Continuous anaerobic acidogenic three-stage enhanced method for cyanobacterial sludge

Abstract

This invention discloses a three-stage enhanced method for continuous anaerobic acid production using cyanobacteria sludge. Cyanobacteria sludge and anaerobic inoculated sludge are added to a continuous stirred tank reactor and started up at 35°C, 120 r / min, and pH 10. After start-up, continuous flow is maintained at a constant flow rate of 10 days HRT, a feed substrate concentration of 10 g VS / L, and equal daily feed and discharge. Once the system reaches steady state, a cellulose-degrading agent, obtained by enriching and acclimating anaerobic sludge using sodium carboxymethyl cellulose as the limiting substrate and washed with buffer solution, is added to the reactor at a dosage of 10% (v / v) of the reactor's working volume. Rhamnose lipids are also added at 0.4 g / g TS based on the total solids of the cyanobacteria sludge. Continuous flow operation continues to obtain a fermentation broth rich in volatile fatty acids. This invention is applicable to continuous anaerobic acid production using cyanobacteria sludge.

Description

Technical Field

[0001] This invention relates to the field of anaerobic fermentation engineering and enhanced operation technology of continuous flow reactors, specifically to a three-stage enhanced method for continuous anaerobic acid production from cyanobacteria using a continuous flow CSTR (Continuous Flow Sterile Reactor) sludge. Background Technology

[0002] The cyanobacterial sludge formed after the harvesting of cyanobacterial blooms is rich in organic components such as proteins and polysaccharides, and has the potential to be converted into platform chemicals such as volatile fatty acids through anaerobic fermentation. However, the cyanobacterial sludge also has a dual mass transfer and hydrolysis barrier composed of an extracellular polymeric substance (EPS) coating layer and a cell wall structural layer, resulting in insufficient substrate dissolution and limited acid production efficiency under continuous flow conditions.

[0003] Batch experiments using bottle-type or serum-bottle-type systems can identify optimal dosing combinations, but they are difficult to evaluate engineering issues such as microbial community maintenance, feed disturbance, mass transfer shear, dosing timing, and long-term steady-state maintenance in continuous flow operations. In particular, when the system is switched from a closed static environment to a continuous stirred tank reactor, whether a single-stage enhancement strategy can still maintain synergistic effects over a long operating period requires verification through a specific process pathway.

[0004] While existing technologies include anaerobic treatment of cyanobacteria, surfactant-assisted cell disruption, bacterial agent-enhanced hydrolysis, and continuous flow anaerobic reactor operation, there is still a lack of targeted technical solutions for organically linking start-up, steady-state operation, and synergistic enhancement in a continuous stirred tank reactor for the specific substrate of cyanobacteria sludge, while taking into account start-up stability, controllable continuous operation, and steady-state synergistic effects after enhancement.

[0005] Therefore, it is necessary to provide a three-stage enhancement method that can effectively transfer the synergistic enhancement conditions obtained from batch experiments to the CSTR continuous flow system, so as to improve the acid production level and engineering implementation stability of cyanobacteria sludge under long-term operating conditions. Summary of the Invention

[0006] Technical problems to be solved The technical problem to be solved by this invention is: to address the issues of slow start-up, limited steady-state concentration, incompatibility of enhancement conditions with continuous reactors, and unstable long-term operation of cyanobacteria sludge in continuous flow anaerobic acid production, and to provide a three-stage enhancement method for continuous flow CSTR cyanobacteria sludge continuous anaerobic acid production.

[0007] Technical solution To solve the above-mentioned technical problems, the present invention adopts the following technical solution: First, cyanobacterial sludge and anaerobic inoculated sludge are added to a continuous stirred tank reactor for startup; after the system establishes an acid-producing functional bacterial community, it is switched to continuous flow steady-state operation; on the basis of steady state, cellulose-degrading bacterial agent and rhamnolipid washed with buffer solution are added for enhancement, so that the synergistic enhancement effect can continue to play a role under continuous flow conditions.

[0008] Preferably, the temperature and stirring speed remain constant during the start-up, stable operation, and enhancement phases, at 35°C and 120 r / min, respectively; preferably, the HRT of the continuous flow phase is 10 days, the feed substrate concentration is 10 g VS / L, the pH is maintained at 10, and the daily feed volume is equal to the daily discharge volume.

