A method for regulating anaerobic digestion bacterial flora and enriching methanosaeta by using low-temperature plasma treatment

Abstract

The application discloses a method for regulating and controlling anaerobic digestion bacteria and enriching methanosaetaceae by using low-temperature plasma treatment. The method comprises the following steps: periodically extracting part of the digestion liquid during the operation of an organic waste anaerobic digestion reactor; discharging and treating the digestion liquid in a low-temperature plasma reaction device, and based on the differential damage of active species generated by discharge to the microbial flora, that is, by using the differences in morphological structure and oxidation stress tolerance between the methanogenic archaea of the methanosaetaceae and free or filamentous competitive microorganisms, the selective enrichment of the methanosaetaceae is realized; then, the obtained sediment is returned to the original anaerobic digestion reactor after being incubated under anaerobic conditions, centrifuged and washed. The above treatment process is repeated, and the methanogenic archaea of the methanosaetaceae are enriched in the reactor, and the method significantly improves the screening performance and anti-load impact capacity of the target bacteria of the anaerobic digestion system.

Description

Technical Field

[0001] This application relates to a method for regulating anaerobic digestive bacteria and enriching methanogenic taenia using low-temperature plasma treatment, belonging to the field of environmental biotechnology. Background Technology

[0002] Anaerobic digestion (AD) technology is an important method for treating organic waste and recovering bioenergy (methane). In AD systems, methanogenic archaea are key microorganisms that ultimately convert organic matter into methane. Among them, *Methanocytoplasma methanogens* (...) Methanosarcina *Methanococcus methanogens* is the most widely known genus of methanogens, possessing three methanogenic pathways: acetate nutrition, hydrogen nutrition, and methyl nutrition. It exhibits significant tolerance to environmental inhibitors such as high organic loading, high ammonia nitrogen, high salinity, and high volatile fatty acids (VFAs). However, in natural environments or conventional anaerobic sludge, *Methanococcus methanogens* is easily competitively inhibited by fermenting bacteria, resulting in a relative abundance of less than 5% in the community, making it difficult for it to establish a dominant position. In aquaponics (AD) systems, hydrolytic acidifying bacteria typically dominate, with a relative abundance of 40-70%. These bacteria play a crucial role in substrate degradation and VFA generation, but their excessive proliferation often leads to system acidification instability. Therefore, selectively reducing excessive acid-producing bacteria while enriching *Methanococcus methanogens* is a key technical bottleneck for improving the stability and methanogenic efficiency of AD systems.

[0003] Currently, the main methods for enriching methanogenic archaea include substrate induction, chemical inhibitor methods, and physical heat treatment. Substrate induction involves long-term culture with a single substrate (such as acetic acid) to screen for target bacteria, but this process takes 30-60 days and has low precision in regulating bacterial community structure. Chemical inhibitor methods (such as antibiotics and BES) have some targeting ability, but chemical reagent residues can easily cause secondary contamination, and it is difficult to selectively reduce competing bacteria. Physical heat treatment is energy-intensive, and high temperatures also inhibit methanogenic archaea; the system requires a long time to recover methanogenic activity after treatment, making it difficult to selectively protect the target bacteria. Summary of the Invention

[0004] The purpose of this invention is to address the technical problems of current methods for enriching methanogenic sarcopentaformis, which generally lack precise control over the bacterial community structure and are prone to secondary contamination due to chemical reagent screening. These problems make it difficult to meet the urgent need for rapid construction of dominant methanogenic bacterial communities in anaerobic digestion systems under steady-state or non-steady-state operating conditions. This invention provides a method for regulating anaerobic digestion bacterial communities and enriching methanogenic sarcopentaformis using low-temperature plasma treatment.

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] This application provides a method for regulating anaerobic digestive bacteria and enriching *Methanococcus methanans* using low-temperature plasma treatment, comprising: taking digestate from an anaerobic digestion reactor, placing it in a low-temperature plasma reaction device, adjusting the discharge voltage, current, and treatment time, and performing discharge treatment; after the discharge treatment, incubating and culturing under anaerobic conditions; after the incubation and culture, removing the supernatant by centrifugation and washing, and returning the precipitate to the original anaerobic digestion reactor; repeating the above steps for recycling according to the reactor's operating status, and finally enriching *Methanococcus methanans* in the anaerobic digestion reactor; wherein the digestate is the digestate obtained after anaerobic digestion of organic waste as a substrate.

[0007] Preferably, the organic waste is selected from at least one of kitchen waste, residual sludge and agricultural organic waste (livestock and poultry manure); the anaerobic digestion conditions are: temperature: 36-50℃, pH: 6.5-8.0, substrate hydraulic retention time: 15-30 days; organic load: 2.0-12.0 g COD / L / d.

[0008] Preferably, the organic waste is inoculated with anaerobic sludge for anaerobic digestion.

[0009] Preferably, the volume of digestate removed from the anaerobic digester is 3%-15% of the original volume of digestate in the anaerobic digester.

