Antibiotic Fermentation Residue Research Articles

Effective removal of tetracycline antibiotics from water by in-situ nitrogen-doped porous biochar derived from waste antibiotic fermentation residues

This study explored the utilization of waste oxytetracycline fermentation residue (OFR), abundant in nitrogen and organic matter, to prepare in-situ nitrogen-doped porous biochar (activated OFR derived biochar, AOBC) through a coupled pyrolysis process, and investigated the removal of tetracycline antibiotics (TCs) from water with the prepared AOBCs. The physicochemical characterization results showed the characterizations of abundant nitrogen functional groups, high specific surface area (1960.38 m2/g), and the high microporosity (61.6%) for AOBCs. At 25 ℃, the maximum adsorption capacity calculated by Langmuir model of AOBC-3-600 for tetracycline hydrochloride (TC), chlortetracycline hydrochloride (CTC), oxytetracycline hydrochloride (OTC), and doxycycline hydrochloride (DOC) reached 473.00, 293.28, 220.61, and 282.72mg/g, respectively. The superior fit of the pseudo-second-order and Langmuir models indicated that the adsorption mechanism involved both the physical effect (pore filling) and chemical interactions (electrostatic attraction, hydrogen bonding, π-π interaction, and Lewis acid-base interaction). Environmental risk assessments substantiated the absence of residual oxytetracycline and antibiotic resistance genes in the prepared biochar, indicating its application safety. This research provides a sustainable route to simultaneously treat the antibiotic contamination in water and rationally utilize antibiotic fermentation residues.

Efficient removal of high concentration dyes from water by functionalized in-situ N-doped porous biochar derived from waste antibiotic fermentation residue

Using biochar for dye wastewater treatment is attracting interest due to its excellent adsorption properties and low costs. In this work, a novel biochar derived from oxytetracycline fermentation residue (functionalized OFR biochar, FOBC) was investigated as a efficient adsorbent for typical dyes removal. At 25 °C, the maximum adsorption capacity calculated by Langmuir model of FOBC-3-600 for methylene blue (MB), malachite green (MG), and methyl orange (MO) reached 643.97, 617.89, and 521.03 mg/g, respectively. The kinetics and isotherm model fitting showed that the chemisorption and physisorption both occurred during the adsorption process. Dyes were efficiently adsorbed through pore filling, electrostatic attraction, π-π interactions, and surface complexation. And the cycling experiment and environmental risk assessment indicated that the FOBC-3-600 had excellent recyclability and utilization safety. Overall, this study provides a practical method to simultaneously treat the dyeing wastewater and utilize the antibiotic fermentation residue.

Efficient Removal of Tetracycline from Water by One-Step Pyrolytic Porous Biochar Derived from Antibiotic Fermentation Residue.

The disposal and treatment of antibiotic residues is a recognized challenge due to the huge production, high moisture content, high processing costs, and residual antibiotics, which caused environmental pollution. Antibiotic residues contained valuable components and could be recycled. Using a one-step controllable pyrolysis technique in a tubular furnace, biochar (OSOBs) was produced without the preliminary carbonization step, which was innovative and time- and cost-saving compared to traditional methods. The main aim of this study was to explore the adsorption and removal efficiency of tetracycline (TC) in water using porous biochar prepared from oxytetracycline fermentation residues in one step. A series of characterizations were conducted on the prepared biochar materials, and the effects of biochar dosage, initial tetracycline concentration, reaction time, and reaction temperature on the adsorption capacity were studied. The experimental results showed that at 298 K, the maximum adsorption capacity of OSOB-3-700 calculated by the Langmuir model reached 1096.871 mg/g. The adsorption kinetics fitting results indicated that the adsorption of tetracycline on biochar was more consistent with the pseudo-second-order kinetic model, which was a chemical adsorption. The adsorption isotherm fitting results showed that the Langmuir model better described the adsorption process of tetracycline on biochar, indicating that tetracycline was adsorbed in a monolayer on specific homogeneous active sites through chemical adsorption, consistent with the kinetic conclusions. The adsorption process occurred on the surface of the biochar containing rich active sites, and the chemical actions such as electron exchange promoted the adsorption process.