[0009] Preferably, the cellulose-degrading bacterial agent is a bacterial solution obtained by enrichment and domestication of anaerobic sludge using sodium carboxymethyl cellulose as the limiting substrate, and is washed with phosphate buffer before use; the bacterial agent is preferably added at 10% (v / v) of the reactor working volume. Rhamnose lipids are preferably added at 0.4 g / g TS based on the total solids of cyanobacteria sludge.

[0010] In this specification, HRT is calculated according to formula (1) HRT=V / Q, where V is the working volume of the reactor and Q is the feed volume per unit time; when HRT is 10 days, the daily feed volume and daily discharge volume are preferably 1 / 10 of the working volume of the reactor.

[0011] In this specification, "reaching stability" means that the total VFA concentration fluctuation does not exceed ±4.68% within 6 consecutive HRTs.

[0012] Beneficial effects The embodiments show that the present invention adopts a three-stage operation path of "start-up-steady-state operation-enhancement", which enables the synergistic enhancement conditions screened in batch experiments to be implemented in a continuous stirred tank reactor, avoiding the adverse effects of directly applying cell wall disruption conditions on the establishment of the microbial community in the early stage of start-up.

[0013] In one set of embodiments, the total VFAs concentration was approximately 3.74 g / L during the stable operation phase and increased to 4.93 g / L during the enhancement phase; the total VFAs yield increased from 0.37 g / g VS to 0.49 g / g VS, and the acetic acid yield increased from 0.22 g / g VS to 0.28 g / g VS, indicating that the present invention can improve the acid production level of cyanobacterial sludge under continuous flow conditions and is beneficial to the directional generation of acetic acid.

[0014] In one set of embodiments, the steady-state concentration of SCOD during the stable operation phase was approximately 6633 mg / L, which increased to approximately 8170 mg / L during the enhanced phase. Cellulase activity increased significantly after the enhanced phase, and acetate kinase activity increased synchronously, indicating that the present invention not only enhances substrate hydrolysis and dissolution but also strengthens the subsequent acetic acid production metabolic flux.

[0015] In one set of embodiments, the hydrophobicity of the algal cell surface decreased from 55.17% to 44.00% after enhancement, and SEM and LSCM showed that the degree of cell structure damage was aggravated; at the same time, the microbial community changed from a diversified structure to a hydrolytic acid-producing functional group dominated by Bacillota, indicating that the present invention can achieve structural cell disruption and functional community remodeling. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the continuous flow CSTR reaction system, in which 1 is the cyanobacteria substrate storage tank, 2 is the feed pump, 3 is the main body of the continuous stirred tank reactor, 4 is the stirring motor and stirring device, 5 is the pH detection and alkali replenishment unit, 6 is the alkali storage tank, 7 is the constant temperature control unit, 8 is the discharge port and collection container, and 9 is the sampling port. Figure 1 For illustrative purposes only.

[0017] Figure 2 This is a graph showing the changes in total VFAs concentration and yield at different stages of the reactor in an embodiment of the present invention. Figure 2 (a) Changes in total VFA concentration and composition. Figure 2 (b) Changes in total VFAs and acetic acid yield.

[0018] Figure 3 This is a graph showing the concentration variations of different components of SMP in the reactor supernatant in an embodiment of the present invention. Figure 3 (a) represents the concentration of soluble protein. Figure 3 (b) represents the concentration of soluble polysaccharides.

[0019] Figure 4 This is a graph showing the changes in SCOD, ammonia nitrogen, and total nitrogen concentrations in the reactor supernatant in an embodiment of the present invention. Figure 4 (a) represents the SCOD concentration. Figure 4 (b) represents the concentrations of ammonia nitrogen and total nitrogen.

[0020] Figure 5 This is a diagram showing the change in enzyme activity during reactor operation in an embodiment of the present invention. Figure 5 (a) represents cellulase activity. Figure 5 (b) is the activity of acetate kinase.

[0021] Figure 6 This is a comparison diagram of the hydrophobicity of algal cell surfaces during the stable operation phase and the enhanced phase in an embodiment of the present invention.

[0022] Figure 7 These are SEM images of algal cells during the stable operation phase and the enhanced operation phase in an embodiment of the present invention. Figure 7 (a) is a sample from the stable operation phase. Figure 7 (b) is the sample for the reinforcement stage.

[0023] Figure 8 These are LSCM images of algal cells during the stable operation and enhanced operation phases in an embodiment of the present invention, where green fluorescence represents proteins, red fluorescence represents α-polysaccharides, and blue fluorescence represents β-polysaccharides.

