A method for coupling enzymatic hydrolysis and microbial enhancement to improve anaerobic acid production from food waste

Abstract

The present invention discloses a method for coupling enzymatic hydrolysis and microbial enhancement to improve anaerobic acid production from food waste, belonging to the technical field of solid waste resource utilization, and comprising the following steps: (1) First, carry out the synergistic enzymatic hydrolysis of cellulase and hemicellulase, adding cellulase and hemicellulase to food waste for enzymatic hydrolysis at the same time; then carry out alkali-heat pretreatment to obtain pretreated food waste; (2) Add Propionibacterium to enhance the process of propionic acid production from food waste: First, carry out the cultivation of Propionibacterium inoculum, and then add Propionibacterium inoculum solution accounting for 20% - 70% of the mass of the inoculated sludge at the initial stage of anaerobic fermentation acid production to enhance the production of propionic acid from food waste. The present invention utilizes the synergistic enzymatic hydrolysis of cellulase and hemicellulase on food waste to degrade cellulose-like refractory organic matter and increase the production of even-carbon volatile fatty acids acetic acid and butyric acid; utilizes Propionibacterium as an exogenous microorganism to enhance the anaerobic fermentation of food waste to generate odd-carbon volatile fatty acid propionic acid.

Description

Technical Field

[0001] The present invention relates to a method for coupling enzymatic hydrolysis and microbial enhancement to improve anaerobic acid production from food waste, mainly a method for using the coupling of enzymatic hydrolysis and microbial enhancement to increase the production of volatile fatty acids in the anaerobic fermentation of food waste; it belongs to the technical field of solid waste resource utilization. Background Art

[0002] Volatile fatty acids (VFAs) are a class of carboxylic acids composed of 2 - 6 carbon atoms; they have wide application values in various fields, such as being used as precursors to synthesize industrial chemical substances (esters, ketones, aldehydes, alcohols) and producing biodegradable plastics (polyhydroxyalkanoates), etc.

[0003] Currently, the technological processes for synthesizing volatile fatty acids are mainly divided into chemical methods and biological methods; in industrial production, volatile fatty acids are mainly produced by oxidizing or carboxylating chemical precursors (such as aldehydes and olefins) generated during traditional petroleum processing, which will cause the consumption of non-renewable petroleum resources and emit carbon dioxide, exacerbating the greenhouse effect; the process of biosynthesizing volatile fatty acids will alleviate the consumption of petrochemical resources, but currently, pure carbon sources such as glucose or sucrose are mostly used as the main raw materials, which also raises questions about whether this technology has a green development prospect. Therefore, to meet the requirements of synthesizing green chemicals and sustainable development, seeking to use organic waste as a raw material to produce volatile fatty acids has become a research hotspot.

[0004] With the continuous advancement of the waste sorting policy in China, the output of kitchen waste shows an increasing trend; kitchen waste in China accounts for about 59% of urban domestic waste, and the output has reached 120-130 million tons per year. As an inexpensive and easily available urban organic solid waste, kitchen waste has attracted much attention as a raw material for the biosynthesis of volatile fatty acids due to its excellent biodegradability and rich organic matter content. Kitchen waste refers to perishable waste such as vegetable leaves, fruit peels and cores, leftovers, and discarded food generated in households; food residues, food processing waste, and waste edible oils generated by enterprises engaged in catering business activities and collective canteens of government agencies, troops, schools, enterprises and institutions during food processing, food service, and unit meal supply activities; and vegetable and fruit waste, rotten meat, meat bones, aquatic products, livestock and poultry offal, etc. generated in farmers' markets and agricultural product wholesale markets; the main components are carbohydrates, proteins, cellulose substances and a small amount of lipids. Using kitchen waste to synthesize volatile fatty acids with high value and wide uses can not only reduce the amount of kitchen waste, but also synthesize volatile fatty acid chemicals with high utilization value to reduce the consumption of petroleum-based raw materials. However, there are a large amount of cellulose substances in the fruit and vegetable waste in kitchen waste. As the main component of the plant cell wall, its crystal structure is firm, and there are problems such as difficult degradation and low yield of synthesized volatile fatty acids during the treatment process.

[0005] At present, in view of the problems of low degradation efficiency of kitchen waste and low yield of volatile fatty acids, some process technologies have been developed to improve the degradation efficiency and acid production performance, such as hot water hydrolysis and enzymatic hydrolysis pretreatment of kitchen waste raw materials, or promoting the anaerobic fermentation and acid production of kitchen waste by changing reaction conditions during the anaerobic fermentation process. These methods all use single means, and the increase in acid production is relatively low, and they do not comprehensively consider how to comprehensively improve the yields of even-carbon and odd-carbon volatile fatty acids.

