CN116622783A

CN116622783A - Anaerobic fermentation method for producing medium-chain fatty acid and simultaneously reducing antibiotic resistance genes by utilizing excess sludge

Info

Publication number: CN116622783A
Application number: CN202310851206.4A
Authority: CN
Inventors: 曹广丽; 周春爽; 刘冰峰
Original assignee: Harbin Institute of Technology
Current assignee: Harbin Institute of Technology
Priority date: 2023-07-12
Filing date: 2023-07-12
Publication date: 2023-08-22

Abstract

The invention relates to the technical field of sludge deep processing. The invention provides an anaerobic fermentation method for producing medium-chain fatty acid and simultaneously reducing antibiotic resistance genes by using excess sludge, which comprises the following steps: (1) settling the sludge, and removing water to obtain concentrated sludge; (2) Adjusting the pH value of the concentrated sludge, and performing alkaline anaerobic fermentation to obtain an alkaline fermentation material of the residual sludge; (3) Mixing the residual sludge alkaline fermentation material with ethanol to obtain a mixture; (4) And mixing the mixture with 2-bromoethyl sodium sulfonate, adjusting the pH value, and performing chain extension anaerobic fermentation to obtain medium-chain fatty acid and simultaneously reducing resistance genes. Compared with the traditional anaerobic fermentation method, the method not only recovers a large amount of high-value chemical medium-chain fatty acid, but also drives the reduction of resistance genes by reducing the horizontal gene transfer frequency, damaging the resistance bacteria and strengthening the phylogenetic development of functional bacteria.

Description

Anaerobic fermentation method for producing medium-chain fatty acid and simultaneously reducing antibiotic resistance genes by utilizing excess sludge

Technical Field

The invention relates to the technical field of sludge deep processing, in particular to an anaerobic fermentation method for producing medium-chain fatty acid and simultaneously reducing antibiotic resistance genes by utilizing surplus sludge.

Background

Antibiotic resistance results in prolonged hospitalization, increased medical costs, and increased mortality, which has become a serious global health threat. The occurrence of Antibiotic Resistance Genes (ARGs) in various environments further exacerbates the widespread spread of drug resistance, and sewage treatment plants serve as an important tie between human activities and the ecosystem, being a hot spot area for ARGs spread. Most ARGs are reported to eventually accumulate in excess activated sludge (WAS), which is considered one of the most important reservoirs of ARGs. Final disposal of WAS (e.g., land application) may lead to the spread of antibiotic resistance in the local ecosystem and increase potential environmental risks. Therefore, it is necessary and important to effectively process WAS to mitigate the spread of ARGs.

Anaerobic fermentation has become a widely used method for stabilizing WAS, and simultaneously, valuable products such as Short Chain Fatty Acids (SCFAs) and methane can be recovered. However, SCFAs are difficult to separate and purify from fermentation broth due to their high solubility, low methane energy density, and strong greenhouse effect. Anaerobic fermentation technology that converts WAS into valuable and readily separable Medium Chain Fatty Acids (MCFAs) is therefore of interest. MCFAs are saturated carboxylic acids containing 6 to 12 carbons, can be used as precursors for biofuel production and biochemical synthesis, and include medicines, antibacterial agents and food additives, and have wide application prospects. It is well known that the spread of ARGs occurs mainly through two processes, moving Gene Elements (MGEs) -mediated Horizontal Gene Transfer (HGT) and Vertical Gene Transfer (VGT) from parent to offspring. Notably, anaerobic fermentation processes for producing MCFAs have the potential to inhibit both horizontal and vertical gene transfer. MCFAs have antibacterial effects by disrupting bacterial cell membranes, potentially reducing MGEs and destroying ARGs hosts and thus reducing HGT and VGT, thereby reducing the spread of ARGs. Furthermore, the phenomenon of ARGs reduction during anaerobic fermentation has been observed in many studies, which is related to the modification of the colony structure, and thus the evolution of colony structure caused by enrichment of functional microorganisms during the anaerobic fermentation to produce MCFAs may be advantageous for the removal of ARGs. In view of the above background, anaerobic fermentation biotechnology utilizing high yields of MCFAs has the potential to curtail ARGs while recovering MCFAs from WAS. In view of the poor biodegradability of WAS, the direct conversion efficiency into MCFAs is low, and the method of producing MCFAs by performing alkaline fermentation on WAS to obtain alkaline fermentation liquor rich in a large amount of SCFAs and then performing chain extension process is adopted, so that the purposes of producing MCFAs by anaerobic fermentation and reducing ARGs are achieved, and the method has important significance for WAS disposal, resource recovery and reduction of environmental health risks.