[0024] Figure 9 This is a relative abundance diagram of bacterial communities at different operating stages in an embodiment of the present invention, wherein... Figure 9 (a) represents the horizontal community composition of the phylum. Figure 9 (b) is composed of horizontal communities. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments. Experimental conditions not specifically described can be performed according to conventional conditions in the art, reagent kit instructions, or conditions explicitly given in this specification.

[0026] Unless otherwise stated, TS refers to Total Solids, VS to Volatile Solids, VFAs to Volatile Fatty Acids, SCOD to Dissolved Chemical Oxygen Demand, HRT to Hydraulic Retention Time, and SMP to Dissolved Microbial Products.

[0027] The stages I, II, and III mentioned in this invention specification correspond to the startup stage, stable operation stage, and enhancement stage, respectively. To avoid ambiguity, the graphic and text numbers and the start and end times of each stage should be consistent before submission.

[0028] In one embodiment, the stable operation phase runs continuously for 64 days to reach a steady state, and the enhancement phase begins on the 84th day, with the entire continuous flow verification cycle reaching 110 days. The above phase days are only examples and do not constitute a limitation of the present invention. The actual phase switching is still based on the steady state criterion.

[0029] Example 1: Raw materials, inoculum and microbial agent The anaerobic inoculated sludge was collected in May 2025 from the granular sludge system of the Xielian Municipal Solid Waste Treatment Plant in Jiangsu Province, China. Its pH, TS, and VS were 7.4±0.1, 140.3±2.70 g / kg, and 90.1±2.28 g / kg, respectively. The cyanobacterial sludge was collected in July 2025 from the Changzhou Taihu Lake Water Environment Research Base. Its dry-basis nutrient composition was: carbohydrates 4.55%, protein 42.73%, lipids 2.38%, and ash 28.87%.

[0030] The cellulose-degrading bacterial agent was derived from a targeted enrichment system using sodium carboxymethyl cellulose as the limiting substrate. The culture medium consisted of: 3 g / L sodium carboxymethyl cellulose, 0.5 g / L ammonium chloride, 0.2 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate heptahydrate, 0.02 g / L calcium chloride, 3.0 g / L sodium bicarbonate, 0.1 g / L L-cysteine, 2 g / L sodium 2-bromoethanesulfonate, 1 mL / L trace element solution, and 1 mL / L vitamin solution. The culture medium was autoclaved at 121℃ for 20 min before use.

[0031] Rhamnollipolipase was analytical grade and purchased from Shanghai Maclean Biotechnology Co., Ltd.; hexadecane was analytical grade C16H34; the acetate kinase (ACK) kit was ACK-1-Y; the laser confocal microscope was TCS SP8; and the field emission scanning electron microscope was SU8600. The main reagents used in this example are shown in Table 1.

[0032] Table 1. Main reagents required for the experiment Example 2: Construction and Operation Scheme of CSTR Reactor A continuous stirred tank reactor is used for continuous anaerobic acid production from cyanobacteria and algal sludge. The reaction system structure is as follows: Figure 1 As shown. The reactor has a working volume of 1.6 L, a total volume of 2 L, a main body material of acrylic, an inner diameter of 5 cm, a liquid height of 19 cm, a paddle stirrer, a stirring shaft speed control of 120 r / min, and is equipped with a constant temperature control unit.

[0033] The reactor operation was divided into three stages: start-up, stable operation, and enhancement. The temperature was maintained at 35℃, the stirring speed at 120 r / min, and the pH was adjusted using NaOH and maintained at 10 in all three stages. The stage switching principle was: once the total VFAs concentration reached stability, the reactor would switch from the current stage to the next; the stability criterion was that the total VFAs concentration fluctuation did not exceed ±4.68% within six consecutive HRTs.

[0034] Sequencing batch fermentation (SBR) is preferred for the start-up phase to facilitate the enrichment and adaptation of acid-producing functional bacteria. During the start-up phase, cyanobacterial sludge and anaerobic inoculated sludge are mixed at a VS mass ratio of 2:1, with an initial substrate dosage of 160 g, a loading volume of 1.6 L, and a substrate VS concentration of 10 g / L. The start-up phase lasts for 20 days, or 2 HRTs. The operating parameters for the three phases are shown in Table 2.

