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• Combined activated carbon supplement and feedstock microwave pretreatment studied. • Joint implementation effective in mitigating antibiotic resistance genes issue. • Average methane yield and CH 4 percentage increased by 10–41% and 6–29%, respectively. • Bacterial genus Treponema and methanogen genus Methanosarcina selectively enriched. • KEGG cluster and genome functional analysis validated enhanced microbial activity. Antibiotic resistance genes (ARGs) stemming from animal manure pose adverse impacts on human health and ecology and consequently cost more to treat the anaerobic digestate after manure fermentation. To jointly reduce the ARGs and improve the methane yield during anaerobic digestion (AD) of chicken manure, microwave pretreatment of manure and activated carbon supplementation were simultaneously applied and evaluated in an anaerobic digester (R1). For comparison, three other digesters, a digester (R2) fed with raw chicken manure without activated carbon, a digester (M1) fed with microwave-pretreated chicken manure and without activated carbon, and a digester (M2) fed with raw chicken manure supplemented with activated carbon, were operated in parallel. The results showed that after 47 days of AD operation, R1 achieved an 87–95% ARGs removal rate, significantly higher than that of R2 (34–58%). As the loading rate was 5.8–7.0 g volatile solids (VS)/L, the average methane yield of R1 was 0.287 L/g VS, which was 1.4-fold, 1.3-fold and 1.1-fold higher than that of R2, M1 and M2, respectively. The increased methane yield was ascribed to the combined effect of feedstock microwave pretreatment and activated carbon supplementation. Pyrosequencing analysis showed that the dominant bacterial phyla were Firmicutes , Bacteroidetes and Proteobacteria while the dominant archaeal genus were Methanosarcina , Methanothrix , Methanolinea , Methanomassiliicoccus , Methanospirillum , Methanoregula and Methanobacterium. The KEGG cluster analysis and functional analysis of genome from archaeal communities demonstrated that the dominant functions enhanced by substrate microwave pretreatment and activated carbon supplementation were linked to amino acid metabolism, carbohydrate metabolism, cell communication, energy metabolism, signaling molecules and interaction, and signal transduction. [ABSTRACT FROM AUTHOR]

This study explored the effect of eight antimicrobials on the efficiency of biogas production in the anaerobic digestion (AD) process of cattle manure. The microbiome involved in AD, presence and number of genes mcrA, MSC and MST specific for Archaea, and antibiotic resistance genes (ARGs) concentration in digestate (D) were examined. Supplementation of antibiotics to substrate significantly lowered biogas production. Amoxicillin caused a 75% decrease in CH4 production in comparison with the control samples. Enrofloxacin, tetracycline, oxytetracycline, and chlortetracycline reduced the amount of biogas produced by 36, 39, 45 and 53%, respectively. High-throughput sequencing of 16S rRNA results revealed that bacteria dominated the Archaea microorganisms in all samples. Moreover, antibiotics led to a decrease in the abundance of the genes mcrA, MSC, MST, and induced an increase in the number of tetracyclines resistance genes. Antibiotics decreased the efficiency of the AD process and lowered the quantity of CH4 obtained, while stimulating an increase in the number of ARGs in D. This work reveals how antimicrobials affect the cattle manure AD process and changes in microbial biodiversity, number of functional genes and ARGs in the digestate due to drugs exposure. It also, provides useful, practical information about the AD process.

This study aimed to evaluate the feasibility of hyperthermophilic anaerobic digestion at 70 °C in the pretreatment of spiramycin fermentation residue. By feeding municipal excess sludge under a solid retention time of 5 days, the hyperthermophilic digester was successfully started up within 3 days from mesophilic digestion by a one-step temperature increase from 35 to 70 °C. MiSeq sequencing showed the fast establishment of thermophilic fermenting bacterial communities in 3 days immediately after the temperature increase, with increases in abundance of Coprothermobacter, Spirochaetaceae_uncultured and Fervidobacterium from <0.001%, 1.06% and <0.001% to 33.77%, 11.65% and 3.42%, respectively. The feasibility of hyperthermophilic digestion for spiramycin residue was evaluated in batch experiments for 7 days. Hyperthermophilic digestion considerably reduced antibiotic concentrations, with removal efficiencies of 55.3% and 99.0% for the spiramycin residue alone and its mixture with hyperthermophilic sludge, respectively. At the same time, the abundances of four macrolide-lincosamide-streptogramin resistance genes were also reduced within 7 days, due to the decrease of their corresponding hosts. These results suggest that hyperthermophilic digestion could easily be started up from mesophilic digestion and might be a suitable pretreatment approach for spiramycin residue. [ABSTRACT FROM AUTHOR]

