Two-step fermentation method for producing PHA (polyhydroxyalkanoate) from organic garbage

Abstract

The invention discloses a two-step fermentation method for producing PHA (polyhydroxyalkanoate) from organic garbage. According to the method, kitchen waste and municipal sludge mixed bacteria are adopted for fermentation (first-step fermentation) to convert organic matters into fermentation liquor mainly containing acetic acid and butyric acid, then cationic polyacrylamide is used for dewatering and conditioning the fermentation sludge, and finally wastewater obtained after anaerobic co-fermentation sludge is treated by a dewatering conditioner is used as a carbon source. The method comprises the following steps of: culturing Cupriavidus necator to synthesize the PHA (fermentation in the second step); according to the method, the accumulation of the PHA content in the growth cells of the Cupriavidus hookworm can be obviously improved by the carbon source type and the nutrient element composition, and the yield of the PHA is up to 73.68%. According to the method, the organic garbage is used as the unique raw material, a carbon source, a nitrogen source and other nutrients do not need to be additionally added for bacterial culture, the cost for producing the PHA through a biological method is greatly reduced, and the method is a technical method for producing high-value products through the organic garbage, and is low in cost, efficient and good in application prospect.

Description

Technical Field

[0001] The invention belongs to the field of solid waste resource utilization and relates to a two-step fermentation method for producing polyhydroxyalkanoates (PHA) by utilizing organic waste. Background Art

[0002] Polyhydroxyalkanoates (PHA) are a type of high molecular weight biopolyester widely found in microorganisms. They are storage materials that provide carbon sources and energy sources inside bacteria. PHA has good biocompatibility, biodegradability, UV stability, bio-tissue compatibility, and special properties such as plastic thermal processing properties, optical activity, moisture resistance, and low air permeability. It can replace ordinary plastics made from petroleum and has great application research value in various fields such as daily life, medicine, industry, and environmental protection. Because PHA products can be biodegraded after being discarded and will not affect the environment, they are more sustainable than traditional chemical synthetic plastics and have become a research hotspot in the field of biomaterials. At present, PHA production mainly relies on microbial fermentation. Among all the fermentation conditions for the synthesis of PHA by microbial fermentation, carbon source is an important factor, and its cost accounts for 28-50% of the total cost. At present, the production process of PHA uses glucose as the carbon source and pure bacteria with the ability to synthesize PHA as the synthesis medium in a biosynthetic reactor. This process not only increases the carbon source cost by half, but also the large-scale use of glucose to synthesize PHA will cause resource competition. Therefore, the use of cheap carbon sources can greatly reduce the production cost of PHA.

[0003] With the acceleration of my country's urbanization process, the total amount of sewage treatment and the amount of food waste have increased year by year. The annual output of residual sludge is as high as 40-60 million tons, and the annual output of food waste is 90 million tons. It is estimated that by 2025, the world will produce 2.2 billion tons of food waste. Residual sludge and food waste contain a large amount of complex organic matter and harmful substances. Therefore, organic solid waste has the dual attributes of pollution and resources. Treatment can not only realize resource utilization, but also reduce pollution to the environment. Summary of the invention

[0004] The object of the present invention is to provide a two-step fermentation method for producing polyhydroxyalkanoate (PHA) by using organic waste. The method realizes PHA production without adding exogenous carbon source, nitrogen source and other nutrients by controlling fermentation conditions, thereby providing a cheap and efficient biological method for PHA production.

[0005] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0006] A two-step fermentation method for producing PHA using organic waste comprises the following steps: mixing food waste and municipal sludge for anaerobic co-fermentation, pre-treating the co-fermentation sludge to obtain anaerobic co-fermentation liquid, and obtaining a fermentation liquid culture medium; activating Copperophilus nebulosa in a basic culture medium, inoculating the activated well-growing bacterial liquid into the fermentation liquid culture medium for culturing, and synthesizing PHA.

[0007] In the above technical solution, further, the anaerobic co-fermentation process includes the following:

[0008] The kitchen waste and municipal sewage sludge are used as substrates, and the anaerobic digestion sludge is used as inoculum sludge. The substrate and the inoculum sludge are mixed, and the organic load is 20-25g / VS for anaerobic fermentation.

[0009] Furthermore, the volatile solid content ratio of the organic waste and the inoculated mud is 20:4-20:3 g / L, and the fermentation time is 10 days.