[0010] Preferably, the low-temperature plasma discharge treatment is carried out in an air or nitrogen atmosphere; the low-temperature plasma reaction device adopts dielectric barrier discharge (DBD) or corona discharge, and the electrode structure adopts needle-plate, wire-plate or coaxial cylindrical type, with a discharge electrode spacing of 1-20 mm.

[0011] Preferably, the power input voltage for the discharge process is 40-140V, the discharge current is 0.5-3A, the discharge time is 1-60min, and the discharge method is multiple intermittent discharges at different processing frequencies.

[0012] Preferably, the incubation temperature is 20-40℃ and the time is 0.5-6 h, and the anaerobic conditions are maintained by filling with nitrogen gas at a flow rate of 0.1-5 L / min.

[0013] Preferably, the centrifugation speed is 2000-10000 rpm and the time is 5-20 min.

[0014] Preferably, after centrifugation, the supernatant is removed, and the mixture is washed 1-3 times, retaining the precipitate, which is then returned to the original anaerobic digester.

[0015] Preferably, the cyclic treatment is performed every 2-20 days until the relative abundance of *Methanococcus methanans* in the system reaches 24% or more.

[0016] The inventive principle of this application is as follows: This application utilizes ROS generated by low-temperature plasma to selectively damage competitive hydrolytic acidifying bacteria (such as Firmicutes) within a bacterial community, thereby adjusting the community structure and releasing niche resources. Its selective screening mechanism stems from the fundamental differences in cell wall composition, morphology, and oxidative stress tolerance between methanogenic archaea of ​​the genus *Methanocytoplasma* and competing microorganisms: the cell walls of competitive hydrolytic acidifying bacteria are primarily composed of peptidoglycan, often exhibiting filamentous or unicellular dispersion, large surface area, and lack of an outer protective layer, making them extremely sensitive to ROS and prone to oxidative rupture and irreversible death; while the outer layer of *Methanocytoplasma* is composed of a highly ordered S-layer, endowing the cell wall with strong mechanical stability and chemical tolerance. Furthermore, its multicellular aggregate structure (1-1000 μm in diameter) is further encased in an EPS protective layer. The synergistic protection of the S-layer and EPS enables *Methanocytoplasma* to exhibit significantly superior tolerance to ROS stress compared to competing bacteria. During the anaerobic incubation phase, *Methanocytosporum methanogenum* gradually restores its cell integrity and metabolic activity through its complete DNA repair system and antioxidant enzyme system, while damaged competing bacteria, unable to repair themselves due to cell structure destruction, continue to die and lyse. A centrifugal washing step removes cell debris and residual ROS, and the enriched precipitated sludge is returned to the original reactor to ensure the rapid recovery and continued dominant proliferation of *Methanocytosporum methanogenum*. The methanogenic archaea of ​​the *Methanocytosporum* genus, gradually enriched through recycling, possess three methanogenic metabolic pathways: acetate nutrition, hydrogen nutrition, and methyl nutrition. These pathways efficiently consume accumulated VFAs and hydrogen in the system, mitigating the risk of acid accumulation and maintaining system pH stability. Furthermore, their significant tolerance to inhibitory factors such as high ammonia nitrogen and high VFAs allows the anaerobic digestion system to exhibit excellent resistance to load shocks and methanogenic efficiency under both steady-state and unsteady-state operating conditions.

[0017] Compared with the prior art, this application has the following beneficial effects: (1) The experimental method of this application is simple and does not require the construction of an additional enrichment culture system. It can be implemented directly in the operation of the existing anaerobic reactor. The microbial community structure in the system is continuously optimized through the circulating side flow treatment. The operation is simple and the engineering adaptability is strong. (2) In this application, plasma can be generated at low temperature, and the temperature conditions are easy to meet; at the same time, the required power is small and the system energy consumption is low. (3) This application adopts the synergistic effect of physical oxidation and biological mechanisms, without the need to add chemical reagents throughout the process, and without secondary pollution. Compared with traditional methods, it is more targeted and more precise in regulation. (4) Significantly improves the shock resistance and treatment efficiency of the anaerobic system. The anaerobic sludge enriched by the method of this application with methanogenic sludge as the dominant bacteria exhibits excellent shock resistance under both steady-state and non-steady-state operating conditions. Attached Figure Description

[0018] Figure 1 The cumulative methane production over a 60-day operating cycle under different processing parameters.

[0019] Figure 2 The dynamic succession of the microbial community structure during the operation of reactor R4-1: (a) phylum-level bacterial and archaea abundance composition; (b) species-level archaea abundance composition.

[0020] Figure 3 Transmission electron microscopy (TEM) results of microbial cells after plasma treatment with different intensities: (a) Blank control group (0V); (b) Moderate intensity treatment group (100V, 0.5h); (c) High intensity treatment group (160V, 0.5h).

