Use of antibiotics in promoting methanogenesis from anaerobic fermentation of cellulose and methods

Abstract

The application discloses application and method of antibiotics in promoting cellulose anaerobic fermentation to produce methane, antibiotics are added in a mixture of cellulose and wastewater sludge, methane is obtained through anaerobic fermentation, in the mixture, the content of cellulose is 0.3-0.4 g per 100 mL of wastewater sludge, the content of antibiotics is 5-15 ug per 100 mL of the mixture; the anaerobic fermentation is cultured in a shaking incubator at 120 rpm for 20 days at a temperature of 37 DEG C; and the antibiotics are tetracycline or azithromycin. Through introducing antibiotics, i.e. tetracycline or azithromycin, in the anaerobic fermentation process with cellulose as a substrate and wastewater sludge as inoculum, the application not only can avoid problems such as high cost and secondary pollution caused by cellulose pretreatment, and simplify production steps, but also can improve methane yield, and fills a blank of antibiotics in promoting cellulose anaerobic fermentation to produce methane.

Description

Technical Field

[0001] This invention relates to the application and method of antibiotics in promoting anaerobic fermentation of cellulose to produce methane. Specifically, it relates to the application of antibiotics in the process of producing methane using cellulose as a substrate and sewage sludge as an inoculum, as well as a method for increasing methane yield using antibiotics, belonging to the field of biofermentation technology. Background Technology

[0002] With the increasing severity of the energy crisis, seeking new clean and renewable energy sources has become one of the important ways to solve such problems. Anaerobic digestion of lignocellulosic biomass is considered an effective method to produce bioenergy and reduce environmental pollution due to its environmental sustainability, abundant supply and low cost. However, due to its complex three-dimensional structure, it is difficult to hydrolyze. Therefore, the methane conversion rate is not ideal when fermenting lignocellulosic biomass to produce methane gas.

[0003] The main components of lignocellulose biomass are cellulose, hemicellulose, and lignin. Cellulose has a higher methane production potential than hemicellulose or lignin. Therefore, finding methods to promote methane production from cellulose is crucial for improving the fermentation efficiency of lignocellulose biomass. Hao Xiaodi et al., in their study "Experimental Study on Enhanced Methane Production from Lignocellulose in Sludge" (Journal of Environmental Engineering, July 2015, Vol. 9, No. 7, pp. 3432-3440), conducted a comparative experiment on the anaerobic fermentation process of sludge with added lignocellulose components. The experiment showed that adding enriched microorganisms to the anaerobic digestion process can effectively enhance the degradation and conversion of cellulose and hemicellulose, increasing methane gas production. However, the abnormally stable chemical structure of lignocellulose in the sludge makes it difficult for easily degradable components such as cellulose and hemicellulose to be released freely and effectively contact the added enriched microorganisms. Therefore, only through pretreatment methods, namely acid, alkali, heat, and ultrasonic pretreatment of the sludge, followed by anaerobic digestion, can the lignocellulose components be effectively degraded and converted, thereby obtaining a higher methane production rate.

[0004] For example, the invention patent with publication number CN115029386A discloses a method for improving the yield and efficiency of methane production from solid-state anaerobic fermentation of agricultural and forestry residues. This method uses a peroxide-alkali coupling system to pretreat agricultural and forestry residues such as straw, cotton stalks, poplar wood, and bamboo. Then, the pretreated products are mixed with livestock and poultry manure for solid-state anaerobic fermentation, which can increase the methane production rate. Compared with untreated lignocellulose raw materials, the cumulative gas production can be increased by 100%. Another invention patent with publication number CN115011639A discloses a method for producing biogas from lignocellulose raw materials. This method involves moistening, chopping, and biologically pretreating the lignocellulose raw materials before primary anaerobic fermentation to produce biogas. The solid residue after fermentation is then dehydrated, alkali solution is added, and after kneading, it is mixed with livestock and poultry manure for secondary fermentation to produce biogas. The resulting biogas slurry is then recycled as inoculum. In this method, the cumulative gas production from lignocellulose raw materials is increased by more than 70% compared with traditional fermentation methods, and the methane volume fraction is increased by 5 percentage points.

