A method for efficiently synthesizing medium-chain fatty acids by dry-wet coupled anaerobic fermentation

Abstract

The present invention belongs to the technical field of resource utilization of agricultural waste, and specifically relates to a method for efficiently synthesizing medium-chain fatty acids by dry-wet coupled anaerobic fermentation. The present invention provides a method for efficiently synthesizing medium-chain fatty acids by dry-wet coupled anaerobic fermentation, which spatially isolates hydrolytic acidifying microorganisms and carbon chain extension microorganisms by partitioning the fermentation device, innovatively uses chitosan and tannic acid as adhesives, fixes hydrolytic acidifying bacteria and lactic acid-producing bacteria on agricultural waste, and realizes efficient hydrolysis and acidification of agricultural waste, and directional production of lactic acid and acetic acid, providing high-quality substrates for the carbon chain extension stage. In addition, the innovation sprays the acidified products produced in the hydrolysis and acidification zone to the carbon chain extension zone by spraying the biogas slurry containing potassium ferrate, and synthesizes medium-chain fatty acids. At the same time, potassium ferrate promotes interspecies electron transfer in the system, further strengthens the carbon chain extension reaction, shortens the fermentation time, and efficiently synthesizes medium-chain fatty acids.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural waste resource utilization, and specifically relates to a method for efficiently synthesizing medium-chain fatty acids through dry-wet coupled anaerobic fermentation. Background Art

[0002] Under the dual pressures of global energy structure transformation and environmental protection, the development and utilization of biomass energy has become a hot topic of research. Crop straw contains a large amount of lignocellulose, which can provide a rich carbon source for anaerobic fermentation microorganisms. In recent years, the use of anaerobic acidification technology to treat straw and extend the carbon chain to synthesize medium-chain fatty acids has attracted widespread attention. The efficient utilization of straw is of great significance for environmental protection, green application of waste and high-value conversion.

[0003] Because it is difficult for carbon chain extension microorganisms to directly utilize macromolecular organic matter in agricultural waste, at present, dry-wet coupled anaerobic fermentation to synthesize medium-chain fatty acids also mostly adopts two-step fermentation synthesis. First, acidifying microorganisms are used to decompose agricultural waste to produce short-chain fatty acids as electron acceptors; short-chain fatty acids are converted into medium-chain fatty acids under the action of carbon chain extension microorganisms. When short-chain fatty acids are used to synthesize medium-chain fatty acids, the oxidation of electron donors and the reduction of electron acceptors are involved. Short-chain fatty acids in the fermentation system are suitable as electron acceptors for carbon chain extension to synthesize even-carbon medium-chain fatty acids. Electron donors are insufficient. Therefore, exogenous electron donors are usually required to be added to the anaerobic fermentation system to maintain a stable carbon chain extension process, which reduces the synthesis efficiency of medium-chain fatty acids. Summary of the invention

[0004] In view of this, the present invention provides a method for efficiently synthesizing medium-chain fatty acids by dry-wet coupled anaerobic fermentation, which does not require the addition of additional electron donors and improves the synthesis efficiency of medium-chain fatty acids.

[0005] In order to solve the above problems, the present invention provides the following technical solutions:

[0006] The present invention provides a method for efficiently synthesizing medium-chain fatty acids by dry-wet coupled anaerobic fermentation, comprising the following steps:

[0007] Mixing agricultural waste with a binder solution to obtain pretreated agricultural waste; the binder is chitosan and tannic acid;

[0008] The pretreated agricultural waste is immersed in a culture solution for culturing to obtain agricultural waste with fixed bacteria; the culture solution includes hydrolytic acidifying bacteria and lactic acid producing bacteria;

[0009] The agricultural waste with fixed bacteria is subjected to dry anaerobic fermentation and wet anaerobic fermentation in a dry-wet coupled anaerobic fermentation device to obtain medium-chain fatty acids;

[0010] The acidified product of the dry anaerobic fermentation is sprayed to the reactor of the wet anaerobic fermentation; the solution used for the spraying includes biogas slurry containing potassium ferrate.

[0011] Preferably, the application form of the adhesive includes a mixture of chitosan and tannic acid; the preparation method of the mixture of chitosan and tannic acid includes: mixing chitosan with acetic acid solution to obtain a chitosan solution, mixing the chitosan solution and tannic acid solution, centrifuging and taking the supernatant, which is the mixture of chitosan and tannic acid.