[0021] Figure 4 Scanning electron microscopy (SEM) results of microbial cells after plasma treatment with different intensities: (a) Blank control group (0V); (b) Moderate intensity treatment group (100V, 0.5h); (c) High intensity treatment group (160V, 0.5h). Detailed Implementation

[0022] To make the technical solution of this application clearer and easier to understand, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0023] Unless otherwise specified, the experimental or testing methods described in the following examples are conventional methods; the reagents and materials described are obtained from conventional commercial sources unless otherwise specified.

[0024] Example 1 To investigate the enrichment efficiency of different types of low-temperature plasma discharge devices on *Sarcoptes methanogens* in the anaerobic digestion system of food waste under side-flow treatment mode, this embodiment explores the selective screening effect of three different types of low-temperature plasma generators—Dielectric Barrier Discharge (DBD), Corona Discharge, and Atmospheric Pressure Plasma Jet (APPJ)—on sludge microorganisms and the long-term enrichment efficiency of *Sarcoptes methanogens* under the same energy consumption level. This aims to clarify the applicability boundaries of different types and determine the most suitable low-temperature plasma generator and configuration for industrial applications.

[0025] (1) Reactor system and device design Four parallel-operated, 5L total mixed anaerobic reactors (CSTRs) were used to treat the collected food waste slurry, with a daily organic loading of approximately 4 g COD / L. The inoculum sludge was sourced and of consistent properties from a long-operational mesophilic anaerobic digester in a municipal wastewater treatment plant; the seed sludge solids content (TS) was adjusted to 16%, and the initial inoculum was *Methanococcus pyogenes* (...). Methanosarcina The relative abundance was <5%; the temperature in the anaerobic reactor was set at 38℃, and the pH was 6.5; nitrogen was introduced to maintain the anaerobic environment at a flow rate of 0.2 L / min; the hydraulic retention time (the average time the material spends in the anaerobic reactor for anaerobic digestion from entry to exit) was 28 days. The experimental cycle was set at 80 days, and the circulation frequency was uniformly set to once every 8 days.

[0026] The reactor grouping design is as follows: R-Control (Control Group): No plasma discharge treatment is performed; normal operation is maintained. R-DBD (Dielectric Barrier Discharge Group): It adopts a coaxial cylindrical dielectric barrier discharge device with a double-tube coaxial structure. The inner electrode is a high-voltage copper rod, the outer electrode is a grounded stainless steel mesh, and a quartz glass tube is installed in the middle as the barrier medium. The discharge gap is 4mm, the working gas is nitrogen, the discharge voltage is 80V, the current is 1A, and the processing time is 0.5h per treatment. R-Corona (Corona Discharge Group): A corona discharge device with a needle-plate electrode structure. It has no dielectric barrier layer and generates local corona discharge by forming a non-uniform strong electric field at the needle tip. The working gas is nitrogen, the discharge voltage is 80V, the current is 1A, and the treatment time is 0.5h per session. R-Jet (Jet Discharge Group): An atmospheric pressure plasma jet device with a needle-ring electrode structure, a flow rate of 2L / min, generates a high-energy plasma plume about 2-3 cm long at the nozzle, which performs localized purging treatment on the sludge surface below. The discharge voltage is 80V, the current is 1A, and the treatment time is 0.5h per session.

[0027] (2) Every 8 days, 200 mL (about 4%) of digestion liquid was taken from each experimental group reactor and placed in the corresponding device for discharge treatment according to the set parameters. After discharge, the digestion liquid treated by discharge was anaerobically incubated at 35°C for 2 h (nitrogen gas was introduced to maintain anaerobic conditions at a gas flow rate of 0.1 L / min), and then centrifuged at 6000 rpm for 10 min. The supernatant was discarded and the precipitate was returned to the original reactor.

[0028] (3) Comparison of ROS generation and distribution characteristics The three devices differ fundamentally in their ROS generation and spatial distribution characteristics, which are the core physical factors determining their screening effectiveness.

[0029] The R-DBD group benefits from the uniform control of discharge by the dielectric barrier layer, which can suppress the formation of a single streamer discharge channel and generate a large number of uniformly distributed micro-discharge filaments within the gap, allowing the digestant flowing through to be uniformly exposed to the high electric field region. Long-lived reactive oxygen species (O2) in the treated digestant are also reduced. 2- With a uniform concentration distribution and an average concentration of 45 mg / L, it can exert equal oxidative stress on all microorganisms, achieving ideal full-coverage and gentle screening.

[0030] Due to the lack of a dielectric barrier layer, the R-Corona group exhibits a high concentration of discharge in the strong electric field region near the needle tip, resulting in some spatial inhomogeneity. The ROS concentration is higher near the needle tip, approximately 55-65 mg / L, decreasing to 15-20 mg / L further away. Although the distribution uniformity is not as good as DBD, with moderate stirring, the overall oxidative stress can still be maintained within the effective screening range, demonstrating a certain selective reduction effect on competitive filamentous bacteria.