Disclosure of Invention

The invention aims to provide an anaerobic fermentation method for producing medium-chain fatty acid by using surplus sludge and reducing antibiotic resistance genes, which can realize reduction of the surplus activated sludge and recovery of valuable biological energy, simultaneously strengthen reduction of the resistance genes and limit propagation and diffusion of the resistance genes in the environment.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an anaerobic fermentation method for producing medium-chain fatty acid and simultaneously reducing antibiotic resistance genes by using excess sludge, which comprises the following steps:

(1) Settling the sludge, and removing water to obtain concentrated sludge;

(2) Adjusting the pH value of the concentrated sludge, and performing alkaline anaerobic fermentation to obtain an alkaline fermentation material of the residual sludge;

(3) Mixing the residual sludge alkaline fermentation material with ethanol to obtain a mixture;

(4) Mixing the mixture with 2-bromoethyl sodium sulfonate, adjusting the pH value, and performing chain extension anaerobic fermentation to obtain medium-chain fatty acid and simultaneously reduce antibiotic resistance genes.

Preferably, the temperature of the sedimentation in the step (1) is 2-6 ℃, and the sedimentation time is 20-28 h.

Preferably, in the step (2), a 4-6M NaOH solution and a 1-3M hydrochloric acid solution are used for adjusting the pH value of the concentrated sludge, and the pH value of the concentrated sludge after adjustment is 9.0-11.0.

Preferably, stirring is performed during the alkaline anaerobic fermentation in the step (2), and the stirring speed is 140-180 rpm.

Preferably, the alkaline anaerobic fermentation in the step (2) is performed for 6-10 days, and the anaerobic fermentation temperature is 34-37 ℃.

Preferably, in the alkaline anaerobic fermentation process in the step (2), the pH value of the concentrated sludge is adjusted every 22-26 hours, and the pH value after each adjustment is 9.0-11.0.

Preferably, the concentration of ethanol in the mixture in the step (3) is 40mmol/L to 500mmol/L.

Preferably, the concentration of the sodium 2-bromoethyl sulfonate in the mixed mixture in the step (4) is 10-11 g/L.

Preferably, in the step (4), a 4-6M NaOH solution and a 1-3M hydrochloric acid solution are used for adjusting the pH value, and the pH value of the mixture after adjustment is 6.5-7.5.

Preferably, the chain extension anaerobic fermentation process in the step (4) is carried out in a shaking table, the rotation speed of the shaking table is 140-180 rpm, the time of the chain extension anaerobic fermentation process is 34-38 d, and the anaerobic fermentation temperature is 34-37 ℃.

The invention provides an anaerobic fermentation method for producing medium-chain fatty acid and simultaneously reducing antibiotic resistance genes by using excess sludge, which comprises the following steps: (1) settling the sludge, and removing water to obtain concentrated sludge; (2) Adjusting the pH value of the concentrated sludge, and performing alkaline anaerobic fermentation to obtain an alkaline fermentation material of the residual sludge; (3) Mixing the residual sludge alkaline fermentation material with ethanol to obtain a mixture; (4) Mixing the mixture with 2-bromoethyl sodium sulfonate, adjusting the pH value, and performing chain extension anaerobic fermentation to obtain medium-chain fatty acid and simultaneously reduce antibiotic resistance genes. The invention establishes a method for producing medium-chain fatty acid by anaerobic fermentation of sludge and simultaneously efficiently reducing antibiotic resistance genes, realizes recycling of solid waste and reduces environmental health risks, and compared with the traditional anaerobic fermentation method, the method has the following advantages that (1) a large amount of medium-chain fatty acid in high-value chemicals is recovered; (2) Reducing the frequency of horizontal gene transfer limits the spread of resistance genes; (3) The produced medium-chain fatty acid has an antibacterial effect, reduces the resistance bacteria and cuts down the resistance genes by damaging the resistance bacteria; (4) The functional bacteria in the anaerobic fermentation process are not potential hosts of resistance genes, so that the phylogenetic development of the functional bacteria also drives the reduction of the resistance genes, and the invention promotes the reduction of high-value chemicals and resistance genes produced by sludge recycling.