[0005] As can be seen from the above, in the method of using sludge or livestock manure as fermentation material to produce methane from fiber raw materials, pretreatment of the fiber raw materials is required. In actual production, although pretreatment can increase the yield of methane gas, the increase in steps will inevitably lead to an increase in production costs and process control difficulties, which is not conducive to industrial conversion. Summary of the Invention

[0006] This invention aims to provide the application and method of antibiotics in promoting anaerobic fermentation of cellulose to produce methane. By introducing antibiotics, namely tetracycline or azithromycin, into the anaerobic fermentation process with cellulose as substrate and wastewater sludge as inoculum, it is possible to not only avoid the high cost and secondary pollution caused by cellulose pretreatment and simplify the production steps, but also increase methane production, thus filling the gap in the application of antibiotics in promoting anaerobic fermentation of cellulose to produce methane.

[0007] This invention is achieved through the following technical solution: a method for increasing methane production from cellulose, wherein antibiotics are added to a mixture of cellulose and wastewater sludge, and methane is obtained through anaerobic fermentation. In the mixture, the cellulose content is 0.3-0.4g per 100mL of wastewater sludge, and the antibiotic content is 0.5-1.5mg per 100mL of the mixture; The anaerobic fermentation was carried out at 37°C in a shaking incubator at 120 rpm for 20 days. The antibiotic is tetracycline or azithromycin.

[0008] The cellulose is microcrystalline cellulose and / or cellulose powder.

[0009] The wastewater sludge comes from a sewage treatment plant and is a wet sludge with a concentration of 1.5%wt obtained after centrifugation and washing at 4°C.

[0010] The centrifugation was controlled at 8000×g.

[0011] The washing process involves thoroughly shaking the water with distilled water, and the washing steps are repeated three times.

[0012] When adding antibiotics to the mixture, the antibiotics are first dissolved in a solvent, then added to the mixture and the concentration of antibiotics in the mixture is adjusted.

[0013] The solvent is ethanol.

[0014] The application of antibiotics in promoting anaerobic fermentation of cellulose to produce methanogens can be achieved using the methods described above.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention introduces antibiotics for the first time in the process of producing methane by anaerobic fermentation of cellulose. This not only avoids the need for pretreatment of cellulose, but also reduces the difficulty and cost of the process and increases the yield of methane produced from cellulose.

[0016] (2) The present invention uses specific antibiotics: tetracycline or azithromycin. In the process of anaerobic methane production with cellulose as substrate and wastewater sludge as inoculum, due to the change in microbial activity, it can promote each stage of the anaerobic production process, especially in the methanogenesis stage.

[0017] (3) In this invention, the production of methane from cellulose with added azithromycin can be increased by 51.94%, and the production of methane from cellulose with added tetracycline can be increased by 34.96%. Attached Figure Description

[0018] Figure 1 This serves as a control for the concentration and timing of antibiotic addition in the reactor.

[0019] Figure 2 Cellulase activity in reactors under different antibiotic pressures.

[0020] Figure 3 The total organic carbon dissolved in the reactor under different antibiotic pressures.

[0021] Figure 4 These are the organic acids produced in the reactor under different treatments.

[0022] Figure 5 Add subphylum-level bacterial diversity for different antibiotics.

[0023] Figure 6The relative abundance of phylum-level bacterial communities (a) and genus-level archaea communities (b) under different antibiotic addition conditions. Detailed Implementation