[0012] Preferably, the hydrolytic acidifying bacteria include Clostridium ( Clostridium sp . ); The lactic acid bacteria include Lactobacillus ( Lactobacillus ).

[0013] Preferably, an inoculum is added during the wet anaerobic fermentation; the inoculum contains microorganisms with carbon chain extension function.

[0014] Preferably, the mass concentration of potassium ferrate in the biogas slurry is 3.0-6.0 g / L; based on the effective volume of the fermentation device used for dry anaerobic fermentation, the amount of biogas slurry added is 10%-30% of the effective volume; and the spraying frequency of the biogas slurry is 4-6 times / d.

[0015] Preferably, the total solid content of the fermentation system of the dry anaerobic fermentation is 15% to 30%.

[0016] Preferably, the fermentation time of the dry anaerobic fermentation is 5 to 15 days; the fermentation time of the wet anaerobic fermentation is 7 to 11 days.

[0017] Preferably, the fermentation temperatures of the dry anaerobic fermentation and the wet anaerobic fermentation are 35-40° C. respectively; and the pH values ​​of the fermentation systems of the dry anaerobic fermentation and the wet anaerobic fermentation are 5.5-6.5 respectively.

[0018] Preferably, aeration is performed during the dry anaerobic fermentation process, and the aeration method includes intermittent aeration; based on the volume of the fermentation system of the dry anaerobic fermentation, the aeration volume each time is 0.1~0.3L oxygen / L, the aeration time is the 1st to 3rd day of the dry anaerobic fermentation, and the aeration interval is 15~30min.

[0019] Preferably, the agricultural waste includes straw; and the straw includes corn straw.

[0020] Beneficial effects of the present invention: The present invention provides a method for efficiently synthesizing medium-chain fatty acids by dry-wet coupled anaerobic fermentation. The technical solution of the present invention creatively uses all-biobased materials of chitosan and tannic acid as adhesives to pretreat agricultural waste, fix hydrolytic acidifying bacteria and lactic acid-producing bacteria on agricultural waste, and achieve efficient hydrolysis and acidification of agricultural waste, and directional production of lactic acid and acetic acid; acetic acid is used as an electron acceptor and lactic acid is used as an electron donor to provide high-quality substrates for the carbon chain extension stage. In addition, the innovation sprays biogas slurry containing potassium ferrate, and the acidified products produced in the hydrolysis and acidification zone enter the carbon chain extension zone through the biogas slurry spray, and further synthesize medium-chain fatty acids after contacting with the bottom carbon chain extension bacteria. At the same time, the presence of potassium ferrate can also promote interspecies electron transfer in the system, further strengthen the carbon chain extension reaction, shorten the fermentation time, and efficiently synthesize medium-chain fatty acids.

[0021] It can be seen that the technical solution provided by the present invention can achieve efficient synthesis of the medium-chain fatty acid caproic acid. The hydrolysis and acidification zone adopts a dry anaerobic fermentation process, and the coupling carbon chain extension zone adopts a wet anaerobic fermentation process. Compared with the traditional anaerobic fermentation carbon chain extension process for synthesizing medium-chain fatty acids, it can increase the raw material processing capacity and the material conversion rate, maintain the stability and control of the production process without adding an external electron donor, and reduce the investment in technology and equipment costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 This is a process flow chart of synthesizing medium-chain fatty acids using dry-wet coupled anaerobic fermentation of crop straw in Example 1. DETAILED DESCRIPTION

[0024] The present invention provides a method for efficiently synthesizing medium-chain fatty acids by dry-wet coupled anaerobic fermentation, comprising the following steps:

[0025] Mixing agricultural waste with a binder solution to obtain pretreated agricultural waste; the binder is chitosan and tannic acid;

[0026] The pretreated agricultural waste is immersed in a culture solution for culturing to obtain agricultural waste with fixed bacteria; the culture solution includes hydrolytic acidifying bacteria and lactic acid producing bacteria;

[0027] The agricultural waste with fixed bacteria is subjected to dry anaerobic fermentation and wet anaerobic fermentation in a dry-wet coupled anaerobic fermentation device to obtain medium-chain fatty acids;

[0028] The acidified product of the dry anaerobic fermentation is sprayed to the reactor of the wet anaerobic fermentation; the solution used for the spraying includes biogas slurry containing potassium ferrate.