[0031] The R-Jet group exhibits extremely strong local heterogeneity, fundamentally different from the previous two devices. The instantaneous ROS concentration in the area directly bombarded by the plume can reach over 200 mg / L, exhibiting strong corrosiveness and potentially causing irreversible cell damage; while the concentration in the edge region far from the bombardment point is only 5-10 mg / L, significantly lower than the effective screening threshold. More critically, plasma jet processing is a gas-phase method, relying on gas-liquid interface mass transfer, resulting in extremely limited effective depth of action. A large amount of sludge remains in the plasma's blind zone, leading to severe uneven overall oxidation pressure and preventing complete coverage of the sludge bulk phase.

[0032] (4) Comparison of system gas production performance The three experimental groups exhibited significant gradient differences in gas production performance. The R-DBD group had a gas production lag period of only 12 hours after each cycle, allowing the target bacterial community to rapidly recover and proliferate, reaching a daily methane production of 0.32 LCH4 / (g COD·d) by day 80. The R-Corona group had a gas production lag period of approximately 18 hours, with a daily methane production of 0.31 LCH4 / (g COD·d) by day 80, performing slightly worse than the DBD group but still significantly better than the control group. The R-Jet group, due to irregular damage to the target bacterial community caused by jet treatment, had a gas production lag period as long as 36 hours after each refluxing, with a daily methane production of only 0.25 LCH4 / (g COD·d) by day 80, almost identical to the control group R-Control, failing to achieve an effective improvement in gas production performance.

[0033] (5) Long-term enrichment effect (metagenomic analysis on day 80) Metagenomic sequencing results on day 80 quantitatively verified the above conclusions. In the R-Control group, the relative abundance of *Methanogenic Micrococcus* remained at only around 8%, with no significant succession in community structure. In the R-Jet group, the relative abundance of *Methanogenic Micrococcus* only increased to 10%, with acetic acid-producing methanogens (… Methanosaeta The relative abundance of *Methanocytosporum* remained at approximately 40%, competing with *Methanocytosporum*, resulting in very limited improvement in the system's bacterial community structure. This fully demonstrates that the non-uniform processing characteristics of jet discharge are fundamentally inadequate to meet the conditions for full-coverage, mild screening. In the R-Corona group, the relative abundance of *Methanocytosporum* increased to 16%, while the competitive filamentous bacteria decreased to approximately 25%, showing a certain enrichment effect. In the R-DBD group, the relative abundance of *Methanocytosporum* reached 23%, confirming the superior performance of the coaxial DBD device in achieving targeted enrichment of *Methanocytosporum*.

[0034] (6) The configuration of the device has a decisive influence on the plasma screening effect, and its core lies in the uniformity of ROS distribution in the sludge bulk phase. Atmospheric pressure plasma jet (APPJ) relies on gas-liquid interface mass transfer, has a limited effective depth, and the ROS distribution is extremely uneven, making it impossible to fully cover the treated sludge bulk phase. At the same time, it has the fundamental defects of local over-oxidation damage to target bacteria and large-scale screening blind zones, which is not conducive to the targeted enrichment of methanogenic sarcococci in anaerobic sludge. Although the uniformity of corona discharge (Corona) is not as good as DBD, it can still achieve effective screening within a certain range by moderate stirring to assist mass transfer, and has certain feasibility. Dielectric barrier discharge (DBD) has the advantage of uniform control of discharge by the dielectric barrier layer, which can implement uniform, mild and full-coverage oxidative stress treatment on the digester flowing through it. It can make full use of the microbial morphological difference mechanism to achieve selective screening and enrichment of methanogenic sarcococci. It is the optimal device configuration for achieving efficient targeted enrichment of methanogenic sarcococci and has the strongest engineering applicability and large-scale promotion potential.

[0035] Example 2 To investigate the optimization of dielectric barrier discharge (DBD) parameters and their targeted enrichment effect on *Methanococcus methanans* in a food waste anaerobic digestion system, this embodiment explores the influence of different plasma discharge voltages and treatment durations on microbial community succession and gas production performance in a food waste anaerobic digestion system, thereby screening out the optimal enrichment parameters.

[0036] (1) The reactor system consists of 13 fully mixed anaerobic reactors (CSTRs) with an effective volume of 5 L each. The inoculum sludge was taken from a long-running mesophilic anaerobic digester in a municipal wastewater treatment plant. The sludge solids content (TS) was adjusted to 16%, and the initial abundance of methanogenic sarcodactylus was <5%. The substrate was the actual collected kitchen waste slurry, which was fed in by a feed pump after impurity removal and crushing. The daily feed load was approximately 10 g COD / L, which is prone to acidification. The temperature in the anaerobic reactor was set at 38℃, and the pH was 6.5. Nitrogen gas was introduced to maintain the anaerobic environment at a flow rate of 0.2 L / min. A coaxial dielectric barrier discharge (DBD) reactor was used with a discharge gap of 4 mm, and the working gas was air.

