Construction method of mainstream short-cut denitrification community aiming at PHA synthesis

Abstract

The invention discloses a construction method of a mainstream short-cut denitrification community with PHA synthesis as a target. According to the method, under mainstream conditions, firstly, dual metabolism selection pressure of aerobic-anoxic alternation and feast-starvation alternation is applied, and resetting of ecological niche of an activated sludge community and directional colonization of Thauera bacteria are rapidly achieved; then, the operation is switched into anoxic banquet-anoxic starvation operation, the PHA synthesis capability of flora is promoted to be remarkably improved by improving the food consumption ratio, and finally, bacterial sludge with high nitrite accumulation rate and ectopic PHA synthesis potential reaching 50% wt or above can be obtained within one month. The community constructed by the method is stable in structure and high in PHA yield, and has good industrial popularization potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of short-cut denitrification, in particular to a method for constructing a main short-cut denitrification community with PHA synthesis as the target. BACKGROUND

[0002] The information disclosed in this Background section is for the purpose of increasing the understanding of the background of the application without admitting that such information forms any part of prior art.

[0003] Polyhydroxyalkanoates (PHA) are a class of biopolymers accumulated in microbial cells. Because they have similar mechanical properties to traditional petroleum-based plastics and can be completely degraded into carbon dioxide (CO2) and water (H2O) in natural environments, they have become a research hotspot in the field of green materials.

[0004] However, the commercialization process of PHA is still constrained by key issues such as high raw material cost, low conversion efficiency, and complex process control in current production technology. Therefore, there is an urgent need to develop new synthesis routes that are economically viable, have high conversion efficiency, and simplify control, in order to promote the large-scale production and application of PHA.

[0005] The patent specification with publication number CN115611416A discloses a method for rapid enrichment of aerobic denitrifying bacteria based on a mixed bacterial community PHA synthesis process. The method uses a sequencing batch operation mode, with a cycle time of 12 hours, including an influent stage (5 minutes), an aerobic abundance stage (2.5 hours), a nitration liquid stage (5 minutes), an aerobic starvation stage (7.5 hours), a standing and sedimentation stage (1.5 hours), and a drainage stage (15 minutes). The operation is maintained at a temperature of 20-25℃, a pH of 7-9, and a sludge age of 10 days, with sludge discharge once a day. After 15 days of operation, the PHA-producing aerobic denitrifying bacterial community enters a stable state. This method can enrich aerobic denitrifying bacteria and combine with the PHA synthesis process, not only accelerating the enrichment speed, but also improving the competitive ability of the bacterial community to improve stability, providing a possible treatment method for nitrate wastewater.

[0006] In recent years, short-cut denitrification, as an efficient and low-cost biological denitrification technology, has gradually gained attention in wastewater treatment. This process uses nitrate as an electron acceptor and reduces it to the nitrite stage. The core functional microorganisms (such as Thiobacillus Thauera ) not only complete denitrification and nitrogen removal, but also exhibit excellent carbon source conversion and storage capacity, providing a new technical possibility for directing organic carbon in wastewater to PHA synthesis.

[0007] However, the existing technology system for producing PHA by short-cut denitrification system is still immature and has obvious limitations: On the one hand, the Thauera bacterial community with high PHA production potential in sludge often has limited abundance and is not dominant in competition with ordinary heterotrophic bacteria, resulting in low overall synthesis efficiency of the system; On the other hand, there is a lack of targeted strengthening strategies for Thauera bacterial carbon source storage metabolic pathways, and the flow direction of carbon sources between denitrification and storage is difficult to accurately control, resulting in insufficient carbon source utilization.

[0008] In addition, the existing methods often regard short-cut denitrification and PHA production as two independent process links, and fail to achieve integrated design and regulation from biological efficient denitrification-colony construction-product synthesis, resulting in a complicated process flow and increased operating cost.