[0024] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0025] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the claimed invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] Antibiotics are secondary metabolites produced by microorganisms (including bacteria, fungi, and actinomycetes) or higher plants and animals during their life processes. They possess antipathogenic or other activities and can interfere with the developmental functions of other living cells. Antibiotics reported for use in anaerobic fermentation (AD) include oxytetracycline, chlortetracycline, cephalexin, sulfadiazine, sulfamethoxazole, enrofloxacin, ciprofloxacin, ofloxacin, norfloxacin, etc. They are commonly used in fermentation processes using waste sludge as a substrate. For example, Andriamanohiarisoamanana et al. studied the inhibitory effects of oxytetracycline and chlortetracycline on methane production in dairy cow manure and investigated the corresponding mechanisms. Other literature indicates that antibiotics have no significant effect on AD performance. For instance, Mitchell et al. demonstrated that sulfadiazine and ampicillin had no effect on the total biogas production of cow manure, with concentrations as high as 280 mg / L and 350 mg / L, respectively. Beneragama et al.'s research showed that cefazolin had no inhibitory effect on methane production in dairy cow manure. Lu et al. studied the long-term effects of cephalexin on methanogenesis in waste activated sludge, demonstrating that after 157 days, methane production increased by 63.8% compared to the control. Zhi and Zhang investigated the effects of several antibiotics (oxytetracycline, sulfadiazine, sulfamethoxazole, enrofloxacin, ciprofloxacin, ofloxacin, and norfloxacin) at concentrations ranging from 0 to 500 mg / L on dewatered sludge AD, showing that 100 mg / L of antibiotics could increase methane production.

[0027] Existing reports indicate that while antibiotics have been used to study their effects on methanogenesis during anaerobic fermentation of wastewater sludge, the results show that some antibiotics may promote anaerobic fermentation of wastewater sludge, while others may inhibit it. However, to date, no reports have been found on the effects of antibiotics on the anaerobic fermentation of cellulose inoculated wastewater sludge.

[0028] The following examples illustrate specific implementations of the present invention. Of course, the scope of protection of the present invention is not limited to the following examples.

[0029] Example 1: A method for methanogenesis by anaerobic fermentation of cellulose using azithromycin Substrate: Microcrystalline cellulose (MCC).

[0030] Inoculum: Wastewater sludge (WSS), centrifuged at 8000×g for 10 min at 4℃, the remaining precipitate was thoroughly shaken and washed with distilled water, the washing step was repeated 3 times, and then the final sludge concentration was adjusted to 1.5% with distilled water.

[0031] Antibiotics: Azithromycin (AZM) was used. AZM was dissolved in an ethanol solution with a concentration greater than 99% to serve as the antibiotic stock solution.

[0032] Anaerobic fermentation: Take a 120 mL fermentation flask, add 0.2 g MCC and 60 mL WSS, then add the antibiotic stock solution, and adjust the final concentration of AZM in the fermentation flask to 15 μg / mL using the antibiotic stock solution. Seal the fermentation flask with a butyl rubber stopper and purge with nitrogen for 5 min to establish anaerobic conditions. Then, incubate the fermentation flask at 37 °C and 120 rpm in a shaking incubator for 20 days, and collect the gas from the fermentation flask.

[0033] Example 2: A method for producing methane through anaerobic fermentation of tetracycline-based cellulose. Substrate: Microcrystalline cellulose (MCC).

[0034] Inoculum: Wastewater sludge (WSS), centrifuged at 8000×g for 10 min at 4℃, the remaining precipitate was thoroughly shaken and washed with distilled water, the washing step was repeated 3 times, and then the final sludge concentration was adjusted to 1.5% with distilled water.

[0035] Antibiotics: Tetracycline (TC) is used. TC is dissolved in an ethanol solution with a concentration greater than 99% to serve as the antibiotic stock solution.

[0036] Anaerobic fermentation: Take a 120 mL fermentation flask, add 0.2 g MCC and 60 mL WSS, then add the antibiotic stock solution, and adjust the final TC concentration in the fermentation flask to 5 μg / mL using the antibiotic stock solution. Seal the fermentation flask with a butyl rubber stopper and purge with nitrogen for 5 min to establish anaerobic conditions. Then, incubate the fermentation flask at 37 °C and 120 rpm in a shaking incubator for 20 days, and collect the gas from the fermentation flask.

[0037] Comparative Example 1: A method for producing methanogens by anaerobic fermentation of cellulose using cephalexin Substrate: Microcrystalline cellulose (MCC).

[0038] Inoculum: Wastewater sludge (WSS), centrifuged at 8000×g for 10 min at 4℃, the remaining precipitate was thoroughly shaken and washed with distilled water, the washing step was repeated 3 times, and then the final sludge concentration was adjusted to 1.5% with distilled water.

[0039] Antibiotic: Cephalexin (CLX), CLX is dissolved in deionized water as the antibiotic stock solution.

