Method for enhancing synthesis of high-salt mixed microflora polyhydroxyalkanoate by using metabolism regulator

Abstract

The invention discloses a method for enhancing synthesis of high-salt mixed microflora polyhydroxyalkanoate by using a metabolism regulator, and belongs to the field of biodegradable plastic synthesis and waste resource recovery. In order to solve the problems of poor activity and the like caused by high-salinity stress of microorganisms in a mixed flora PHA synthesis process under high salinity, a metabolism regulator is added in an enrichment process of the PHA-producing mixed flora and a PHA synthesis process, so that the enrichment efficiency of the PHA-producing mixed flora under a high-salinity environment and the mass synthesis of PHA are improved. When the duration ratio of the satiation stage to the starvation stage of the system is stabilized to be 0.2-0.3 or below, it is considered that the enrichment requirement of the PHA-producing mixed flora is met, and when the maximum PHA synthesis capacity is enhanced and is kept stable, it is considered that PHA synthesis under high salinity is effectively improved. According to the method, efficient enrichment of the PHA-producing mixed flora in the high-salinity environment can be achieved, meanwhile, the PHA yield under the high-salinity environment is increased, and a new thought is provided for efficient resource recycling of salt-containing waste in the form of PHA.

Description

Technical Field

[0001] The invention belongs to the field of biodegradable plastic synthesis and waste resource recovery, and specifically relates to a method for enhancing the synthesis of high-salt mixed bacterial community polyhydroxyalkanoate by utilizing a metabolic regulator. Background Art

[0002] Polyhydroxyalkanoate (PHA) is a green and environmentally friendly plastic synthesized by microorganisms. It has the characteristics of high quality, durability and biodegradability, and has become an ideal substitute for traditional petroleum-based plastics. The mixed bacterial community PHA synthesis process will become the research focus in the field of PHA synthesis in the future because it can utilize complex waste carbon sources and does not require sterilization. With the development of my country's food, pharmaceutical, petroleum and other industries, more and more salt-containing wastes are produced, and a large amount of organic matter rich in them needs to be recycled. Recycling them in the form of PHA is a very promising way to recycle waste resources. The mixed bacterial community PHA synthesis process is the best choice for recycling organic matter in saline waste. However, high salinity (salinity>1%) has always been an important limiting factor for microbial diversity, abundance and activity, resulting in low utilization of organic matter and even causing the collapse of the microbial system. Therefore, successfully enriching PHA synthesis mixed bacterial communities under high salinity and increasing PHA production at the same time has become a key problem in the resource utilization of saline waste. Summary of the invention

[0003] In view of the problem that the PHA production mixed bacterial community enrichment system is unstable and PHA synthesis is inhibited under high salinity, the present invention provides a method for enhancing the synthesis of polyhydroxyalkanoate by high-salinity mixed bacterial community using a metabolic regulator. By adding an appropriate amount of metabolic regulator to the PHA production mixed bacterial community enrichment system, the stable operation of the PHA production mixed bacterial community enrichment system under high salinity environment is achieved, and the PHA production under high salinity is increased.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A method for enhancing the synthesis of polyhydroxyalkanoate by a high-salt mixed bacterial community using a metabolic regulator comprises the following steps:

[0006] Step 1: Run a sequencing batch reactor to enrich the PHA-producing mixed bacterial community using a "Feast-Famine" enrichment mode. The stage in which the microorganisms utilize carbon sources to synthesize PHA is the Feast stage. After the carbon source is fully utilized, the stage in which the microorganisms utilize PHA to survive is the Famine stage. The salinity of the substrate I in the sequencing batch reactor is 1%-3%. During the operation, a metabolic regulator is added to the sequencing batch reactor in stages or throughout the entire process.

[0007] Step 2: using the sludge discharged from the sequencing batch reactor as seed sludge in the PHA synthesis device, adding substrate II, and adding a metabolic regulator to the PHA synthesis device during the PHA synthesis process;

[0008] The metabolic regulator includes a combination of one or more of betaine, ectoine, trehalose, glutamic acid, and mannitol.

[0009] Furthermore, the concentration of the metabolic regulator in the sequencing batch reactor is 100-250 mg / L, and the maximum dosage should not exceed 250 mg / L. The concentration of the metabolic regulator in the PHA synthesis device is 100-250 mg / L.