Antibiotic fermentation residue for biohydrogen production: Inhibitory mechanisms of the inherent antibiotic

Antibiotic fermentation residue, which is generated from the microbial antibiotic production process, has been a troublesome waste faced by the pharmaceutical industry. Dark fermentation is a potential technology to treat antibiotic fermentation residue in terms of renewable H2 generation and waste management. However, the inherent antibiotic in antibiotic fermentation residue may inhibit its dark fermentation performance, and current understanding on this topic is limited. This investigation examined the impact of the inherent antibiotic on the dark H2 fermentation of Cephalosporin C (CEPC) fermentation residue, and explored the mechanisms from the perspectives of bacterial communities and functional genes. It was found that CEP-C in the antibiotic fermentation residue significantly inhibited the H2 production, with the H2 yield decreasing from 17.2 mL/g-VSadded to 12.5 and 9.6 mL/g-VSadded at CEP-C concentrations of 100 and 200 mg/L, respectively. CEP-C also prolonged the H2-producing lag period. Microbiological analysis indicated that CEP-C remarkably decreased the abundances of high-yielding H2-producing bacteria, as well as downregulated the genes involved in hydrogen generation from the“pyruvate pathway” and“NADH pathway”, essentially leading to the decline of H2 productivity. The present work gains insights into how cephalosporin antibiotics influence the dark H2 fermentation, and provide guidance for mitigating the inhibitory effects.

Metagenomics reveals the resistance patterns of electrochemically treated erythromycin fermentation residue

Erythromycin fermentation residue (EFR) represents a typical hazardous waste produced by the microbial pharmaceutical industry. Although electrolysis is promising for EFR disposal, its microbial threats remain unclear. Herein, metagenomics was coupled with the random forest technique to decipher the antibiotic resistance patterns of electrochemically treated EFR. Results showed that 95.75% of erythromycin could be removed in 2 hr. Electrolysis temporarily influenced EFR microbiota, where the relative abundances of Proteobacteria and Actinobacteria increased, while those of Fusobacteria, Firmicutes, and Bacteroidetes decreased. A total of 505 antibiotic resistance gene (ARG) subtypes encoding resistance to 21 antibiotic types and 150 mobile genetic elements (MGEs), mainly including plasmid (72) and transposase (52) were assembled in EFR. Significant linear regression models were identified among microbial richness, ARG subtypes, and MGE numbers (r2=0.50–0.81, p< 0.001). Physicochemical factors of EFR (Total nitrogen, total organic carbon, protein, and humus) regulated ARG and MGE assembly (%IncMSE value = 5.14-14.85). The core ARG, MGE, and microbe sets (93.08%-99.85%) successfully explained 89.71%–92.92% of total ARG and MGE abundances. Specifically, gene aph(3′)-I, transposase tnpA, and Mycolicibacterium were the primary drivers of the resistance dissemination system. This study also proposes efficient resistance mitigation measures, and provides recommendations for future management of antibiotic fermentation residue.

Rapid and efficient removal of multiple heavy metals from diverse types of water using magnetic biochars derived from antibiotic fermentation residue

Pyrolysis is a promising method to treat antibiotic fermentation residue (AFR), a hazardous waste in China, with the benefits of detoxification and resource recycling. However, the application of the AFR-derived biochar has been limited yet, restricting the use of pyrolysis to treat AFR. Herein, for the first time, we reported the use of magnetic biochars derived from vancomycin fermentation residue to rapidly and efficiently co-adsorb multiple heavy metals from diverse types of water with complex matrices. The biochar prepared at 700 °C (labeled as VBC700) exhibited high affinity and selectivity for multiple heavy metals, especially for Ag(I), Hg(II), Pb(II), and Cu(II). The kinetics for Ag(I), Hg(II), and Pb(II) were ultrafast with an equilibrium time of only 5 min, while those for Cu(II) were relatively slower. The maximum adsorption capacity calculated from the Langmuir model for Ag(I), Hg(II), Pb(II), and Cu(II) reached 177.4, 105.9, 387.1, 124.5 mg/g, respectively, which were superior to much previously reported adsorbents. Impressively, Na(I), K(I), Ca(II), Mg(II), and salinity did not affect the capture of these heavy metals, and thus >99% of Ag(I), Pb(II), and Cu(II) were concurrently removed from complex water matrices including seawater, which has rarely been reported before. Furthermore, VBC700 remained high adsorption performance at pH ≥ 3. The adsorption mechanisms included ion exchange, precipitation, and inner-sphere complexation. Overall, the results demonstrate that VBC700 would be an excellent adsorbent to co-capture multiple heavy metals from diverse types of water, highlighting the feasibility of using pyrolysis to achieve a win-win goal for AFR management and heavy metal pollution control.