[0040] Anaerobic fermentation: Take a 120 mL fermentation flask, add 0.2 g MCC and 60 mL WSS, then add the antibiotic stock solution, and adjust the final concentration of CLX in the fermentation flask to 15 μg / mL using the antibiotic stock solution. Seal the fermentation flask with a butyl rubber stopper and purge with nitrogen for 5 min to establish anaerobic conditions. Then, incubate the fermentation flask at 37 °C and 120 rpm in a shaking incubator for 20 days, and collect the gas from the fermentation flask.

[0041] Comparative Example 2: Blank Control Substrate: Microcrystalline cellulose (MCC).

[0042] Inoculum: Wastewater sludge (WSS), centrifuged at 8000×g for 10 min at 4℃, the remaining precipitate was thoroughly shaken and washed with distilled water, the washing step was repeated 3 times, and then the final sludge concentration was adjusted to 1.5% with distilled water.

[0043] Anaerobic fermentation: Take a 120 mL fermentation flask, add 0.2 g MCC and 60 mL WSS, and then add an equal amount of ethanol as AZM and TC. Seal the fermentation flask with a butyl rubber stopper and purge with nitrogen for 5 min to establish anaerobic conditions. Then, incubate the fermentation flask at 37 °C and 120 rpm in a shaking incubator for 20 days, and collect the gas from the fermentation flask.

[0044] (I) Analysis of Methane Production For the gases collected in Examples 1, 2, Comparative Example 1, and Comparative Example 2 above, the methane yield was calculated using the Gompertz model, as shown in equation (1) below: B (t) = B0exp {- exp [ 1 + μ m e (λ – t) / B0]} (1) in, B (t) The cumulative methane production over time t (mL / g-VS); B0 represents the maximum methane production (mL / g-VS). μ m Maximum methane yield (mL / (g-VS•d)); λ is the lag time (d) for methanogenesis. t represents the digestion time (d); e is the Euler number 2.718.

[0045] Calculations showed that during the 20-day anaerobic fermentation period, compared with the control group, methane production increased by 51.94% and 34.96% in the examples with added AZM and TC, respectively, while methane production decreased by 23.95% in the example with added CLX. Specifically, the addition of AZM promoted all stages of anaerobic cellulose fermentation (hydrolysis, acid / acetic acid production, and methanogenesis), while the addition of TC mainly played a positive role in the methanogenesis stage.

[0046] (II) Analysis of antibiotic concentrations Antibiotic concentration analysis was performed according to the methods of Example 1, Example 2, Comparative Example 1, and Comparative Example 2. The antibiotic concentrations were as follows: CTRL (CLX) group: CLX was 0 μg / mL; CLX group: CLX concentration was 15 μg / mL. CTRL (TC / AZM) group: TC and AZM were 0 μg / mL; TC group: TC was 5 μg / mL; AZM group: AZM concentration was 15 μg / mL.

[0047] like Figure 1 As shown, the methane production in the CTRL (TC / AZM) group was significantly higher than that in the CTR (CLX) group, indicating that the use of ethanol can convert acetic acid and H2 during the acetogenesis stage, and further produce methane during the methanogenesis stage. The methane production in the CTRL (CLX) group was higher than that in the CLX group, indicating that CLX did not promote methane production. The methane production in the AZM and TC groups was higher than that in the CTRL (TC / AZM) group, indicating that AZM and TC have a promoting effect on methane production, and the promoting effect of AZM is significantly greater than that of TC.

[0048] In addition, from Figure 1 As shown, after 20 days of anaerobic fermentation, the addition of AZM and TC can promote the anaerobic fermentation of cellulose to produce methane, increasing the yield by 51.94% and 34.96% respectively compared with the control.

[0049] (III) Cellulase Activity Analysis Following the methods described in Examples 1, 2, Comparative Examples 1 and 2, 0.5 mL of the supernatant after centrifugation of the fermentation broth was taken as a sample for cellulase activity analysis, and the cellulase activity analysis was performed according to the method of Hua, et al (2014).