[0006] Therefore, it has become an urgent task to explore a method of coupling enzymatic hydrolysis and microbial enhancement to improve the anaerobic fermentation and acid production of kitchen waste and comprehensively increase the production of odd-carbon and even-carbon volatile fatty acids. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for synergistically enzymatically hydrolyzing kitchen waste with cellulase and hemicellulase and then coupling the addition of Propionibacterium to enhance the anaerobic fermentation, hydrolysis and acidification of kitchen waste to produce volatile fatty acids. For kitchen waste raw materials, the synergistic action of cellulase and hemicellulase is used to degrade cellulose-like refractory organic matter and increase the production of even-carbon volatile fatty acids acetic acid and butyric acid in the subsequent anaerobic fermentation and acid production process; during the anaerobic fermentation and acid production process of kitchen waste, Propionibacterium is added as an exogenous microorganism to increase the production of odd-carbon volatile fatty acid propionic acid in the anaerobic fermentation and acid production process, providing a method for enhancing the anaerobic fermentation of kitchen waste to produce volatile fatty acids that is convenient to operate, environmentally friendly, stable and effective.

[0008] The above object of the present invention is achieved by the following technical solutions:

[0009] A method for coupling enzymatic hydrolysis and microbial enhancement to improve anaerobic acid production from food waste, comprising the following steps:

[0010] (1) Process steps of synergistic enzymatic hydrolysis with cellulase and hemicellulase

[0011] ① Synergistic enzymatic hydrolysis with cellulase and hemicellulase

[0012] Using cellulase and hemicellulase as enzymatic hydrolysis additives and adding them simultaneously to food waste with a solids content of 5 wt% to 40 wt%. The addition amount of cellulase is 100 - 150 U / g VS 厨余 (U: the amount of enzyme that can convert 1 μmol of substrate in 1 minute, VS 厨余 : volatile solids in food waste), and the addition amount of hemicellulase is 200 - 800 U / g VS 厨余 ; Carry out synergistic enzymatic hydrolysis at 35 - 55 °C for 12 - 48 h to complete the enzymatic hydrolysis process;

[0013] ② Alkali - heat pretreatment

[0014] Adjust the pH of the food waste after enzymatic hydrolysis to 7 - 9 and carry out alkali treatment for 4 - 8 h; carry out heat treatment at 80 - 120 °C for 1 - 3 h to obtain the food waste raw material after pretreatment;

[0015] (2) Process for adding Propionibacterium to enhance anaerobic fermentation of food waste to produce propionic acid, including the following steps:

[0016] ① Culture method of Propionibacterium inoculum

[0017] Inoculate Propionibacterium into a stoppered serum bottle containing sterilized medium, fill it with high - purity nitrogen to ensure an anaerobic environment in the bottle, seal it with a rubber stopper, and after culturing, adjust the OD 600 value of the Propionibacterium bacterial liquid to 1.0 - 2.0 with sterile water as the inoculation bacterial liquid;

[0018] ② Method for adding Propionibacterium to enhance propionic acid production from food waste

[0019] Configure the anaerobic fermentation acid - producing substrate from the food waste raw material pretreated in step (1) and anaerobic inoculated sludge according to the volatile solids mass ratio of 2:1 - 9:1. Use Propionibacterium acidipropionici as an exogenous microorganism, and add the Propionibacterium inoculation bacterial liquid accounting for 20% - 70% of the mass of the anaerobic inoculated sludge at the initial stage of the reaction. Carry out an anaerobic hydrolysis - acidification reaction of food waste for 6 - 10 days at 25 - 35 °C to enhance propionic acid production and enhance the production of volatile fatty acids from food waste.

[0020] Preferably, in step (1) ①, the kitchen waste includes kitchen waste or perishable organic waste generated from waste sorting.

[0021] Preferably, in step (1) ①, the solid content of the kitchen waste is 5% to 15% by weight.

[0022] Preferably, in step (1) ①, the addition amount of cellulase is 110 - 130 U / g VS 厨余 , and the addition amount of hemicellulase is 400 - 600 U / g VS 厨余 ; the enzymatic hydrolysis temperature is 40 - 50 °C, and the enzymatic hydrolysis time is 18 - 36 h. The cellulase and hemicellulase are commercially available products.