[0010] Furthermore, the co-fermentation sludge is pretreated to obtain anaerobic co-fermentation liquid, specifically comprising: placing the co-fermentation sludge in a container and stirring to mix the sludge; then adding an organic coagulant thereto and continuing to stir to fully mix the organic coagulant and the co-fermentation sludge, and when flocculent sludge appears, stirring until clear mud and water are separated and the supernatant is clarified, centrifuging, taking the supernatant and filtering it through a membrane to obtain the fermentation liquid, and storing it at 4°C for later use.

[0011] Furthermore, the organic coagulant is cationic polyacrylamides (CPAM), which has a molecular weight of 8-10 million, an ionicity of 30-35%, and a molecular formula of C 3X H 15X N X O X The dosage of organic coagulant is about 1‰-5‰ of the volume of co-fermentation sludge.

[0012] Furthermore, the fermentation broth medium is obtained by mixing the fermentation broth and water in a volume ratio of 1:5-1:10. Preferably, the volume ratio is 1:9.

[0013] Furthermore, the hookworm copper-greedy bacteria is Cupriavidus necator DSM428, which is from the China Microbiological Culture Collection Management Center, with the number 1.7092.

[0014] Furthermore, the activated bacterial liquid is inoculated into the basic culture medium at an inoculation rate of 0.8-1.2% (v%) for subculture; then the bacterial bodies in the logarithmic growth phase are added to the fermentation medium at an inoculation rate of 1% to produce PHA.

[0015] Furthermore, the basic culture medium contains: 10 g / L peptone, 3 g / L beef extract and 5 g / L sodium chloride, and the pH is 7.0.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects:

[0017] The present invention adopts mixed bacterial fermentation of kitchen waste and municipal sludge (first step fermentation) to convert organic matter into a fermentation liquid mainly composed of acetic acid and butyric acid, and then dehydrates and conditions the fermented sludge with cationic polyacrylamide, and finally uses the wastewater of anaerobic co-fermentation sludge after being treated with a dehydration conditioner as a carbon source to culture and synthesize PHA by hookworm greedy copper bacteria (Cupriavidus.necator) (second step fermentation). In this method, the metabolic intermediates produced by organic waste fermentation have the advantages of no corrosiveness, no change in filtrate properties, good dehydration performance, no secondary pollution, and the ability to provide nutrients for bacterial growth, and the carbon source type and nutrient element composition therein can significantly improve the cumulative PHA content in the growth cells of hookworm greedy copper bacteria, and the PHA yield is as high as 73.68%. This method uses organic waste as the only raw material, does not need to add carbon source, nitrogen source and other nutrients for bacterial culture, greatly reduces the cost of biological production of PHA, is a cheap, efficient, good application prospect, and a technical method for producing high-value products using organic waste. In addition, the resource utilization method of restaurant kitchen waste and municipal sludge provided by the present invention can not only solve the environmental pollution problems such as wastewater and waste gas caused by improper treatment of organic solid waste, but also restaurant kitchen waste and municipal sludge are widely available and inexpensive, and using them as carbon sources can reduce the production cost of PHA. Among them, using cationic polyacrylamide as a dehydrating agent to treat sludge can not only maintain the stability of water quality before and after treatment, but also does not cause secondary pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the growth curve of Hookworm Cuprix in basal medium and fermentation broth medium.

[0019] Figure 2 Comparison of PHA content in microbial cells in different culture media under the same culture conditions. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0021] The present invention uses a high-efficiency bacterium to synthesize polyhydroxyalkanoates from food waste and municipal sludge fermentation liquid, and the used strain is Cupriavidus necator (DSM428), CGMCC No. 1.709. The method uses the fermentation liquid medium obtained by anaerobic co-fermentation of food waste and municipal sludge by the Cupriavidus necator as a carbon source, and efficiently synthesizes PHA without adding any external carbon source, nitrogen source and other nutrient additives. The method includes the following steps:

[0022] 1. First, anaerobic co-fermentation of kitchen waste and municipal sludge is carried out;

[0023] 2. Pre-treating the co-fermentation sludge to obtain anaerobic co-fermentation liquid and preparing the fermentation liquid culture medium;

[0024] 3. Activate the copper-greedy bacteria of hookworm in the basic culture medium, inoculate the activated bacteria with good growth into the fermentation medium for cultivation, and synthesize PHA.

[0025] Specific:

[0026] Step 1: Convert the degradable organic matter in the organic waste into a fermentation liquid with acetic acid and butyric acid as the main components (i.e. the first fermentation step)

[0027] Step 1-1: The volatile solid contents of the food waste, municipal sludge and inoculated sludge are approximately 82, 9.76 and 23.14 g / L, respectively.

