Method for anaerobic acid production of blue-green algae sludge by synergistic effect of rhamnolipid and cellulose-degrading microbial agent

Abstract

This invention discloses a method for anaerobic acid production from cyanobacterial sludge using a synergistic effect of rhamnolipin and cellulose-degrading bacteria. The cyanobacterial sludge is mixed with anaerobic inoculated sludge to form a fermentation system, with the total solids (TS) of the substrate being 14% and the volatile solids (VS) of the system being 10 g / L. A cellulose-degrading bacteria agent, obtained from anaerobic sludge enrichment and domestication using sodium carboxymethyl cellulose as the limiting substrate and washed with buffer solution, is added at a dosage of 10% of the fermentation system volume. Rhamnolipin at 0.1–0.5 g / g TS based on the total solids of the cyanobacterial sludge is added, along with a methane inhibitor. The system pH is adjusted to 10, and the headspace is purged with nitrogen for 5 min. Anaerobic fermentation is then carried out at 37°C and 120 r / min for 10 days to obtain a fermentation broth rich in volatile fatty acids.

Description

Technical Field

[0001] This invention relates to the field of anaerobic fermentation and resource utilization of organic solid waste, specifically to a method for anaerobic acid production from cyanobacterial mud using a synergistic effect of rhamnose glycolipid and cellulose-degrading bacteria. Background Technology

[0002] The large amounts of cyanobacterial sludge formed after a cyanobacterial bloom are characterized by high water content, rich organic matter, easy putrefaction and foul odor, and the potential release of algal toxins. Timely and effective harmless treatment and resource utilization of cyanobacterial sludge are crucial steps in the management of lake eutrophication. Anaerobic fermentation can convert cyanobacterial sludge into volatile fatty acids (VFAs) and other platform compounds, which have resource utilization value.

[0003] However, cyanobacterial sludge is not a typical biodegradable substrate. The outer layer of cyanobacterial cells typically contains an outer layer mainly composed of extracellular polymers, while the inner layer contains structural layers composed of cellulose, hemicellulose, etc., forming a double barrier that is detrimental to substrate hydrolysis. This double barrier restricts enzyme contact with the substrate, hinders the release of intracellular organic matter, and makes the hydrolysis stage the rate-limiting step in the anaerobic acid production process of cyanobacterial sludge.

[0004] Existing technologies include methods such as alkali treatment, heat treatment, ultrasonic treatment, surfactant treatment, exogenous bacterial enhancement, or combinations thereof to promote anaerobic fermentation of algae or sludge; there are also disclosures on the preparation of volatile fatty acids using cyanobacteria in algal blooms, and the use of rhamnose glycolipids to enhance the hydrolysis and acidification of residual sludge. However, for cyanobacterial sludge, a substrate with a dual barrier of extracellular polymer coating and cellulose / hemicellulose structural layer, existing technologies still lack specific technical pathways for simultaneously improving extracellular polymer stripping, cell wall hydrolysis, and subsequent acidification under relatively mild conditions.

[0005] Therefore, it is necessary to provide an anaerobic acid production method that is more suitable for cyanobacteria sludge as a specific substrate, so as to improve the hydrolysis efficiency and volatile fatty acid generation level of cyanobacteria sludge without relying on high-energy-consuming physicochemical pretreatment. Summary of the Invention

[0006] Technical problems to be solved The technical problem to be solved by this invention is: to address the issue that the extracellular polymer coating layer and cellulose / hemicellulose structural layer of cyanobacteria mud form a double barrier, which leads to limited anaerobic fermentation hydrolysis and low acid production efficiency, a method for anaerobic acid production of cyanobacteria mud using rhamnolipid and cellulose-degrading bacteria in synergy is provided.

[0007] Technical solution To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A fermentation system is formed by mixing cyanobacterial sludge with anaerobic inoculated sludge; a cellulose-degrading bacterial agent washed with buffer solution is added to the system, preferably a bacterial solution obtained by enrichment and domestication of anaerobic sludge using sodium carboxymethyl cellulose as the limiting substrate; rhamnolipids are added based on the total solids of the cyanobacterial sludge, and a methane inhibitor is added; the pH of the system is adjusted to 10, the headspace of the system is purged with nitrogen for 5 min, and then anaerobic fermentation is carried out at 37℃ and 120 r / min for 10 days to obtain a fermentation broth rich in volatile fatty acids.