[0029] As an practicable manner, the present invention mixes agricultural waste with an adhesive solution to obtain pretreated agricultural waste; the adhesive is chitosan and tannic acid; the pretreatment method preferably includes: sterilizing the crushed agricultural waste to obtain sterilized agricultural waste; mixing and immersing the sterilized agricultural waste in a sterilized adhesive solution to obtain pretreated agricultural waste. As an practicable manner, the adhesive is applied in the form of a mixture of chitosan and tannic acid. The length of the crushed agricultural waste in the present invention is 1 to 2 cm; the present invention does not specifically limit the crushing method, and conventional methods can be used. The sterilization method in the present invention is high-pressure steam sterilization; there is no specific limitation on the parameters of the high-pressure steam sterilization, and conventional methods can be used. The preparation method of the chitosan and tannic acid mixed solution of the present invention comprises the following steps: mixing chitosan with an acetic acid solution to obtain a chitosan solution, mixing the chitosan solution with a tannic acid solution, centrifuging, and taking the supernatant to obtain a chitosan and tannic acid mixed solution; the concentration of the chitosan solution is 10 mg / mL-20 mg / mL, preferably 10 mg / mL; the concentration of acetic acid in the acetic acid solution is 1%-2% (g / mL), preferably 1% (g / mL); the mass volume ratio of the chitosan and acetic acid solution is 1g:(50-100)mL, preferably 1g:100mL; the mass concentration of the tannic acid solution is 0.5%-2%, preferably 2%; the mixing volume ratio of the chitosan solution and the tannic acid solution is 4:1; the centrifugation temperature is room temperature, the centrifugation speed is 5000 rpm, and the centrifugation time is 10min. The present invention soaks the sterilized agricultural waste in a sterilized chitosan and tannic acid solution. After the soaking is completed, the agricultural waste is taken out and rinsed with sterile physiological saline, and the first treated agricultural waste is obtained after suction filtration; the mass volume ratio of the sterilized agricultural waste to the sterilized chitosan and tannic acid mixture is 1g: (5-10) mL, preferably 1g: (6-7) mL. The soaking process is accompanied by stirring; the soaking time is 15-60min, preferably 40min. The stirring speed is 100-300r / min, preferably 100r / min. After soaking in the chitosan and tannic acid mixture, the soaking and stirring cause the chitosan fixed in the agricultural waste to undergo a cross-linking reaction with the tannic acid, so that the chitosan and tannic acid are both fixed on the agricultural waste. After soaking, the agricultural waste is taken out and rinsed with sterile physiological saline. The function of the rinsing in the present invention is to remove the unbound chitosan and tannic acid mixture. The number of rinsing in the present invention is 5-10 times, preferably 5 times. Chitosan and tannic acid are both naturally occurring substances with good biocompatibility, biodegradability, and environmental friendliness. Good biocompatibility will not have a significant negative impact on the activity of functional bacteria.The adhesive is a cross-linking reaction between bacteria and the functional groups on the surface of straw to form a stable structure, thereby achieving the fixation of bacteria on the straw. By adding chitosan and tannic acid adhesives, the amino groups on the surface of the functional bacteria react with the amino groups, hydroxyl groups and other functional groups on the surface of the straw to form covalent bonds, which helps to firmly fix the bacteria. It has high stability, little effect on the activity of functional bacteria, and can improve the utilization efficiency of functional bacteria. This strategy can not only achieve the immobilization of functional bacteria on the surface of straw, improve the adhesion ability and utilization efficiency of microorganisms to the substrate, but also achieve the gradual degradation of the adhesive during the fermentation process through the reasonable selection of adhesives, avoiding potential pollution to the environment. In addition, compared with some synthetic adhesives, the cost of chitosan and tannic acid may be lower, and more suitable for large-scale application.