[0009] Therefore, exploring a colony construction method that can co-enrich Thauera bacteria and strengthen their PHA synthesis metabolism is urgently needed to promote the cost reduction and efficiency improvement of PHA production and the upgrading of wastewater treatment resource utilization. SUMMARY

[0010] In view of the technical bottlenecks introduced in the background art, the present application provides a method for constructing a mainstream short-cut denitrification colony with PHA synthesis as the target. The method uses an SBR reactor to apply double metabolic selection pressure of alternating oxygen deficiency and feast-hunger to reset the ecological niche of the activated sludge colony and Thauera directly colonize the bacteria, and then improve the reactor micro-food ratio to significantly promote the PHA synthesis capacity of the bacterial community, and finally obtain a sludge with an ex situ PHA synthesis potential of more than 50%wt. The colony structure constructed by the method is stable, has high PHA yield, and has good potential for industrialization.

[0011] The specific technical solutions are as follows: A method for constructing a mainstream short-cut denitrification colony with PHA synthesis as the target, comprising the steps of: (1) Resetting the ecological niche: inoculating activated sludge into an SBR (sequencing batch reactor) reactor, pumping in mainstream nitrification unit effluent and carbon source, and controlling aeration and stirring to apply double metabolic selection pressure of alternating oxygen deficiency and feast-hunger to pretreat the activated sludge colony, and under the condition of SRT (sludge retention time) = 2-5 d (for example, 2.5 d, etc.), screening out autotrophic bacteria and heterotrophic bacteria that do not have the ability to store PHA under oxygen deficiency and oxygen deficiency in the original colony; (2) Colonization Thauera bacteria: switching the reactor operation mode to anoxic feast-anoxic hunger mode (i.e. anoxic long stirring), and controlling COD VFA / NO3- -N (volatile fatty acid COD / nitrate nitrogen) and SRT, targeted enhancement of stepwise denitrification phenotype Thauera Bacterial proliferation occurs when both the nitrate nitrogen removal rate and nitrite nitrogen accumulation rate in the effluent are stable above 80%. Thauera The bacteria successfully colonized. (3) Enhance the potential for PHA synthesis: Promote the synthesis of PHA by increasing the food-to-microbe ratio in the reactor. Thauera The community is integrated with bacteria as the core until the ectopic aerobic PHA synthesis potential of the bacterial mud reaches more than 50%wt, and the construction of the target community is completed. The ectopic aerobic PHA synthesis potential of the bacterial mud refers to the maximum amount of PHA accumulation in the bacterial mud cells when both carbon source and oxygen are unrestricted.

[0012] In the method for constructing a mainstream short-range denitrification community targeting PHA synthesis, the preferred activated sludge inoculation concentration in step (1) is 1.5~2.5 gVSS L. -1 For example, 2 gVSS L -1 wait.

[0013] In the method for constructing a mainstream short-range denitrification community with PHA synthesis as the target, in step (1), the preferred dual metabolic selection pressure of feast-starvation alternation and aerobic-hypoxia alternation is aerobic feast-aerobic starvation, aerobic feast-hypoxia starvation, or hypoxia feast-aerobic starvation.

[0014] Further preferably, in the aerobic feast-aerobic starvation process, the aerobic feast stage includes the influent of the main nitrification unit and a carbon source, and the COD... VFA The range is 50~500 mg COD L. -1 (e.g., 168 mg COD L) -1 (etc.), by controlling the dissolved oxygen concentration to be higher than 1.0 mg L / L through aeration. -1 After stirring, the sediment settles and is drained. Stirring is restarted while aeration begins, entering the aerobic starvation stage.

[0015] Further preferably, in the aerobic feast-anoxic starvation process, the aerobic feast stage involves adding the influent and carbon source from the main nitrification unit, and the COD... VFA The range is 50~500 mg COD L. -1 (e.g., 168 mg COD L) -1 (etc.), by controlling the dissolved oxygen concentration to be higher than 1.0 mg L / L through aeration. -1 After stirring, the water settles and is drained, followed by an anaerobic starvation stage. The effluent from the main nitrification unit is then added, along with NO3. - -N ranges from 20 to 70 mgN L -1 (e.g., 60 mgN L) -1 (etc.), continue stirring.

[0016] Further preferably, in the anoxic feast-aerobic starvation, the main stream nitrification unit effluent and carbon source are added in the anoxic feast stage, and the COD VFA / NO3 - -N is in the range of 2.5-6.0:1 (for example, 2.8:1, etc.), the water is drained after stirring and sedimentation, the stirring is started again while the dissolved oxygen concentration is controlled to be higher than 1.0 mg / L by aeration -1 .

