Method for producing acid by anaerobic co-fermentation of blue-green algae sludge pretreated by bovine rumen fluid

Abstract

This invention discloses a method for anaerobic co-fermentation of cyanobacterial sludge to produce acid through pretreatment with bovine rumen fluid. 6–12 g of bovine rumen fluid is added to every 100 mL of the cyanobacterial sludge-water mixture, and the mixture is pretreated at 35–39°C for 12–24 h. Without removing or deactivating the bovine rumen fluid, it is mixed with inoculum sludge, and the pH of the system is adjusted to 9.5–10.5. After replacing oxygen with an inert gas, anaerobic fermentation is carried out at 35–37°C, maintaining a stirring speed of 120–150 rpm. This method can promote the dissolution of proteins and carbohydrates in cyanobacterial sludge and increase the yield of acetic acid and volatile fatty acids, making it suitable for the resource utilization of cyanobacterial sludge and the preparation of alternative carbon sources.

Description

Technical Field

[0001] This invention relates to the fields of cyanobacterial resource utilization, anaerobic fermentation, and wastewater treatment alternative carbon source preparation technology, and particularly to a method for anaerobic co-fermentation of cyanobacterial sludge pretreated with bovine rumen fluid to produce acid. Background Technology

[0002] The large amounts of cyanobacterial sludge formed after cyanobacterial blooms are rich in proteins and polysaccharides, possessing the potential to be converted into volatile fatty acids (VFAs) through anaerobic fermentation. These VFAs can serve as an external carbon source for subsequent biological denitrification processes in wastewater, thus realizing the resource utilization of cyanobacterial sludge. However, the relatively dense cell wall structure of cyanobacteria and the fact that their cell surfaces are typically coated with extracellular polymeric substances (EPS) limit substrate dissolution and slow hydrolysis rates, thereby restricting the acid production efficiency of anaerobic fermentation of cyanobacterial sludge.

[0003] Existing enhancement methods mainly include physical or chemical approaches such as ultrasound, homogenization, heat treatment, and acid / alkali treatment. While these methods can improve substrate accessibility to some extent, they typically suffer from high energy consumption, high operating costs, and the potential introduction of additional reagents or interference with subsequent microbial processes. Furthermore, in engineering algae harvesting or algae-water separation scenarios, cyanobacterial sludge may carry flocculant residues, further leading to floc aggregation and enhanced EPS barriers, thus exacerbating mass transfer limitation.

[0004] Bovine rumen fluid contains abundant cellulose-degrading bacteria, protein-hydrolyzing bacteria, and related complex enzyme systems, enabling it to degrade complex organic substrates. However, current technologies mainly focus on the promoting effect of rumen fluid on substrates such as straw, distiller's grains, or ordinary sludge. For cyanobacterial sludge, a substrate with a special cell wall and EPS barrier, especially the synergistic enhancement mode of "pretreatment with bovine rumen fluid followed by anaerobic co-fermentation without removing or deactivating the bovine rumen fluid," there is still a lack of targeted process construction and long-term continuous flow validation.

[0005] Therefore, it is necessary to provide a bio-enhancement method that can target the characteristics of cyanobacterial sludge substrates, take into account both batch acid production performance and continuous flow stability, so as to improve the efficiency of acid production from anaerobic fermentation of cyanobacterial sludge and improve the feasibility of the process. Summary of the Invention

[0006] Technical problems to be solved This invention aims to address the problems of insufficient substrate dissolution, limited hydrolysis, and low acid production efficiency caused by algal cell walls and extracellular polymer barriers during the anaerobic fermentation of cyanobacterial sludge. It provides a pretreatment and co-fermentation synergistic method using bovine rumen fluid as a biofortifier to improve the hydrolysis and acidification efficiency of cyanobacterial sludge under milder conditions and enhance the stability of continuous flow operation.