[0023] Preferably, in step (1) ②, the pH value for alkali treatment of the kitchen waste after enzymatic hydrolysis is 7.5 - 8.5, and the alkali treatment time is 5 - 7 h; the heat treatment temperature is 90 - 110 °C, and the heat treatment time is 1.5 - 2.5 h.

[0024] Preferably, in step (2) ①, the Latin name of the Propionibacterium is Propionibacterium acidipropionici, the preservation number is CGMCC 1.2232, the preservation unit is the China General Microbiological Culture Collection Center, and its isolation source is Swiss cheese.

[0025] Preferably, in step (2) ①, the OD 600 range of the inoculation bacterial solution of the Propionibacterium is 1.3 - 1.8.

[0026] Preferably, in step (2) ①, the OD 600 of the inoculation bacterial solution of the Propionibacterium is 1.5.

[0027] Preferably, in step (2) ②, the ratio of the pre - treated kitchen waste raw material to the anaerobic inoculated sludge is 3:1 - 5:1 in terms of the mass ratio of volatile solids, the addition amount of the inoculation bacterial solution of the Propionibacterium is 20% - 40% of the mass of the anaerobic inoculated sludge, the reaction temperature is 30 - 33 °C, and the reaction time is 7 - 9 days.

[0028] The method for calculating the volatile solid content in the kitchen waste raw material and the anaerobic inoculated sludge: Determine the solid content (% by weight) and the ash content (% by weight) respectively according to GB 5009.3 - 2016 and GB 5009.4 - 2016, and the difference between the solid content and the ash content represents the volatile solid content (% by weight).

[0029] Among them, the anaerobic inoculated sludge is taken from the IC reactor (internal - circulation anaerobic reactor) in sewage treatment.

[0030] Preferably, in item ① of step (2), the composition of the sterilized culture medium is: 10 g / L of trypticase, 5 g / L of yeast extract, 10 g / L of sodium lactate; the pH is 7.0 - 7.2; the propionic acid bacteria are cultured at 29 - 32 °C for 1.5 - 2.5 days.

[0031] Beneficial effects:

[0032] Advantages of the present invention: 1. Using cellulase and hemicellulase to synergistically enzymatically hydrolyze kitchen waste to degrade cellulose-like organic matter and increase the production of even-carbon volatile fatty acids during the subsequent anaerobic fermentation acid production process. Among them, the degradation rates of cellulose and hemicellulose after enzymatic hydrolysis reach 40.03% and 32.91% respectively; during the anaerobic fermentation acid production process, the production of acetic acid in even-carbon volatile fatty acids can be increased by 56.17%, and the production of butyric acid can be increased by 108.30%. 2. Using propionic acid bacteria as exogenous microorganisms to strengthen the production of odd-carbon volatile fatty acid propionic acid in the volatile fatty acids generated from kitchen waste, and the production of propionic acid is increased by 79.57% compared with that without propionic acid bacteria strengthening. Description of the drawings

[0033] Figure 1 is the process schematic diagram of the present invention;

[0034] Figure 2 is the SEM diagram of the enzymatic hydrolysis of kitchen waste in Examples 1 - 5 of the present invention;

[0035] Figure 3 is the influence of different enzymatic hydrolysis conditions of the present invention on the total VFAs generated from kitchen waste;

[0036] Figure 4 is the influence of different enzymatic hydrolysis conditions of the present invention on the production of acetic acid and butyric acid from kitchen waste;

[0037] Figure 5 is the influence of the addition amount of propionic acid bacteria of the present invention on the total VFAs generated from kitchen waste;

[0038] Figure 6 is the influence of the addition amount of propionic acid bacteria of the present invention on the production of propionic acid from kitchen waste;

[0039] Figure 7 is the influence of the addition time of propionic acid bacteria of the present invention on the total VFAs generated from kitchen waste;

[0040] Figure 8 is the influence of the addition time of propionic acid bacteria of the present invention on the production of propionic acid from kitchen waste. Detailed implementation manners

[0041] The present invention will be further described below in conjunction with specific embodiments.

[0042] The advantages and features of the present invention will become clearer with the description. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that without departing from the spirit and scope of the present invention, modifications or substitutions can be made to the details and forms of the technical solutions of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention.

[0043] The methods used in the embodiments are all conventional methods unless otherwise specified. All percentage concentrations are mass percentage concentrations, and the solvents in all culture media are distilled water.