Drawings

FIG. 1 shows the concentration variation of SCFAs in the residual sludge alkaline fermentation broth (WASAFL) after WAS alkaline anaerobic fermentation;

FIG. 2 shows the evolution of MCFAs concentration produced during chain extension anaerobic fermentation with ethanol as electron donor, WASAFL as electron acceptor and inoculum;

FIG. 3 shows changes in Intracellular ARGs (iARGs), extracellular ARGs (eARGs) and total resistance genes (tARGs, i.e., the sum of iARGs and eARGs) abundance after WAS, WASAFL and 6 sets of chain extended anaerobic fermentation;

FIG. 4 shows changes in abundance of Intracellular MGEs (iMGEs), extracellular MGEs (eMGEs) and total MGEs (tMGEs, i.e., the sum of iMGEs and eMGEs) after WAS, WASAFL and 6 sets of chain extended anaerobic fermentation;

FIG. 5 is a Pearson correlation and linear regression of the effect of the chain extension anaerobic fermentation process on intracellular and extracellular 16S rDNA abundance (a) and between MCFAs and extracellular 16S rDNA (b), absolute abundance of iARGs (c) and absolute abundance of eARGs (d);

FIG. 6 shows the microbial community distribution (a) at WAS, WASAFL and chain extension anaerobic fermentation reactor R4 sample genus level (total abundance at position 25) and the co-occurrence network analysis of ARGs and genus characteristics (b);

FIG. 7 shows the relationship (a) between the abundance of different factors MCFAs, bacterial communities, iMGEs, eARGs and iARGs and the direct, indirect and total effects (b) derived based on PLS-PM, described by the partial least squares path model (PLS-PM).

Detailed Description

(1) Settling the sludge, and removing water to obtain concentrated sludge;

In the present invention, the temperature of the sedimentation in the step (1) is preferably 2 to 6 ℃, more preferably 4 ℃, and the time of the sedimentation is preferably 20 to 28 hours, more preferably 24 hours.

In the present invention, in the step (2), the pH of the concentrated sludge is adjusted preferably to 9.0 to 11.0, more preferably to 10.0, by using a NaOH solution of 4 to 6M and a hydrochloric acid solution of 1 to 3M, even more preferably a NaOH solution of 5M and a hydrochloric acid solution of 2M.

In the present invention, stirring is preferably performed during the alkaline anaerobic fermentation in the step (2), and the stirring speed is preferably 140 to 180rpm, more preferably 160rpm.

In the present invention, the time of the alkaline anaerobic fermentation in the step (2) is preferably 6 to 10d, more preferably 8d, and the temperature of the anaerobic fermentation is preferably 34 to 37 ℃, more preferably 35 to 36 ℃.

In the present invention, the pH of the concentrated sludge is preferably adjusted every 22 to 26 hours, more preferably every 24 hours, during the alkaline anaerobic fermentation in the step (2), and the pH after each adjustment is preferably 9.0 to 11.0, more preferably 10.0.

In the present invention, the concentration of ethanol in the mixture in the step (3) is 40mmol/L to 500mmol/L, more preferably 47mmol/L, 93mmol/L, 187mmol/L, 280mmol/L, 373mmol/L, 467mmol/L.

In the present invention, the concentration of sodium 2-bromoethyl sulfonate in the mixed mixture in the step (4) is preferably 10 to 11g/L, more preferably 10.5g/L.

In the present invention, in the step (4), a NaOH solution of 4 to 6M and a hydrochloric acid solution of 1 to 3M are preferably used, a NaOH solution of 5M and a hydrochloric acid solution of 2M are more preferably used, and the pH of the mixture after adjustment is preferably 6.5 to 7.5, and more preferably 7.0.

In the present invention, the chain extension anaerobic fermentation process in step (4) is preferably carried out in a shaking table, the rotation speed of the shaking table is preferably 140 to 180rpm, more preferably 160rpm, the time of the chain extension anaerobic fermentation process is preferably 34 to 38d, more preferably 36d, and the temperature of the anaerobic fermentation is preferably 34 to 37 ℃, more preferably 35 to 36 ℃.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

(1) Precipitating and concentrating the residual sludge for 24 hours at the temperature of 4 ℃ and removing water to obtain concentrated sludge;

(2) Each reactor having a working volume of 1200mL received 800mL of concentrated sludge, and the pH of the concentrated sludge was adjusted to 10.0 with 5M NaOH solution. Nitrogen was introduced into the reactor for 5min to remove residual oxygen, and the reactor was sealed and then subjected to alkaline anaerobic fermentation at 35 ℃ at a stirring speed of 160rpm. Every 24 hours, the pH was adjusted to 10.0. Samples were collected periodically to measure the SCFAs produced. When the SCFAs yield no longer increases, obtaining residual sludge alkaline fermentation material (WASAFL);