Erythromycin fermentation residue exposure induces a short-term wave of antibiotic resistance in a soil-lettuce system

The pattern of antibiotic resistance assembly and their unclear transfer in a soil-lettuce system render the treated erythromycin fermentation residue (EFR) land application risky. Herein, the antibiotic resistance genes (ARGs), mobile gene elements, and microbial communities were examined under erythromycin stress at three stages of lettuce growth. Erythromycin exhibited degradation rates of 99.4 % in soils for 60 d, with little uptake in the seedling tissues, reaching a 0.11–0.71 bioconcentration factor range. The EFR application rate must be limited <1 % to avoid human exposure risk. The diversity, biotic networks complexity, and edaphic ARG abundances of the rhizospheric microbial communities increased at the early stage, but returned to the control levels at the mature stage. The Planomicrobium and Pseudomonas bacterial genera were important biotic factors for erythromycin variation. Thirty-three MLSB genes (macrolide, lincosamide, and streptogramin B) conferring resistance to erythromycin were detected in soil, but only two endophytic ARGs (mphA-01 and ermX) were identified, with members of the Microvirga genus being the potential hosts. Partial least-squares path modeling suggested that erythromycin concentration was the main factor for endophytic ARGs evolution. This study highlighted the leaf endophytic ARG emergence and potential exposure human risks majorly caused by the drug traces in antibiotic fermentation residues.

Speciation Evolution and Phase Migration of Phosphorus and Nitrogen during Subcritical Hydrothermal Recycling of Antibiotic Fermentation Residue

Speciation Evolution and Phase Migration of Phosphorus and Nitrogen during Subcritical Hydrothermal Recycling of Antibiotic Fermentation Residue

Effective CO2 capture by in-situ nitrogen-doped nanoporous carbon derived from waste antibiotic fermentation residues

It is of great environmental benefit to rationally dispose of and utilize antibiotic fermentation residues. In this study, oxytetracycline fermentation residue was transformed into an in-situ nitrogen-doped nanoporous carbon material with high CO2 adsorption performance by low-temperature pyrolysis pre-carbonization coupled with pyrolytic activation. The results indicated the activation under mild conditions (600 °C, KOH/OC = 2) was able to increase micropores and reduce the loss of in-situ nitrogen content. The developed microporous structure was beneficial for the filling adsorption of CO2, and the in-situ nitrogen doping in a high oxygen-containing carbon framework also strengthened the electrostatic adsorption with CO2. The maximum CO2 adsorption reached 4.38 mmol g−1 and 6.40 mmol g−1 at 25 °C and 0 °C (1 bar), respectively, with high CO2/N2 selectivity (32/1) and excellent reusability (decreased by 4% after 5 cycles). This study demonstrates the good application potential of oxytetracycline fermentation residue as in-situ nitrogen-doped nanoporous carbon materials for CO2 capture.

Production of medium chain fatty acids from antibiotic fermentation residuals pretreated by ionizing radiation: Elimination of antibiotic resistance genes

The propagation of antibiotic resistance genes (ARGs) restricts the application of antibiotic fermentation residues (AFRs). This study investigated medium chain fatty acids (MCFA) production from AFRs, focusing on the effect of ionizing radiation pretreatment on the fates of ARGs. The results indicated that ionizing radiation pretreatment not only stimulated the MCFA production, but also inhibited the proliferation of ARGs. Radiation at 10–50 kGy decreased ARGs abundances by 0.6–21.1% at the end of fermentation process. Mobile genetic elements (MGEs) exhibited higher resistance to ionizing radiation, radiation over 30 kGy was required to suppress the proliferation of MGEs. Radiation at 50 kGy achieved an adequate inhibition to MGEs, and the degradation efficiency was 17.8–74.5% for different kinds of MGEs. This work suggested that ionizing radiation pretreatment could be a good option to ensure the safer application of AFRs by eliminating the ARGs and preventing the horizontal gene transfer of ARGs.

