Method for accelerating starting of organic garbage fermentation and enhancing directional production of butyric acid by using biological agent

Abstract

The invention discloses a method for accelerating the starting of organic waste fermentation and enhancing the directional production of butyric acid by using a biological agent, which comprises the following steps of: adding the biological agent prepared from clostridium butyricum in a specific growth state into an anaerobic fermentation system of organic waste (such as a mixed substrate of kitchen waste and municipal sludge); the hydrolysis efficiency of organic wastes can be effectively enhanced, the ratio of butyric acid in a product is remarkably increased, and quick start of an organic waste acid production fermentation system and quick and efficient production of butyric acid are realized. By means of the method, reduction treatment of the urban organic waste can be efficiently achieved, meanwhile, the urban organic waste is converted into high-value organic matter, and therefore the effect of recycling the organic waste is achieved. The technology has further research, development and application prospects in the fields of urban organic waste reduction treatment, high-value organic matter conversion and development and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of organic waste resource utilization, and relates to a method for accelerating the fermentation of organic waste and strengthening the directional production of butyric acid by using a biological bacterial agent. Background Art

[0002] The harmless treatment and resource utilization of municipal sludge and kitchen waste is an important challenge in current waste management. With the acceleration of urbanization, the amount of these organic wastes has increased dramatically. If scientific and effective treatment measures are not taken in time, they will pose a threat to the environment and public health. Traditionally, municipal sludge and kitchen waste are often disposed of by landfill, incineration, etc., but such methods not only waste resources seriously, but also produce greenhouse gases and secondary pollution, making it difficult to achieve true resource utilization.

[0003] Anaerobic fermentation technology is a technology that uses anaerobic microbial communities to decompose organic matter and produce biogas in an oxygen-free environment. This technology has been widely used in the treatment of organic solid waste, especially in waste degradation, resource recovery and energy generation. In this process, organic matter is first decomposed into simple organic acids, which can be used as chemical raw materials or in industries such as agriculture and food processing. At the same time, anaerobic fermentation technology has shown good results in the treatment of agricultural waste, kitchen waste, sludge and other industrial organic wastes, especially in reducing greenhouse gas emissions and reducing the cost of hazardous waste treatment. It has broad application prospects.

[0004] However, in the application of anaerobic fermentation acid production technology, it often requires long-term microbial domestication and adaptation to achieve excellent acid production efficiency. During the fermentation startup process, a large number of pollutants that are not treated up to standard are also produced and time is wasted. On this basis, the balance of the microbial community will also affect the type and quantity of acid. The fermentation products of the adaptive community are often dominated by evenly distributed volatile fatty acids, which limits its subsequent high-value utilization. Among the many volatile fatty acids, butyric acid, as an important chemical raw material and energy precursor, is widely used in the fields of food, medicine and biomaterials. Butyric acid can be produced by exogenous addition of bacterial agents that can produce butyric acid, and directed production through bio-enhancement. However, how to optimize fermentation conditions to increase butyric acid production is still a key problem in current research. Exploring the efficient and low-cost conversion of organic waste using short-chain fatty acid-producing strains such as Clostridium tyrobutyricum will provide a new way for the resource utilization of organic waste and promote waste management to develop in an environmentally friendly and sustainable direction. Summary of the invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art, select Clostridium tyrobutyricum in a specific growth state to prepare a bacterial agent according to the special property of Clostridium tyrobutyricum that it first accumulates biomass and then produces butyric acid in the culture medium, add the bacterial agent to a garbage anaerobic fermentation reactor, and provide a method for accelerating the fermentation of organic garbage and strengthening the directional production of butyric acid using a biological bacterial agent. The method can promote the rapid fermentation of organic garbage such as food garbage and sewage treatment plant sludge, and simultaneously efficiently and directionally produce butyric acid, thereby realizing the high-value resource conversion of organic garbage.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0007] A method for accelerating the fermentation of organic waste and strengthening the directional production of butyric acid by using a biological agent, wherein Clostridium butyricum is cultured in a strengthened clostridial culture medium to obtain a Clostridium butyricum bacterial liquid in a specific growth state as a biological strengthening agent, and is added to an anaerobic fermentation system constructed by organic waste, thereby accelerating the directional production of butyric acid.

[0008] In the above technical solution, further, the Clostridium tyrobutyricum was purchased from Guangdong Microbiological Culture Collection Center (GDMCC: 1.262).

[0009] Furthermore, the strain was cultured in a reinforced Clostridium medium, the composition of which was: 5 g / L glucose, 10 g / L tryptone, 3 g / L yeast extract, 1 g / L soluble starch, 10 g / L beef extract, 0.5 g / L L-cysteine ​​hydrochloride, 5 g / L sodium chloride and 3 g / L sodium acetate. The pH was 6.8 and sterilized at 121°C for 20 min. 1 M HCl and 1 M NaOH were used to adjust the pH of the medium.