[0037] (2) Experimental group design: To determine the optimal discharge intensity, different voltage gradients and treatment times were set as shown in Table 1. The reactor operation cycle was 60 days, and the cycle treatment frequency was uniformly set to once every 10 days.

[0038] Table 1 Experimental group design for different combinations of voltage and discharge treatment time

[0039] (3) Every 10 days, take 200 mL (about 4%) of digestion liquid from each reactor and put it into the DBD device for discharge according to the set parameters. After the discharge is completed, the digestion liquid treated by the discharge is anaerobically incubated at 35°C for 2 hours, and then centrifuged at 5000 rpm for 10 minutes to discard the supernatant and return it to the original reactor.

[0040] (4) Based on the system performance monitoring data of the 60-day operation cycle, the experiment showed three typical stage succession characteristics.

[0041] The first stage was the screening and adaptation period (0-20 days): During this stage, the reactors in each group showed significant performance differentiation. While the control group R0 and the low-voltage treatment groups R1 and R2 maintained steady-state operation, their gas production performance did not show a significant improvement; whereas the gas production of the high-voltage treatment group R6 showed a continuous downward trend. In contrast, the medium- and high-voltage treatment groups R3, R4, and R5 began to show performance advantages, with their daily methane production increasing by more than 20% compared to the control group on day 20, preliminarily verifying the positive effect of low-temperature plasma-directed screening on the enrichment of methanogenic bacteria.

[0042] The second phase, the stabilization and enhancement period (days 21-50), saw further widening of performance differences among the groups. By day 40, due to prolonged exposure to high organic loads of food waste, both the control group R0 and the low-voltage treatment group R1 experienced ammonia nitrogen accumulation (concentration exceeding 3000 mg / L) and slight acidification, with gas production fluctuating and decreasing to 0.15-0.20 L CH4 / (gCOD·d). Group R2 maintained steady-state operation, with gas production fluctuating between 0.20-0.28 L CH4 / (gCOD·d). Group R6 experienced continuous ammonia nitrogen accumulation and significant system acidification, with gas production dropping to 0.05-0.12 L CH4 / (gCOD·d). It is noteworthy that groups R3-1, R4-1, and R5-1 exhibited extremely strong system stability. Despite the increase in ammonia nitrogen concentration, the gas production remained stable at 0.28-0.35 L CH4 / (g COD·d). Groups R3-2, R4-2, and R5-2 also showed strong system stability, with gas production remaining stable at 0.24-0.33 L CH4 / (g COD·d).

[0043] The third phase, the shock resistance challenge and long-term performance verification period (50-60 days), involved increasing the organic loading rate (OLR) of all reactors by 30% on day 51 to assess the system's ultimate tolerance. Results showed that systems R0, R1, and R6 completely destabilized, with pH plummeting below 5.5 and gas production ceasing entirely, exhibiting typical acidification collapse characteristics. While group R2 experienced a slight pH decrease, it still maintained gas production, demonstrating a certain buffering capacity. Groups R3, R4, and R5, however, exhibited excellent shock resistance, maintaining a stable pH within the optimal range of 7.2-7.5 and a gas production rate of 0.25-0.35 L CH4 / (g COD·d).

[0044] After 60 days of continuous operation, the cumulative methane production of each reactor group showed significant differences, such as... Figure 1 As shown, the cumulative yield of R0 was only 125.5 L, while the low-voltage treatment groups R1 and R2 yielded 126.2-130.1 L and 154.2-162.2 L respectively, showing limited improvement. The medium- and high-voltage treatment groups exhibited significant advantages. The cumulative yield of group R4-1 reached the highest value of 188.4 L, an increase of 50.1% compared to the control group; the cumulative yields of groups R3, R4, and R5 were all in the range of 169-188 L, significantly higher than the control group. This significant improvement in gas production performance is attributed to the successful targeted enrichment of *Methanococcus methanans* by moderate plasma treatment. However, the cumulative yield of the excessively high-voltage treatment group R6 was only 124-125 L, even slightly lower than the control group. Considering gas production efficiency, system stability, and economics (energy consumption and processing time cost), the optimal process parameters were ultimately determined to be group R4-1 (discharge voltage 100V, processing time 0.5h). After the load shock, the system pH only fluctuated briefly before quickly recovering and stabilizing to 7.2-7.5. Furthermore, at the end of the experiment on day 60, the system maintained a methanogenic level of 0.34 L CH4 / (g COD·d), with a cumulative methane production of 188.4 L, fully demonstrating excellent steady-state anti-interference ability and long-term operational stability.