[0050] The results are as follows Figure 2 As shown, CTR (CLX) is the CLX control, with no antibiotics added, only an equal volume of deionized water added; CLX is the control with CLX added; CTRL (TC / AZM) is the TC and AZM control, with no antibiotics added, only an equal volume of ethanol added; TC is the control with TC added; AZM is the control with AZM added. Figure 2 It was found that during the entire 20-day anaerobic fermentation process, the cellulase activity of samples with added TC and AZM was higher than that of the control group, which is consistent with the methane yield results, indicating that the addition of TC and AZM can enhance cellulase activity to a certain extent and further promote cellulose hydrolysis. Compared with the control, the samples with added CLX had lower cellulase activity, indicating that the addition of CLX reduces cellulase activity.

[0051] (iv) DOC concentration analysis Following the methods described in Examples 1, 2, Comparative Examples 1 and 2, 30 mL of the supernatant sample after centrifugation of the fermentation broth was taken for DOC concentration analysis. The DOC concentration analysis was performed using a total organic carbon analyzer (TOC-VCPH, Japan).

[0052] The results are as follows Figure 3 As shown, CTR (CLX) is the CLX control, with no antibiotics added, only an equal volume of deionized water added; CLX is the control with CLX added; CTRL (TC / AZM) is the TC and AZM control, with no antibiotics added, only an equal volume of ethanol added; TC is the control with TC added; AZM is the control with AZM added. Figure 3 It was observed that after 20 days of fermentation, the DOC concentration in all reactors increased significantly due to the hydrolysis of MCC. In particular, the accumulated DOC in AZM was 24.55% higher than the control group (p = 0.003 << 0.05), indicating that AZM promotes the hydrolysis of MCC and its methane yield. The DOC content in TC on day 20 was similar to that of the control group, indicating that TC addition had no significant effect on cellulose hydrolysis. Conversely, the DOC concentration in CLX was 17.50% lower than the control, consistent with the trend of methane production in CLX.

[0053] (v) Organic acid analysis Following the methods described in Examples 1, 2, 1, and 2, 1 mL of sample was taken for organic acid content analysis using a gas chromatograph (GC-2030, Shimadzu, Japan).

[0054] The results are as follows Figure 4As shown, CTR (CLX) is the CLX control, with no antibiotics added, only an equal volume of deionized water added; CLX is the control with CLX added; CTRL (TC / AZM) is the TC and AZM control, with no antibiotics added, only an equal volume of ethanol added; TC is the control with TC added; AZM is the control with AZM added. Figure 4 It was found that, compared with the control, the concentration of organic acids in the TC-added samples was the highest among all samples, indicating that TC can promote the production of organic acids to some extent and further increase the methane yield of cellulose. Compared with the control, the organic acid yield in the AZM-added samples was lower, but the highest methane yield was detected in the presence of AZM, suggesting that AZM mainly increases methane yield in the methanogenesis stage by promoting the acetic acid-to-methane pathway. For CLX, the organic acid yield was generally lower than that of the control, which is consistent with the methane yield results.

[0055] (vi) Microbial community analysis Following the methods described in Examples 1, 2, 1, and 2, 15 mL of fermentation broth samples were taken for microbial community analysis, and the analysis was performed according to the method of Zhu et al. (2021).

[0056] The results are as follows Figure 5 and Figure 6 As shown, CTR (CLX) is the CLX control, with no antibiotics added and only an equal volume of deionized water added; CLX is the addition of CLX; CTRL (TC / AZM) is the TC and AZM control, with no antibiotics added and only an equal volume of ethanol added; TC is the addition of TC; AZM is the addition of AZM.

[0057] Depend on Figure 5The relative abundance (RA) of Proteobacteria was highest in CTRL(TC / AZM) among all reactors, indicating that ethanol addition promotes Proteobacteria growth, likely due to the conversion of ethanol to acetic acid. The RA of Proteobacteria in TC and AZM was lower than that in CTRL(TC / AZM), suggesting that the addition of TC and AZM negatively impacted Proteobacteria growth. This may be one reason for the lower acetic acid content observed in AZM compared to the control. Importantly, AZM yielded the highest RA for Firmicutes (29.79%), followed by TC (27.00%). Methane production was positively correlated with the RA of Firmicutes, consistent with the highest methane yield observed in AZM. A similar phenomenon was observed in TC. Furthermore, the RA of Bacteroides was higher in both AZM and TC than in CTRL(TC / AZM), supporting the results of higher cellulase activity and methane production obtained in AZM and TC. The highest RA (5.38%) of Cholecystoblasts was detected in AZM, which also contributed to the anaerobic fermentation of cellulose. Similarly, the higher RA of actinomycetes in TC compared to the control may be one reason for its higher methane production. The higher RA of chlamydia observed in CLX may be one reason for its lower methane production than the control.