We evaluated the chronic impact of oxytetracycline (OTC) on performance and antibiotic resistance development during the mesophilic anaerobic digestion (AD) of antibiotic-containing biomass. Mesophilic AD was conducted in a completely stirred tank reactor by constantly feeding municipal excess sludge spiked with increasing concentrations of OTC (0–1000 mg L −1 ) under a solid retention time of 20 days over a period of 265 days. Results showed that methane generation of mesophilic AD was inhibited when the OTC concentration in digested sludge was increased to around 18,000 mg kg −1 (OTC dose, 1000 mg L −1 ), due to the inhibition of fermenting and acidogenic bacteria. Metagenomic sequencing and high-throughput quantitative PCR analysis demonstrated that tetracycline resistance genes were the most dominant type (38.47–43.76%) in the resistome, with tetG , tetX , tetM , tetR , tetQ , tetO , and tetL as the dominant resistant subtypes throughout the whole experimental period. The relative abundance of these tet genes increased from 2.10 × 10 −1 before spiking OTC (OTC concentration in digested sludge, 8.97 mg kg −1 ) to 2.83 × 10 −1 ( p  < 0.05) after spiking OTC at a dose of 40 mg L −1 (OTC concentration in digested sludge, 528.52 mg kg −1 ). Furthermore, mobile genetic elements, including integrons, transposons, and plasmids, were also enriched with the increase in OTC dose. Based on partial canonical correspondence analysis, the contributions of horizontal (mobile element alteration) and vertical (bacterial community shift) gene transfer to antibiotic resistome variation were 29.35% and 21.51%, respectively. Thus, considering the inhibition of hydrolytic acidification and enrichment of antibiotic resistome, mesophilic AD is not suggested to directly treat the biomass containing OTC concentration higher than 200 mg L −1 . [ABSTRACT FROM AUTHOR]

As antibiotic resistance continues to spread globally, there is growing interest in the potential to limit the spread of antibiotic resistance genes (ARGs) from wastewater sources. In particular, operational conditions during sludge digestion may serve to discourage selection of resistant bacteria, reduce horizontal transfer of ARGs, and aid in hydrolysis of DNA. This study applied metagenomic analysis to examine the removal efficiency of ARGs through thermophilic and mesophilic anaerobic digestion using bench-scale reactors. Although the relative abundance of various ARGs shifted from influent to effluent sludge, there was no measureable change in the abundance of total ARGs or their diversity in either the thermophilic or mesophilic treatment. Among the 35 major ARG subtypes detected in feed sludge, substantial reductions (removal efficiency >90 %) of 8 and 13 ARGs were achieved by thermophilic and mesophilic digestion, respectively. However, resistance genes of aadA, macB, and sul1 were enriched during the thermophilic anaerobic digestion, while resistance genes of erythromycin esterase type I, sul1, and tetM were enriched during the mesophilic anaerobic digestion. Efflux pump remained to be the major antibiotic resistance mechanism in sludge samples, but the portion of ARGs encoding resistance via target modification increased in the anaerobically digested sludge relative to the feed. Metagenomic analysis provided insight into the potential for anaerobic digestion to mitigate a broad array of ARGs. [ABSTRACT FROM AUTHOR]

Waste activated sludge (WAS) from wastewater treatment plants is an important reservoir of antibiotic resistance genes (ARGs). The fate of ARGs in this process was not revealed previously. The present study applied metagenomic approach to examine the occurrence and fate of ARGs in thermophilic alkaline fermentation followed by mesophilic anaerobic digestion (TM), by comparison with mesophilic alkaline fermentation followed by mesophilic anaerobic digestion (MM) and one-stage mesophilic anaerobic digestion (M) process. The removal efficiency of two-stage anaerobic digestion (AD) to total ARGs is higher than that of one-stage AD. The hydrogen and methane production stages of two-stage AD processes have dissimilar impact on the fate of ARGs. Macrolide, lincosamide, and streptogramin (MLS) resistance genes were enriched, especially in the hydrogen production reactors of TM and MM processes. Statistical analysis of metagenomic profiles analysis suggested that bac A may be the differential ARG subtype of two-stage AD process. ARG-like sequences encoding antibiotic efflux pump, antibiotic inactivation and antibiotic target alteration mechanisms were identified as the dominant ARGs resistance mechanisms in all samples. Procrustes analysis showed that microbial community composition structured the resistome. Co-occurrence patterns between ARGs and microbial phylogeny revealed that 26 bacterial species might be potential hosts of 94 ARG subtypes. ga1 • Two-stage anaerobic digestion caused a more great elimination of the total ARGs. • MLS were enriched in hydrogen production reactor of two-stage anaerobic digestion. • bac A may be the differential ARG subtype of two-stage anaerobic digestion. • Microbial community composition determined the ARGs composition. [ABSTRACT FROM AUTHOR]