[0008] Preferably, the amount of the cellulose-degrading microbial agent added is 10% of the fermentation system volume; preferably, the amount of rhamnolipin added is 0.1-0.5 g / g TS, more preferably 0.3-0.5 g / g TS, and even more preferably 0.4 g / g TS.

[0009] Preferably, the total solids (TS) of the substrate in the fermentation system is 14%, and the volatile solids (VS) of the system are 10 g / L; the ratio of substrate to inoculum is 2:1 based on the VS mass ratio; the methane inhibitor is sodium 2-bromoethanesulfonate; and the fermentation is carried out in batches in 250 mL serum bottles.

[0010] Preferably, the enrichment culture medium for the cellulose-degrading bacterial agent comprises: 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.

[0011] Preferably, the methane inhibitor and rhamnolipin are added before fermentation begins and thoroughly mixed with the fermentation system; the headspace of the system is purged with nitrogen for 5 minutes before fermentation to establish an anaerobic environment.

[0012] Unless otherwise expressly defined, the term "synergistic" in this specification is used to describe the combined treatment system formed by the combined use of rhamnolipin and cellulose-degrading bacteria, and is not required to be limited by statistical interaction terms.

[0013] Beneficial effects Examples show that the present invention improves substrate accessibility and promotes the migration of organic matter from the solid phase to the liquid phase in cyanobacterial sludge by combining the weakening effect of rhamnolipids on the extracellular polymer coating layer with the enzymatic hydrolysis of the cell wall structural layer by cellulose-degrading bacteria.

[0014] Examples show that, under the preferred conditions of this invention, the SCOD peak value of the RL4 group reached 9511 mg / L, which was 44% higher than that of the blank control; the PFO model rate constant k1 was reduced from 1.45 d in the CK1 group. -1 Increased to 1.93 d -1 The total VFA concentration reached 4.87 g / L, and the acetic acid yield reached 0.24 g / gVS, indicating that the method can effectively enhance the hydrolysis and acid production process of cyanobacteria sludge.

[0015] Examples show that the present invention not only causes an increase in soluble proteins and polysaccharides in SMP and a decrease in proteins and polysaccharides in TB-EPS, but also causes changes in indicators such as cellulase, glucanase, protease, CAT and GSH, as well as changes in the relative abundance of functional genes such as ackA, pta, celA and pilA, providing support for the mechanism of action of the technical solution.

[0016] Examples show that the present invention can be used for the resource utilization of cyanobacteria sludge, and the operating conditions are relatively mild, suitable for anaerobic acid production by cyanobacteria sludge. Attached Figure Description

[0017] Figure 1 The results show the SCOD changes of cyanobacterial sludge under different concentrations of rhamnolipin. Figure 1 (a) shows the curve of SCOD concentration changing over time. Figure 1 (b) shows the pseudo-first-order dynamic fitting results of SCOD accumulation for groups CK1 and RL4.

[0018] Figure 2 The results of acid production from anaerobic fermentation of cyanobacterial sludge under different concentrations of rhamnolipin are shown. Figure 2 (a) is a graph showing the change in acetic acid yield. Figure 2 (b) is a graph showing the changes in total VFAs concentration.

[0019] Figure 3 This is a graph showing the distribution changes of proteins and polysaccharides in different groups of samples after 1 day of reaction. Figure 3 (a) represents the concentration of soluble proteins in SMP. Figure 3 (b) represents the concentration of soluble polysaccharides in SMP. Figure 3 (c) represents the concentration of soluble protein in EPS. Figure 3 (d) represents the concentration of soluble polysaccharides in EPS.

[0020] Figure 4 The image shows the three-dimensional fluorescence spectra of different EPS components in groups CK1, CK2, and RL4 after 1 day of reaction.

[0021] Figure 5The graph shows the changes in ammonia nitrogen, total nitrogen, and pH at the fermentation endpoint under different concentrations of rhamnolipid. Figure 5 (a) represents the concentrations of ammonia nitrogen and total nitrogen. Figure 5 (b) is the pH value.

[0022] Figure 6 The graph shows the changes in the activity of enzymes related to antioxidant stress and oxidative damage indicators under different concentrations of rhamnolipid.

[0023] Figure 7 The graph shows the changes in the activities of cellulase, glucanase, and protease under different concentrations of rhamnolipid.

[0024] Figure 8 This is a graph showing the relative abundance changes of key functional genes in different experimental groups.