[0010] Furthermore, the substrate I includes a carbon source containing volatile fatty acids, a nitrogen source, a phosphorus source and other nutrient elements; the substrate II includes a carbon source containing volatile fatty acids. The carbon source containing volatile fatty acids includes any proportion combination of small molecular volatile fatty acids such as acetic acid, propionic acid, butyric acid and valeric acid; or sludge acid-producing liquid, kitchen waste acid-producing liquid, etc. The nitrogen source is NH4Cl, the phosphorus source is KH2PO4, and other nutrient elements include MgSO4, CaCl2, thiourea and trace elements, and the trace elements are provided by EDTA, H3BO3, CoCl2, CuSO4, FeCl3·6H2O, ZnSO4·7H2O, MnCl2·H2O and Na2MoO4·2H2O.

[0011] Furthermore, in step 1, the sludge retention time (SRT) in the sequencing batch reactor is 10 days, the hydraulic retention time (HRT) is 24 hours, and the operation cycle is 12 hours.

[0012] Furthermore, in step 1, the salinity of substrate Ⅰ is determined by Na + Mg 2+ , Ca 2+ , K + One or more ions are provided.

[0013] Furthermore, in step 1, the influent load range of the sequencing batch reactor is 1000-2500 mg COD / L / d, and its maximum influent load should not exceed 2500 mg COD / L / d.

[0014] Furthermore, in step 1, the temperature of the sequencing batch reactor is maintained at 15-25° C. by heating with a heating belt.

[0015] Furthermore, in step one, aeration is introduced into the sequencing batch reactor through an aeration device, and the aeration flow rate is controlled by a flow controller to maintain the dissolved oxygen concentration of the sequencing batch reactor at 7±1 mg / L in the "satiety" stage; when the "hunger" / "satiety" stage duration ratio (F / F ratio) in the sequencing batch reactor is less than 0.2-0.3, it is considered that the enrichment system can efficiently enrich the PHA-producing mixed bacterial community.

[0016] Furthermore, in step 2, a pulse feeding method is used to add substrate II to the PHA synthesis device. Specifically, after adding substrate II to the PHA synthesis device, the reaction is carried out for 1.5 hours, and then the sedimentation is carried out for 0.5 hours to separate the mud and water. After the supernatant is discharged, substrate II is continued to be added for reaction, and the reaction and precipitation are continued for 5-6 times to obtain the maximum PHA yield.

[0017] Furthermore, in step 2, the concentration of substrate II is 3000-4500 mg COD / L.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention utilizes the metabolic regulation function of a metabolic regulator to enhance the biosynthesis of PHA in a high-salt environment. On the one hand, the metabolic regulator used in the present invention can maintain the intracellular and extracellular osmotic pressure balance of PHA-producing bacteria, prevent them from being dehydrated and dying in a high-salt environment, and is conducive to the enrichment of PHA-producing microorganisms in a high-salt environment; on the other hand, the metabolic regulator used in the present invention promotes the absorption of nutrients and the discharge of metabolites by increasing the permeability of the cell membrane, and at the same time, by enhancing the expression of electron transfer-related genes, it improves the metabolic activity of microorganisms, enhances the utilization of organic matter and the synthesis of PHA by PHA-producing bacteria. The synergistic effect of these characteristics of the metabolic regulator enables PHA-producing bacteria to still efficiently synthesize PHA in a high-salt environment. Therefore, the method proposed in the present invention of enhancing the synthesis of PHA by a high-salt mixed bacterial community using a metabolic regulator not only solves the technical problems of slow enrichment of PHA-producing microorganisms and low PHA synthesis efficiency in a high-salt environment, but also demonstrates the unique advantages of the metabolic regulator used in the present invention in new field applications. This invention provides a new idea for the resource recovery of salt-containing waste in the form of PHA. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a schematic diagram of an experimental device of the present invention;

[0021] Figure 2 is the change in the F / F ratio during the enrichment process;

[0022] Figure 3 is the change in maximum PHA production during the enrichment process;

