone (10-16.1 g/L). Close to the highest value reported in all the literature at present. Under the condition of jointly adding chloroform and calcium carbonate, the obvious promotion effect on the synthesis of the caproic acid is embodied.

The scanning electron microscope of the experimental group with different dosages (0.5,1,5 per thousand) of chloroform and different dosages of calcium carbonate (5,10,15g/L) in combination with chloroform (0.5 per thousand) is shown in fig. 8.

The difference between the first three groups of floras which are independently added with chloroform with different concentrations and the floras which are jointly added with chloroform-carbonic acid is mainly reflected in the bacterial form through comparison in the field of an electron scanning microscope. It can be obviously seen that the ratio of microorganisms in the mixed flora presenting coarse form is higher under the combined adding condition, and similar form microorganisms are reported in the research of producing hexanoic acid by other mixed flora and are considered to be microorganisms in the production state of hexanoic acid. These forms of caproic acid bacteria may be more viable as described by Barker et al for Clostridium kluyveri. In the mixed flora caproic acid production environment, a large amount of practice also finds that the caproic acid bacteria in the form of stout are more active in fermentation performance, and the caproic acid bacteria in the form of lean bacteria are difficult to produce caproic acid efficiently.

Based on the fermentation performance of the anaerobic mixed flora of each group, the experiment further analyzes the adding concentration of the chloroform and the flora structure of the experimental group of the combined adding calcium carbonate-chloroform, the species level difference is shown in figure 9, and the strain information is shown in table 2.

TABLE 2 sequencing to identify the species level of the microorganism (some OTUs are genus level)

According to the heat map and the species information, the relative abundance of the caproic acid bacterium Clostridium kluyveri can be effectively improved in a mixed bacterium system through chloroform, and the relative abundance of each group reaches more than 30.9%. The relative abundance of the caproic acid bacteria is obviously higher than that of the caproic acid bacteria found in the early experiments, which indicates that the leading position of the caproic acid bacteria can be really consolidated by introducing a proper amount of chloroform, and the caproic acid bacteria are proved to be not very sensitive to the chloroform.

In addition, the relative abundance of caproic acid bacteria in the combined addition of chloroform and calcium carbonate is improved to 38.9 percent, 49.3 percent and 43.1 percent, and is obviously (p is less than 0.05) higher than 31.5 percent, 30.9 percent and 35.6 percent of that in the independent addition of chloroform (0.5,1,5 per thousand). It is demonstrated that the introduction of calcium carbonate promotes the higher relative abundance of caproic acid bacteria among mixed flora under the precondition that chloroform effectively limits the competitive pathway. Compared with single-machine chloroform, the combined feeding can better improve the abundance of the caproic acid bacteria, and is related to the biological effect of calcium carbonate, (1) on the premise that the competitive path is weakened, the inorganic carbon source provided for the mixed flora can be effectively utilized by the caproic acid bacteria, and the proliferation of the caproic acid bacteria is greatly improved. (2) The calcium carbonate is weak in alkalinity, and a slightly alkaline environment is probably formed around the calcium carbonate, so that a favorable local environment is provided for relieving feedback inhibition of caproic acid, and the proliferation of caproic acid bacteria is facilitated.

Comparative example 1: blank group

The fermentation mixture finally obtained in example 1 was directly fermented without adding chloroform and calcium carbonate, and the results are shown in Table 3, except for the same conditions as in example 1.

Comparative example 2: calcium carbonate is added separately

The fermentation was carried out by referring to the method of example 3 except that 10g/L calcium carbonate was added without adding chloroform, and the other conditions were the same as in example 1, and the results are shown in Table 3.