[0025] Figure 9 Microscopic images of algal cells stained with trypan blue from groups CK1, CK2, and RL4.

[0026] Figure 10 The graph shows the changes in extracellular lactate dehydrogenase activity under different concentrations of rhamnolipid.

[0027] Figure 11 The graph shows the changes in ATP content on day 1 and day 10 for different experimental groups.

[0028] Figure 12 Plots showing the relative abundance of bacterial communities at the phylum and genus levels for different treatment groups. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments. Experimental conditions not specifically described are generally performed according to conventional conditions in the art, reagent instructions, or conditions explicitly given in this specification.

[0030] Unless otherwise stated, TS refers to total solids, VS to volatile solids, SCOD to dissolved chemical oxygen demand, VFAs to volatile fatty acids, SMP to soluble microbial products, LB-EPS to loosely bound extracellular polymers, TB-EPS to tightly bound extracellular polymers, BES to sodium 2-bromoethanesulfonate, PBS to phosphate buffer, and PFO to pseudo-first-order kinetics; percentages of bacterial agent dosage are volume fractions, and percentages of substrate TS are mass fractions.

[0031] In this specification, "cyanobacterial mud" refers to a mud-like substance formed by collecting, concentrating, dehydrating, or settling cyanobacteria blooms in eutrophic freshwater lakes; "cellulose-degrading bacterial agent" is preferably a functional bacterial agent existing in the form of bacterial liquid; "synergistic" preferably refers to a composite treatment method formed by the combined use of rhamnolipid and the cellulose-degrading bacterial agent.

[0032] Example 1: Raw materials, inoculum and reagents 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%. The cyanobacterial sludge had a moisture content of approximately 90%. After sampling, it was aliquoted into sealed bags or centrifuge tubes, degassed, and frozen at -20℃. Before use, it was slowly thawed at 4℃ or rapidly thawed at room temperature. No pretreatment was performed before feeding.

[0033] Rhamnollipids are analytical grade reagents with the chemical formula C. 32 H 58 O 13 The purity is ≥95%; the cellulose-degrading bacterial agent is derived from the bacterial culture obtained during the previous domestication; indicators such as lactate dehydrogenase, ATP, glucanase, alkaline protease, CAT, MDA, and GSH can be detected using commercial kits. Unless otherwise specified, the experimental water is ultrapure water; the rhamnolipids mentioned can be conventional commercial products in this field.

[0034] The suppliers of the aforementioned raw materials and reagents do not constitute a limitation on the present invention; in this embodiment, the relevant reagent kits and reagents can be conventional commercial products in the art.

[0035] In this embodiment, the real-time quantitative PCR instrument is QuantStudio 3, the fluorescence spectrophotometer is F-7000, and the biological microscope is CX31RTSF; the above instrument models are only examples used in the embodiment and do not constitute a limitation on the present invention.

[0036] Example 2: Preparation of Cellulose-Degrading Bacterial Agent The cellulose-degrading bacterial agent was obtained as follows: an enrichment medium was prepared using sodium carboxymethyl cellulose as the limiting substrate. The medium composition was: 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 pH of the medium was the natural pH, and it was autoclaved at 121℃ for 20 min before use. The enrichment culture temperature was 30℃. The trace element solution and vitamin solution could use a conventional formula for anaerobic microbial culture. Before culture, the headspace of a 250 mL serum bottle was purged with high-purity nitrogen for 5 min to establish an anaerobic environment.

[0037] Anaerobic sludge was inoculated into the above-mentioned culture medium for anaerobic enrichment and acclimatization. Five subculture rounds were performed, with a single round culture time of 10 days and an inoculation ratio of 10% (V / V). The endpoint of bacterial enrichment was determined by the near-complete depletion of CMC-Na, the cessation of acid production, and a relatively stable pH. Before addition, the resulting bacterial solution was washed with phosphate-buffered saline (PBS) to remove residual culture medium. The washing was performed three times, followed by centrifugation at 5000 r / min for 10 min.