[0023] In the figure, 1, sequencing batch reactor, 2, PHA synthesis device, 1-1, stirring paddle Ⅰ, 1-2, aeration pump Ⅰ, 1-3, aeration tube Ⅰ, 1-4, dissolved oxygen online monitoring device, 1-5, pH online monitoring device, 1-6, heating belt, 1-7, temperature control device, 1-8, carbon source storage barrel Ⅰ, 1-9, other nutrient storage barrels, 1-10, feed pump, 1-11, sludge pump, 1-12, drainage pump, 2-1, stirring paddle Ⅱ, 2-2, aeration pump Ⅱ, 2-3, aeration tube Ⅱ, 2-4, carbon source storage barrel Ⅱ. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be clearly and completely described below in conjunction with the drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0025] The present invention aims at the problem of poor activity of microorganisms in the mixed bacterial community PHA synthesis process under high salinity due to high salinity stress. The present invention selects metabolic regulators to improve the enrichment efficiency of the PHA-producing mixed bacterial community and the large-scale synthesis of PHA under high salinity environment. The specific method is: adding an appropriate amount of metabolic regulator to the high-salinity PHA-producing mixed bacterial community enrichment system, the system satiation and hunger stage duration ratio is stable below 0.2-0.3, which is considered to meet the enrichment requirements of the PHA-producing mixed bacterial community, and its maximum PHA synthesis capacity is enhanced and remains stable, which is considered to have achieved an effective improvement in PHA synthesis under high salinity. The present invention can achieve efficient enrichment of PHA-producing mixed bacterial communities under high salinity environments, while improving PHA production under high salinity, providing a new idea for efficient resource recovery of salt-containing waste in the form of PHA.

[0026] The schematic diagram of the experimental device of the present invention is as follows Figure 1As shown, it includes a sequencing batch reactor 1 and a PHA synthesis device 2, wherein the sequencing batch reactor 1 is provided with a stirring paddle Ⅰ1-1 for stirring the substrate Ⅰ, an aeration pump Ⅰ1-2 aerates the interior of the sequencing batch reactor through an aeration pipe Ⅰ1-3, and an online dissolved oxygen monitoring device 1-4 and an online pH monitoring device 1-5 are used to measure the dissolved oxygen concentration and pH in the sequencing batch reactor; a heating belt 1-6 is provided on the outer side wall of the bottom of the sequencing batch reactor 1, and the heating belt 1-6 is electrically connected to the temperature control device 1-7 The sequencing batch reactor 1 is connected to maintain the temperature inside the sequencing batch reactor; the side wall of the sequencing batch reactor 1 is provided with a feed port, a mud discharge port and a drain port, the carbon source storage barrel Ⅰ1-8 and other nutrient storage barrels 1-9 are connected to the feed port through pipeline Ⅰ, and a feed pump 1-10 is provided on the pipeline Ⅰ; the mud discharge port is connected to the PHA synthesis device 2 through pipeline Ⅱ, and a mud discharge pump 1-11 is provided on the pipeline Ⅱ, and the drain port is connected to pipeline Ⅲ for drainage, and a drainage pump 1-12 is provided on the pipeline Ⅲ. The interior of the PHA synthesis device 2 is provided with a stirring paddle Ⅱ2-1 for stirring the substrate Ⅱ, and an aeration pump Ⅱ2-2 aerates the interior of the PHA synthesis device through an aeration pipe Ⅱ2-3, and the carbon source storage barrel Ⅱ2-4 is connected to the PHA synthesis device through pipeline Ⅳ.

[0027] The PHA-producing mixed bacterial community enrichment reaction adopts a sequencing batch operation mode, and the PHA synthesis adopts a batch operation mode. The metabolic regulator is added to the feed of the PHA-producing mixed bacterial community enrichment reaction and the PHA synthesis reaction.

[0028] Embodiment 1:

[0029] An application example of a method for enhancing the synthesis of polyhydroxyalkanoate by high-salt mixed bacterial flora using betaine is as follows:

[0030] (1) Two sequencing batch reactors (SBR_A and SBR_B) were operated simultaneously to enrich the PHA-producing mixed bacterial community, with SBR_A serving as the control group. The operating temperature of the two SBRs was 25±2℃, the SRT was 10d, the HRT was 24h, the salinity was 1.8±0.2%, and the volumetric load was 1600±200mg COD / L / d;

[0031] (2) The substrate carbon sources of both SBRs included acetic acid, propionic acid, n-butyric acid, and n-valeric acid (molar ratio of 2:1:6:1). In addition to the carbon source, the feed also contained 367 mg / L NH4Cl, 105 mg / L KH2PO4, 100 mg / L thiourea, 100 mg / L CaCl2, 250 mg / L MgSO4, and 1 mL / L trace elements (the trace elements were 40

[0032] The feed was composed of 1 mol / L EDTA, 0.3 mg / L H3BO3, 0.3 mg / L CoCl2·6H2O, 0.06 mg / L CuSO4·5H2O, 3 mg / L FeCl3·6H2O, 0.24 mg / L ZnSO4·7H2O, 0.24 mg / L MnCl2·H2O and 0.12 mg / L Na2MoO4·2H2O), and the feed pH was adjusted to 7.0±0.5 using 1 mol / L NaOH. In addition, the feed of SBR_B also contained 200 mg / L anhydrous betaine;