[0035] BES at a concentration of 3 g / L was added to the reaction system to inhibit methanogenesis and direct the accumulation of VFAs by metabolic flux.

[0036] During the stable operation phase, the reactor was switched to continuous flow mode with a heating time (HRT) of 10 days and a feed substrate concentration of 10 g VS / L. The daily feed and discharge volumes were equal to maintain a constant reactor working volume. The corresponding daily feed and discharge rates were 160 mL, and the feeding frequency was once daily, using a manual timed feeding method.

[0037] The intensification phase begins after the stable operation phase reaches a steady state. Following the optimal combination determined in batch experiments, cellulose-degrading bacteria and rhamnolipin are added to the reactor. The bacteria dosage is 10% (v / v) of the reactor's working volume, and the rhamnolipin dosage is 0.4 g / g TS based on the total solids of the cyanobacteria sludge. The agents are added synchronously with the feed, and the addition is continuous throughout the entire Phase III operation until the end of the experiment.

[0038] Table 2. Three-stage operating parameters Example 3: Detection and Analysis Methods The concentrations of total VFAs and their components were determined by gas chromatography; SCOD was determined by potassium dichromate digestion; ammonia nitrogen and total nitrogen were determined by Nessler's reagent method and potassium persulfate oxidation method, respectively; soluble protein and soluble polysaccharide were determined by Lowry-Folin method and phenol-sulfuric acid method, respectively. The main instruments used in this example are shown in Table 3.

[0039] Table 3 Main instruments required for the experiment Cellulase activity was determined using the filter paper enzymatic method: crude enzyme solution was reacted with filter paper substrate at 50℃ for 1 h, and the amount of reducing sugar produced was measured at 540 nm using the DNS method. Acetylkinase activity was determined using a kit method (kit number ACK-1-Y). One unit of enzyme activity was defined as 1 nmol of NADH consumed per gram of tissue per minute. ACK activity was calculated as ACK (nmol / min / g fresh weight) = [ΔA × V total ÷ (ε × d) × 10]. 9 ]÷[W×(V sample ÷ V sample total)]÷T=536×ΔA÷W.

[0040] Cell surface hydrophobicity was determined using the MATH method: 10 mL of the algal solution was centrifuged at 8000 rpm for 10 min at 4℃, the supernatant was discarded, and the algal cells were washed with PBS and resuspended. The OD680 was adjusted to 0.8–1.0. 4 mL of the resuspended algal solution was taken, 1 mL of hexadecane was added, the mixture was vortexed for 3 min, and allowed to stand at room temperature for 30 min. The lower aqueous phase was then aspirated to measure the OD680. Three parallel samples were set up for each group. Hydrophobicity was calculated as H (%) = (1 - OD1 / OD0) × 100%, where OD0 is the initial absorbance of the algal solution before the addition of hexadecane, and OD1 is the absorbance of the lower aqueous phase after separation with hexadecane.

[0041] The SEM analysis method was as follows: Samples from Stage II and Stage III were lyophilized, fixed with glutaraldehyde for 24 h, washed three times with PBS, and then subjected to a gradient dehydration process using 50%, 70%, 80%, 90%, and 100% ethanol, 10 min per step. After gold sputtering, the samples were observed under a scanning electron microscope. The SEM instrument used was a SU8600; the accelerating voltage was 5 kV, the working distance was 8.9 mm, and the magnification was 8000x.

[0042] The LSCM analysis method was as follows: Phase II and Phase III samples were fixed with 2.5% glutaraldehyde for 24 h; then, the samples were subjected to multiplex fluorescence staining with 50 μL of fluorescein isothiocyanate (1 g / L), 100 μL of concanavalin A (250 mg / L), and 100 μL of Carcorflur (300 mg / L) to characterize proteins, α-polysaccharides, and β-polysaccharides, respectively. The LSCM instrument was a TCS SP8; the detection wavelengths for proteins, α-polysaccharides, and β-polysaccharides were 481–520 nm, 561–580 nm, and 400–435 nm, respectively.

[0043] For microbial community analysis, samples from phases I, II, and III were subjected to 16S rRNA high-throughput sequencing. DNA extraction and sequencing were performed by a third-party sequencing platform, with 338F / 806R amplification primers, Illumina MiSeq as the sequencing platform, and SILVA 138 and RDP 11.5 as the database versions.