[0007] Technical solution To solve the above-mentioned technical problems, the present invention provides the following technical solution: A method for pretreating cyanobacterial sludge with bovine rumen fluid through anaerobic co-fermentation to produce acid includes the following steps: (1) Provide cyanobacterial sludge, bovine rumen fluid and inoculation sludge; (2) Add bovine rumen fluid to the cyanobacterial sludge, such that the amount of bovine rumen fluid added is 6% to 12% (w / v), wherein the 6% to 12% (w / v) is calculated based on adding 6 to 12 g of bovine rumen fluid to every 100 mL of cyanobacterial sludge-water mixture, and pretreat at 35 to 39°C for 12 to 24 h; (3) Without removing or deactivating the bovine rumen fluid, the pretreated mixture is mixed with the inoculated sludge, the pH of the system is adjusted to 9.5-10.5, and after replacing the oxygen with an inert gas, anaerobic fermentation is carried out at 35-37°C. (4) Maintain a stirring speed of 120-150 rpm during the fermentation process to obtain a fermentation broth rich in volatile fatty acids.

[0008] The volatile fatty acids preferably include acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid.

[0009] Preferably, the amount of bovine rumen fluid added is 8% to 12% (w / v), more preferably 10% (w / v).

[0010] Preferably, the pretreatment time is 24 hours.

[0011] Preferably, the inoculation ratio of inoculated sludge to cyanobacterial sludge is 1:5, by volume.

[0012] Preferably, the inoculated sludge is heat-treated at 80°C for 5 hours before use.

[0013] Preferably, in the continuous operation implementation, the cyanobacterial sludge is further subjected to mechanical pretreatment and operated in a continuous stirred tank reactor with a hydraulic retention time of 14 days; the continuous operation includes, in sequence, a start-up phase, an organic load increase phase, a mechanical pretreatment phase, and a bovine rumen fluid addition phase.

[0014] Beneficial effects (1) Since the bovine rumen fluid pretreatment in step (2) can act on the cyanobacterial cell wall and EPS barrier, it can reduce the particle size of cyanobacterial sludge and promote the release of intracellular organic matter, thereby improving the accessibility of substrate organisms.

[0015] (2) Since the anaerobic co-fermentation is carried out in step (3) without removing or losing the bovine rumen fluid, it is more beneficial to improve the yield of acetic acid and the yield of total VFAs than pretreatment or co-fermentation alone.

[0016] (3) Within the parameter window of the present invention, a higher acid production efficiency can be obtained when the dosage is preferably 10% (w / v), and the main acid composition remains stable.

[0017] (4) In the continuous flow implementation, mechanical pretreatment and the addition of bovine rumen fluid form a composite enhancement, which is beneficial to maintaining a high acetic acid ratio and a relatively stable total VFA concentration.

[0018] (5) This invention provides an feasible technical path for the resource utilization of cyanobacterial sludge and the preparation of subsequent alternative carbon sources. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of a CSTR device, where 1 is the pretreatment CSTR reactor, 2 is the feed pump, 3 is the CSTR reactor, 4 is the stirring motor, 5 is the stirring shaft and stirring paddle, 6 is the heating jacket, 7 is the pH adjustment port, 8 is the sampling port, 9 is the discharge port, 10 is the gas outlet pipe, and 11 is the gas collection bag.

[0020] Figure 2 The figure shows the effect of bovine rumen fluid pretreatment on the physicochemical properties and dissolution-release behavior of algal sludge. Figure 2 (a) is a graph showing the variation in particle size distribution. Figure 2 (b) is a graph showing the change in the dissolution rate of organic substrates.

[0021] Figure 3 The figure shows the effect of bovine rumen fluid pretreatment on the EPS of algal sludge. Figure 3 (a) and Figure 3 (b) is a diagram showing the changes in extracellular polymer components at different levels.

[0022] Figure 4 The figure shows the effects of bovine rumen fluid dosage and treatment method on the yield and composition of VFA from cyanobacterial fermentation. Figure 4 (a) Figure 4 (b) Figure 4 (c) and Figure 4 (g) is a graph showing the changes in acetic acid production under different treatments. Figure 4 (d) Figure 4 (e) Figure 4 (f) and Figure 4 (h) is a graph showing the changes in total VFAs production under different treatment methods. Figure 4 (i) is a graph showing the percentage changes of various VFAs.

[0023] Figure 5 This is a diagram showing the operation of the CSTR reactor, where... Figure 5 (a) and Figure 5 (b) is a graph showing the changes in protein and carbohydrate content in the effluent at each stage. Figure 5 (c) is a graph showing the changes in VFA concentration in the reactor.