[0044] As Figure 1 shown, the kitchen waste is crushed and dehydrated to obtain kitchen waste with a solid content of 5 wt% - 15 wt%. The enzymatic hydrolysis is completed by using the synergistic enzymatic hydrolysis process of the cellulase and hemicellulase of the present invention, and then it is subjected to alkali heat treatment to obtain the pre-treated kitchen waste; then the pre-treated kitchen waste is subjected to microbial enhanced anaerobic fermentation hydrolysis to produce volatile fatty acids.

[0045] The method for optimizing the synergistic enzymatic hydrolysis process of the cellulase and hemicellulase of the present invention includes the following steps: First, use cellulase and hemicellulase as enzymatic hydrolysis additives and add them simultaneously to the kitchen waste with a solid content of 5 wt% - 40 wt%. The addition amount of cellulase is 100 - 150 U / g VS 厨余 , and the addition amount of hemicellulase is 200 - 800 U / g VS 厨余 ; carry out synergistic enzymatic hydrolysis for 12 - 48 h under the condition of 35 - 55 °C; after enzymatic hydrolysis by the present invention, the cellulose content in the kitchen waste is degraded from 29.25 ± 0.53% to 17.55 ± 1.47%, and the hemicellulose content is degraded from 15.80 ± 0.77% to 10.60 ± 0.38%; then adjust the pH of the kitchen waste to 7 - 9 and carry out alkali treatment for 4 - 8 h; carry out heat treatment at 80 - 120 °C for 1 - 3 h; after anaerobic fermentation hydrolysis and acidification, detect its acid production effect. The yield of volatile fatty acids generated from the kitchen waste after enzymatic hydrolysis by the present invention can be increased by 61.61% compared with that without enzymatic hydrolysis, the acetic acid yield is increased by 56.17% compared with that without enzymatic hydrolysis, and the butyric acid yield is increased by 108.30% compared with that without enzymatic hydrolysis;

[0046] The enzymatic hydrolysis process of the present invention is a synergistic treatment method of cellulase and hemicellulase. The cellulase and hemicellulase are simultaneously added into the kitchen waste with a preferred solid content of 5 wt% - 15 wt% and mixed evenly; during enzymatic hydrolysis, the preferred contents of cellulase and hemicellulase are 110 - 130 U / g VS 厨余 and 400 - 600 U / g VS 厨余; The preferred enzymatic hydrolysis temperature is 40 - 50 °C, and the preferred enzymatic hydrolysis time is 18 - 36 h; the preferred pH value for alkaline treatment of kitchen waste is 7.5 - 8.5, and the time is 5 - 7 h; the preferred heat treatment temperature is 90 - 110 °C, and the time is 1.5 - 2.5 h.

[0047] The method for optimizing the process of enhancing anaerobic fermentation of kitchen waste to produce propionic acid by adding Propionibacterium in the present invention includes the following steps: First, culture Propionibacterium. Inoculate Propionibacterium into a stoppered serum bottle containing a sterilized medium (10 g / L of trypticase peptone, 5 g / L of yeast extract, 10 g / L of sodium lactate; pH is 7.0 - 7.2), fill it with high-purity nitrogen to ensure an anaerobic environment in the bottle, seal it with a rubber stopper, and culture it at 30 °C for 2 days; Use sterile water to adjust the OD 600 value of the bacterial liquid to 1.0 - 2.0 as the inoculated bacterial liquid. Then, configure the anaerobic fermentation acid-producing substrate with the pretreated kitchen waste raw material and anaerobic inoculated sludge according to the volatile solid mass ratio of 2:1 - 9:1. Use Propionibacterium (Propionibacterium acidipropionici, CGMCC 1.2232) as an exogenous microorganism, and add the inoculated bacterial liquid of Propionibacterium (CGMCC 1.2232) accounting for 20% - 70% of the mass of the inoculated sludge at the initial stage of the reaction to carry out anaerobic hydrolysis and acidification of kitchen waste, strengthening the production of propionic acid. After being strengthened by the microorganism of the present invention, the yield of volatile fatty acids generated by anaerobic hydrolysis and acidification of kitchen waste can reach 41.16 ± 1.49 g / L, and the yield of propionic acid can reach 8.79 ± 0.29 g / L. Compared with that without being strengthened by Propionibacterium, the yield of propionic acid increases by 79.57%.

[0048] After culturing Propionibacterium in the present invention, the preferred OD 600 range of the inoculated bacterial liquid of Propionibacterium is between 1.3 and 1.8; when hydrolyzing and acidifying kitchen waste to produce volatile fatty acids, the preferred pretreated kitchen waste raw material and anaerobic inoculated sludge are configured as the anaerobic fermentation acid-producing substrate according to the volatile solid mass ratio of 3:1 - 5:1. The preferred addition amount of Propionibacterium is 20% - 40% of the mass of the anaerobic inoculated sludge, and the optimal addition time of Propionibacterium is to carry out strengthening at the initial stage of the reaction.