[0010] Further, 1-5 ml of bacterial liquid (ie, 1-5% (v / v)) was taken from the activated bacterial liquid culture medium and inoculated into a sterile anaerobic culture bottle containing 100 ml of enhanced Clostridium culture medium, and cultured at 37° C. for 48 hours.

[0011] Furthermore, based on the special property of Clostridium tyrobutyricum that it first accumulates biomass and then produces butyric acid in the culture medium, Clostridium tyrobutyricum bacterial liquid cultured for 12-24 hours was selected as the bio-enhancing bacterial agent to ensure sufficient bacterial volume and that butyric acid had not yet accumulated in the bacterial liquid.

[0012] Furthermore, municipal sludge and food waste are mixed to construct an anaerobic fermentation system.

[0013] The municipal sludge and the kitchen waste are mixed in proportion and pre-treated, and the pre-treatment includes homogenization and oil removal, to obtain mixed waste. The proportion is a volume ratio, preferably 8:2 to 6:4.

[0014] Furthermore, the mixed garbage is poured into a continuous flow anaerobic fermentation tank for fermentation, the temperature is set at 35 degrees, the organic load of the reactor is 10-15g COD / L, and the hydraulic retention time is 5-8 days.

[0015] The Clostridium tyrobutyricum agent in a specific growth state is used to enhance the efficiency of organic waste hydrolysis and directional acid production. It is most preferred that the Clostridium tyrobutyricum bacterial solution cultured for 18 hours is added to the reaction system at the beginning of the reaction.

[0016] Furthermore, the minimum effective dosage of Clostridium tyrobutyricum liquid is about 1% of the volume of the effective reaction system.

[0017] Furthermore, the addition time of Clostridium tyrobutyricum liquid is at least one hydraulic retention time.

[0018] The culture stage of Clostridium tyrobutyricum and the dosage of Clostridium tyrobutyricum in the reaction system are critical to whether the reaction system can be quickly started to produce butyric acid. When the culture time is less than 12 hours, even if the dosage of the bacterial solution in the reaction system is increased, the system cannot be quickly started to produce butyric acid.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects:

[0020] The present invention utilizes the special property of Clostridium tyrobutyricum that it first accumulates biomass and then produces butyric acid in the culture medium, and obtains Clostridium tyrobutyricum liquid in a specific growth state as a bio-enhanced bacterial agent by controlling the culture conditions of Clostridium tyrobutyricum. In this state, the Clostridium tyrobutyricum liquid has accumulated active bacteria but has not yet begun to produce butyric acid. The addition of the above-mentioned bacterial agent realizes rapid fermentation of organic waste and directional production of butyric acid. Compared with the system of organic waste fermentation without bio-enhanced, the bio-enhanced method of the present invention effectively improves the substrate hydrolysis efficiency of the organic waste fermentation system by 10%, and the concentration of butyric acid produced accounts for 50% of the total volatile organic acid concentration, and the butyric acid concentration can reach up to 6.5g / L. The bacterial agent preparation and addition scheme of the present invention are key technical parameters for ensuring the effectiveness of organic waste fermentation bio-enhanced, and the technology has the prospect of further research and development and application in the fields of organic waste reduction and high-value organic matter conversion. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Growth curves and butyrate production of Clostridium tyrobutyricum in fortified Clostridial medium.

[0022] Figure 2 Cell morphology of Clostridium tyrobutyricum culture after 18 hours.

[0023] Figure 3 This is the experimental device used in the embodiments of the present invention.

[0024] Figure 4 The hydrolysis efficiency of the substrate during the startup of the anaerobic fermentation system with (A, B) and without (C, D) microbial agents.

[0025] Figure 5 Butyric acid production concentration of substrate during the startup of anaerobic fermentation system with (A, B) and without (C, D) microbial inoculants. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0027] The method of the present invention using a biological agent to accelerate the fermentation of organic waste and produce butyric acid in a targeted manner can rapidly improve the butyric acid production capacity of the system by adding the biological agent, and is described by the following example:

[0028] The Clostridium butyricum strain purchased from Guangdong Microbial Culture Collection Center (GDMCC: 1.262) was used as the raw material for preparing the bacterial agent.

[0029] The anaerobic sludge from an anaerobic fermentation plant in Xiaoshan, Hangzhou was used as the inoculum sludge.

[0030] Municipal sludge from a sewage treatment plant in Hangzhou and kitchen waste collected from the cafeteria of the Yungu Campus of West Lake University were used as fermentation substrates.