[0028] Step 1-2: The anaerobic co-fermentation batch experiment of food waste and municipal sludge is used to prepare the fermentation liquid, which includes the following steps: food waste and municipal sludge are selected as substrates, anaerobic digestion mud is used as inoculation mud, the volume ratio is 1:1, the working volume is 500mL, the organic load is about 20-25g / VS, and the anaerobic batch experiment is carried out, and the reaction time is 10 days; Step 2: The fermentation liquid is conditioned with a cationic flocculant to remove solid particles and microorganisms therein.

[0029] Step 2-1: Using fermentation mud to prepare fermentation liquid includes the following steps:

[0030] 200mL of sludge was measured and placed in a 500mL beaker, and the sludge was manually stirred to mix; then, an organic coagulant was added to the beaker and continued to be manually stirred to fully mix the organic coagulant and the fermented sludge. When flocculent sludge appeared, it was slowly stirred until the mud and water were clearly separated and the supernatant was relatively clear, and then centrifuged at 5000rmp / min for 5 minutes. Then, the supernatant after centrifugation was filtered through a 0.45 micron filter membrane to obtain the fermentation liquid, which was stored at 4°C for later use. The organic coagulant was cationic polyacrylamides (CPAM) (molecular weight of 8-10 million, ionicity of 30-35%, and molecular formula of C 3X H 15X N X O X ), the dosage of organic coagulant is about 1‰-5‰ of the sludge volume;

[0031] Step 2-2: The fermentation liquid culture medium comprises fermentation liquid: water = 1:9 (v / v), and the fermentation liquid is the supernatant of the anaerobic fermentation mud after being treated with an organic coagulant.

[0032] Step 2-3: Using fermentation liquid as carbon source, wherein the carbon source is mainly volatile fatty acids (Volatile Fatty Acids, VFAs), with a content of about 1.5-2g / L. The present invention performs anaerobic co-fermentation on organic waste, and utilizes volatile fatty acids, an important product of the acidification stage, as a substrate for synthesizing biomass PHAs. The co-fermentation of municipal sludge and kitchen waste can well make up for the deficiencies of each fermentation separately, achieve synergistic effects, significantly improve the performance of the fermentation system, and increase the output of VFAs. Using volatile fatty acids produced by anaerobic co-fermentation as raw materials can effectively reduce the production cost of PHA, and also realize the resource utilization of organic waste.

[0033] Step 3: Using the fermentation liquid of the anaerobic co-fermentation sludge treated with a dehydration conditioner as a carbon source, Cupriavidus necator is cultured to synthesize PHA (i.e., the second step of fermentation)

[0034] Step 3-1: activation and cultivation of the hookworm copper bacteria strain, comprising the following steps:

[0035] After disinfecting the surface of the ampoule with 75% alcohol absorbent cotton, heat the top of the ampoule with a flame, drip a small amount of sterile water onto the top of the heated ampoule to break it, and knock down the broken ampoule with tweezers; use a sterile pipette to draw about 0.3 ml of a suitable basic culture medium and drip it into the ampoule, gently shake it to dissolve the freeze-dried bacteria and make it suspended, draw all the bacterial suspension, transplant it into the basic culture medium, and culture it at 30°C and 150r / min for 36-48h for activation; inoculate the activated bacterial liquid into the basic culture medium at an inoculation rate of 0.8-1.2% (v%) for subculture; then take the bacteria in the logarithmic growth period and add it to the fermentation medium at an inoculation rate of 1% to culture and produce PHA.

[0036] The basic culture medium contains: 10g / L peptone, 3g / L beef extract and 5g / L sodium chloride, and the pH value is 7.0.

[0037] Step 3-2: After culturing for 18 hours, pretreat the bacterial solution and determine the PHA content.

[0038] The pretreatment includes taking an appropriate amount of bacterial liquid into a centrifuge tube, centrifuging at 8000r / min for 5 minutes, freeze-drying and then derivatizing the sample. The freeze-dried sample is pre-esterified and the PHA content is detected by gas chromatography.

[0039] The ratio of polyhydroxyalkanoates to cell dry weight was measured after culturing Cupriavidus necator (DSM428) in basal medium and fermentation medium for 18 hours, and the ratio was about 1.2-1.4% in basal medium, the PHA yield was about 0.09-0.1 g / L, and about 47.8-73.68% in fermentation medium. When PHA was synthesized using fermentation broth as a carbon source, the PHA yield was 3 g / L-5 g / L.