Environmental factors induced macrolide resistance genes in composts consisting of erythromycin fermentation residue, cattle manure, and maize straw

With the growing concerns about antibiotic resistance, it is more and more important to prevent the environmental pollution caused by antibiotic fermentation residues. In this study, composted erythromycin fermentation residue (EFR) with the mixture of cattle manure and maize straw at ratios of 0:10 (CK), 1:10 (T1), and 3:10 (T2) explores the effects on physicochemical characteristics, mobile genetic elements (MGEs), and antibiotic resistance genes (ARGs). Results reflected that the addition of EFR reduced the carbon/nitrogen ratio of each compost and improved the piles' temperature, which promoted the composting process. However, the contents of Na+, SO42-, and erythromycin were also significantly increased. After 30days of composting, the degradation rates of erythromycin in CK, T1, and T2 were 72.7%, 20.3%, and 37.1%, respectively. Meanwhile, the total positive rates for 26 detected ARGs in T1 and T2 were 65.4%, whereas that of CK was only 23.1%. Further analysis revealed that ARGs responsible for ribosomal protection, such as ermF, ermT, and erm(35), dominated the composts of T1 and T2, and most were correlated with IS613, electrical conductivity (EC), nitrogen, and Zn2+. Above all, adding EFR helps to improve the nutritional value of composts, but the risks in soil salinization and ARG enrichment caused by high EC and erythromycin content should be further investigated and eliminated.

Production of medium chain fatty acids from fermentation of antibiotic residuals: Fate of antibiotic resistance genes

The potential of antibiotic resistance genes (ARGs) amplification restricts the biological recovery of antibiotic fermentation residues (AFRs) through two-stage anaerobic fermentation. This study explored the fate of ARGs during the fermentation of AFRs that comprising of acidification and chain elongation (CE). Results showed that with the alteration of fermentation process from acidification to CE, microbial richness was significantly increased, total abundance of ARGs was slightly decreased by 1.84%, and the significant negative correlations between ARGs and microbes were increased, implied the inhibitory effect of CE microbes to ARGs amplification. However, the total abundance of mobile genetic elements (MGEs) was increased by 24.5%, indicating that the potential of gene horizontal transfer of ARGs increased. This work suggested that two-stage anaerobic fermentation could effectively restrict the ARGs amplification, but more concerns are needed for the long-term dissemination of ARGs.

Ciprofloxacin degradation performances and mechanisms by the heterogeneous electro-Fenton with flocculated fermentation biochar

Antibiotic fermentation residue flocculated by polymeric ferric sulfate (PFS) has been classified as a “hazardous waste” in China. In this study, it was recycled into antibiotic fermentation residue biochar (AFRB) by pyrolysis and used as a heterogeneous electro-Fenton (EF) catalyst for ciprofloxacin (CIP) degradation. The results show that PFS was reduced to Fe0 and FeS during pyrolysis, which was beneficial for the EF process. The AFRB with mesoporous structures exhibited soft magnetic features, which were convenient for separation. CIP was completely degraded within 10 min by the AFRB-EF process at an initial concentration of 20 mg/L. Increasing the working current and catalyst dosage within a certain range could improve the degradation rate. ·OH and O2·- were the dominant reactive oxygen species that played critical roles for CIP degradation. The antibacterial groups of CIP have been destroyed by the heterogeneous electro-Fenton process and its toxicity was negligible. The AFRB showed satisfactory performance, even though it was recycled five times. This study provide new insights into the resourceful treatment of antibiotic fermentation residues.

Pyrolysis of Ca/Fe-rich antibiotic fermentation residues into biochars for efficient phosphate removal/recovery from wastewater: Turning hazardous waste to phosphorous fertilizer

Ca/Fe-rich antibiotic fermentation residues (AFRs), a type of hazardous waste, can be regarded as recyclable biomass and metal resources. However, concurrent detoxification and reutilization of biomass and metals resources from AFRs have never been reported before. In this study, Ca/Fe-rich vancomycin fermentation residues were pyrolyzed into biochar to adsorb phosphate for the first time. The residual vancomycin and antibiotic resistance genes were completely decomposed during pyrolysis. The resultant Ca/Fe-rich biochar exhibited excellent performance at adsorbing phosphate without further modifications. The process had rapid kinetics and a maximum adsorption capacity of 102 mg P/g. Ca and Fe were the active sites, whereas different mechanisms were observed under acidic and alkaline conditions. Surprisingly, HCO3− enhanced phosphate adsorption with an increase of adsorption capacity from 43.9 to 71.0 mg/g when HCO3− concentration increased from 1 to 10 mM. Furthermore, actual wastewater could be effectively treated by the biochar. The phosphate-rich spent biochar significantly promoted seed germination (germination rate: 96.7 % vs. 80.0 % in control group, p < 0.01) and seedling growth (shoot length was increased by 57.9 %, p < 0.01) due to the slow release of bioavailable phosphate, and thus could be potentially used as a phosphorous fertilizer. Consequently, the hazardous waste was turned into phosphorous fertilizer, with the additional benefits of detoxifying AFRs, reutilizing biomass and metal resources from AFRs, controlling phosphate pollution, and recovering phosphate from wastewater.