[0033] (3) The operation F / F of two SBRs is as follows Figure 2 As shown, the F / F ratios of SBR_A and SBR_B were stabilized below 0.05 on the 13th and 10th days of operation, respectively, both meeting the enrichment requirements of the PHA-producing mixed bacterial community;

[0034] (4) The sludge discharged from the two SBRs was used as seed sludge for two PHA synthesis units, and the PHA synthesis was carried out by pulse feeding (reaction 1.5 h, precipitation 0.5 h). The feed of the two PHA synthesis units only contained carbon source with a concentration of 3200 mg / L (the molar ratio of acetic acid, propionic acid, butyric acid and valeric acid was 2:1:6:1). In addition, the feed of the PHA synthesis unit corresponding to SBR_B also contained 200 mg / L of anhydrous betaine. The feed was fed 5 times to ensure the maximum PHA yield.

[0035] (5) The maximum PHA synthesis capacity of the sludge from the two SBRs was tested on the 0th, 10th, 30th, 60th, 80th, and 100th days of operation. The results are as follows: Figure 3 As shown in the figure, on the 10th day, the maximum PHA yields of SBR_A and SBR_B were 0.293±0.016 and 0.467±0.003 g PHA / g VSS, respectively, and the maximum PHA yield increased by 59% after adding betaine. On the 30th day, the PHA yields of the two SBRs basically reached the maximum value, and the maximum PHA yields of SBR_A and SBR_B were 0.452±0.014 and 0.586±0.023 g PHA / gVSS, respectively, which increased by 30% after adding betaine. From the 30th day to the 100th day, the maximum PHA yield of the SBR with added betaine was 30-40% higher than that of the SBR without added betaine.

[0036] (6) The addition of betaine allowed the maximum PHA production to approach the peak value more quickly and could maintain high PHA production while operating stably for up to 70 days, which is an effective method to increase PHA synthesis in mixed bacterial communities under high-salinity environments.

[0037] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for enhancing the synthesis of polyhydroxyalkanoate by a high-salt mixed bacterial community using a metabolic regulator, characterized in that: The following steps are involved: Step 1: Run a sequencing batch reactor to enrich the PHA-producing mixed bacterial community using a "starvation-satiation" enrichment mode, wherein the salinity of substrate I in the sequencing batch reactor is 1%-3%; during the operation, a metabolic regulator is added to the sequencing batch reactor in stages or throughout the entire process; Step 2: using the sludge discharged from the sequencing batch reactor as seed sludge in the PHA synthesis device, adding substrate II, and adding a metabolic regulator to the PHA synthesis device during the PHA synthesis process; The metabolic regulator includes a combination of one or more of betaine, ectoine, trehalose, glutamic acid, and mannitol.

2. The method according to claim 1, characterized in that: The concentration of the metabolic regulator in the sequencing batch reactor is 100-250 mg / L, and the concentration of the metabolic regulator in the PHA synthesis device is 100-250 mg / L.

3. The method according to claim 1, characterized in that: The substrate I comprises a carbon source containing volatile fatty acids, a nitrogen source, a phosphorus source and other nutrient elements; the substrate II comprises a carbon source containing volatile fatty acids.

4. The method according to claim 1, characterized in that: In step 1, the sludge retention time in the sequencing batch reactor is 10 days, the hydraulic retention time is 24 hours, and the operation cycle is 12 hours.

5. The method according to claim 1, characterized in that: In step 1, the salinity of substrate Ⅰ is determined by Na + Mg 2+ , Ca 2+ , K + One or more ions are provided.

6. The method according to claim 1, characterized in that: In step 1, the influent load range of the sequencing batch reactor is 1000-2500 mg COD / L / d.

7. The method according to claim 1, characterized in that: In step 1, the temperature of the sequencing batch reactor is maintained at 15-25°C.

8. The method according to claim 1, characterized in that: In step 1, the dissolved oxygen concentration of the sequencing batch reactor in the "satiation" stage is maintained at 7±1 mg / L through an aeration device; the duration ratio of the "hunger" / "satiation" stage is less than 0.2-0.

3.

9. The method according to claim 1, characterized in that: In step 2, substrate II is added to the PHA synthesis device by pulse feeding. Specifically, after adding substrate II to the PHA synthesis device, react for 1.5 hours, then precipitate for 0.5 hours to separate mud and water, discharge the supernatant and continue to add substrate II for reaction, continue 5-6 reactions and precipitations to obtain the maximum PHA yield.

10. The method according to claim 1, characterized in that: In step 2, the concentration of substrate II is 3000-4500 mg COD / L.

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