[0024] Figure 6 The Venn diagram shows the species in the four stages of the reactor, where S1, S2, S3, and S4 correspond to stages I, II, III, and IV, respectively.

[0025] Figure 7 This diagram shows the changes in microbial phylum levels during the four stages of the reactor, where A1, A2, A3, and A4 correspond to stages I, II, III, and IV, respectively.

[0026] Figure 8 This diagram shows the changes in microbial genera at the four stages of the reactor, where A1, A2, A3, and A4 correspond to stages I, II, III, and IV, respectively. Detailed Implementation

[0027] Example 1: Preparation of Experimental Materials The cyanobacterial sludge was collected from an algae-water separation station in Wujin District, Changzhou City. The total solids (TS) content of the cyanobacterial sludge was approximately 10%, and the volatile solids (VS) content was approximately 7.8%. Bovine rumen fluid was collected from fresh rumen contents at a slaughterhouse in Yixing City, Jiangsu Province. After filtration through four layers of gauze, it was immediately incubated at 39℃ under anaerobic conditions for later use; its TS content was 18.5±0.25 g / L, and its VS content was 11.65±0.38 g / L. The inoculum sludge had a TS content of 6.36±0.33 g / L and a VS content of 5.22±0.14 g / L; before inoculation, the inoculum sludge was heat-treated at 80℃ for 5 h to inhibit methanogenic activity while retaining acidogenic activity. The cyanobacteria collection season was from June to August in summer, with Microcystis aeruginosa being the main algae species. The inoculated sludge was taken from the anaerobic expanded granular sludge bed (EGSB) reactor of Wuxi Huilian Thermal Power Co., Ltd. Before inoculation, larger particles and impurities were removed using a 60-mesh sieve, and the concentrated sludge was then heat-treated in a water bath at 80℃ for 5 h to selectively inhibit methanogenic bacteria while retaining acid-producing activity. All inoculation ratios mentioned in this article are based on volume ratios.

[0028] Example 2: Screening of Bovine Rumen Fluid Dosage and Treatment Method Batch fermentation experiments were conducted in 250 mL fermentation flasks. In this example, the inoculum ratio (inoculation sludge: cyanobacterial sludge) was 1:5, by volume. Bovine rumen fluid was added to the cyanobacterial sludge system at four gradients: 6%, 8%, 10%, and 12% (w / v), and the following three treatment methods were set up: (1) Pretreatment only: The cyanobacterial sludge was first mixed with bovine rumen fluid and pretreated at 35°C for 24 h; after the pretreatment was completed, air was circulated for 4 h to inactivate the rumen microorganisms before proceeding to the subsequent fermentation.

[0029] (2) Co-fermentation group only: Blue-green algae sludge is fermented directly with bovine rumen fluid without pretreatment.

[0030] (3) Pretreatment + co-fermentation group: The cyanobacterial sludge was pretreated with bovine rumen fluid at 35°C for 24 h, and then directly entered the subsequent anaerobic fermentation without inactivation treatment.

[0031] Before fermentation, the pH of each group was adjusted to 10 and nitrogen was used to purge for 5 minutes to create an anaerobic environment. Fermentation was then carried out at 35℃ and 150 rpm for 16 days. 2 mL of fermentation broth samples were collected daily to determine the concentration and composition of VFAs.

[0032] like Figure 4 As shown, the acid production performance of the pretreatment + co-fermentation group was superior to that of the pretreatment-only group and the co-fermentation-only group. At a dosage of 10% (w / v), the acetic acid yield of the pretreatment + co-fermentation group reached 0.304 g / g VS, and the total VFAs yield reached 0.519 g / g VS; correspondingly, the acetic acid yield of the pretreatment-only group was 0.284 g / g VS, and the total VFAs yield was 0.486 g / g VS; while the co-fermentation-only group had an acetic acid yield of 0.219 g / g VS and a total VFAs yield of 0.400 g / g VS. With further increases in bovine rumen fluid dosage, the increase in acid production tended to plateau. Figure 4 (i) shows that acetic acid was the dominant acid type in all treatment groups, accounting for approximately 55% to 60% of the total VFAs.