[0030] As an practicable manner, pretreated agricultural waste is obtained, and the present invention soaks the pretreated agricultural waste in a culture solution for culture to obtain agricultural waste with fixed bacteria. The culture solution described in the present invention is a mixture of a hydrolytic acidifying bacteria culture solution and a lactic acid producing bacteria culture solution; the volume ratio of the hydrolytic acidifying bacteria culture solution and the lactic acid producing bacteria culture solution when mixed is (1~2):1. The present invention does not specifically limit the number of live bacteria in the hydrolytic acidifying bacteria culture solution and the lactic acid producing bacteria culture solution during the mixing, as long as they are mixed according to the volume ratio. The hydrolytic acidifying bacteria described in the present invention is Clostridium ( Clostridium sp.), any strain of Clostridium can implement the technical solution of the present invention. In the embodiment of the present invention, the number of the Clostridium strain is CICC 24702. The lactic acid-producing bacteria of the present invention are Lactobacillus ( Lactobacillus The lactobacillus of the present invention is Lactobacillus plantarum ( Lactobacillus plantarum ), any plant lactobacillus can realize the technical scheme of the present invention. In the embodiment of the present invention, the plant lactobacillus is numbered as CICC 21790. The present invention does not specifically limit the preparation method of the hydrolysis and acidification bacterial culture solution, and a conventional method can be used. The present invention does not specifically limit the preparation method of the lactic acid bacteria culture solution, and a conventional method can be used. The load amount of the agricultural waste soaked in the culture solution of the present invention is 40~50g / L. In a specific embodiment of the present invention, the load amount is 40, 45 or 50g / L. The load amount is the mass of agricultural waste added to each liter of culture solution. The soaking temperature of the present invention is 37°C, and the soaking time is 12~48h, preferably 24h. The present invention fixes microorganisms with hydrolysis and acidification functions and lactic acid production functions on agricultural waste by soaking, and the zoning setting of the fermentation process can generate electron donors and electron acceptors in a directional manner, thereby increasing the yield of electron donors and electron acceptors without additional addition.

[0031] As an practicable manner, the agricultural waste of the present invention preferably includes straw; the straw preferably includes corn straw; the present invention has no special limitation on the source and composition of the straw, and conventional straw can be used.

[0032] As an implementable method, the pretreated agricultural waste is subjected to dry anaerobic fermentation and wet anaerobic fermentation in a dry-wet coupled anaerobic fermentation device to obtain medium-chain fatty acids. The dry anaerobic fermentation is a batch dry anaerobic fermentation, and the wet anaerobic fermentation is a continuous wet anaerobic fermentation. The dry-wet coupled anaerobic fermentation device of the present invention comprises a dry fermentation device and a wet fermentation device, which are the upper and lower parts of the dry-wet coupled anaerobic fermentation device. Dry anaerobic fermentation is carried out in the dry fermentation device, and the dry fermentation device is recorded as a hydrolysis and acidification zone. Wet anaerobic fermentation is carried out in the wet anaerobic fermentation device, and the wet fermentation device is recorded as a carbon chain extension zone. Any conventional dry-wet coupled anaerobic fermentation device can implement the technical solution of the present invention.

[0033] In the prior art, the medium-chain fatty acids and electron donors formed during fermentation have toxic effects on acidifying microorganisms, which also reduce the degree of hydrolysis and acidification of agricultural waste to a certain extent, and reduce the synthesis efficiency of medium-chain fatty acids. The present invention is based on a dry-wet coupled anaerobic fermentation device. The dry fermentation reactor (i.e., the hydrolysis and acidification zone) and the wet fermentation reactor (i.e., the carbon chain extension zone) in the anaerobic fermentation reactor spatially isolate the hydrolysis and acidification microorganisms and the carbon chain extension microorganisms. By separating the hydrolysis and acidification zone from the carbon chain extension zone, microorganisms with different functions are placed in a suitable growth environment, and the two types of microorganisms can be more effectively controlled to be in the best fermentation conditions. At the same time, the toxicity of medium-chain fatty acids and electron donors to acidifying microorganisms is avoided, and the influence of the difference in growth conditions of different functional organisms on the material conversion efficiency is avoided, the degradation degree of agricultural waste is improved, the raw material processing capacity is increased, the material conversion rate is improved, and the stable control of the fermentation process is maintained without adding electron donors, reducing the technology and equipment costs invested, and increasing the yield of medium-chain fatty acids.

[0034] As an practicable manner, the present invention preferably mixes the pretreated agricultural waste with water to obtain a fermentation system, and the present invention performs dry anaerobic fermentation in a dry fermentation reactor (i.e., a hydrolysis and acidification zone) to obtain an acidified product. The present invention does not specifically limit the parameters of the mixing, and a conventional method can be used. The initial total solid content of the fermentation system of the present invention is 15% to 30%; in a specific embodiment of the present invention, the initial total solid content of the fermentation system is 15%, 20%, 25% or 30%. The total solid content is set to promote the growth and metabolic activity of fixed microorganisms in agricultural waste, which helps to increase the yield of fermentation products. The pH value of the fermentation system of the present invention is 5.5 to 6.5, preferably 5.5. The temperature of the dry anaerobic fermentation of the present invention is 35°C to 40°C, preferably 37 to 38°C; the fermentation time of the dry anaerobic fermentation of the present invention is 5 to 15 days, preferably 9 to 11 days.