[0049] Example 1

[0050] The kitchen waste with a solid content of 11.2% (kitchen waste generated from a certain community) was not enzymatically hydrolyzed.

[0051] Example 2

[0052] Enzymatically hydrolyze kitchen waste with cellulase, and the steps are as follows:

[0053] Using cellulase (commercially available) as an enzymatic hydrolysis additive, it was added to food waste with a solids content of 11.2% (food waste generated from a certain community in Example 1), and the addition amount of cellulase was 120 U / g VS 厨余 ; It was enzymatically hydrolyzed at 45 °C for 24 h to complete the enzymatic hydrolysis process.

[0054] Example 3

[0055] Hydrolyzing food waste with hemicellulase, the steps are as follows:

[0056] Using hemicellulase (commercially available) as an enzymatic hydrolysis additive, it was added to food waste with a solids content of 11.2% (food waste generated from a certain community in Example 1), and the addition amount of hemicellulase was 500 U / g VS 厨余 ; It was enzymatically hydrolyzed at 45 °C for 24 h to complete the enzymatic hydrolysis process.

[0057] Example 4

[0058] Hydrolyzing food waste with cellulase and hemicellulase in sequence (double enzymes in sequence), the steps are as follows:

[0059] (1) Using cellulase (commercially available) as an enzymatic hydrolysis additive, it was added to food waste with a solids content of 11.2% (food waste generated from a certain community in Example 1), and the addition amount of cellulase was 120 U / g VS 厨余 ; It was enzymatically hydrolyzed at 45 °C for 24 h to obtain semi-hydrolyzed food waste;

[0060] (2) Then, using hemicellulase (commercially available) as an enzymatic hydrolysis additive, it was added to the above semi-hydrolyzed food waste, and the addition amount of hemicellulase was 500 U / g VS 厨余 ; It was enzymatically hydrolyzed at 45 °C for 24 h to complete the enzymatic hydrolysis process.

[0061] Example 5

[0062] Hydrolyzing food waste with cellulase and hemicellulase in synergy (double enzymes in synergy), the steps are as follows:

[0063] Using cellulase (commercially available) and hemicellulase (commercially available) as enzymatic hydrolysis additives and adding them simultaneously to food waste with a solids content of 11.2% (food waste generated from a certain community in Example 1), the addition amount of cellulase was 120 U / gVS 厨余 , and the addition amount of hemicellulase was 500 U / g VS 厨余 ; It was enzymatically hydrolyzed in synergy at 45 °C for 24 h to complete the enzymatic hydrolysis process.

[0064] Examples 1-5 set up non-enzymatic hydrolysis (Example 1) and four different enzymatic hydrolysis methods for food waste, namely single cellulase enzymatic hydrolysis (Example 2), single hemicellulase enzymatic hydrolysis (Example 3), sequential enzymatic hydrolysis of cellulase and hemicellulase (Example 4), and simultaneous enzymatic hydrolysis of cellulase and hemicellulase (Example 5).

[0065] In the experiments of the present invention, the determination of cellulose and hemicellulose contents adopted the Van Soest washing fiber analysis method. As Figure 2 shown, it is the SEM images of food waste after enzymatic hydrolysis in Examples 1-5 of the present invention, where: a is Example 1; b is Example 2; c is Example 3; d is Example 4; e is Example 5.

[0066] In Example 1 (a), there were fine granular substances on the surface of the food waste, with a complete and dense structure, and the organic matter was not easily utilized by microorganisms; in Example 2 (b), part of the cell wall on the surface of the food waste was broken, the structure changed significantly, and the porosity increased, which was beneficial to the hydrolysis of organic matter; in Example 3 (c), the surface of the food waste was smooth and the structure was intact, and the hydrolysis effect was not ideal when treated with hemicellulase; in Example 4 (d), a large area of the cell wall on the surface of the food waste was broken, holes were generated inside, and the specific surface area increased, which was beneficial to the hydrolysis of organic matter; in Example 5 (e), on the basis of a large area of fracture on the surface layer of the food waste, part of the organic matter had been hydrolyzed, and the crystalline fiber substances were exposed, and its structure was more conducive to the utilization of microorganisms in the anaerobic fermentation process.