[0038] Anaerobic enrichment and acclimatization culture was conducted using sodium carboxymethyl cellulose (CMC-Na) as the limiting substrate and anaerobic sludge as the inoculum. The specific procedure was as follows: Anaerobic sludge was inoculated into the enrichment and acclimatization medium at a 10% (V / V) inoculum and cultured at 30℃ and 120 r / min. Acclimatization was carried out by gradually increasing the concentration of CMC-Na as the carbon source and gradually decreasing the concentration of glucose. Subculturing was performed every 10 days, with each subculturing and transfer constituting one round of acclimatization, up to the 5th round. In preliminary experiments, a 10-day cycle ensured that the substrate CMC-Na was fully consumed, preventing further acid production and maintaining a relatively stable pH. When the pH did not decrease significantly, the culture was stopped, and the culture medium was transferred to fresh enrichment and acclimatization medium at a 10% (V / V) inoculum. The changes in the concentration of different carbon sources during acclimatization are shown in Table 1.

[0039] Table 1. Variations in concentration of different carbon sources The washing buffer used was phosphate-buffered saline (PBS), specifically formulated as follows: 0.27 g potassium dihydrogen phosphate (KH₂PO₄), 1.42 g disodium hydrogen phosphate (Na₂HPO₄), and 8.5 g sodium chloride (NaCl), dissolved in approximately 800 mL of distilled water. The pH was measured using a pH meter and adjusted to 7.4 with NaOH or HCl, then brought to a final volume of 1 L. The wash was performed three times, with each resuspension in PBS followed by centrifugation at 5000 rpm for 10 min.

[0040] Example 3: Construction of a batch anaerobic acid production system To investigate the effect of the combined use of cellulose-degrading bacteria and rhamnolipin on anaerobic acid production from cyanobacterial sludge, batch fermentation was conducted using 250 mL serum bottles. The substrate total concentration (TS) was 10%, the final system saturation concentration (VS) was 10 g / L, and the cellulose-degrading bacteria dosage was 10% of the fermentation system volume. The substrate to inoculum ratio was 2:1 based on the VS mass ratio. The anaerobic bottle volume was 120 mL, and the head empty volume was approximately 130 mL.

[0041] The methane inhibitor BES was added to the fermentation system at a final concentration of 3 g / L. After mixing, the pH was adjusted to 10 with NaOH, followed by headspace purging with nitrogen for 5 min. The system was then anaerobic fermented at 37℃ and 120 r / min for 10 days. Three replicates were set up for each group.

[0042] In this embodiment, a blank control group CK1 (without cellulose-degrading bacteria and without rhamnolipin), a control group CK2 (with 10% bacteria but without rhamnolipin), and experimental groups RL1 to RL5 (all with 10% bacteria, and rhamnolipin dosages of 0.1, 0.2, 0.3, 0.4, and 0.5 g / g TS, respectively) were set up; where RL represents the experimental group with rhamnolipin added in addition to the cellulose-degrading bacteria. Specific groupings are shown in Table 2.

[0043] Table 2 Detailed Experimental Grouping Example 4: Detection and Analysis Methods During fermentation, 2 mL of fermentation broth was collected daily. Immediately after collection, the sample was centrifuged at 10,000 rpm for 10 min and filtered through a 0.45 μm filter membrane. The supernatant was used to determine SCOD, VFAs, ammonia nitrogen, total nitrogen, soluble protein, and soluble polysaccharides. VFAs were determined by gas chromatography: 0.5 mL of the supernatant was further filtered through a 0.22 μm filter membrane, acidified with 0.5 mL of 25% phosphoric acid, and immediately detected. The chromatographic column was HP-INNOWax, the carrier gas was high-purity nitrogen at a flow rate of 1.5–2.0 mL / min, the injection port temperature was 250℃, the detector (FID) temperature was 250℃, and the temperature program was: 80℃ for 2 min, increased to 105℃ without holding, then increased to 205℃ for 2 min. The external standard curve range was 10–1000 mg / L.

[0044] SCOD was determined using the potassium dichromate digestion method; ammonia nitrogen was determined using the Nessler's reagent method; total nitrogen was determined using the potassium persulfate oxidation method; soluble protein was determined using the Lowry-Folin method; soluble polysaccharides were determined using the phenol-sulfuric acid method; and pH was measured using a pH meter. The relative deviation of parallel samples for routine indicators was controlled within 5%–10%, and the relative standard deviation of biological indicators was controlled within 20%. The results were characterized using the average value of parallel samples.