(3) The Chain Extension (CE) process to produce MCFAs with ethanol as electron donor, SCFAs in WASAFL as electron acceptor and WASAFL as inoculum was performed in 330mL serum bottles. 200ml of LWAFFL was added to each serum bottle, and 6 sets of reactors R1, R2, R3, R4, R5, R6 were each set up by adding ethanol at different concentrations, 47mmol/L, 93mmol/L, 187mmol/L, 280mmol/L, 373mmol/L, 467mmol/L, respectively, and in addition, 10.5g/L of sodium 2-bromoethyl sulfonate was added to each serum bottle, respectively, in order to suppress the formation of methane, i.e., the conversion of acetic acid, carbon dioxide, and hydrogen into methane. The pH was adjusted to 7.0 using 5M sodium hydroxide and 2M hydrochloric acid. The headspace was purged with nitrogen for 5min. Each serum bottle was then closed with a butyl rubber stopper and packed with an aluminum cap. Finally, anaerobic fermentation was carried out in an air bath shaker at 35℃and 160rpm until the MCFAs concentration no longer increased and ethanol was no longer consumed. All experiments were performed in triplicate.

Test examples

1. SCFAs, MCFAs and alcohol detection methods:

the fermentation broth was tested for the content of SCFAs (acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid and n-valeric acid), MCFAs (caproic acid and caprylic acid) and alcohols (ethanol, propanol, butanol and hexanol) using a gas chromatograph (model HP 7890) manufactured by agilent, america. The sample treatment method comprises the following steps: the fermentation broth was filtered with a 0.45 μm filter, then 1mL of the filtered sample was added to a chromatographic vial and acidified with 100 μl of formic acid, 1.0 μl each. The test conditions of the gas chromatograph were: the hydrogen flame detector, chromatographic column was 30m x 0.25mm DB WAX polyethylene glycol capillary column, carrier gas was nitrogen, sample inlet and detector temperatures were 220 ℃ and 240 ℃, respectively, the column temperature program was from 70 ℃ to 170 ℃ in 20 ℃ per minute increments, and after 5 minutes, the column temperature program was increased to 240 ℃ again in 20 ℃ per minute increments.

2. Detection of ARGs

100mL of the sludge sample was taken, centrifuged at 8000rpm for 5min at 4℃and the supernatant was filtered with a 0.22 μm sterile membrane. After freeze-drying the filtrate (FD-1C-50, biocool), the resulting powder was used for extracellular DNA extraction. The pellet and residual cellular particles on the filter were collected for intracellular DNA extraction. The collected samples were extracted for intracellular and extracellular DNA using the soil genomic DNA extraction kit (tengen). The extracted DNA samples were used for subsequent gene quantification analysis. Using StepOneGlus ^TM Real-time PCR systems quantitate gene abundance, and 9 representative ARGs, including sulfonamide (sul 1 and sul 2), macrolide (ermF, ereA and mefA), aminoglycoside (strB), tetracyclines (tetG and tetX) and β -lactam (blaSHV) resistance genes were quantitated. Meanwhile, 2 MGEs, including a class I integrant gene (intI 1) and a conjugated transposon gene (tn 916) as well as a 16S rDNA gene were also tested. The content of extracellular ARGs (eARGs) and Intracellular ARGs (iARGs) in the sludge sample was calculated by normalizing the ARGs copy number to the dry weight per gram of sludge (ARGs copies/g dw).

3. Microbial community analysis

The microbial community distribution was determined by analysis of amplicon sequencing in the V3-V4 region of the 16S rRNA gene. Samples were taken from WAS, WASAFL and 6 chain extended anaerobic fermentation MCFAs production reactors, respectively. 338F-806R primers were amplified by the thermocycler PCR system (GeneAmp 9700, ABI, USA). The amplified products were sequenced on an Illumina MiSeq platform (Illumina, san Diego, USA) from Majorbio Bio-Pharm Technology co.ltd. And in order to obtain an optimal sequence, splicing the obtained sequencing data by using Flash software. The operational classification units (OTUs) were clustered based on a 97% recognition threshold using Uparse software and the sample sequences were ranked to ensure the correctness of the subsequent data analysis. The RDP Classifier is adopted to conduct classification analysis on the representative OTU sequence, and the Silva database is used for classification labeling. The raw sequence data is stored in the national center for biotechnology information (NCBI, no.SRP438415).