[0035] As an practicable manner, the present invention performs dry anaerobic fermentation in a dry fermentation reactor (i.e., a hydrolysis and acidification zone), and the dry anaerobic fermentation is accompanied by microaerobic fermentation aeration during the process, and the aeration method of the microaerobic fermentation of the present invention may include intermittent aeration. Based on the volume of the fermentation system of the dry anaerobic fermentation, the aeration amount of each intermittent aeration of the present invention is 0.1~0.3L oxygen / L, preferably 0.25L oxygen / L. The time of the intermittent aeration of the present invention is the 1st to 3rd day of the dry anaerobic fermentation. The interval time between the two aerations of the present invention is 15~30min. In a specific embodiment of the present invention, the interval time between the two aerations is 15min, 20min, 25min or 30min. The present invention regulates the hydrolysis and acidification zone by coupling microaerobic, pH and temperature, and has the following advantages: microaerobic can realize pre-heating of the fermentation system, promote rapid heating of the fermentation system, improve fermentation efficiency and cellulose decomposition efficiency of agricultural waste; suitable pH value can create the best living and metabolic environment for hydrolysis and acidification microorganisms, promote the hydrolysis and acidification of organic matter in agricultural waste by microorganisms, convert more insoluble organic matter into soluble matter, and increase the concentration of substrates that can be used by carbon chain extension microorganisms in the carbon chain extension stage; within the suitable temperature range, the metabolic reaction speed of hydrolysis and acidification microorganisms can be accelerated, and higher temperature may increase the content of volatile fatty acids in hydrolysis and acidification products and reduce the molecular weight, so that they are easier to be used by carbon chain extension in the subsequent anaerobic fermentation carbon chain extension process, thereby improving the efficiency of the entire anaerobic fermentation system and the yield of medium-chain fatty acids.

[0036] As an practicable manner, the acetic acid concentration in the acidified product is greater than 4 g / L and the lactic acid concentration is greater than 4 g / L. In the present invention, the acidified product obtained by the dry anaerobic fermentation is sprayed into a wet fermentation reactor; the solution used for the spraying includes a biogas slurry containing potassium ferrate. The wet fermentation reactor of the present invention is recorded as a carbon chain extension zone. An inoculum containing microorganisms having a carbon chain extension function has been added to the carbon chain extension zone of the present invention. The inoculum of the present invention may include sludge, and the sludge is taken from granular sludge of a sewage treatment plant. The microorganisms having a carbon chain extension function contained in the sludge; the microorganisms having a carbon chain extension function may include members of the genus Hexanoic acid bacteria ( Caproiciproducens ) and / or Clostridium ( Clostridium_sensu_stricto ). The sludge of the present invention may be domesticated before application, and the domestication method may include: mixing the sludge with lactic acid and fermenting. Based on the volume of the sludge, the concentration of the lactic acid of the present invention is 10g / L; the fermentation is performed until the caproic acid concentration in the fermentation system reaches 3-4g / L; the fermentation temperature is 38°C. The role of the domestication is to increase the abundance of microorganisms with carbon chain extension function in the sludge.

[0037] As an practicable manner, the solution for spraying application of the present invention includes biogas slurry containing potassium ferrate. The present invention does not specifically limit the preparation method of the biogas slurry, and conventional biogas slurry can be used. The mass concentration of potassium ferrate in the biogas slurry is 3.0~6.0g / L; based on the effective volume of the fermentation device used in the hydrolysis and acidification zone, the amount of biogas slurry added is 10%~30% of the effective volume. In a specific embodiment of the present invention, the amount of biogas slurry added is 10%, 15%, 20%, 25% or 30% of the effective volume. The spraying frequency of the biogas slurry of the present invention is 4~6 times / d, preferably 5 times / d. The effective volume of the fermentation device of the present invention is 80% of the volume of the fermentation device, such as a 5L fermentation tank, and the effective volume of the fermentation tank is 4L. The present invention innovatively sprays biogas slurry containing potassium ferrate, and the acidified products produced in the hydrolysis and acidification zone enter the carbon chain extension zone through the biogas slurry spray, and further synthesize medium-chain fatty acids after contacting with the bottom carbon chain extension bacteria. At the same time, the presence of potassium ferrate can also promote interspecies electron transfer in the system, further strengthen the carbon chain extension reaction, shorten the fermentation time and efficiently synthesize medium-chain fatty acids.