[0045] To characterize the changes in different EPS components, the fermentation broth was first centrifuged, and the supernatant was filtered through a 0.45 μm filter to obtain SMP. The centrifuged precipitate was resuspended in PBS, incubated in a 50℃ water bath for 10 min and vortexed for 1 min, then centrifuged at 8000 rpm for 10 min. The supernatant was collected and filtered to obtain LB-EPS. The remaining sediment was resuspended in PBS again, incubated in a 80℃ water bath for 30 min, then centrifuged at 8000 rpm for 15 min. The supernatant was collected and filtered to obtain TB-EPS. Subsequently, the polysaccharide and protein concentrations were determined using the phenol-sulfuric acid method and the Lowry-Folin method, respectively. 3D-EEM characterization was performed using a Hitachi F-7000 fluorescence spectrophotometer. The detection wavelength range was 250–550 nm, the scan interval was 5 nm, and the scan rate was 2400 nm / min. The PBS used was phosphate buffered saline at pH 7.4.

[0046] To study the hydrolysis kinetics of algal sludge, this invention uses a pseudo-first-order (PFO) model to fit the changes in SCOD. The PFO model is shown in equation (1): ln(q e -q t )=lnq e -k1t Where, q e q represents the SCOD value at equilibrium, in mg / L.t k is the SCOD value at time t; k1 is the PFO model rate constant, in d. -1 .

[0047] To analyze the relative abundance of functional genes, centrifuged sediment samples were collected at the end of the experiment, and genomic DNA was extracted from 500 mg samples using a soil DNA extraction kit. Genes related to hydrolysis and transformation (ompF, pilA, and celA), and genes related to acetic acid synthesis (pta and ackA) were selected as targets. qPCR reactions were performed on a QuantStudio 3 real-time quantitative PCR instrument, with a total volume of 10 μL, including 5 μL of SYBR mixture, 0.4 μL of forward primer, 0.4 μL of reverse primer, 0.2 μL of ROX, 3 μL of H2O, and 1 μL of DNA template. The template DNA concentration was 10 ng / μL; the cycling program and annealing temperature were set according to the primer and kit instructions. Primer sequences for each target gene are shown in Table 3.

[0048] qPCR analysis was performed using a real-time quantitative PCR system. The 16S rRNA gene was used as an internal reference gene. The relative abundance of the target gene was analyzed by normalizing the changes in Ct values.

[0049] To evaluate the physiological state of algal cells, samples CK1, CK2, and RL4 were collected on day 10 for trypan blue staining analysis. Simultaneously, extracellular lactate dehydrogenase (LDH) activity and ATP content were measured. LDH is a lactate dehydrogenase and can serve as an indicator of cell membrane damage.

[0050] To evaluate the level of antioxidant stress, the mud-water mixture was centrifuged at 10,000 rpm for 10 min to remove the supernatant. 0.1 g of sludge was placed in a 2 mL centrifuge tube, 1 mL of PBS was added, and the tube was sonicated on ice for 5 min. It was then centrifuged again at 10,000 rpm for 10 min. The supernatant was collected and the levels of CAT, MDA, and GSH were measured using a kit. The sonication program was 20% power, 3 s working time / 10 s interval, for a total working time of 6 min.

[0051] Microbial community analysis was performed using 16S rRNA high-throughput sequencing of fermentation samples from groups CK1, CK2, and RL4. The amplification primers were 338F / 806R, the sequencing platform was Illumina MiSeq, and the data analysis platform was the Majorbio I-Sanger cloud platform. The databases used were SILVA 138 and RDP 11.5. Samples were collected on day 10 after fermentation ended, and one representative sample from each group was used for sequencing analysis. Data processing was performed using SPSS 27.0 and Origin 2021.

[0052] Table 3 qPCR primer sequences Example 5: Comparative Example and Results of the Examples Comparative Example 1 was CK1 group, without the addition of cellulose-degrading bacteria and rhamnolipin; Comparative Example 2 was CK2 group, with only the addition of cellulose-degrading bacteria; Examples 1 to 5 correspond to RL1 to RL5 groups respectively, that is, with the addition of 10% cellulose-degrading bacteria, 0.1, 0.2, 0.3, 0.4 and 0.5 g / g TS of rhamnolipin were added respectively.

[0053] Table 4 Comparison of Key Performance Indicators (CK1, CK2 and RL4) Hydrolysis and acid production results like Figure 1 As shown, within 0–2 days after the start of fermentation, the SCOD concentration in each group accumulated rapidly. On day 1, the system basically entered the hydrolysis-dominated stage, and after reaching its peak, the SCOD gradually transitioned to dynamic equilibrium. As the rhamnolipin dosage increased from 0 to 0.4 g / g TS, the SCOD peak value generally showed an upward trend. When the rhamnolipin dosage was further increased to 0.5 g / g TS, the gain was no longer significant, indicating that excessively high dosage did not further improve the hydrolysis effect. Taking group RL4 as an example, its SCOD peak value reached 9511 mg / L, which was 44% higher than the blank control group.