4. Other analyses

By calculating the spin scale correlation coefficient, determining the co-occurrence relationship of ARGs with bacteria, and visualizing the results using Gephi (0.9.2) software, only statistically significant (p < 0.05) and strongly correlated (Spearman's rho > 0.70) correlations are shown. In each network, the node size is proportional to the number of connections. The use of partial least squares path model (PLS-PM) reveals the effect of different factors on ARGs abundance (R package, plspm), blue for positive effects and orange for negative effects. The goodness of fit of the model was 0.6703. Significance levels are expressed as P <0.05, P <0.01, respectively.

Results:

FIG. 1 shows the concentration variation of SCFAs in the residual sludge alkaline fermentation broth (WASAFL) after WAS alkaline anaerobic fermentation; at 8 days of fermentation, the concentration of SCFAs produced reached a stable level, indicating that the reaction was substantially complete. The total SCFAs in the WASAFL obtained at this time were 4873mg/L and carboxylic acids of predominantly 2 to 5 carbon atoms, including acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid and n-valeric acid.

FIG. 2 shows the evolution of MCFAs concentration produced during chain extension anaerobic fermentation with ethanol as electron donor, WASAFL as electron acceptor and inoculum; chain Extension (CE) reactors R1, R2, R3, R4, R5, R6 were set up with ethanol concentrations of 47mmol/L, 93mmol/L, 187mmol/L, 280mmol/L, 373mmol/L, 467mmol/L, respectively, the CE process produced two types of MCFAs, n-hexanoic acid and n-octanoic acid, and reached a stable level at 36 days of fermentation. As the ethanol concentration increased, the total accumulated MCFAs increased from 1202mg COD/L for R1 to 15089mg COD/L for R4. However, as the ethanol concentration continues to increase, the cumulative yield of MCFAs decreases, down to 11348mg COD/L in R6. It should be noted that ethanol damages the cell membrane, resulting in leakage of the cell contents, and thus the decrease in MCFAs production in R5 and R6 may be related to the high concentration of ethanol affecting microbial activity, and the study resulted in relatively high MCFAs production by WAS anaerobic fermentation.

FIG. 3 shows changes in Intracellular ARGs (iARGs), extracellular ARGs (eARGs) and total resistance gene abundance (tARGs, i.e., the sum of iARGs and eARGs) after anaerobic fermentation of WAS, WASAFL and 6 groups CE; the removal rate of tARGs by alkaline anaerobic fermentation is 58.98%. The removal rates for the tARGs by CE processes R1, R2, R3, R4, R5 and R6 reached 52.45%, 71.61%, 73.98%, 81.15%, 78.35% and 80.68%, respectively. Compared with the alkaline anaerobic fermentation process, the subsequent CE process has obviously higher tARGs removal rate. The removal rate of tARGs by the CE process is consistent with the variation of the productivity of MCFAs, and the removal rate is increased and then decreased with the increase of the concentration of ethanol. For example, the highest yield of MCFAs produced R4 also had the highest removal of tARGs. The absolute abundance of iards decreases from 10.37log copies/g dw of untreated WAS to 9.98log copies/g dw of WASAFL. It is worth mentioning that the removal rate of iards by CE process is much higher than alkaline fermentation. In R1, R2, R3, R4, R5 and R6, the absolute abundance of iARGs decreased to 9.65log, 9.42log, 9.38log, 9.23log, 9.29log and 9.24log copies/g dw, respectively. The results show that the CE process not only can recover energy from organic solid waste, but also can reduce the environmental risk brought by ARGs.

In contrast to the trend of variation in the abundance of iARGs, absolute abundance of iARGs increases after alkaline fermentation and CE treatment. The absolute abundance of eARGs increased from 5.30log copies/g dw of untreated WAS to 6.19log copies/g dw of WASAFL. This is because alkaline conditions (ph=10) destroy bacteria, promote solubilization of sludge, and result in an increase in the abundance of eARGs. Notably, the abundance of eARGs increased more after CE treatment, reaching 7.10log, 7.68log, 7.93log, 7.98log, 7.94log and 8.00log copies/g dw in R1, R2, R3, R4, R5 and R6, respectively, which may be related to damage to the produced MCFAs against the bacterial strain. Despite the increased abundance of eARGs, eARGs are readily biodegradable and have a short half-life, and thus there is limited spread of eARGs transformation to ARGs. In addition, the abundance of eARGs is about 2 orders of magnitude lower than that of iARGs, the abundance of tARGs is mainly dominated by iARGs, so that the abundance of tARGs after WAS is treated by the method is obviously reduced, and the technology for producing MCFAs by anaerobic fermentation is a very promising method for reducing ARGs.