[0017] Preferably, in the method for constructing the main stream short-cut denitrification community with PHA synthesis as the target, in step (1), the feast stage time is less than 1 / 6 of the total length of each typical cycle. In the present application, one typical cycle includes the feast stage, the sedimentation stage and the starvation stage.

[0018] Preferably, in the method for constructing the main stream short-cut denitrification community with PHA synthesis as the target, in step (2), the COD VFA / NO3 - -N is controlled in the range of 2.5-6.0:1, for example, 2.8:1, etc.

[0019] Preferably, in the method for constructing the main stream short-cut denitrification community with PHA synthesis as the target, in step (2), the SRT is controlled in the range of 2-40 d, for example, 2.5 d, etc.

[0020] Preferably, in the method for constructing the main stream short-cut denitrification community with PHA synthesis as the target, in step (3), the food microorganism ratio is in the range of 1.5-3.5 gCOD -1 gVSS -1 d -1 , for example, 2.5 gCOD -1 gVSS -1 d -1 , etc.

[0021] Compared with the prior art, the present application has the following beneficial effects: The method for constructing the main stream short-cut denitrification community with PHA synthesis as the target in the present application first realizes the reset of the ecological niche of the activated sludge community and the directional colonization of the bacteria by applying the double metabolic selection pressure of aerobic-anoxic alternation and feast-starvation alternation under the main stream condition Thauera ; then the operation is switched to anoxic feast-anoxic starvation, and the PHA synthesis capacity of the bacterial community is promoted by increasing the food microorganism ratio, so that the bacterial sludge with a high nitrite accumulation rate and a heterotrophic PHA synthesis potential of more than 50%wt can be obtained within one month.

[0022] The community constructed by the method in the present application has stable structure and high PHA yield, and has good industrialization promotion potential. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure 1. Nitrate removal rate (NaRE) and nitrite accumulation rate (NiAE) of activated sludge in continuous flow for Example 1.

[0024] Figure 2 Figure 2. Microbial community structure of the starting activated sludge S0 and the sludge N1_4 after cultivation stabilization for Example 1.

[0025] Figure 3 Figure 3. Ex situ PHA content of the starting activated sludge S0 and the sludge N1 after cultivation stabilization for Example 1.

[0026] Figure 4 Figure 4. Nitrate removal rate (NaRE) and nitrite accumulation rate (NiAE) of activated sludge in continuous flow for Example 2.

[0027] Figure 5 Figure 5. Microbial community structure of the starting activated sludge S0 and the sludge N2_4 after cultivation stabilization for Example 2.

[0028] Figure 6 Figure 6. Ex situ PHA content of the starting activated sludge S0 and the sludge N2 after cultivation stabilization for Example 2.

[0029] Figure 7 Figure 7. Nitrate removal rate (NaRE) and nitrite accumulation rate (NiAE) of activated sludge in continuous flow for Example 3.

[0030] Figure 8 Figure 8. Microbial community structure of the starting activated sludge S0 and the sludge N3_4 after cultivation stabilization for Example 3.

[0031] Figure 9 Figure 9. Ex situ PHA content of the starting activated sludge S0 and the sludge N3 after cultivation stabilization for Example 3. DETAILED DESCRIPTION

[0032] The application will be further described with reference to the drawings and specific examples. It should be understood that these examples are intended to illustrate the application and are not intended to limit the scope of the application.

[0033] The following examples were carried out under the conditions indicated, unless otherwise stated.