[0040] Example 1: Fermentation of organic waste to produce fermentation liquid with acetic acid and butyric acid as the main components 1.1 Take kitchen waste from the school cafeteria, remove bones and other difficult-to-crush materials, and then use a meat grinder to crush other materials and mix them thoroughly. Municipal sludge was taken from the primary sedimentation mud of a sewage treatment plant, and anaerobic digestion mud (thermal hydrolysis anaerobic digestion mud from a sewage treatment plant) was used as inoculation mud. The volume ratio of substrate to inoculation mud is 1:1, the working volume is 500ml, and the organic load is about 20g / VS. Batch experiments are carried out, and the specific parameter ratios are shown in Table 1 below:

[0041] Table 1: Batch experimental parameters for anaerobic co-fermentation of food waste and municipal sludge

[0042]

[0043] Take samples after anaerobic fermentation reaction, take 50 mL of samples from each experimental group and put them in a centrifuge tube and place them at 4°C for later use.

[0044] 1.2 Weigh 2g of cationic polyacrylamides (CPAM, molecular weight of 8-10 million, ionicity of 30-35%, molecular formula of C3XH15XNXOX) and dissolve it in 100mL of deionized water. Dissolve it fully under the action of a magnetic stirrer (stir for about 2h). Prepare 2% (v / v) polyacrylamide sludge flocculant (2% (v / v) CPAM).

[0045] 1.3 Take 50mL of the fermented mud and put it into a 250mL beaker, place it on a magnetic stirrer, stir slowly and mix thoroughly, add 2% CPAM in small amounts and multiple times until there is obvious separation of mud and water and the supernatant is clear, then stop adding CPAM. Pour the mud and water mixture into a 50mL centrifuge tube, centrifuge at 5000rmp for 5min, take the supernatant and filter it through a 0.45μm filter membrane, then store it at 4℃ for later use.

[0046] Example 2: Synthesis of PHA from food waste and municipal sludge fermentation liquid using Cupriavidus necator (DSM428)

[0047] In order to explore the efficiency of high-efficiency bacteria in producing PHA using cheap carbon sources, anaerobic fermentation of kitchen waste and municipal sludge was used, and the fermented sludge was deeply dehydrated using a cationic polyacrylamide organic flocculant. The supernatant was taken as a culture medium to culture Cupriavidus necator to produce PHA, which achieved the reduction and harmlessness of organic solid waste and the process of converting waste into high-value products. The strain selected in the present invention is Cupriavidus necator (DSM428), purchased from the China General Microbial Culture Collection Management Center, No. 1.7092.

[0048] 2.1 Take 30 mL of the supernatant after the membrane in a clean bench and dilute it to 300 mL (i.e. dilute it 10 times with deionized water) as the fermentation medium for culturing the hookworm copper bacteria;

[0049] 2.2 Take the bacterial solution in the logarithmic growth period (the bacterial solution of hookworm greedy copper bacteria cultured for 14 hours after subculture) and inoculate it into the fermentation medium at an inoculation rate of 1% (v%). Six 150mL conical flasks were used, each containing 50mL of liquid. The culture was carried out at 30°C and a shaking speed of 150r / min. Among them, three bottles (numbered 10d-1, 10d-2, and 10d-3) were used to determine the growth curve of the strain in the fermentation medium (2mL of bacterial solution was taken in the clean bench every 2 hours and the absorbance was measured at 600nm until 48 hours). Its growth curve is as follows Figure 1As shown, it can be seen that the growth rate of Hookworm Cuprifolia in the fermentation broth medium is similar to that in the LB basic medium.

[0050] 2.3 After culturing the remaining 3 bottles (numbered 10d-4, 10d-5, 10d-6) for 18 hours, take all the bacterial solution (50mL) into a 50mL centrifuge tube in a clean bench, centrifuge at 8000rmp for 5 minutes in a high-speed refrigerated centrifuge, pour out the supernatant solution, and then wash the precipitate with 2mL of ultrapure water, use a pipette to repeatedly blow and resuspend, centrifuge at 8000rmp for 5 minutes, and discard the supernatant. The obtained precipitate was frozen in a -80℃ refrigerator for 24 hours, and the frozen bacteria were placed in a freeze dryer for freeze drying for 48 hours, and then crushed to obtain bacterial powder.