TABLE 3

As can be seen from Table 3, the single 0.5 per mill chloroform treatment only increases the caproic acid yield by 1.2g/L and the caproic acid bacteria abundance by 26.1% compared with the blank group; compared with a blank group, the single 10g/L calcium carbonate treatment increases the abundance of caproic acid bacteria by 8 percent, and the yield of caproic acid is reduced by 4.65 g/L; compared with a blank group, the combined treatment of 0.5 thousandth chloroform and 10g/L calcium carbonate increases the caproic acid yield by 6.12g/L and increases the caproic acid bacteria abundance by 39.8 percent, and the effect is superior to the sum of the single treatment of 0.5 thousandth chloroform and the single treatment of 10g/L calcium carbonate, which shows that the two are mutually supported and supplemented in the aspect of enhancing the caproic acid production and have a synergistic effect.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for producing hexanoic acid by fermentation is characterized in that chloroform and calcium carbonate are added into fermentation mixed liquor for anaerobic fermentation; the adding amount of the chloroform is 0.1-2 per mill of the mass volume concentration of the fermentation mixed solution; the addition amount of the calcium carbonate is 5-25g/L relative to the mass volume concentration of the fermentation mixed liquor; the fermentation mixed liquor is prepared by taking inoculated sludge fermentation liquor and naturally acidified fermentation liquor as raw materials; the preparation method of the naturally acidified fermentation liquor comprises the following steps: adding water into fruit and vegetable wastes to obtain a fermentation system with total solid concentration TS of 7-10% and volatile solid VS content of 80-120 g-VS/L, and then fermenting the fermentation system under the conditions of temperature of 34-36 ℃, pH of 4.0-4.5 and rotation speed of 12-60 rpm to obtain a naturally acidified fermentation liquid; the preparation method of the inoculated sludge fermentation liquor comprises the following steps: adding water into fruit and vegetable wastes to obtain a fermentation system with total solid concentration TS of 7-10% and volatile solid VS content of 80-120 g-VS/L, inoculating anaerobic sludge into the fermentation system with an inoculum size of 50-100 g-VS/L, and fermenting the fermentation system under the conditions of temperature of 34-36 ℃, pH of 4.0-4.5 and rotation speed of 12-60 rpm to obtain inoculated sludge fermentation liquor.

2. The method of claim 1, wherein the fermentation mixture is a fermentation broth comprising both ethanol and a carboxylic acid.

3. The method according to claim 2, wherein the mass ratio of ethanol to carboxylic acid is (3-5): 1.

4. the method of claim 2, wherein the carboxylic acids are acetic acid, butyric acid and lactic acid.

5. The method of claim 1, wherein the fruit and vegetable waste comprises sugar.

6. The method as claimed in claim 5, wherein the sugar content of the fruit and vegetable waste is not less than 8%.

7. A method for improving the abundance of caproic acid bacteria in a fermentation mixed solution is characterized in that chloroform and calcium carbonate are added into the fermentation mixed solution for anaerobic fermentation; the adding amount of the chloroform is 0.1-2 per mill of the mass volume concentration of the fermentation mixed solution; the addition amount of the calcium carbonate is 5-25g/L relative to the mass volume concentration of the fermentation mixed liquor; the fermentation mixed liquor is prepared by taking inoculated sludge fermentation liquor and naturally acidified fermentation liquor as raw materials; the preparation method of the naturally acidified fermentation liquor comprises the following steps: adding water into fruit and vegetable wastes to obtain a fermentation system with total solid concentration TS of 7-10% and volatile solid VS content of 80-120 g-VS/L, and then fermenting the fermentation system under the conditions of temperature of 34-36 ℃, pH of 4.0-4.5 and rotation speed of 12-60 rpm to obtain a naturally acidified fermentation liquid; the preparation method of the inoculated sludge fermentation liquor comprises the following steps: adding water into fruit and vegetable wastes to obtain a fermentation system with total solid concentration TS of 7-10% and volatile solid VS content of 80-120 g-VS/L, inoculating anaerobic sludge into the fermentation system with an inoculum size of 50-100 g-VS/L, and fermenting the fermentation system under the conditions of temperature of 34-36 ℃, pH of 4.0-4.5 and rotation speed of 12-60 rpm to obtain inoculated sludge fermentation liquor.