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601200.9730Metal resistance-related genes are differently expressed in response to copper and zinc ion in six Acidithiobacillus ferrooxidans strains. Metal resistance of acidophilic bacteria is very significant during bioleaching of copper ores since high concentration of metal is harmful to the growth of microorganisms. The resistance levels of six Acidithiobacillus ferrooxidans strains to 0.15 M copper and 0.2 M zinc were investigated, and eight metal resistance-related genes (afe-0022, afe-0326, afe-0329, afe-1143, afe-0602, afe-0603, afe-0604, and afe-1788) were sequenced and analyzed. The transcriptional expression levels of eight possible metal tolerance genes in six A. ferrooxidans strains exposed to 0.15 M Cu(2+) and 0.2 M Zn(2+) were determined by real-time quantitative PCR (RT-qPCR), respectively. The copper resistance levels of six A. ferrooxidans strains declined followed by DY26, DX5, DY15, GD-B, GD-0, and YTW. The zinc tolerance levels of six A. ferrooxidans strains exposed to 0.2 M Zn(2+) from high to low were YTW > GD-B > DY26 > GD-0 > DX5 > DY15. Seven metal tolerance-related genes all presented in the genome of six strains, except afe-0604. The metal resistance-related genes showed different transcriptional expression patterns in six A. ferrooxidans strains. The expression of gene afe-0326 and afe-0022 in six A. ferrooxidans strains in response to 0.15 M Cu(2+) showed the same trend with the resistance levels. The expression levels of genes afe-0602, afe-0603, afe-0604, and afe-1788 in six strains response to 0.2 M Zn(2+) did not show a clear correlation between the zinc tolerance levels of six strains. According to the results of RT-qPCR and bioinformatics analysis, the proteins encoded by afe-0022, afe-0326, afe-0329, and afe-1143 were related to Cu(2+) transport of A. ferrooxidans strains.201425023638
615210.9653Identification of Bacillus megaterium and Microbacterium liquefaciens genes involved in metal resistance and metal removal. Bacillus megaterium MNSH1-9K-1 and Microbacterium liquefaciens MNSH2-PHGII-2, 2 nickel- and vanadium-resistant bacteria from mine tailings located in Guanajuato, Mexico, are shown to have the ability to remove 33.1% and 17.8% of Ni, respectively, and 50.8% and 14.0% of V, respectively, from spent petrochemical catalysts containing 428 ± 30 mg·kg(-1) Ni and 2165 ± 77 mg·kg(-1) V. In these strains, several Ni resistance determinants were detected by conventional PCR. The nccA (nickel-cobalt-cadmium resistance) was found for the first time in B. megaterium. In M. liquefaciens, the above gene as well as the czcD gene (cobalt-zinc-cadmium resistance) and a high-affinity nickel transporter were detected for the first time. This study characterizes the resistance of M. liquefaciens and B. megaterium to Ni through the expression of genes conferring metal resistance.201627210016
348220.9646Metagenomic profiling of ARGs in airborne particulate matters during a severe smog event. Information is currently limited regarding the distribution of antibiotic resistance genes (ARGs) in smog and their correlations with airborne bacteria. This study characterized the diversity and abundance of ARGs in the particulate matters (PMs) of severe smog based on publicly available metagenomic data, and revealed the occurrence of 205 airborne ARG subtypes, including 31 dominant ones encoding resistance to 11 antibiotic types. Among the detectable ARGs, tetracycline, β-lactam and aminoglycoside resistance genes had the highest abundance, and smog and soil had similar composition characteristics of ARGs. During the smog event, the total abundance of airborne ARGs ranged from 4.90 to 38.07ppm in PM(2.5) samples, and from 7.61 to 38.49ppm in PM(10) samples, which were 1.6-7.7 times and 2.1-5.1 times of those in the non-smog day, respectively. The airborne ARGs showed complicated co-occurrence patterns, which were heavily influenced by the interaction of bacterial community, and physicochemical and meteorological factors. Lactobacillus and sulfonamide resistance gene sul1 were determined as keystones in the co-occurrence network of microbial taxa and airborne ARGs. The results may help to understand the distribution patterns of ARGs in smog for the potential health risk evaluation.201829751438