[0051] 2.4 Take the above-obtained 5 mg cell dry weight (Cell Dry Weight, CDW) in a 15 ml Corning centrifuge tube, add 1 mL of chloroform and 1 mL of acidified methanol (85% methanol; 15% H2SO4 v / v), incubate at 105°C for 2 h, and then quickly cool with running water; then add 1 mL of chloroform containing 0.2% (v / v) methyl benzoate and 1 mL of deionized water for washing, manually shake the test tube for 30 seconds, let it stand for 10 minutes to separate the phases, discard the aqueous phase, transfer the organic phase to a 2 mL centrifuge tube, add 1 mL of deionized water, shake the centrifuge tube to mix the phases, centrifuge at 5000 r / min for 2 minutes, and finally transfer about 0.5 mL of PHBV sample from the organic phase (bottom) to a gas chromatography (GC) bottle.

[0052] 2.5 The gas chromatograph used for the determination of PHA was Agilent 7890A GC, the chromatographic column was Agilent 125-3237DB-FFAP capillary column (30mX0.32mmX0.25um), the standard sample was Poly (3HB-co-HV), the PHV content was 8mol%, the PHB content was 92mol% (Sigma, cat NO.80181, 403105). Chromatographic conditions: injection port 250℃, carrier gas was N2, pressure 5psi, flow rate 40mL / min, split ratio was 10:1. Column box: initial temperature 40℃, maintained for 1min, then increased to 140℃ at a speed of 110℃ / min, maintained for 2min, detector: FID detector, temperature 250℃, H2 flow rate 35mL / min, air flow rate 350mL / min. The gas chromatogram of the product (PHA) obtained by the method of the present invention shows that the PHB peak is at 5.728 min and the PHV peak is at 6.184 min. The gas chromatogram is used to analyze the ability of the strain to synthesize PHA by calculating the PHA content and PHA yield after 18 h of cultivation.

[0053] 2.6. The PHA content of CuCl2 in the fermentation medium was 47.85%-73.68%, which was significantly higher than that of the microbial cells cultured in synthetic medium under the same culture conditions. Figure 2 shown.

[0054] 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 two-step fermentation method for producing PHA using organic waste, characterized in that: The method comprises the following steps: mixing food waste and municipal sludge for anaerobic co-fermentation, pre-treating the co-fermentation sludge to obtain anaerobic co-fermentation liquid, and obtaining a fermentation liquid culture medium; activating hookworm copper-grabbing bacteria in a basic culture medium, inoculating the activated bacterial liquid into the fermentation liquid culture medium for culturing, and synthesizing PHA.

2. The two-step fermentation method for producing PHA using organic waste according to claim 1, characterized in that: The anaerobic co-fermentation process comprises the following: The kitchen waste and municipal sewage sludge are used as substrates, and the anaerobic digestion sludge is used as inoculum sludge. The substrate and the inoculum sludge are mixed, and the organic load is 20-25g / VS for anaerobic fermentation.

3. The two-step fermentation method for producing PHA using organic waste according to claim 2, characterized in that: The volatile solid content ratio of the organic waste and the inoculated mud is 20:4-20:3 g / L, and the fermentation time is 10 days.

4. The two-step fermentation method for producing PHA using organic waste according to claim 1, characterized in that: The pretreatment of the co-fermentation sludge to obtain the anaerobic co-fermentation liquid specifically includes: placing the co-fermentation sludge in a container and stirring to mix the sludge; then adding an organic coagulant thereto and continuing to stir to fully mix the organic coagulant and the co-fermentation sludge, and when flocculent sludge appears, stirring until clear mud and water are separated and the supernatant is clarified, centrifuging, taking the supernatant and filtering the membrane to obtain the fermentation liquid, and storing it at 4°C for later use.

5. The two-step fermentation method for producing PHA using organic waste according to claim 4, characterized in that: The organic coagulant is cationic polyacrylamide CPAM, with a molecular weight of 8-10 million and an ionicity of 30-35%. The dosage of the organic coagulant is 1‰-5‰ of the volume of the co-fermented sludge.

6. The two-step fermentation method for producing PHA using organic waste according to claim 1, characterized in that: The fermentation liquid culture medium is obtained by mixing fermentation liquid and water in a volume ratio of 1:5-1:

10.

7. The two-step fermentation method for producing PHA using organic waste according to claim 1, characterized in that: The hookworm copper-greedy bacteria is Cupriavidus necator DSM428, which is from the China Microbiological Culture Collection Management Center and is numbered 1.7092.

8. The two-step fermentation method for producing PHA using organic waste according to claim 1, characterized in that: The activated bacterial liquid is inoculated into the basic culture medium at a volume ratio of 0.8-1.2% for subculture; then the bacterial bodies in the logarithmic growth phase are added into the fermentation medium at a volume ratio of 1% to produce PHA.

Images