[0044] Example 4: Three-stage operation results The following results are derived from the same long-term CSTR test, and the key performance indicators are shown in Table 4. Unless otherwise specified, Phase I, Phase II, and Phase III correspond to the startup phase, stable operation phase, and enhancement phase, respectively.

[0045] Table 4 Key Performance Indicators of CSTR in Three Stages (a) Acid production performance and substrate dissolution like Figure 2As shown, the reactor was initially started up using a sequencing batch reactor (SBR). In stage I, as the acid-producing bacteria gradually accumulated, the total VFAs concentration rapidly increased to a peak of 4.11 g / L, followed by a plateau. After switching to continuous flow, the total VFAs concentration in stage II stabilized at approximately 3.74 g / L, indicating that the system had established a dynamic balance between input and output. In one embodiment, the system met the predetermined steady-state criterion after 64 days of continuous operation and switched to stage III on day 84. According to available data, acetic acid and butyric acid were the main acid components in stage II, with proportions of approximately 60.08% and 18.74%, respectively. After adding cellulose-degrading bacteria and rhamnolipids in the enhancement stage, the total VFAs concentration further increased to approximately 4.93 g / L, the total VFAs yield increased from 0.37 g / gVS to 0.49 g / gVS, and the acetic acid yield increased from 0.22 g / gVS to 0.28 g / gVS.

[0046] like Figure 3 As shown, SMP analysis results indicated that soluble protein and soluble polysaccharides rapidly increased in the initial stage of initiation, reaching peak values ​​of 2028 mg / L and 566 mg / L, respectively. Upon entering stage II, the system reached a lower and more stable equilibrium level, with soluble protein at approximately 677 mg / L and soluble polysaccharides at approximately 393 mg / L. Upon entering stage III, soluble protein experienced a brief increase and formed a new, higher steady state; its instantaneous peak value was approximately 1522 mg / L. Soluble polysaccharides, after a brief peak of approximately 483 mg / L, stabilized at approximately 413 mg / L, indicating that the enhanced treatment promoted further release of intracellular and extracellular organic components.

[0047] like Figure 4 As shown, SCOD peaked at 6678 mg / L in Phase I and stabilized at approximately 6633 mg / L in Phase II; after fortification, it rapidly increased and stabilized at approximately 8170 mg / L. Nitrogen speciation data indicated that in Phase I, ammonia nitrogen increased from 327 mg / L to 523 mg / L, and total nitrogen slowly increased from 689 mg / L to 774 mg / L; in Phase II, ammonia nitrogen and total nitrogen stabilized at approximately 511 mg / L and 754 mg / L, respectively, and in Phase III, they further increased to approximately 581 mg / L and 780 mg / L.

[0048] (II) Enzymatic response and changes in interfacial properties like Figure 5As shown, cellulase activity gradually increased from 2.83 U / mL on day 10 to 7.31 U / mL on day 40 in Phase I, indicating that the enrichment of hydrolytic functional bacteria had been achieved in the initiation phase; the average value in Phase II was approximately 5.63 U / mL; and in the enhancement phase, it continued to increase significantly after day 90, reaching a peak of 14.18 U / mL on day 110. Acetylkinase activity showed a similar trend to cellulase activity, stabilizing at 90–93 nmol / min / g in the later stages of Phase I, and then rising to a peak of 129.11 nmol / min / g in Phase III, indicating that the enhancement treatment simultaneously improved both hydrolytic capacity and acetic acid production flux.

[0049] like Figure 6 As shown, the MATH results indicated that the mean hydrophobicity of algal cell surface in stage II was 55.17%, ranging from 51% to 60%, with a median of 54.5%; while in stage III, the mean decreased to 44.00%, narrowing the range to 41% to 49%, with a median of 43.5%. These results suggest that the EPS coating on the cell surface was further weakened after enhancement, leading to a remodeling of surface properties and a tendency for exposure.

[0050] (III) Morphological and Microbial Community Evidence like Figure 7 and Figure 8 As shown, SEM results revealed that in Phase II samples, cell surfaces exhibited depressions, shrinkage, and localized pores. In Phase III samples, the number of intact cells further decreased, with residues consisting mainly of severely damaged cell wall fragments or recalcitrant organic matter. LSCM results showed that after enhancement, green fluorescence (protein) and red fluorescence (α-polysaccharide) were enhanced and shifted from concentrated intracellular distribution to diffuse peripheral distribution, while blue fluorescence (β-polysaccharide) was significantly weakened, indicating further damage to the cell wall fibrous skeleton accompanied by leakage of intracellular substances.