[0033] Example 3: Substrate dissolution and EPS reconstruction analysis Using untreated cyanobacterial sludge as a control, the effects of bovine rumen fluid pretreatment on particle size, soluble organic matter release, and extracellular polymeric substance composition were investigated. Particle size was determined using laser particle size analysis to measure the median particle size (D50); soluble protein was determined using the Lowry-Folin method; soluble carbohydrates were determined using the phenol-sulfuric acid method; and EPS from different grades were separated using thermal extraction.

[0034] like Figure 2 and Figure 3As shown, after pretreatment with bovine rumen fluid, the median particle size D50 of the cyanobacterial sludge decreased from 80.64 μm to 63.89 μm; the ratio of soluble protein to total protein increased by approximately 142%, and the ratio of soluble carbohydrates to total carbohydrates increased by approximately 197%. Simultaneously, the protein and polysaccharide contents in TB-EPS and LB-EPS decreased, while the corresponding components in S-EPS increased, indicating that bovine rumen fluid can disrupt the physical barrier of the algal sludge and promote the release of organic substrates into the liquid phase.

[0035] Example 4: Long-term operation verification of continuous flow CSTR like Figure 1 As shown, long-term operation verification was conducted in a continuous stirred tank reactor. The reactor operated at 35°C, with a stirring speed of 120 rpm and a hydraulic retention time of 14 days, in a semi-continuous mode, with an effective volume of 4 L and feeding once a day. The experiment lasted for 168 days and can be divided into four stages: start-up stage (stage I), organic load increase stage (stage II), mechanical pretreatment stage (stage III), and bovine rumen fluid addition stage (stage IV). In stage I, the organic load was 5 g VS / L; after entering stage II on day 42, the organic load was increased to 10 g VS / L; after entering stage III on day 84, mechanical pretreatment was implemented on the fed cyanobacterial sludge; after entering stage IV on day 126, 10% (w / v) bovine rumen fluid was added to the cyanobacterial sludge while maintaining mechanical pretreatment, with the same measurement basis as before. Each stage was run until the VFA concentration in the system was basically stable.

[0036] like Figure 5 As shown, simply increasing the organic loading rate resulted in a system acetic acid yield of 0.224 g / g VS and a total acid yield of 0.449 g / g VS. After introducing mechanical pretreatment, the acetic acid yield increased to 0.293 g / g VS and the total acid yield to 0.544 g / g VS. With the addition of bovine rumen fluid while maintaining mechanical pretreatment, the system acetic acid yield increased to 0.317 g / g VS and the total acid yield to 0.617 g / g VS. In stage IV, the acetic acid concentration was maintained at approximately 3.8 g / L, the total VFA concentration was maintained at approximately 7.4 g / L, and the acetic acid content was maintained at 50%–60%. Compared to stage III, stage IV showed an increase in acetic acid yield and total acid yield of approximately 8.2% and 13.4%, respectively, indicating a synergistic enhancing effect between bovine rumen fluid and mechanical pretreatment.

[0037] Example 5 Microbial Community Analysis 16S rRNA high-throughput sequencing analysis was performed on samples from different operating stages of the continuous flow reactor. The sequencing platform was Illumina MiSeq, with primers 338F / 806R corresponding to the amplified regions. Annotation was performed using the Silva database, and the OTU clustering similarity threshold was 0.97. Figures 6 to 8 As shown, the community diversity decreased and the functional dominant bacteria increased during the addition of bovine rumen fluid. Among them, the proportion of Firmicutes (Bacillota) increased, and genera related to hydrolysis, acid production, and syntrophic metabolism, such as Clostridium and Syntrophobacter, were enriched. This indicates that bovine rumen fluid not only improves substrate accessibility, but also promotes the evolution of the fermentation system to a highly efficient acid-producing state through community reconstruction. Figure 6 S1, S2, S3, and S4 in the text and Figure 7 , Figure 8 A1, A2, A3, and A4 in the diagram correspond to stages I, II, III, and IV, respectively.