[0058] like Figure 6 As shown, the diversity and abundance of archaea changed with the addition of AZM, TC, and CLX. Specifically, *Methanobacterium* was found to be the dominant genus in all samples. However, compared to the control, AZM yielded a significantly reduced *Methanobacterium* RA. Considering the highest methane yield observed in AZM, it is speculated that the addition of AZM primarily promotes methane production from cellulose via the acetic acid-producing pathway. Furthermore, the highest RA of *Methanobacterium* genus was observed in AZM (5.26%), which, as acetic acid-producing methanobacteria, may be one of the main reasons for the highest methane yield detected in AZM. *Methanococcus methanans* also had the highest RA in AZM, another positive factor for anaerobic fermentation of cellulose, as the RA of *Methanococcus methanans* is positively correlated with methane yield during anaerobic fermentation. The higher RA of *Methanococcus* obtained in TC compared to the control may also contribute to its higher methane yield. Compared to CTRL (CLX), the CLX reactor showed a significantly increased *Methanococcus* RA, therefore, less methane was produced in CLX compared to CTRL (CLX).

[0059] Figure 5In the table, Others refers to other species, Spirochaetes to Spirochetes, Chloroflexi to Chloroflexi, Caldiserica to Chlamydia, Actinobacteria to Actinobacteria, Fibrobacteres to Fibrobacteres, Acidobacteria to Acidobacteria, Patescibacteria to Pasteurella, Bacteroidetes to Bacteroidetes, Firmicutes to Firmicutes, and Proteobacteria to Proteobacteria.

[0060] Figure 6 In the text, "Others" refers to other species, "Methanocorpusculum" refers to methanogenic bacteria, "Methanosphaerula" refers to methanogenic cocci, "Methanomethylovorans" refers to methyl-eating methanogenic bacteria, "Methanospirillum" refers to methanogenic spirilla, "Methanocellaceae" refers to methanogenic bacteria, "Methanosarcina" refers to methanogenic octopus, "Methanobrevibacter" refers to methanogenic short bacilli, "Methanomassiliicoccus" refers to methanogenic cocci, "Methanosaeta" refers to methanogenic bacteria, and "Methanobacterium" refers to methanogenic bacteria.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for promoting anaerobic fermentation of cellulose to produce methane, characterized in that: Methane is obtained by adding antibiotics to a mixture of cellulose and wastewater sludge and then subjecting it to anaerobic fermentation. In the mixture, the cellulose content is 0.3-0.4g per 100mL of wastewater sludge, the antibiotic content is 0.5-1.5mg per 100mL of the mixture, and the concentration of wet sludge in the wastewater sludge is 1.5%wt. The anaerobic fermentation was carried out at 37°C in a shaking incubator at 120 rpm for 20 days. The antibiotic is tetracycline or azithromycin.

2. The method according to claim 1, characterized in that: The cellulose is microcrystalline cellulose and / or cellulose powder.

3. The method according to claim 1, characterized in that: The wastewater sludge comes from a sewage treatment plant and is obtained by centrifugation and washing at 4°C.

4. The method according to claim 3, characterized in that: The centrifugation was controlled at 8000×g.

5. The method according to claim 3, characterized in that: The washing process involves thoroughly shaking the water with distilled water, and the washing steps are repeated three times.

6. The method according to claim 1, characterized in that: When adding antibiotics to the mixture, the antibiotics are first dissolved in a solvent, then added to the mixture and the concentration of antibiotics in the mixture is adjusted.

7. The method according to claim 6, characterized in that: The solvent is ethanol.

8. The application of antibiotics in promoting anaerobic fermentation of cellulose to produce methanogens, characterized by: The method described in any one of claims 1 to 7 shall be used.

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