[0034] Example 1: The sludge concentration was 2.0 g VSS L -1Activated sludge was inoculated into the SBR reactor, and the low SRT (Strain Time) was controlled at 2.5 days. After operating in an aerobic feast-aerobic starvation mode for 5 days, the reactor was switched to an anoxic long-stirring mode (SRT=2.5 days). Once the effluent nitrate nitrogen removal rate and nitrite nitrogen accumulation rate were both stabilized above 80%, the feed-to-microbe ratio in the reactor was controlled at 2.5 g COD. -1 gVSS -1 d -1 Add COD during the aerobic peak stage. VFA The concentration was 168 mg COD L. -1 The nitrification unit's influent is treated with aeration to maintain a dissolved oxygen concentration above 1.0 mg / L. -1 After reacting for 30 minutes, the sediment was drained, and stirring was restarted while aeration began, initiating an aerobic starvation phase that lasted for 105 minutes. The feast phase lasted less than 1 / 6 of the total duration of each typical cycle. The long, anoxic stirring phase controlled COD. VFA / NO3 - -N is 2.8:1.

[0035] like Figure 1 As shown, the system's nitrogen removal rate (NaRE) and nitrogen accumulation rate (NiAE) rapidly increased and stabilized at approximately 95% and 90%, respectively. From... Figure 2 The community structure confirms that Thauera The bacteria have successfully colonized, and autotrophic bacteria and heterotrophic bacteria that lack both aerobic and anaerobic PHA storage capabilities in the original community have been effectively screened out. Using this bacterial sludge for extrinsic aerobic PHA synthesis, the PHA dry weight content of the sludge reached 60%. Figure 3 As shown.

[0036] Example 2: The initial inoculum concentration in the SBR reactor was 2.0 g VSS L. -1 The activated sludge was set with a SRT of 2.5 days and operated for 5 days in an aerobic feast-anoxic starvation mode. COD was added during the aerobic feast period. VFA The concentration was 168 mg COD L. -1 The influent to the nitrification unit is controlled to have dissolved oxygen greater than 1.0 mg / L. -1 After reacting for 30 minutes, the sediment was drained; this was followed by a period of hypoxia and starvation, during which NO3 was supplemented. - -N concentration is 60 mgN L -1 The effluent from the nitrification unit was continuously stirred for 105 min under anoxic conditions; the feast phase lasted less than 1 / 6 of the total duration of each typical cycle. Then, the reactor was switched to anoxic long-stirring mode with a SRT of 2.5 days, during which COD was controlled. VFA / NO3 -After achieving a -N ratio of 2.8:1 and stabilizing the effluent nitrate removal rate and nitrite accumulation rate above 80%, the feed-to-microbe ratio in the reactor was adjusted to maintain at 2.5 gCOD. -1 gVSS -1 d -1 .

[0037] System performance such as Figure 4 As shown, after treatment with this model, NaRE and NiAE rapidly increased and eventually stabilized at high levels of 97% and 90%, respectively. Microbial community analysis results. Figure 5 show, Thauera The fungus has become the dominant genus, confirming its successful colonization, while autotrophic bacteria and heterotrophic bacteria in the original community that lack both aerobic and anaerobic PHA storage capabilities have been effectively eliminated. Further utilization of this fungal sludge for extrinsic aerobic PHA synthesis yielded a PHA dry weight content of 66%. Figure 6 ).

[0038] Example 3: The inoculum sludge concentration in the SBR reactor was 2.0 gVSS L. -1 SRT was controlled at 2.5 days. The system operated for 5 days under anoxic-aerobic starvation mode, with COD added during the anoxic period. VFA / NO3 - The effluent from the nitrification unit with a nitrogen-to-hydrogen ratio of 2.8:1 was discharged after 30 minutes of reaction, followed by a period of aerobic starvation, during which dissolved oxygen was maintained above 1.0 mg / L through aeration. -1 The process lasted 105 minutes; the feast phase lasted less than 1 / 6 of the total duration of each typical cycle. Afterward, the reactor was switched to anoxic long-stirring mode with a SRT of 2.5 days, during which COD was controlled. VFA / NO3 - After achieving a nitrogen-to-nitrate ratio of 2.8:1 and ensuring that both the nitrate removal rate and nitrite accumulation rate in the effluent remained stable above 80%, the feed-to-microbe ratio in the reactor was controlled at 2.5 gCOD. -1 gVSS -1 d -1 .