FIG. 4 shows changes in Intracellular MGEs (iMGEs), extracellular MGEs (eMGEs) and total MGEs (tMGEs, i.e., the sum of iMGEs and eMGEs) after anaerobic fermentation of WAS, WASAFL and 6 groups CE. HGT by MGEs is considered an important model of ARGs transmission, and the potential of HGT in a system can be assessed by exploring the abundance of the major MGEs (intI 1 and tn 916). After alkaline fermentation, absolute abundance of tmgas increased from 9.36log copies/g dw to 9.61log copies/g dw of WAS, while subsequent CE treatment significantly reduced tmgas abundance, with the remaining MGEs in the 6-group reactors ranging from 8.52log copies/g dw to 8.85log copies/g dw. This suggests that the CE process can reduce HGT frequency of ARGs by reducing MGEs abundance compared to alkaline fermentation, with a higher potential for ARGs reduction. Gene transfer requires ATP, and the resulting MCFAs cause the bacteria to consume ATP to maintain intracellular pH, suggesting that CE processes may inhibit HGT by inhibiting energy supply, and thus HGT limitation is one of the reasons for reduced ARGs abundance during CE fermentation. Consistent with the ARGs abundance changes, the abundance of emgas is also about 2 orders of magnitude lower than that of imgas, so that the abundance of tmgas is predominantly dominated by imgas.

FIG. 5 is a Pearson correlation and linear regression of the effect of the chain extension anaerobic fermentation process on intracellular and extracellular 16S rDNA abundance (a) and between MCFAs and extracellular 16S rDNA (b), absolute abundance of iARGs (c) and absolute abundance of eARGs (d); analysis of the decrease in iarg abundance and the increase in iarg abundance suggests that alkaline fermentation and CE processes lead to damage and death of resistant bacteria, which is one of the factors affecting arg s distribution. Basic fermentation (ph=10) has been shown to cut ARGs and subsequent CE processes have more pronounced ARGs removal efficacy, so the present invention focused on analyzing the efficacy and mechanism of subsequent CE anaerobic fermentation processes for ARGs cut. The analysis of the damage resistance bacteria is shown in FIG. 5a, which shows the result of CEFollowing the process, intracellular 16S rDNA abundance decreased, indicating bacterial damage and reduced biomass. Previous studies showed that the abundance of ARGs correlates with the abundance of the 16S rDNA gene, presumably the biomass reduction drives the decrease in the abundance of ARGs. The release of extracellular 16S rDNA as an indicator of cell membrane integrity increased by 1.37log copies/g dw after alkaline fermentation and 0.72log, 1.40log, 1.52log, 1.56log, 1.48log and 1.49log copies/g dw in R1, R2, R3, R4, R5 and R6, respectively, after CE treatment, indicating a more intense bacterial damage by the CE process. Previous studies have shown that undissociated MCFAs are toxic to microorganisms and are considered to be a good antimicrobial agent. Consistent with the law that the concentration of MCFAs increases with the increase of the concentration of ethanol and then decreases, the concentration of undissociated hexanoic acid in R4 is the highest. In addition, MCFAs release anions (RCOO ^- Ions) have toxic effects by affecting targeted replication and metabolic functions, the longer the carbon chain the stronger the antimicrobial activity. Interestingly, through correlation analysis, a significant positive correlation was found between MCFAs and extracellular 16S rDNA (fig. 5 b), indicating that MCFAs driven bacterial damage. Furthermore, MCFAs are significantly inversely related to iARGs and MCFAs are significantly positively related to eARGs (fig. 5c and d). Thus, given the toxic effects of MCFAs on bacteria, it is speculated that damage to ARGs hosts by MCFAs results in a decrease in ARGs abundance, and that the higher the concentration of MCFAs, the more significant the decrease in ARGs.