[0038] The temperature of the wet anaerobic fermentation of the present invention is 35-40°C, preferably 37°C; in a specific embodiment of the present invention, the fermentation temperature is 35, 36, 37, 38, 39 or 40°C. The fermentation time of the wet anaerobic fermentation of the present invention is 7-11 days. In a specific embodiment of the present invention, the fermentation time can be 7, 8, 9, 10 or 11 days. The pH value of the fermentation system of the wet anaerobic fermentation of the present invention is 5.5-6.5.

[0039] In the prior art, if high solid content anaerobic fermentation is used in the hydrolysis and acidification stage, the donor content in the system can be increased while increasing the fermentation raw material processing capacity. However, the mass transfer and heat transfer limitations caused by the high solid content condition affect the contact between the microorganisms and the agricultural waste substrate, reducing the degree of hydrolysis and acidification of the agricultural waste, resulting in low concentrations of synthesized electron donors and electron acceptors. Therefore, the present invention creatively uses a fully bio-based adhesive based on chitosan and tannic acid to pretreat the straw, thereby reducing the concentration of hydrolysis and acidification bacteria. Clostridium sp. (Clostridium) and lactic acid-producing bacteria Lactobacillus (Lactobacillus) is fixed on the straw, and at the same time, through microaerobic, pH and temperature coupling regulation, efficient hydrolysis and acidification of the straw is achieved, the raw material processing capacity and substrate degradation degree are increased, lactic acid and acetic acid are produced in a directional manner, and high-quality substrates are provided for the carbon chain extension stage, solving the technical problem of insufficient electron donors and acceptors in the carbon chain extension process. In addition, by separating the hydrolysis and acidification zone from the carbon chain extension zone, different functional microorganisms are all in a suitable growth environment, avoiding the influence of differences in growth conditions of different functional organisms on the material conversion efficiency. At the same time, the biogas slurry containing potassium ferrate is sprayed. The presence of potassium ferrate can also promote inter-species electron transfer in the system, solve the problem of poor matching between electron donors and acceptors, and improve the synthesis efficiency of medium-chain fatty acids. The present invention uses a dry-wet coupled anaerobic fermentation device to provide a metabolic environment suitable for the growth of functional microorganisms at different stages, reduce other competitive metabolic pathways, have a large raw material processing capacity, and a high substrate utilization rate, and can continuously provide high-quality substrates for the carbon chain extension zone.

[0040] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] Lactobacillus plantarum Lactobacillus plantarum For CICC 21790, Clostridium spp. Clostridium sp . It is CICC 24702.

[0042] The preparation method of CICC 21790 culture medium is as follows: CICC 21790 is inoculated into MRS medium and cultured at 37° C. for 7 days, and the pH during the culture process is 6.2-6.5.

[0043] The composition of MRS medium is as follows: each liter of medium contains 10.0 g of casein peptone (trypsin digestion), 10.0 g of beef extract powder, 5.0 g of yeast extract powder, 20.0 g of glucose, 1.0 g of Tween 80, 5.0 g of sodium acetate, 2.0 g of triammonium citrate, 2.0 g of KH2PO4, 0.2 g of MgSO4·7H2O, 0.05 g of MnSO4·H2O, and 15.0 g of agar.

[0044] The preparation method of CICC 24702 culture medium is as follows: CICC 24702 is inoculated into a culture medium and cultured at 37°C for 7 days; the composition of the culture medium is as follows: each 1L of the culture medium contains 10.0 g of yeast extract powder, 5.0 g of sodium acetate, 0.2 g of MgSO4·7H2O, 0.23 g of KH2PO4, 0.31 g of K2HPO4, 2.5 g of NaHCO3 and 0.5 g of (NH4)2SO4.

[0045] The sludge is sourced from anaerobic granular sludge from a sewage treatment plant. The sludge is domesticated by adding lactic acid to the anaerobic granular sludge and fermenting it in a fermentation tank; the concentration of lactic acid is 10 g / L based on the volume of the sludge, and the fermentation is carried out until the caproic acid concentration in the fermentation system reaches 3-4 g / L; the fermentation temperature is 38°C. The domesticated sludge is the sludge with carbon chain extension function. Sludge domestication is to increase the abundance of microorganisms with carbon chain extension function.

[0046] Example 1

[0047] The mixed solution of chitosan and tannic acid was recorded as chitosan-tannic acid mixed solution.