[0038] Example 6 Quality Control and Testing Methods To ensure the feasibility of the process, the present invention preferably employs the following detection method: (1) Total solids (TS) and volatile solids (VS) were determined according to standard methods; (2) VFAs were determined by gas chromatograph equipped with flame ionization detector (FID), which can be GC-2030 (Shimadzu, Japan); total VFAs were calculated as the sum of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid; the chromatographic column was a DB-FFAP capillary column (30 m × 0.25 mm × 0.25 μm), the initial temperature was 50℃ and held for 1 min, then the temperature was increased to 200℃ at a rate of 10℃ / min and held for 1 min, the carrier gas flow rate was 1.0 mL / min, the injection volume was 1 μL, splitless injection was used, and 5 to 7 series of mixed standard solutions of different concentrations were prepared using acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid standards, and an external standard curve was established with the concentration of each component as the abscissa and the peak area as the ordinate; (3) EPS was extracted using the thermal extraction method, and S-EPS, LB-EPS and TB-EPS were analyzed separately; (4) Particle size was determined using a laser particle size analyzer, and D50 represents the median particle size; (5) Statistical analysis should be performed in at least 3 parallel replicates. ANOVA should be used to determine the significance of the difference. p<0.05 is considered to be statistically significant.

[0039] The above embodiments are used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. For those skilled in the art, equivalent substitutions or conventional adjustments made to process parameters, testing conditions, and operating modes without departing from the concept of the present invention should all fall within the scope of protection of the present invention.

Claims

1. A method for pretreating cyanobacterial sludge with bovine rumen fluid through anaerobic co-fermentation to produce acid, characterized in that, Includes the following steps: S1. Provide cyanobacterial sludge, bovine rumen fluid, and inoculated sludge; S2. Add bovine rumen fluid to the cyanobacterial sludge, such that the amount of bovine rumen fluid added is 6%–12% (w / v), wherein the 6%–12% (w / v) is calculated based on adding 6–12 g of bovine rumen fluid per 100 mL of cyanobacterial sludge-water mixture, and pretreat at 35–39°C for 12–24 h; S3. Without removing or deactivating the bovine rumen fluid in step S2, the pretreated mixture obtained in step S2 is mixed with the inoculated sludge, the pH of the system is adjusted to 9.5-10.5, and after replacing the oxygen with an inert gas, anaerobic fermentation is carried out at 35-37℃. S4. During the anaerobic fermentation process in step S3, maintain the stirring speed at 120-150 rpm to obtain a fermentation broth rich in volatile fatty acids.

2. The method according to claim 1, characterized in that, The dosage of the bovine rumen fluid is 8% to 12% (w / v), and its measurement standard is the same as that in claim 1.

3. The method according to claim 2, characterized in that, The dosage of bovine rumen fluid was 10% (w / v).

4. The method according to claim 1, characterized in that, The preprocessing time in step S2 is 24 hours.

5. The method according to claim 1, characterized in that, The inoculation ratio of the inoculated sludge to the cyanobacterial sludge in step S3 is 1:5, by volume.

6. The method according to claim 1, characterized in that, In step S3, nitrogen gas is used to flush the air for 5 minutes to create an anaerobic environment.

7. The method according to claim 1, characterized in that, The inoculated sludge was heat-treated at 80°C for 5 hours before inoculation.

8. The method according to claim 1, characterized in that, The bovine rumen fluid was filtered through four layers of gauze and then kept warm at 39°C under anaerobic conditions for later use.

9. The method according to claim 1, characterized in that, Mechanical pretreatment is performed on the cyanobacterial sludge prior to step S2 to improve substrate accessibility.

10. The method according to claim 9, characterized in that, The method is operated in a semi-continuous manner in a continuous stirred tank reactor with a hydraulic retention time of 14 days, a reaction temperature of 35°C, and a stirring speed of 120 rpm. The continuous operation includes a start-up stage, an organic load enhancement stage, a mechanical pretreatment stage, and a bovine rumen fluid addition stage. The organic load in the start-up stage is 5 g VS / L, the organic load in the organic load enhancement stage is 10 g VS / L, and the bovine rumen fluid addition stage continues to add the amount of bovine rumen fluid described in claim 3 to the cyanobacterial sludge based on the mechanical pretreatment.

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