FIG. 6 shows the microbial community distribution (a) at WAS, WASAFL and chain extension anaerobic fermentation reactor R4 sample genus level (total abundance at position 25) and the co-occurrence network analysis of ARGs and genus characteristics (b); as can be seen from fig. 6a, the horizontal community structure of the CE process is significantly changed, and the abundance of functional bacteria clostridium_sendu_stricto_1, clostridium_sendu_stricto_12 and the like are increased. As shown in fig. 6b, the co-occurrence network is composed of 83 nodes and 271 sides, and in each network, the size of the node is proportional to the number of connections. Co-occurrence network analysis between ARGs and bacterial genera has been widely used to emphasize complex pattern of interrelations and potential host analysis between them. Co-occurrence networks focus on and predict the association between the ARGs subtype (ARGs-ARGs) and the association between the ARGs subtype and the genus (ARGs-bacteria).

In the ARGs-ARGs association, coexistence patterns of different ARGs subtypes are observed. For example, aminoglycosides (strB) and macrolides (ereA) are different ARGs subtypes that show co-occurring events. This is probably because ARGs share the same potential host bacteria, i.e. strB and ereA are both associated with the norankjf __ ns9_marie_group. Notably, there is no significant correlation of intI1 with other ARGs subtypes, tn916 only has a significant correlation with mefA, suggesting that MGEs mediate ARGs with very low frequency. This is consistent with the reduced potential for HGT due to reduced abundance of MGEs during CE, helping to reduce the risk of transfer of ARGs between different microorganisms during CE.

In the ARGs-bacteria association, the sul2 gene is presumed to be the hub ARGs (i.e., high nodes with a large number of junctions), 6 junctions with bacteria, and 2 junctions with ARGs (blaSHV and tetX), suggesting that the sul2 gene has a broad range of potential hosts. After CE treatment, the relative abundance of the potential host of main sul2 (Dokdonella, unclassified _f __ Blastocatellaceae, norank _f __ ns9_marine_ group, unclassified _f __ Comamonadaceae, norank _f __ normal_o __ normal_c __ SJA-28) decreased, especially the total potential hosts of tetX (Rhodoferax, unclassified _f __ Xanthobacteria and unclassified_f __ blastcatellace) decreased after CE treatment, which may be responsible for the decrease in tetX abundance. It is speculated that the decrease in the abundance of potential host bacteria is related to the damage of the resistant bacteria by the produced MCFAs and the phylogenetic development of the functional bacteria. Interestingly, CE functional microorganisms (i.e., dechloromonas, clostridium _sensu_stricto_1, clostridium_sensu_stricto_12, and clostridium_sensu_stricto_13) were not significantly associated with ARGs, indicating that enrichment of functional bacteria contributed to the reduction of ARGs. This suggests that CE process functional bacterial phylogenetic drives changes in community structure, reducing ARGs abundance.

FIG. 7 shows the relationship (a) between the abundance of different factors MCFAs, bacterial communities, iMGEs, eARGs and iARGs and the direct, indirect and total effects (b) derived based on PLS-PM, described by the partial least squares path model (PLS-PM). Given that the distribution of tARGs abundance is mainly affected by changes in iARGs abundance, the effect of MCFAs, bacterial communities, MGEs and eARGs on iARGs was evaluated by PLS-PM in order to analyze deeply the relevant mechanisms of CE process reduction iARGs (fig. 7 a). PLS-PM indicated that MCFAs had a significant positive effect on eARGs (P < 0.05), probably due to damage to ARGs hosts by undissociated MCFAs, resulting in increased eARGs abundance. MCFAs also have a very significant positive effect on the bacterial community (P < 0.01), which is related to the damage of the host ARGs by MCFAs production during CE and the enrichment of functional microorganisms. Furthermore, the bacterial community has a direct and strong negative impact on iARGs and imags, which is related to reduced bacterial abundance in potential hosts of ARGs and MGEs and enrichment of CE functional microorganisms in non-potential hosts. MCFAs have a weak positive effect on iMGEs, while bacterial communities have a strong negative effect on iMGEs. Thus, MCFAs may lead to decreased abundance of imoges by affecting bacterial communities. The iMGEs have no positive effect on iARGs, indicating that iMGEs induce HGT with very low frequency. The eARGs had no positive effect on iARGs, again indicating that even if the eARGs were released they were not obtained by the cells, and therefore did not affect the abundance of iARGs. These results indicate that changes in the structure of the community during MCFAs production are one of the important pathways for decreasing the abundance of iARGs. Furthermore, MCFAs have a strong negative indirect effect on iARGs and bacterial communities have a strong negative direct effect on iARGs based on PLS-PM analysis (fig. 7 b). This suggests that MCFAs affect iards primarily by affecting bacterial communities. Furthermore, eARGs and imgs have no positive effect on iards, suggesting that CE process producing MCFAs systems limit their risk of spreading iards. Comparing the total effect of factors on iards, the decrease in iards abundance during CE is mainly affected by MCFAs and bacterial community composition.