In situ N, O co-doped porous carbon derived from antibiotic fermentation residues as electrode material for high-performance supercapacitors.

With the widespread use of antibiotics, the safe utilization of waste antibiotic fermentation residues has become an urgent issue to be resolved. In this study, in situ N, O co-doped porous carbon was prepared using fresh oxytetracycline fermentation residue under the mild activation of the green activator K2CO3. The optimal sample exhibited a 3D grid carbon skeleton structure, excellent specific surface area (SBET = 948 m2 g-1), and high nitrogen and oxygen content (N = 3.42 wt%, O = 14.86 wt%). Benefiting from its developed morphology, this sample demonstrated excellent electrochemical performance with a high specific capacitance of 310 F g-1 at a current density of 0.5 A g-1 in the three-electrode system. Moreover, it exhibited superior cycling stability with only a 5.32% loss of capacity after 10 000 cycles in 6 M KOH aqueous electrolyte. Furthermore, the symmetric supercapacitor prepared from it exhibited a maximum energy density of 7.2 W h kg-1 at a power density of 124.9 W kg-1, demonstrating its promising application prospects. This study provided a green and facile process for the sustainable and harmless treatment of antibiotic fermentation residues.

Sustainable on-farm strategy for the disposal of antibiotic fermentation residue: Co-benefits for resource recovery and resistance mitigation

Antibiotic fermentation residue is a key issue for the sustainable operation of pharmaceutical companies, and its improper disposal may cause antibiotic resistance transfer in the environment. However, little is known about the resource recycling strategy of this pharmaceutical waste. Herein, we used hydrothermal spray-dried (HT+SD) and multi-plate dryer (MD) methods to produce bio-organic fertilizers and applied them to an internal recycling model of a field trial. The concentrations of antibiotics (penicillin, cephalosporin, and erythromycin) in the bio-fertilizer, wastewater, and exhaust gas were in the range of 0.002–0.68 mg/kg, ≤ 0.35 ng/mL, and 0.03–0.89 ng/mL, respectively. The organic matter and total nitrogen, phosphorus, and potassium contents were approximately 80% and 10%, respectively. The soil bacterial community was similar among the fertilizer treatments in the same crop cultivation. A total of 233 antibiotic resistance genes (ARGs) and 43 mobile genetic elements (MGEs) were detected, including seven Rank I ARGs and five Rank II ARGs. Random forest analysis showed that gene acc(3)-Via and plasmid trb-C were biomarkers, for which the resistance and the transfer mechanisms were antibiotic inactivation and conjugation, respectively. The results imply that AFR recycling disposal mode is a promising prospect for pharmaceutical waste management.

Application potential of antibiotic fermentation residue for co-combustion with coal: Thermal behavior, gaseous products, and kinetics

Co-combustion is an effective measure to achieve high efficiency of fuel and energy and promote the reduction of pollutants. As a high-protein, low-fiber, and energy-bearing biomass, antibiotic fermentation residues have the potential to be co-fired with coal. The behavior of co-combustion is studied by TG/DSC-MS method and the result shows that the effects of interaction between AFR and coal are presented in the burn process. The addition of AFR reduces ignition temperature by 25% − 45%, reduces burnout temperature by 2.7%-7.4% and improves the stability of combustion. Moreover, the normalized analysis of gaseous products shows that the emission of NH3 and NO are promoted, while NO2 and SO2 are inhibited. Kinetic analysis shows that co-combustion leads to the increase of apparent activation energy. Random nucleation and growth mechanism can well describe the combustion process, and the kinetic index decreases with the reaction process.