[0031] Specific:

[0032] Step 1: Cultivate Clostridium butyricum to obtain Clostridium butyricum in a specific growth state as a bio-enhancing bacterial agent.

[0033] 1-1: Prepare the fortified Clostridium culture medium, including: 5g / L glucose, 10g / L tryptone, 3g / L yeast extract, 1g / L soluble starch, 10g / L beef extract, 0.5g / L L-cysteine ​​hydrochloride, 5g / L sodium chloride and 3g / L sodium acetate. pH 6.8, sterilize at 121℃ for 20min. Use 1M HCl and 1M NaOH to adjust the pH of the culture medium.

[0034] 1-2: Take a loop of bacteria from the preserved plate and inoculate it into a sterile anaerobic culture bottle filled with fortified Clostridium liquid culture medium. Place the inoculated culture bottle in a constant temperature incubator and incubate at 37°C for 24 hours. Check the growth of the bacteria to ensure that the bacteria are growing well;

[0035] From the activated bacterial culture medium, 5 ml (5% v / v) of bacterial culture was taken and inoculated into a sterile anaerobic culture bottle containing 100 ml of enhanced Clostridium culture medium, and cultured at 37° C. for 48 hours.

[0036] 1-3: The bacteria were sampled every 6 hours, and the OD600 value of the fermentation broth was measured to analyze the growth characteristics of the strain; the supernatant of the fermentation broth was filtered through a 0.22 um filter membrane, and the filtrate was used to measure the butyric acid production at the corresponding time point.

[0037] 1-4: Butyric acid in the supernatant of the fermentation broth was determined using Waters UPLC H-Class, with a mobile phase of 0.1% formic acid solution in ultrapure water and 0.1% formic acid solution in methanol as solvent, a chromatographic column of BEH C18 1.7um 2.1*100mm, and an injection volume of 5μm.

[0038] 1-5: According to Figure 1 The bacterial accumulation curve and butyrate growth curve shown in the figure confirm that the bioaugmentation is performed with a bacterial solution cultured for 18 hours to ensure sufficient bacterial mass and that butyrate has not yet accumulated in the bacterial solution.

[0039] 1-6: Microbial cell technology instrument (QUANTOM-Tx TM ) The total number of Clostridium tyrobutyricum in the bacterial agent cultured for 18 hours was recorded to be 3.89×10e8 copies / ml, the number of viable bacteria was 2.22×10e8 copies / ml, and the cell morphology of the viable bacteria was as follows Figure 2 shown.

[0040] Step 2: Mix municipal sludge and kitchen waste in proportion to build an anaerobic fermentation system

[0041] 2-1: Pretreatment of kitchen waste for oil removal: Put the fresh kitchen waste into a 5L measuring cylinder and let it stand for 1 hour, then separate and remove the floating oil from the kitchen waste.

[0042] 2-2: Homogenization pretreatment of food waste: First, use a meat grinder to grind the food waste into a paste, then dilute the paste with water, and then use a wall breaking machine to continue stirring and crushing the diluted food waste for 1 minute, and finally store it at minus 20 degrees to prevent premature acidification.

[0043] 2-3: Preparation of mixed substrates of food waste and municipal sludge: Two mixed substrates were prepared according to the volume ratio of municipal sludge to food waste of 8:2 and 6:4, respectively. The final mixed substrates of food waste, municipal sludge and two ratios were prepared (Table 1).

[0044] Table 1 Composition and physicochemical properties of organic waste used in this example

[0045]

[0046] 2-4: The mixed garbage is placed in a continuous flow anaerobic fermentation tank for fermentation. The temperature is set to 35 degrees. The organic load of the reactor is 12g COD / L / day. The hydraulic retention time is 7.5 days. The reaction system is as follows: Figure 3 shown.

[0047] Step 3: Use Clostridium tyrobutyricum in a specific growth state to enhance the efficiency of organic waste hydrolysis and directional acid production.

[0048] 3-1: The above-mentioned Clostridium tyrobutyricum cultured for 18 hours was added to the reaction system (8:2 mixed substrate and 6:4 mixed substrate reaction system) respectively. At the same time, PBS solution was added to the same reaction system as a control group.

[0049] The specific experimental parameters are shown in Table 2.

[0050] Table 2. Experimental parameters

[0051]

[0052] 3-2: The dosage of Clostridium tyrobutyricum solution is about 1% of the effective reaction system, that is, 180 ml of Clostridium tyrobutyricum solution is added exogenously to groups A and B every day. 180 ml of PBS solution is added exogenously to groups C and D every day as a blank control. In addition, groups A and C add 240 ml of mixed matrix with a volume ratio of 8:2 every day, and groups B and D add 240 ml of mixed matrix with a volume ratio of 6:4 every day. Anaerobic sludge is discharged in equal volumes every day to maintain an effective reaction volume of 1.8L. After each feeding and sludge discharge, nitrogen is used to blow off to keep the reactor in an anaerobic state.