[0048] 1. Treat corn stalks before fermentation begins

[0049] The processing process of corn straw is as follows: (1) Using corn straw as fermentation raw material, wash the straw, crush it to a length of 1 cm as an immobilized carrier, and then sterilize it in a high-pressure steam sterilizer for use; (2) Dissolve chitosan in acetic acid solution with a mass concentration of 2%, the mass volume ratio of chitosan to acetic acid solution is 1 g:100 mL, stir evenly, and obtain a 10 mg / mL chitosan solution; (3) Dissolve tannic acid in sterile saline, stir evenly, and obtain a tannic acid solution with a mass concentration of 2%; (4) Mix the chitosan solution and tannic acid solution in a volume ratio of 4:1, centrifuge at room temperature at a speed of 5000 rpm, and centrifuge for 10 min to obtain the supernatant, which is the chitosan-tannic acid mixed solution; (5) Soak the sterilized straw in the chitosan-tannic acid mixed solution, the mass volume ratio of the sterilized straw to the chitosan-tannic acid solution is 1 g:6 mL, stir for 40 min, and centrifuge for 10 min. r / min; allow chitosan to fully adhere to the surface of the straw, remove the straw, and rinse it with sterile saline to remove the unbound chitosan-tannic acid mixture. Allow tannic acid to cross-link with chitosan, remove the straw, rinse it with sterile saline, and filter it for later use; (5) Place the pretreated straw in a sterile container, add CICC21790 culture solution and CICC 24702 culture solution, the volume ratio of CICC 21790 culture solution to CICC 24702 culture solution is 1:1, and allow the straw to be fully immersed in the culture solution. The loading amount is 40g / L (40g / L means adding 40g of straw to each liter of culture solution), and culture it at 37℃ for 24h to fix CICC 21790 and CICC 24702 on the straw.

[0050] 2. The fermentation device consists of two layers, the upper layer is the hydrolysis and acidification zone, and the lower layer is the carbon chain extension zone. The effective volume of the hydrolysis and acidification zone is 5L. The hydrolysis and acidification zone adopts a sequencing batch dry anaerobic fermentation process, and the carbon chain extension zone adopts a continuous wet anaerobic fermentation process.

[0051] In the hydrolysis and acidification zone, the corn stalks treated in step 1 were used as raw materials, water was added to adjust the total solid content of the fermentation system to 30%, and fermentation was carried out at a fermentation temperature of 38° C. The pH value of the fermentation system in the hydrolysis and acidification zone was adjusted to a constant value of 5.5 using 0.5 M hydrochloric acid and 0.5 M sodium hydroxide.

[0052] During the first three days of fermentation in the hydrolysis and acidification zone, aeration was performed every 15 minutes, and the aeration volume was 0.25L oxygen / L based on the volume of the fermentation system of dry anaerobic fermentation. On the 7th day of fermentation, the concentrations of acetic acid and lactic acid in the hydrolysis and acidification products reached 8.03g / L and 4.43g / L, respectively. At this time, the acidified products produced in the hydrolysis and acidification zone were sprayed into the carbon chain extension zone of the fermentation device through the biogas slurry containing 6.0 g / L potassium ferrate. The spraying frequency was 4 times / d, and the spraying was performed every 6 hours. The spraying volume was 20% of the effective volume of the hydrolysis and acidification zone. Before the reaction in the hydrolysis and acidification zone was started, the domesticated sludge was connected to the bottom of the carbon chain extension zone, and the sludge addition amount was 1 / 3 of the effective volume of the hydrolysis and acidification zone. The spray liquid enters the carbon chain extension zone and contacts with the carbon chain extension bacteria to undergo anaerobic fermentation to extend the carbon chain and synthesize medium-chain fatty acids. The carbon chain extension zone is equipped with a stirring device. The stirring mode is continuous stirring with a stirring frequency of 200r / min. The fermentation time in the carbon chain extension zone is shortened to 11d. The caproic acid concentration in the system reaches 6.73g / L, the heptanoic acid concentration is 0.03g / L, and the octanoic acid concentration is 0.21g / L.

[0053] Comparative Example 1

[0054] Same as Example 1, except that potassium ferrate was not added to the sprayed biogas slurry in step 2. The caproic acid concentration in the carbon chain extension zone discharge on the 15th day of fermentation reached a peak of 4.91 g / L, the heptanoic acid concentration was 0.02 g / L, and the octanoic acid concentration was 0.18 g / L.