[0045] (4) The microbial community structure of R0 and the optimal experimental group R4-1 (100V, 0.5h) at different operating stages was thoroughly tracked and analyzed using 16S rRNA gene amplicon high-throughput sequencing technology. Figure 2At the initial stage of the experiment (day 0), the microbial communities of both groups inoculated with sludge exhibited typical bacterial-dominated community characteristics. As the operating time progressed, the community succession trajectories of the two reactors showed significant differentiation. In R0, the community structure changed slowly and limitedly throughout the entire 60-day operating cycle. By day 20, the total relative abundance of *Methanococcus methanans* only slightly increased to 5.64%; by day 40, it increased slightly to 6.26%; even at the experimental endpoint of day 60, its total relative abundance only reached 5.38%, almost unchanged from the initial state. Meanwhile, bacterial phyla remained at a high abundance in group R0, and no substantial community reconstruction occurred in the system. Figure 2 (b)

[0046] In contrast, the community structure of group R4-1 underwent a significant reversal under plasma-directed screening. By day 20, the phylum Halobacteria ( Halobacteriota The relative abundance of halophilia (Salmonella) gradually increased from an initial 4.12% to 12.82% by day 20, more than three times the initial value; it further climbed to 21.61% by day 40; and by day 60, the experimental endpoint, the abundance of halophilia (Salmonella) was [missing information]. Halobacteriota The total relative abundance eventually stabilized at 25.72%, approximately five times higher than the 5.04% of the control group (R0). Figure 2 (a). At the species level, *Methanococcus faecalis* ( Methanosarcina flavescens The relative abundance of *Methanococcus thermophilus* increased from an initial 4.04% to 23.69% on day 60. Methanosarcina thermophila The percentage of halophilic bacteria increased from 0.096% to 2.08%, and the combined percentage of these two groups in the phylum Hallobacterium ( Halobacteriota The vast majority of the total abundance ( Figure 2 (b) confirmed the targeted enrichment effect of plasma treatment on *Methanococcus* spp. Meanwhile, competitive bacterial communities in group R4-1 were continuously suppressed, with Firmicutes (… Firmicutes The percentage decreased from 12.47% to 9.25%, and the percentage of *Vibrio vulgaris* (a type of fungus) decreased. Chloroflexota The percentage decreased from 58.43% to 38.07%. Figure 2 (a) This significant community differentiation reveals the core mechanism of plasma-directed screening: through the selective culling of competitive bacteria and the targeting of methanogenic occulta ( Methanosarcina Through targeted enrichment of *Methanococcus methanans*, group R4-1 successfully constructed a highly streamlined microecological network with *Methanococcus methanans* as the absolute core. The system retained an appropriate amount of symbiotic bacteria, such as symbiotic bacteria (…). Synergistota ): 2.02%, Dethiobacterium ( Desulfobacterota : 0.16%, Spirochete ( Spirochaetota : 1.07%. It possesses interspecies electron transfer (DIET) function, and interacts with *Methanococcus methanans* (…). Methanosarcina ) to form a stable symbiotic relationship Figure 2(a) This unique microbial ecological structure is the microbiological basis for the R4-1 group to maintain efficient gas production and excellent stability under high load shocks, while the control group R0, due to the lack of effective microbial community optimization, eventually collapsed under load shocks.

[0047] (5) 60 days of operation confirmed that improper voltage settings of 10V and 160V could not achieve the purpose of enriching *Methanococcus methanans*. Regular treatment at 40V-130V (every 10 days) maintained system stability even under load shocks and efficiently enriched *Methanococcus methanans*. DBD voltage of 100V, treatment time of 0.5h, and interval of 10 days were the optimal parameters for achieving long-term stable enrichment. The relative abundance of *Methanococcus methanans* in the reactor increased from the initial 4.24% and stabilized at 25.77%.

[0048] Example 3 Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) observations of the cell morphology of anaerobic digested sludge microorganisms under low-temperature plasma (DBD) treatment of different intensities. This embodiment aims to use electron microscopy to intuitively reveal the differential damage mechanism of different plasma treatment intensities on competitive filamentous bacteria and methanogenic taenia from a cellular morphological perspective, providing direct morphological evidence for the selective screening principle of this application.

[0049] (1) Experimental design and sample preparation During the operation of Example 2, samples from the blank control group (R0, 0V), the moderate intensity treatment group (R4-1, 100V, 0.5h), and the high intensity treatment group (R6-1, 160V, 0.5h) were taken at the end of incubation after a single discharge treatment and observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

[0050] (2) Observation of cell morphology in different treatment groups Transmission electron microscopy (TEM) observations revealed that the three groups of samples exhibited distinctly different cellular structural characteristics. Figure 3 In the blank control group (R0, 0V), the microbial cells were healthy and intact. The competitive bacteria showed continuous, smooth cell walls and clear inner membrane structures, while the *Methanococcus methanans* cells had regular outlines and abundant contents, confirming the normal competitive state between the competitive and target bacteria under plasma-free conditions. Figure 3 (a). The moderate intensity treatment group (R4-1, 100V, 0.5h) exhibited ideal differential damage characteristics: the number of competing bacteria in the field of view was significantly reduced, the remaining filamentous bacteria were broken and twisted, the cell walls were damaged, and the contents were leaked; in stark contrast, the multicellular aggregates of *Methanococcus methanans* were intact, and although the outer EPS layer had some oxidative damage, the inner cocci remained round and plump, the cell membranes were intact, and the metabolic activity was maintained. Figure 3 (b) The high-intensity treatment group (R6-1, 160V, 0.5h) showed indiscriminate and devastating damage, with the field of view filled with a large number of cell debris and broken hyphae. Even the aggregates of *Methanococcus methanans* protected by EPS underwent severe disintegration, and the surface of the individual cocci showed deep pit-like collapse, shriveling, and even tearing. The cell membrane integrity was completely lost, confirming that the excessively high intensity ROS of 160V had exceeded the EPS buffer limit and caused irreversible lethal damage to all microorganisms. Figure 3 (c)