[0067] After enzymatic hydrolysis of food waste using single cellulase and hemicellulase, the contents of cellulose and hemicellulose were still higher than 26% and 14%; among them, after simultaneous enzymatic hydrolysis of cellulase and hemicellulase, compared with non-enzymatic hydrolysis, cellulose hydrolysis was 40.03% and hemicellulose hydrolysis was 32.91%; and compared with the enzymatic hydrolysis in Examples 2, 3, and 4, the degradation rates of recalcitrant organic matter were all improved.

[0068] Table 1 Degradation effects of enzymatic hydrolysis of food waste on cellulose and hemicellulose

[0069]

[0070] Then, the pH of the food waste after enzymatic hydrolysis in Examples 1-5 was adjusted to 8 and treated with alkali for 6 h; heat treatment was carried out at 100 °C for 2 h to obtain the pretreated food waste raw material. The pretreated food waste was used as the raw material and anaerobic inoculated sludge (commercially available) to configure the anaerobic fermentation acidogenic substrate according to the volatile solid mass ratio of 4:1, and the hydrolysis acidification reaction was carried out to detect the volatile fatty acid yield:

[0071] The solid content and ash content of food waste and anaerobic inoculum sludge were determined according to the methods of GB 5009.3-2016 and GB 5009.4-2016, respectively. The difference between the solid content and ash represented the volatile solid content. The volatile solid content of food waste was 10.20 wt%, and the volatile solid content of anaerobic inoculum sludge was 3.19 wt%.

[0072] Example 1: The yield of volatile fatty acids generated from food waste without enzymatic hydrolysis was 22.47 ± 1.18 g / L, among which the acetic acid yield was 9.77 ± 0.20 g / L and the butyric acid yield was 7.27 ± 0.19 g / L.

[0073] Example 2: After only cellulase treatment of food waste, the yield of volatile fatty acids increased by 34.23% compared with that without enzymatic hydrolysis (Example 1), among which the acetic acid yield increased by 36.40% and the butyric acid yield increased by 77.70%.

[0074] Example 3: After only hemicellulase treatment of food waste, the yield of volatile fatty acids increased by 29.43% compared with that without enzymatic hydrolysis (Example 1), among which the acetic acid yield increased by 29.44% and the butyric acid yield increased by 34.52%.

[0075] Example 4: After food waste was first treated with cellulase and then with hemicellulase, the yield of volatile fatty acids increased by 42.45% compared with that without enzymatic hydrolysis (Example 1), among which the acetic acid yield increased by 52.83% and the butyric acid yield increased by 55.17%.

[0076] Example 5: When food waste was treated with cellulase and hemicellulase simultaneously, the yield of volatile fatty acids increased by 61.61% compared with that without enzymatic hydrolysis (Example 1), among which the acetic acid yield increased by 56.17% and the butyric acid yield increased by 108.30%.

[0077] As Figure 3 shown, it is the influence of different enzymatic hydrolysis conditions of the present invention on the anaerobic fermentation of food waste to produce volatile fatty acids; as Figure 4 shown, it is the influence of different enzymatic hydrolysis conditions of the present invention on the anaerobic fermentation of food waste to produce even-carbon volatile fatty acids acetic acid and butyric acid; it can be seen that compared with Example 1, the simultaneous treatment with cellulase and hemicellulase has the best promotion effect on the yields of volatile fatty acids, acetic acid and butyric acid produced by food waste, and there is a certain increase in the yield of volatile fatty acids compared with other enzymatic hydrolysis methods in Examples 2, 3 and 4.

[0078] Examples 6-10

[0079] Adjust the pH of the enzymatically hydrolyzed kitchen waste prepared in Example 5 to 8 and perform alkali treatment for 6 h; perform heat treatment at 100 °C for 2 h to obtain the pretreated kitchen waste raw material. The pretreated kitchen waste is used as a raw material and anaerobic inoculated sludge (commercially available) to prepare an anaerobic fermentation acidogenic substrate according to a volatile solid mass ratio of 4:1 for anaerobic hydrolysis and acidogenesis; and set up an experiment without Propionibacterium enhancement and adding 10%, 30%, 50%, and 70% of the Propionibacterium inoculation broth (OD 600 = 1.5) of the mass of the anaerobic inoculated sludge at the beginning of the reaction for Propionibacterium enhancement experiment.