[0054] PFO fitting was performed on the SCOD data of CK1 and RL4 groups to obtain R0 2 The values ​​are 0.982 and 0.971 respectively; the corresponding k1 values ​​are 1.45 d. -1 and 1.93 d -1 This indicates that the hydrolysis rate of algal sludge is significantly accelerated under the preferred conditions of this invention. Key performance indicators for each group are shown in Table 4.

[0055] like Figure 2 As shown, with increasing rhamnolipin dosage, the acetic acid yield and total VFA concentration generally showed a trend of first increasing and then decreasing. The acetic acid yield in group RL4 reached 0.24 g / gVS, which was 71.4% and 60% higher than that in groups CK1 and CK2, respectively; the total VFA concentration reached 4.87 g / L, higher than 1.98 g / L in group CK1 and 2.25 g / L in group CK2. Group RL5 did not show further increases, suggesting that excessive rhamnolipin may have an adverse effect on anaerobic bacterial activity.

[0056] EPS migration and 3D-EEM characterization Using samples fermented for 1 day as the analytical object, such as Figure 3As shown, the concentrations of soluble protein and polysaccharide in SMP of group RL4 were 3300 mg / L and 286 mg / L, respectively, which were 47.65% and 83.3% higher than those of group CK2 (2235 mg / L and 156 mg / L). Meanwhile, the protein concentration in TB-EPS of group RL4 decreased from 968 mg / L in group CK2 to 400 mg / L, and the polysaccharide concentration decreased from 50.56 mg / L to 6.24 mg / L, indicating that tightly bound EPS was weakened and some of it migrated to LB-EPS or the supernatant.

[0057] like Figure 4 As shown, the 3D-EEM results of different EPS components in groups CK1, CK2, and RL4 revealed that the main fluorescent regions in each group were concentrated in the aromatic protein region and the soluble microbial byproduct region. Proteins and biodegradable components were more prominent in the supernatant of group RL4, while LB-EPS and TB-EPS showed the opposite migration trend to the liquid phase, further supporting the conclusion that rhamnolipids promote the transfer of solid-phase organic matter to the liquid phase.

[0058] Nitrogen form and endpoint pH like Figure 5 As shown, at day 1 of fermentation, the ammonia nitrogen concentration in group RL4 reached 229 mg / L and the total nitrogen concentration reached 450 mg / L, both higher than the control group. This indicates that the combined use of rhamnolipin and cellulose-degrading bacteria helps release nitrogenous organic matter and convert it into dissolved nitrogen. At the end of fermentation, the pH of group CK1 decreased from 10 to approximately 9.1, while the pH decrease was more significant in the groups with added bacteria and rhamnolipin, with group RL4 decreasing to 6.7, indicating a more complete acidification process.

[0059] Antioxidant stress and hydrolytic enzyme activity like Figure 6 As shown, the CAT activity and GSH content in the RL4 group were approximately 5.4 times and 6.22 times that of the control group, respectively, and the MDA content increased by 1.83 times, indicating that the algal cells under the combined treatment experienced stronger oxidative stress and membrane structure damage.

[0060] like Figure 7 As shown, when only the cellulose-degrading bacterial agent was added, the cellulase activity was approximately 1.63 times that of the control group. However, after further addition of rhamnolipids, the cellulase activity increased with increasing dosage, with the RL4 group showing an activity approximately 6.15 times that of the control group. The glucanase activity in the RL4 group was approximately 2.66 times that of the control group, and the protease activity was approximately 4.2 times that of the control group, indicating that rhamnolipids improved the utilization efficiency of the cellulose-degrading bacterial agent on algal cell walls and intracellular components by improving substrate accessibility.

[0061] Functional genes and cellular physiological state like Figure 8As shown, with the increase of rhamnolipid concentration, the relative abundance of ackA and pta generally increased, suggesting an enhanced potential metabolic capacity for the conversion of acetyl-CoA to acetic acid; the increase in the relative abundance of celA indicates an enhanced ability to hydrolyze cellulose, and the increase in the relative abundance of pilA indicates an improved ability of cell attachment and interfacial interaction; while the decrease in the relative abundance of ompF suggests that the structure of the system's microbial community is shifting towards a functional community that is more conducive to hydrolysis and acid production.