Metagenomic insights into microorganisms and antibiotic resistance genes of waste antibiotic fermentation residues along production, storage and treatment processes

Antibiotic fermentation residue (AFR) is nutrient-rich solid waste generated from fermentative antibiotic production process. It is demonstrated that AFR contains high-concentration of remaining antibiotics, and thus may promote antibiotic resistance development in receiving environment or feeding farmed animals. However, the dominate microorganisms and antibiotic resistance genes (ARGs) in AFRs have not been adequately explored, hampering understanding on the potential antibiotic resistance risk development caused by AFRs. Herein, seven kinds of representative AFRs along their production, storage, and treatment processes were collected, and multiple methods including amplicon sequencing, metagenomic sequencing, and bioinformatic approaches were adopted to explore the biological characteristics of AFRs. As expected, antibiotic fermentation producer was found as the predominant species in raw AFRs, which were collected at the outlet of fermentation tanks. However, except for producer species, more environment-derived species persisted in stored AFRs, which were temporarily stored at a semi-open space. Lactobacillus genus, classified as Firmicutes phylum and Bacilli class, became predominant bacterial taxa in stored AFRs, which might attribute to its tolerance to high concentration of antibiotics. Results from metagenomic sequencing together with assembly and binning approaches showed that these newly-colonizing species (e.g., Lactobacillus genus) tended to carry ARGs conferring resistance to the remaining antibiotic. However, after thermal treatment, remaining antibiotic could be efficiently removed from AFRs, and microorganisms together with DNA could be strongly destroyed. In sum, the main risk from the AFRs was the remaining antibiotic, while environment-derived bacteria which tolerate extreme environment, survived in ARFs with high content antibiotics, and may carry ARGs. Thus, hydrothermal or other harmless treatment technologies are recommended to remove antibiotic content and inactivate bacteria before recycling of AFRs in pharmaceutical industry.

Enhanced medium-chain fatty acids production from Cephalosporin C antibiotic fermentation residues by ionizing radiation pretreatment

Antibiotic fermentation residues (AFRs) have been classified as hazardous waste in China. Anaerobic fermentation may be a good approach for AFRs treatment, through which value-added chemicals could be obtained simultaneously. This study firstly explored medium-chain fatty acids (MCFAs) production from AFRs through two-stage anaerobic fermentation, and gamma radiation was adopted for AFRs pretreatment. The results showed that both antibiotics removal and MCFAs production from AFRs were significantly promoted by gamma radiation pretreatment. No residual Cephalosporin C (CEP-C) was detected in gamma radiation treated groups after fermentation. Highest MCFAs concentration of 90.55 mmol C/L was obtained in 50 kGy treated group, which was 2.22 times of the control group. Genera that were positively correlated with MCFAs production were enriched in gamma radiation treated groups, like genus Paraclostridium, Terrisporobacter, Caproiciproducens and Sporanaerobacter, while genera that were negatively correlated with MCFAs production were diminished during the chain elongation process, like genus Bacteroides and NK4A214_group. Enzymes analysis suggested that the promoted MCFAs production was induced by the enrichment of functional enzymes involved in Acetyl-CoA formation and RBO pathway. This work suggested that gamma radiation pretreatment and two-stage anaerobic fermentation could achieve the dual benefits of AFRs treatment and value-added chemicals recovery.

Production of medium-chain fatty acids by co-fermentation of antibiotic fermentation residue with fallen Ginkgo leaves

The co-fermentation of antibiotic fermentation residues (AFRs) and fallen Ginkgo leaves at C/N ratios of 10–60 was conducted for medium-chain fatty acids (MCFA) production. It was found that a proper C/N ratio could largely promote the MCFA accumulation. Group with C/N ratio of 50 exhibited highest MCFA production of 133.14 mmol C/L, which was 42 %-121 % higher than the other groups. Through the co-fermentation, substrate condition was optimized with rich micro-nutrients in AFRs and abundant polysaccharides in Ginkgo leaves, the hydrolysis of leaves was promoted by the active microbes in AFRs, and the predominance of CE microbes was also stimulated with the dilution of AFRs. The increased C/N ratio significantly affected the SCFA producers like genus Escherichia Shigella and Proteiniphilum, and enriched CE microbes like genus Romboutsia, Eubacterium and Clostridium_sensu_stricto_12. Functional enzymes analysis showed that both reverse β oxidation and fatty acid biosynthesis pathways were strengthened with the increased C/N ratio.