This study explored the effect of eight antimicrobials on the efficiency of biogas production in the anaerobic digestion (AD) process of cattle manure. The microbiome involved in AD, presence and number of genes mcrA, MSC and MST specific for Archaea, and antibiotic resistance genes (ARGs) concentration in digestate (D) were examined. Supplementation of antibiotics to substrate significantly lowered biogas production. Amoxicillin caused a 75% decrease in CH4 production in comparison with the control samples. Enrofloxacin, tetracycline, oxytetracycline, and chlortetracycline reduced the amount of biogas produced by 36, 39, 45 and 53%, respectively. High-throughput sequencing of 16S rRNA results revealed that bacteria dominated the Archaea microorganisms in all samples. Moreover, antibiotics led to a decrease in the abundance of the genes mcrA, MSC, MST, and induced an increase in the number of tetracyclines resistance genes. Antibiotics decreased the efficiency of the AD process and lowered the quantity of CH4 obtained, while stimulating an increase in the number of ARGs in D. This work reveals how antimicrobials affect the cattle manure AD process and changes in microbial biodiversity, number of functional genes and ARGs in the digestate due to drugs exposure. It also, provides useful, practical information about the AD process. [ABSTRACT FROM AUTHOR]

The fate of diverse antibiotic resistance genes (ARGs) in anaerobic digestion (AD) system under different temperature remains largely unknow. This study employed the shotgun metagenomic sequencing and 16S rRNA amplicon sequencing approaches and investigated the shifts of composition and abundance of more than 1360 ARGs, as well as microbial community, in a temperature-raising AD system (from 35 °C to 55 °C) treating municipal sewage sludge. Results suggested that the six ARGs, inlcuding mac B, tet A(58), bcr A, evg S, mtr A, and ole C, were predominated in digested sludge. More than 407ARG subtypes (30%) could be largely reduced under high temperature condition. Thermophilic AD (55 °C) provided a better removal for tetracycline, macrolide, penam, fluoroquinolone, acridine dye, and peptide resistance genes when compared to mesophilic AD (35 °C). The removal rates of these genes reached 29.7%–32.3%. In addition, at the end of thermophilic digestion process, the relative abundance of Firmicutes increased (from 18.22% to 74.89%), while Proteobacteria and Chloroflexi both decreased, from 36.39% to 6.80% and 20.29%–2.64%. This study further proposed the underlying mechanisms of effective elimination of ARGs under high temperature by: (1) the reduction of genes encoding antibiotic efflux pump, (2) the promotion of sludge biomass degradation rate, (3) the loss of microbial community diversity in AD systems, and (4) the inactivation of multidrug-resistant bacteria. The significance of current study is in characterizing the fate of ARGs across AD process, which allows to correlate diverse genetic properties with digesters' operational condition. Results provided an insight into the manipulation of AD technology (e.g. temperature) to efficiently remove ARGs from municipal sewage sludge. • Fate of ARGs in a temperature-raising anaerobic digester was investigated. • More than 1360 ARG subtypes were identified by metagenomics approach. • Effective reduction of ARGs under thermophilic condition was confirmed. • Underlying mechanisms of ARGs removal were proposed. [ABSTRACT FROM AUTHOR]

In this work, we investigated the impact of iron nanoparticle, including magnetite nanoparticles (Fe 3 O 4 NPs) and nanoscale zero-valent iron (nZVI), on the anaerobic digestion (AD) performance. Moreover, the evolutions of antibiotic resistance genes (ARGs), class 1 integrons-integrase (intI 1) and potential hosts of ARGs were also investigated. The optimal addition of Fe 3 O 4 NPs and nZVI to promote methane production was 0.5 g/L and 1 g/L, which led to 22.07% and 23.02% increase in methane yield, respectively. The degradation rate of organic matter was also enhanced with the addition of Fe 3 O 4 NPs or nZVI. The results of high-throughput sequencing showed that the reactors with iron NPs exhibited significant differences in microbial community structure, compared to the reactors with the non‑iron NPs. Iron NPs have caused the relative abundance of the dominant bacteria (Proteobacteria, Firmicutes and Actinobacteria) generally decreased, while the dominant archaea (Euryarchaeota) increased in AD sludge. Quantitative PCR results revealed that iron NPs accelerated the reductions in total absolute abundance of ARGs, especially a beta-lactamase resistance encoded gene (bla OXA). Network analysis displayed that the attenuation of ARGs was mainly attributed to the decline of potential hosts (Proteobacteria, Firmicutes and Actinobacteria). Meanwhile, environmental factors (such as pH, soluble chemical oxygen demand and heavy metals) were also strongly correlated with ARGs. Unlabelled Image • Iron NPs promoted methane production and organic matter degradation in anaerobic digestion. • Iron NPs changed microbial communities and reduced microbial diversity in anaerobic digestion. • The absolute abundance of total ARGs decreased in the anaerobic digestion with iron NPs. • Proteobacteria , Firmicutes and Actinobacteria were potential hosts of ARGs. • The potential host of ARGs was the main driving factor for the fate of ARGs and intI 1. [ABSTRACT FROM AUTHOR]