[0051] Scanning electron microscopy (SEM) observations further validated the above TEM conclusions at the level of cell surface morphology. Figure 4 In the blank control group, filamentous bacteria intertwined to form a dense network, with smooth and plump surfaces, dominating the field of view. The EPS layer on the surface of *Methanococcus methanans* aggregates was intact, and the overall morphology was healthy. Figure 4 (a) In the moderate intensity treatment group, the main field of view transformed into dense clusters of *Methanococcus methanans* with a diameter of 500-800 μm. The internal cocci were round and plump without collapse, and some clusters showed rough marks on their surface (damage to the EPS protective layer). The remaining small amount of filamentous bacteria were broken and twisted, which strongly confirms that moderate intensity ROS achieves ideal targeted enrichment by precisely utilizing the inherent morphological differences between filamentous bacteria (large specific surface area, lack of EPS protection) and *Methanococcus methanans* aggregates (EPS buffer protection). Figure 4 (b) In the high-intensity treatment group, all microorganisms in the field of view showed severe damage. The originally dense aggregates of *Methanococcus methanans* completely disintegrated, and cell fragments were diffusely distributed, making it impossible to identify intact cell morphology. Figure 4 (c) is in complete agreement with the macroscopic results of the gas production collapse of the high-voltage treatment group in Example 2.

[0052] The aforementioned TEM and SEM multi-scale morphological evidence corroborates each other, systematically verifying the core technical principle of selective screening through microbial morphological differences in this application, and providing intuitive cellular morphological basis for determining the optimal discharge parameters (100V, 0.5h).

[0053] Example 4 To investigate the effect of discharge cycle frequency on the enrichment and maintenance of *Methanococcus sanguinalis* in the anaerobic digestion system of food waste, this embodiment, based on a voltage of 100V and a single discharge treatment time of 0.6h, further explores the long-term effects of discharge cycle interval (frequency) on microbial community succession and system stability, aiming to find the optimal balance between screening pressure and bioremediation.

[0054] The specific steps are as follows: (1) Five parallel CSTR reactors were used, with an effective volume of 5L and a daily feed organic load of 8 g COD / L. The substrate was kitchen waste slurry, and the inoculated sludge was the same as in Example 1. The experimental period was set to 60 days. The temperature in the anaerobic reactor was set to 37℃ and the pH to 6.5. Nitrogen gas was introduced to maintain the anaerobic environment at a flow rate of 0.2 L / min. The same low-temperature plasma generator was used for all reactors, and the discharge parameters (voltage 100V, time 0.6h, discharge volume 6%) were kept consistent.

[0055] Group settings: R0 (control group): No discharge treatment was performed.

[0056] R1 (single group): The liquid treatment is cancelled and a discharge cycle is performed only on the day the experiment starts (day 0), following the same steps as in Example 2, and no further treatment is performed thereafter.

[0057] R2 (High-frequency group): The digestive fluid is taken out every 1 day and circulated once according to the steps described in Example 2.

[0058] R3 (Medium Frequency Group): Every 10 days, the digestive fluid is taken out and circulated once according to the steps described in Example 2.

[0059] R4 (low-frequency group): Every 30 days, the digestive fluid is taken out and circulated once according to the steps described in Example 2.

[0060] (2) Dynamic monitoring and analysis of gas production performance During the experiment, the daily methane production and pH changes of each reactor were continuously monitored, and the results showed a significant frequency dependence: ① Ineffectiveness and rebound phenomenon (R0 and R1): R0 (control): Severe acidification (pH < 6.2) occurred on day 30, and the system collapsed.

[0061] R1 (Single): Although gas production increased slightly in the first 15 days after treatment, performance began to decline from day 20. Analysis suggests that while a single screening eliminated some surface bacteria, it did not completely remove deep-seated competing bacteria. Methanosaeta In the absence of subsequent inhibitory pressure, the cells rapidly proliferated and rebounded, eventually leading to gradual acidification of the R1 group on day 40.

[0062] ② Cumulative damage effect (R2): This group performed reasonably well in the initial stage (days 0-5), but after the fourth cycle, its gas production began to decline continuously. By day 60, its daily methane production was only 30% of that of group R3. The 1-day interval was shorter than the physiological cycle required for *Methanococcus methanans* to repair the EPS protective layer and oxidative damage to the cell membrane. Frequent plasma shocks caused cumulative cytotoxicity, leading to suppression of the target bacterial population's activity.