[0080] Propionibacterium inoculum culture method: Inoculate Propionibacterium (China Center for Microbial Culture Collection, the Latin name of the Propionibacterium is Propionibacterium acidipropionici, CGMCC 1.2232, the original number is DSM20272, the strain source is DSMZ, the direct source country is Germany, the preservation time is July 14, 1998, other preservation numbers = ATCC4875 = NCIMB8070, non-model strain, culture temperature 30 °C, medium 0286, other culture conditions are anaerobic, and its isolation source is Swiss cheese) into a stoppered serum bottle containing sterilized medium, fill with high-purity nitrogen to ensure an anaerobic environment in the bottle, seal with a rubber stopper, and culture at 30 °C for 2 days. Use sterile water to adjust the OD 600 value of the Propionibacterium broth to 1.5 as the inoculation broth for microbial enhancement experiment; the composition of the sterilized medium is: trypticase peptone 10 g / L, yeast extract 5 g / L, sodium lactate 10 g / L; pH is 7.0 - 7.2.

[0081] Example 6: The yield of volatile fatty acids generated from kitchen waste without Propionibacterium enhancement is 37.68 ± 2.16 g / L; the yield of propionic acid is 4.89 ± 0.16 g / L.

[0082] As Figure 5 shown, it is the effect of the addition amount of Propionibacterium in the present invention on the total VFAs generated from kitchen waste. It can be seen that after adding 10%, 30%, 50%, and 70% of the Propionibacterium inoculation broth of the sludge mass in Examples 7, 8, 9, and 10, the yields of volatile fatty acids are 36.12 ± 1.36, 41.16 ± 1.49, 34.09 ± 2.88, and 32.93 ± 1.47 g / L respectively; as Figure 6As shown, it is the influence of the addition amount of Propionibacterium in the present invention on the production of propionic acid from kitchen waste. The propionic acid yields are 5.81±0.04, 8.79±0.29, 7.96±0.59 and 7.41±0.07 g / L respectively. Among them, in Example 8, after adding the Propionibacterium inoculum solution accounting for 30% of the mass of anaerobic inoculated sludge, the volatile fatty acid yield increased the most. The volatile fatty acid yield increased by 9.21% compared with Example 6 without microbial enhancement, and was higher than other Propionibacterium addition conditions; the propionic acid yield increased by 79.57% compared with Example 6, and adding Propionibacterium in an amount exceeding 30% of the mass of anaerobic inoculated sludge did not further significantly increase the propionic acid yield.

[0083] Examples 11 - 12

[0084] Perform anaerobic fermentation hydrolysis acidogenesis reaction on the pretreated kitchen waste, as Figure 7 and 8 shown, which are respectively the influence of the addition time of Propionibacterium in the present invention on the production of total VFAs and propionic acid from kitchen waste; Examples 11 and 12 are respectively the experiments of adding Propionibacterium accounting for 30% of the mass of anaerobic inoculated sludge only at 3 days and 6 days of the reaction. The volatile fatty acid yields are 41.16±2.04 and 35.05±1.24 g / L respectively; the propionic acid yields are 4.90±0.15 and 5.28±0.32 g / L. Combining with Example 8, it can be seen that adding Propionibacterium at the initial stage of kitchen waste hydrolysis acidogenesis (day 0) has the best effect on improving the volatile fatty acid yield and propionic acid yield.

[0085] In the method of the present invention, the enzymatic hydrolysis method adopted is to add cellulase and hemicellulase simultaneously, so that the two enzymes produce a synergistic treatment effect to improve the degradation efficiency of kitchen waste and increase the production of even-carbon volatile fatty acids acetic acid and butyric acid; further, Propionibacterium is added during the anaerobic fermentation acidogenesis process of kitchen waste to increase the production of odd-carbon volatile fatty acid propionic acid during the anaerobic fermentation acidogenesis process, and finally realize the comprehensive improvement of the volatile fatty acid yield generated by the anaerobic fermentation of kitchen waste by the coupling method of enzymatic hydrolysis and microbial enhancement.