Summarizing: the above results demonstrate that the anaerobic fermentation technology reduces the spread and diffusion of antibiotic resistance while recovering the medium chain fatty acids of high value chemicals. Compared with the traditional anaerobic fermentation, the method has the following characteristics and advantages: (1) recovering a substantial amount of medium chain fatty acids of the high value chemical; (2) Reducing the abundance of MGEs reduces HGT frequency and thus reduces the abundance of ARGs; (3) The produced MCFAs have a destructive effect on the resistant bacteria, thereby reducing the abundance of the resistant bacteria and the resistant genes; (4) Functional bacteria enriched in the CE process are not potential hosts of ARGs, driving the decrease in the abundance of iARGs. In conclusion, the invention promotes the reduction of high-value chemicals and resistance genes produced by sludge recycling.

From the above examples, the present invention provides an anaerobic fermentation method for producing medium-chain fatty acids while reducing antibiotic resistance genes by using surplus sludge, comprising the steps of: (1) settling the sludge, and removing water to obtain concentrated sludge; (2) Adjusting the pH value of the concentrated sludge, and performing alkaline anaerobic fermentation to obtain an alkaline fermentation material of the residual sludge; (3) Mixing the residual sludge alkaline fermentation material with ethanol to obtain a mixture; (4) Mixing the mixture with 2-bromoethyl sodium sulfonate, adjusting the pH value, and performing chain extension anaerobic fermentation to obtain medium-chain fatty acid and simultaneously reduce antibiotic resistance genes. The invention establishes a method for efficiently reducing antibiotic resistance genes while producing medium-chain fatty acid by anaerobic fermentation of sludge, realizes recycling of solid waste and reduces environmental health risks, and has the following advantages compared with the traditional anaerobic fermentation method, (1) a large amount of medium-chain fatty acid in high-value chemicals is recovered; (2) Reducing the frequency of horizontal gene transfer limits the propagation and diffusion of the resistance genes (3) to produce medium chain fatty acids with antibacterial effect, reducing the resistance bacteria and reducing the resistance genes by damaging the resistance bacteria; (4) The functional bacteria in the anaerobic fermentation process are not potential hosts of resistance genes, so that the phylogenetic development of the functional bacteria also drives the reduction of the resistance genes, and the invention promotes the reduction of high-value chemicals and resistance genes produced by sludge recycling.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. An anaerobic fermentation method for producing medium-chain fatty acid and reducing antibiotic resistance genes by using excess sludge is characterized by comprising the following steps:

(1) Settling the sludge, and removing water to obtain concentrated sludge;

2. The method according to claim 1, wherein the sedimentation temperature in step (1) is 2 to 6 ℃ and the sedimentation time is 20 to 28 hours.

3. The method according to claim 2, wherein the pH of the concentrated sludge is adjusted to 9.0 to 11.0 by using a NaOH solution of 4 to 6M and a hydrochloric acid solution of 1 to 3M in the step (2).

4. The process according to claim 3, wherein stirring is carried out during the alkaline anaerobic fermentation in step (2), and the stirring speed is 140-180 rpm.

5. The process according to claim 4, wherein the alkaline anaerobic fermentation in step (2) is carried out for a period of 6 to 10 days and the anaerobic fermentation is carried out at a temperature of 34 to 37 ℃.

6. The method according to claim 5, wherein the pH value of the concentrated sludge is adjusted every 22 to 26 hours during the alkaline anaerobic fermentation in the step (2), and the pH value after each adjustment is 9.0 to 11.0.

7. The method according to claim 6, wherein the concentration of ethanol in the mixture in the step (3) is 40mmol/L to 500mmol/L.

8. The method according to claim 7, wherein the concentration of sodium 2-bromoethyl sulfonate in the mixed mixture in the step (4) is 10 to 11g/L.

9. The method according to claim 8, wherein in the step (4), the pH of the mixture is adjusted to 6.5 to 7.5 by using a NaOH solution of 4 to 6M and a hydrochloric acid solution of 1 to 3M.

10. The process according to any one of claims 1 to 9, wherein the chain extension anaerobic fermentation process in step (4) is carried out in a shaker at a speed of 140 to 180rpm for a period of 34 to 38d and at a temperature of 34 to 37 ℃.