[0063] ③ Sawtooth wave effect (R4): The gas production curves of this group exhibited significant sawtooth-like fluctuations. Gas production increased within 5-10 days after each treatment, but gradually declined over the following 20 days. The 30-day interval was too long, providing a window for competing filamentous bacteria to recover and proliferate. Plasma could not completely remove the already regenerated network of contaminating bacteria in a single treatment, resulting in an unstable, tug-of-war state in the system.

[0064] ④ Steady-state reinforcement effect (R3 10 days / time): The performance was optimal. No acidification occurred throughout the process, and the daily methane production steadily increased and eventually stabilized at 0.31 L CH4 / (gCOD · d). Furthermore, no significant hysteresis in gas production was observed after each cycle.

[0065] (3) Microbial community endpoint analysis (day 60) Analysis of the digestive fluid at the end of the experiment verified the selective effect of frequency on the microbial community structure: R1 (single): The relative abundance of *Methanococcus methanans* dropped to 12%, while filamentous bacteria ( Methanosaeta The abundance rebounded to 35%, confirming that the single enrichment effect is reversible and unsustainable.

[0066] R2 (high frequency): Although there are fewer contaminants, the total biomass (VSS) is low, and ATP activity assays show that the overall metabolic level of the microorganisms is limited.

[0067] R4 (low frequency): The community structure is also mixed, with the abundance of *Methanococcus methanans* at 15%, and about 30% of filamentous bacteria remaining.

[0068] R3 (mid-frequency): The relative abundance of *Methanococcus methanans* was as high as 24%. Microscopic examination revealed that the bacterial clusters in this group had the densest structure and the largest particle size. The EPS layer surrounding the clusters was of moderate thickness, which provided protection without hindering mass transfer.

[0069] (4) The discharge frequency is the key parameter that determines the long-term enrichment stability. A single treatment cannot establish a long-term mechanism. High-frequency disturbance (1 day) will cause fatigue damage to the target bacteria. Low-frequency treatment (30 days) will cause the competing bacteria to recover. The cycle frequency of once every 10 days is the optimal process point. It can provide continuous screening pressure to effectively suppress competing bacteria, and also give the target bacteria sufficient physiological self-repair window, thus achieving the best balance between screening efficiency and microbial activity.

[0070] The above description is merely a preferred embodiment of this application and is not intended to limit this application in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from this application, and these improvements and additions should also be considered within the scope of protection of this application.

Claims

1. A method for regulating anaerobic digestive bacteria and enriching *Methanococcus methanans* using low-temperature plasma treatment, characterized in that... include: The digestate was taken out of the anaerobic digester and placed in a low-temperature plasma reactor. The discharge voltage, current and processing time were adjusted to carry out discharge treatment. After the discharge treatment is completed, the mixture is incubated and cultured under anaerobic conditions. After the incubation and culture are completed, the supernatant is removed by centrifugation and washing, and the precipitate is returned to the original anaerobic digestion reactor. Depending on the reactor's operating status, the above steps are repeated for cyclic processing, ultimately enriching *Methanococcus sanguinalis* in the anaerobic digestion reactor; the digestate is obtained from the anaerobic digestion of organic waste as a substrate; wherein... The volume of digestate removed from the anaerobic digester is 1%-20% of the original volume of digestate in the anaerobic digester. The discharge voltage for the discharge treatment is 40-140V, the discharge current is 0.5-3A, the discharge treatment time is 1-60min, and the discharge method is single discharge or multiple discharges. The incubation temperature is 20-40℃, and the time is 0.5-6 h; The cyclic treatment is performed every 2-20 days until the relative abundance of *Methanococcus methanans* in the anaerobic digester reaches 24% or more.

2. The method according to claim 1, characterized in that, The organic waste is selected from at least one of kitchen waste, residual sludge and agricultural organic waste; the anaerobic digestion conditions are: temperature: 36-50℃, pH: 6.5-8.0, substrate hydraulic retention time: 15-30 days; organic load: 2.0-12.0 g COD / L / d.

3. The method according to claim 1, characterized in that, The organic waste is inoculated with anaerobic sludge for anaerobic digestion.

4. The method according to claim 1, characterized in that, The low-temperature plasma discharge treatment is carried out in an air or nitrogen atmosphere; the low-temperature plasma reaction device adopts dielectric barrier discharge or corona discharge, and the electrode structure adopts needle-plate, wire-plate or coaxial cylindrical type, with a discharge electrode spacing of 1-20 mm.

5. The method according to claim 1, characterized in that, The anaerobic conditions are maintained by introducing nitrogen gas at a flow rate of 0.1-5 L / min.

6. The method according to claim 1, characterized in that, The centrifugation speed is 2000-10000 rpm, and the time is 5-20 min.

Images