[0062] like Figure 9 and Figure 10 As shown, the trypan blue staining area in group RL4 was enlarged and the cell morphology was irregular. The extracellular LDH activity reached 40 nmol / min / g fresh weight, which was about 4 times that of group CK1, indicating that the cell membrane integrity was significantly damaged.

[0063] In this embodiment, the LDH enzyme activity is calculated using the following formula: LDH = 146.4 × (ΔA - 0.0037) ÷ W, where W is the fresh weight of the sample in g; and LDH is measured in nmol / min / g fresh weight.

[0064] like Figure 11 As shown, on day 1, the ATP content of groups RL3, RL4 and RL5 was 4.66 times, 6.88 times and 6.79 times that of group CK1, respectively, indicating that the release of substrate after algal cell rupture promoted the overall metabolic activity of the fermentation system; by day 10, the ATP in each group had dropped to a low level, indicating that the contents of algal cells had been further utilized.

[0065] Microbial community structure like Figure 12 As shown, at the phylum level, the relative abundance of Firmicutes in groups CK1, CK2, and RL4 was approximately 5.9%, 9.1%, and 9.1%, respectively; Bacteroidetes decreased to 9.4% in group RL4; and Chlorophyta maintained a high proportion in all groups. At the genus level, the relative abundance of norank_f__Anaerolineaceae in groups CK1, CK2, and RL4 was 23.9%, 21.0%, and 26.7%, respectively, with the highest value in group RL4; Proteiniphilum decreased to 3% in group RL4; and Candidatus_Caldatribacterium remained relatively stable, suggesting an enrichment trend of functional bacteria related to cellulose degradation and acid production in the system after combined enhancement.

[0066] In summary, the preferred embodiment of the present invention is RL4 group, where the rhamnolipin dosage is 0.4 g / g TS and the cellulose-degrading bacterial agent dosage is 10%.

[0067] Industrial applicability The method described in this invention uses cyanobacteria sludge as a specific substrate and enhances the hydrolysis and acid production efficiency of cyanobacteria sludge through the combined use of rhamnolipin and cellulose-degrading bacteria. This improves efficiency without relying on high-energy-consuming physicochemical pretreatment and is suitable for batch anaerobic acid production from cyanobacteria sludge, preparation of volatile fatty acids, and related resource utilization. It should be noted that any equivalent substitutions and modifications made by those skilled in the art without departing from the concept of this invention should fall within the scope of protection of this invention.

Claims

1. A method for anaerobic acid production from cyanobacterial sludge using a synergistic effect of rhamnolipid and cellulose-degrading bacteria, characterized in that, The process includes the following steps: (1) Mixing cyanobacterial sludge with anaerobic inoculated sludge to form a fermentation system, such that the total solids (TS) of the substrate in the fermentation system is 14% and the volatile solids (VS) of the system is 10 g / L; (2) Adding a cellulose-degrading bacterial agent washed with buffer solution to the fermentation system, wherein the cellulose-degrading bacterial agent is a bacterial solution obtained by enrichment and domestication of anaerobic sludge with sodium carboxymethyl cellulose as the limiting substrate, and the amount added is 10% of the volume of the fermentation system; (3) Adding rhamnolipin to the fermentation system according to the total solids of cyanobacterial sludge, with an addition amount of 0.1-0.5 g / g TS, and adding a methane inhibitor; (4) Adjusting the pH of the fermentation system to 10, purging the headspace of the system with nitrogen for 5 min, and then anaerobic fermenting for 10 days at 37℃ and 120 r / min to obtain a fermentation broth rich in volatile fatty acids.

2. The method according to claim 1, characterized in that, The dosage of rhamnolipin is 0.3–0.5 g / gTS.

3. The method according to claim 2, characterized in that, The dosage of rhamnolipin is 0.4 g / g TS.

4. The method according to claim 1, characterized in that, The methane inhibitor is sodium 2-bromoethanesulfonate, with a final concentration of 3 g / L.

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 fermentation was carried out in batches in 250 mL serum bottles.

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, The cellulose-degrading bacterial agent described in step (2) is washed three times with phosphate buffer before addition to remove culture medium residue.

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