[0053] 3-3: The addition time of Clostridium tyrobutyricum solution is 7 consecutive days, which is about 1 hydraulic retention time, and the overall fermentation start-up time is 15 days, which is two hydraulic retention times.

[0054] 3-4: The daily sludge samples were stored at 4°C. The anaerobic sludge was centrifuged at 4°C and 10,000 rpm for 10 min. The supernatant was filtered using a 0.22 μm PES filter membrane to obtain the anaerobic sludge supernatant. The sludge indicators tested included soluble COD, pH, lactic acid, and volatile organic acids, which were used to evaluate the acid production effect.

[0055] 3-5: Dissolved COD and total COD using COD Cr The content of 4-nitropropene was determined by the reagent method (Lianhua Technology LH-DE-100, China).

[0056] 3-6: COD result display ( Figure 4), with the concentration of soluble COD in the bio-augmentation group (A, B) being higher than that in the control group (C, D), indicating that the method of the present invention effectively improves the substrate hydrolysis efficiency during the startup of the organic waste anaerobic fermentation reactor.

[0057] 3-7: The sludge was centrifuged at 12000rpm and 4℃ for 10 minutes using an Eppendorf Centrifuge 5810R centrifuge. The supernatant was filtered through a 0.45μm filter membrane to obtain the dissolved sample. The volatile fatty acids in the dissolved sample were determined using Waters UPLC H-Class, with a mobile phase of 0.1% formic acid solution in ultrapure water and 0.1% formic acid solution in methanol as the solvent, a chromatographic column of BEH C18 1.7um 2.1*100mm, and an injection volume of 5μm.

[0058] 3-8: Volatile fatty acid results ( Figure 5 ) showed that the production of butyric acid in the bioaugmentation group (A, B) was significantly earlier than and higher than that in the control group (C, D), indicating that the bioaugmentation effect of Clostridium tyrobutyricum can rapidly improve the butyric acid production capacity during the reactor startup process and significantly reduce the reactor startup and adaptation time.

[0059] The above-described embodiments are only some of the preferred solutions of the present invention, but they are not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A method for accelerating the fermentation of organic waste and strengthening the directional production of butyric acid by using a biological agent, characterized in that: By culturing Clostridium butyricum in an enhanced clostridial culture medium, a Clostridium butyricum liquid in a specific growth state is obtained as a biological enhanced bacterial agent, which is added to an anaerobic fermentation system constructed by organic waste, thereby achieving accelerated start-up of directional butyric acid production.

2. The method for accelerating the fermentation of organic waste and strengthening the directional production of butyric acid by using a biological agent according to claim 1, characterized in that: The composition of the fortified Clostridium culture medium is: 5 g / L glucose, 10 g / L tryptone, 3 g / L yeast extract, 1 g / L soluble starch, 10 g / L beef extract, 0.5 g / L L-cysteine ​​hydrochloride, 5 g / L sodium chloride and 3 g / L sodium acetate, pH 6.8, and sterilization at 121°C for 20 minutes.

3. The method for accelerating the fermentation of organic waste and strengthening the directional production of butyric acid by using a biological agent according to claim 1, characterized in that: The specific growth state is: selecting bacterial liquid cultured for 12-24 hours as the biological enhancement bacterial agent.

4. The method for accelerating the fermentation of organic waste and strengthening the directional production of butyric acid by using a biological agent according to claim 1, characterized in that: The anaerobic fermentation system is obtained by mixing and pre-treating municipal sludge and restaurant kitchen waste in proportion.

5. The method for accelerating the fermentation of organic waste and strengthening the directional production of butyric acid by using a biological agent according to claim 4, characterized in that: The pretreatment includes homogenization and oil removal.

6. The method for accelerating the fermentation of organic waste and strengthening the directional production of butyric acid by using a biological agent according to claim 1, characterized in that: In the anaerobic fermentation system, the temperature is set at 35 degrees, the organic load of the reactor is 10-15 gCOD / L, the hydraulic retention time is 5-8 days, and the bio-enhancing agent is added to the reaction system at the beginning of the reaction.

7. The method for accelerating the fermentation of organic waste and strengthening the directional production of butyric acid by using a biological agent according to claim 1, characterized in that: The dosage of bacterial solution is 1% of the volume of the effective reaction system.

8. The method for accelerating the fermentation of organic waste and strengthening the directional production of butyric acid by using a biological agent according to claim 1, characterized in that: The bacterial solution addition time should be at least one water retention time.

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