[0051] like Figure 9 As shown, microbial community analysis revealed that at the phylum level, the relative abundance of Bacillota continuously increased from 15.4% in Stage I, reaching over 90% in Stage III, indicating that continuous flow operation and enhanced strategies significantly targeted the hydrolysis-producing functional bacterial community. At the genus level, Sporosarcina was significantly enriched in Stage III, and genera such as Schnuerera, Tissierella, and Erysipelothrix also showed an upward trend, indicating the formation of a functional community structure centered on hydrolysis-producing acid in the reactor.

[0052] In summary, the three-stage operation mode can transfer the synergistic enhancement conditions screened out in the bottle experiment to the continuous flow CSTR system and achieve sustainable acid production enhancement under long-term operation conditions.

[0053] Industrial applicability The method described in this invention is applicable to continuous-flow directional acid production scenarios using high-water-content organic substrates such as cyanobacteria-based sludge that require structural cell disruption. This method, through a phased operation strategy of "start-up-steady-state operation-enhancement," improves the SCOD dissolution level, total VFAs yield, and acetic acid yield of cyanobacteria-based sludge while balancing microbial community establishment, continuous operation stability, and subsequent enhancement. It has demonstrated good engineering adaptability and continuous operation application value in a 110-day continuous-flow operation validation. The above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention; all equivalent substitutions and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A three-stage enhanced method for continuous anaerobic acid production from cyanobacteria-based sludge, characterized in that, The process includes the following steps: S1, Start-up stage: Blue-green algae sludge and anaerobic inoculated sludge are added to a continuous stirred tank reactor and started up in a batch process at 35℃, 120 r / min, and pH 10; S2, Stable operation stage: While maintaining 35℃, 120 r / min, and pH 10, the reactor is switched to continuous flow operation with a hydraulic retention time (HRT) of 10 days, a feed substrate concentration of 10 g VS / L, and the daily feed volume equal to the discharge volume; S3, Enhancement stage: After stabilization is achieved in step S2, cellulose-degrading bacteria and rhamnolipids, washed with buffer solution, are added to the reactor. The cellulose-degrading bacteria are a bacterial solution obtained from anaerobic sludge enrichment and acclimatization using sodium carboxymethyl cellulose as the limiting substrate, and the dosage is 10% (v / v) of the reactor's working volume. The rhamnolipids are added at a dosage of 0.4 g / g based on the total solids of the blue-green algae sludge. TS, and under the condition that the daily feed volume is equal to the discharge volume, the continuous flow operation is continued to obtain a fermentation broth rich in volatile fatty acids.

2. The method according to claim 1, characterized in that, In steps S1 to S3, the pH of the reactor is maintained at 10 by adding alkali solution.

3. The method according to claim 1, characterized in that, Step S1 adopts a sequential batch start-up method to enrich acid-producing functional bacteria and establish a stable fermentation environment.

4. The method according to claim 1, characterized in that, Both steps S2 and S3 are operated continuously under the condition of daily equal volume feeding and discharging to maintain a constant working volume of the reactor.

5. The method according to claim 1, characterized in that, The enrichment culture medium for the cellulose-degrading bacterial agent includes: sodium carboxymethyl cellulose 3 g / L, ammonium chloride 0.5 g / L, potassium dihydrogen phosphate 0.2 g / L, magnesium sulfate heptahydrate 0.1 g / L, calcium chloride 0.02 g / L, sodium bicarbonate 3.0 g / L, L-cysteine ​​0.1 g / L, sodium 2-bromoethanesulfonate 2 g / L, trace element solution 1 mL / L, and vitamin solution 1 mL / L.

6. The method according to claim 5, characterized in that, The enrichment medium was sterilized by autoclaving at 121°C for 20 minutes before use.

7. The method according to claim 1, characterized in that, The cellulose-degrading bacterial agent in step S3 is washed with phosphate buffer before addition to remove culture medium residue.

8. The method according to claim 1, characterized in that, The cyanobacterial sludge is taken from the collection or dehydrated concentrate of cyanobacteria blooming in freshwater eutrophic lakes.

9. The method according to claim 1, characterized in that, Step S3 begins after the total volatile fatty acid concentration in step S2 has fluctuated by no more than ±4.68% over six consecutive HRTs.

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