[0086] The specific embodiments described above have further detailed the purpose, technical solution and beneficial effects of the present invention, but do not limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for coupling enzymatic hydrolysis and microbial enhancement to improve anaerobic acid production from food waste, comprising the following steps: (1) Process steps of synergistic enzymatic hydrolysis of cellulase and hemicellulase ① Synergistic enzymatic hydrolysis of cellulase and hemicellulase Using cellulase and hemicellulase as enzymatic hydrolysis additives and adding them simultaneously to food waste with a solids content of 5 wt% to 40 wt%, the addition amount of cellulase is 100 - 150 U / g VS 厨余 , and the addition amount of hemicellulase is 200 - 800 U / g VS 厨余 ; Co-enzymatic hydrolysis is carried out at 35 - 55 °C for 12 - 48 h to complete the enzymatic hydrolysis process; ② Alkali thermal pretreatment Adjust the pH of the food waste after enzymatic hydrolysis to 7 - 9 and perform alkali treatment for 4 - 8 h; perform heat treatment at 80 - 120 °C for 1 - 3 h to obtain the pretreated food waste raw material; (2) Process for enhancing propionic acid production by anaerobic fermentation of food waste by adding Propionibacterium, comprising the following steps: ① Cultivation method of Propionibacterium inoculum Inoculate Propionibacterium into a stoppered serum bottle containing sterilized medium, fill it with high-purity nitrogen to ensure an anaerobic environment inside the bottle, seal it with a rubber stopper, and after culturing, use sterile water to adjust the OD 600 value of the Propionibacterium bacterium solution to 1.0 - 2.0 as the inoculation bacterium solution; ② Method for enhancing propionic acid production from food waste by Propionibacterium Configure the anaerobic fermentation acid - producing substrate with the pretreated food waste raw material in step (1) and anaerobic inoculated sludge according to the volatile solid mass ratio of 2:1 - 9:

1. Use Propionibacterium as the exogenous microorganism, and add the Propionibacterium inoculum solution accounting for 20% - 70% of the mass of the anaerobic inoculated sludge at the initial stage of the reaction. Carry out the anaerobic fermentation hydrolysis and acidification reaction of food waste for 6 - 10 days at 25 - 35 °C to enhance propionic acid production and enhance the production of volatile fatty acids from food waste; In step (2) ①, the Latin name of the Propionibacterium is Propionibacterium acidipropionici , with the preservation number of CGMCC 1.2232 and the preservation unit being the China General Microbiological Culture Collection Center.

2. The method for enhancing anaerobic acid production from food waste by coupling enzymatic hydrolysis and microbial intensification according to claim 1, characterized in that: In step (1) ①, the food waste includes food waste generated from garbage classification or perishable organic waste.

3. The method for improving anaerobic acid production of food waste by coupling enzymatic hydrolysis and microbial enhancement according to claim 2, characterized in that: In step (1) ①, the solid content of the food waste is 5 wt% - 15 wt%.

4. The method for enhancing anaerobic acid production of kitchen waste by coupling enzymatic hydrolysis and microbial enhancement according to claim 1, characterized in that: In step (1) ①, the addition amount of cellulase is 110 - 130 U / g VS 厨余 , the addition amount of hemicellulase is 400 - 600 U / g VS 厨余 ; the enzymatic hydrolysis temperature is 40 - 50 °C, and the enzymatic hydrolysis time is 18 - 36 h.

5. The method for enhancing anaerobic acid production of food waste by coupling enzymatic hydrolysis and microbial enhancement according to claim 1, characterized in that: In step (1) ②, the pH value of the food waste after enzymatic hydrolysis for alkali treatment is 7.5 - 8.5, and the alkali treatment time is 5 - 7 h; the heat treatment temperature is 90 - 110 °C, and the heat treatment time is 1.5 - 2.5 h.

6. The method for enhancing anaerobic acid production from food waste by coupling enzymatic hydrolysis and microbial enhancement according to claim 1, wherein: In step (2) ①, the OD of the inoculum of Propionibacterium acidipropionici 600 ranges from 1.3 to 1.

8.

7. The method for enhancing anaerobic acid production from food waste by coupling enzymatic hydrolysis and microbial intensification according to claim 6, wherein: In step (2) ①, the OD of the inoculum of Propionibacterium acidipropionici 600 = 1.

5.

8. The method for enhancing anaerobic acid production of food waste by coupling enzymatic hydrolysis and microbial enhancement according to claim 1, characterized in that: In step (2) ②, the ratio of the pretreated food waste raw material to the anaerobic inoculated sludge is a volatile solid mass ratio of 3:1 - 5:1, the addition amount of the Propionibacterium inoculum solution is 20% - 40% of the mass of the anaerobic inoculated sludge, the reaction temperature is 30 - 33 °C, and the reaction time is 7 - 9 days.

9. The method for enhancing anaerobic acid production from food waste by coupling enzymatic hydrolysis and microbial enhancement according to claim 1, characterized in that: In step (2) ①, the composition of the sterilized culture medium is: trypticase 10 g / L, yeast extract 5 g / L, sodium lactate 10 g / L; the pH is 7.0 - 7.2; the Propionibacterium is cultured at 29 - 32 °C for 1.5 - 2.5 days.

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