[0039] from Figure 7 The system performance curves show that the system can start up quickly and reach a highly efficient and stable short-range denitrification state. Its NaRE and NiAE levels rapidly increase in a short period and eventually stabilize at around 95% and 90%, respectively. Community analysis was performed on the sludge during the stabilization period. Figure 8 The results showed Thauera The bacteria were specifically enriched, demonstrating the effectiveness of this method in targeted screening of target functional bacteria and removal of non-target bacteria. Ex-situ aerobic PHA synthesis was performed using this bacterial sludge, achieving a PHA dry weight content of 56%. Figure 9 ).

[0040] It is to be understood that even though numerous characteristics and embodiments of the application have been set forth in the foregoing disclosure, the details can be varied without departing from the application, which is defined by the claims.

Claims

1. A method for constructing a mainstream short-cut denitrifying community targeting PHA synthesis, characterized by, The method comprises the steps of: (1) resetting the ecological niche: inoculating activated sludge into the SBR reactor, pumping in the effluent of the main stream nitrification unit and carbon source, controlling aeration and stirring to exert feast-famine alternation and aerobic-anoxic alternation double metabolic selection pressure on the activated sludge community for pretreatment, and screening out autotrophic bacteria and heterotrophic bacteria without the ability of storing PHA under aerobic and anoxic conditions in the original community under the condition of SRT=2-5 d; (2) colonization Thauera bacteria: reactor operation mode switching to anoxic feast-anoxic famine mode, regulating COD VFA / NO3 - -N and SRT, and the Thauera bacteria proliferation, when the effluent nitrate nitrogen removal rate and nitrite nitrogen accumulation rate are both stable at more than 80% Thauera bacteria colonization is successful; (3) Enhance the potential for PHA synthesis: Promote the synthesis of PHA by increasing the food-to-microbe ratio in the reactor. Thauera The community is integrated with bacteria as the core until the ectopic aerobic PHA synthesis potential of the bacterial mud reaches more than 50%wt, and the construction of the target community is completed. The ectopic aerobic PHA synthesis potential of the bacterial mud refers to the maximum amount of PHA accumulation in the bacterial mud cells when both carbon source and oxygen are unrestricted.

2. The construction method of claim 1, wherein, In step (1), the activated sludge was inoculated at a concentration of 1.5 to 2.5 g VSS L -1 .

3. The construction method of claim 1, wherein, In step (1), the double metabolic selection pressure of feast-famine alternation and aerobic-anoxic alternation is aerobic feast-aerobic famine, aerobic feast-anoxic famine or anoxic feast-aerobic famine.

4. The construction method according to claim 3, characterized in that, In the aerobic feast-aerobic famine, the influent and carbon source are added into the mainstream nitrification unit in the aerobic feast stage, and the COD VFA range is 50~500 mgCOD L -1 The dissolved oxygen concentration is controlled to be higher than 1.0 mg / L by aeration -1 After stirring and sedimentation, the water is discharged, and the stirring is started again while aeration enters the aerobic famine stage; The feast of oxygen - anoxic famine, feast of oxygen stage to join the mainstream nitrification unit influent and carbon source and COD VFA The range is 50 ~ 500 mgCOD L -1 , by aeration control dissolved oxygen concentration is higher than 1.0 mg L -1 , after stirring and sedimentation drainage, followed by anoxic famine stage, join the mainstream nitrification unit effluent and NO3 - -N range is 20 ~ 70 mgN L -1 , continue to stir; In the feast-famine of anoxia-aerobic, the main stream nitrification unit effluent and carbon source are added in the feast stage of anoxia, and the COD VFA / NO3 - -N is in the range of 2.5~6.0:1, after stirring, the water is drained by sedimentation, and the stirring is started again while the dissolved oxygen concentration is controlled to be higher than 1.0 mg / L by aeration -1 .

5. The construction method according to claim 1, 3 or 4, characterized in that, In step (1), the feast stage time is less than 1 / 6 of the total length of each typical cycle.

6. The construction method of claim 1, wherein, In step (2), COD VFA / NO3 - The -N regulation range is 2.5-6.0:

1.

7. The construction method of claim 1, wherein, In step (2), the SRT regulation range is 2-40 d.

8. The construction method of claim 1, wherein, In step (3), the food microorganisms ratio is in the range of 1.5-3.5 gCOD -1 gVSS -1 d -1 .

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