[0055] Comparative Example 2

[0056] The same as Example 1, except that untreated corn stalks, CICC 21790 culture solution and CICC24702 culture solution were directly added to the fermentation device for hydrolysis and acidification, and the addition amount of the two culture solutions was 10% of the effective volume of the hydrolysis and acidification zone. The caproic acid concentration in the carbon chain extension zone discharge on the 15th day of fermentation reached a peak value of 4.37 g / L, the heptanoic acid concentration was 0.01 g / L, and the octanoic acid concentration was 0.09 g / L.

[0057] In summary, the technical solution provided by the present invention can increase the synthesis amount of medium-chain fatty acids and shorten the synthesis time of medium-chain fatty acids.

[0058] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for efficiently synthesizing medium-chain fatty acids by dry-wet coupled anaerobic fermentation, characterized in that: The following steps are involved: Mixing agricultural waste with a binder solution to obtain pretreated agricultural waste; the binder is chitosan and tannic acid; The pretreated agricultural waste is immersed in a culture solution for culture to obtain agricultural waste with fixed bacteria; the culture solution includes hydrolytic acidifying bacteria and lactic acid producing bacteria; the hydrolytic acidifying bacteria is Clostridium ( Clostridium sp . ) strain, the number of the Clostridium strain is CICC 24702; the lactic acid-producing bacteria is plant lactobacillus ( Lactobacillus ), the strain number of the plant lactobacillus is CICC 21790; The agricultural waste with fixed bacteria is subjected to dry anaerobic fermentation in a dry-wet coupled anaerobic fermentation device, and the acidified product of the dry anaerobic fermentation is sprayed into a reactor of wet anaerobic fermentation for wet anaerobic fermentation to obtain medium-chain fatty acids; The solution used for spraying includes biogas slurry containing potassium ferrate; Before the spraying, an inoculum is added to the reactor of the wet anaerobic fermentation; the inoculum contains microorganisms with carbon chain extension function; the inoculum is sludge; the sludge is anaerobic granular sludge from a sewage treatment plant; The sludge is first domesticated before inoculation, and the domestication method is: lactic acid is added to the anaerobic granular sludge and fermented in a fermentation tank; the concentration of lactic acid is 10g / L based on the volume of the sludge, and fermentation is performed until the caproic acid concentration in the fermentation system reaches 3-4g / L.

2. The method according to claim 1, characterized in that The application form of the adhesive includes a mixture of chitosan and tannic acid; the preparation method of the mixture of chitosan and tannic acid includes: mixing chitosan with acetic acid solution to obtain a chitosan solution, mixing the chitosan solution and tannic acid solution and centrifuging to obtain a supernatant to obtain a mixture of chitosan and tannic acid.

3. The method according to claim 1, characterized in that The mass concentration of potassium ferrate in the biogas slurry is 3.0-6.0 g / L; based on the effective volume of the fermentation device used for dry anaerobic fermentation, the amount of biogas slurry added is 10%-30% of the effective volume; the spraying frequency of the biogas slurry is 4-6 times / d.

4. The method according to claim 1 or 3, characterized in that: Based on the effective volume of the fermentation device for dry anaerobic fermentation, the amount of biogas slurry added is 20% of the effective volume.

5. The method according to claim 1, characterized in that: The total solid content of the fermentation system of the dry anaerobic fermentation is 15% to 30%.

6. The method according to claim 1, characterized in that The fermentation time of the dry anaerobic fermentation is 5 to 15 days; the fermentation time of the wet anaerobic fermentation is 7 to 11 days.

7. The method according to claim 1 or 6, characterized in that: The fermentation time of the dry anaerobic fermentation is 7 days.

8. The method according to claim 1 or 6, characterized in that: The fermentation temperatures of the dry anaerobic fermentation and the wet anaerobic fermentation are 35-40° C. respectively; the pH values ​​of the fermentation systems of the dry anaerobic fermentation and the wet anaerobic fermentation are 5.5-6.5 respectively.

9. The method according to claim 1, characterized in that: Aeration is performed during the dry anaerobic fermentation process, and the aeration method includes intermittent aeration; based on the volume of the dry anaerobic fermentation system, the aeration volume each time is 0.1-0.3L oxygen / L, the aeration time is the 1st to 3rd day of the dry anaerobic fermentation, and the aeration interval is 15-30min.

10. The method according to claim 1, characterized in that The agricultural waste includes straw; the straw includes corn straw.

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