810830.9645Insights into the beneficial effects of woody peat for reducing abundances of antibiotic resistance genes during composting. Antibiotic resistance genes (ARGs) in manure endangered human health, while heavy metals in manure will pose selective pressure on ARGs. This study explored the effects on ARGs of adding woody peat during composting at different ratios (0 (CK), 5% (T1), and 15% (T2)). After composting, the relative abundances of 8/11 ARGs were 6.97-38.09% and 10.73-54.31% lower in T1 and T2, respectively, than CK. The bioavailable Cu content was 1.40% and 18.40% lower in T1 and T2, respectively, than CK. Network analysis showed that ARGs, mobile genetic elements (MGEs), and metal resistance genes possessed common potential host bacteria, such as Streptococcus, Dietzia, and Corynebacterium_1. Environmental factors, especially bioavailable Cu, and MGEs accounted for 80.75% of the changes in the abundances of ARGs. In conclusion, 15% Woody peat is beneficial to decrease the bioavailable Cu content and weaken horizontal gene transfer for controlling the spread of ARGs during composting.202134534940
810940.9639The fate of antibiotic resistance genes and their influential factors in swine manure composting with sepiolite as additive. Manures are storages for antibiotic resistance genes (ARGs) entering the environment. This study investigated the effects of adding sepiolite at 0%, 2.5%, 5%, and 7.5% (CK, T1, T2, and T3, respectively) on the fates of ARGs during composting. The relative abundances (RAs) of the total ARGs in CK and T3 decreased by 0.23 and 0.46 logs, respectively, after composting. The RAs of 10/11 ARGs decreased in CK, whereas they all decreased in T3. The reduction in the RA of the total mobile genetic elements (MGEs) was 1.26 times higher in T3 compared with CK after composting. The bacterial community accounted for 47.93% of the variation in the abundances of ARGs. Network analysis indicated that ARGs and MGEs shared potential host bacteria (PHB), and T3 controlled the transmission of ARGs by reducing the abundances of PHB. Composting with 7.5% sepiolite is an effective strategy for reducing the risk of ARGs proliferating.202235063626
39950.9637Identification of genes conferring arsenic resistance to Escherichia coli from an effluent treatment plant sludge metagenomic library. The majority of bacteria elude culture in the laboratory. A metagenomic approach provides culture-independent access to the gene pool of the whole bacterial community. A metagenomic library was constructed from an industrial effluent treatment plant sludge containing about 1.25 Gb of microbial community DNA. Two arsenic-resistant clones were selected from the metagenomic library. Clones MT3 and MT6 had eight- and 18-fold higher resistance to sodium arsenate in comparison with the parent strain, respectively. The clones also showed increased resistance to arsenite but not to antimony. Sequence analysis of the clones revealed genes encoding for putative arsenate reductases and arsenite efflux pumps. A novel arsenate resistance gene (arsN) encoding a protein with similarity to acetyltransferases was identified from clone MT6. ArsN homologues were found to be closely associated with arsenic resistance genes in many bacterial genomes. ArsN homologues were found fused to putative arsenate reductases in Methylibium petroleiphilum PM1 and Anaeromyxobacter dehalogenans 2CP-C and with a putative arsenite chaperone in Burkholderia vietnamiensis G4. ArsN alone resulted in an approximately sixfold higher resistance to sodium arsenate in wild-type Escherichia coli W3110.200919016868
810660.9637Reducing antibiotic resistance genes, integrons, and pathogens in dairy manure by continuous thermophilic composting. This study explored the effects of composting using three temperature regimes, namely, insufficient thermophilic composting (ITC), normal thermophilic composting (NTC), and continuous thermophilic composting (CTC), on antibiotic resistance genes (ARGs), integrons, and human pathogenic bacteria (HPB), as well as the mechanisms involved. The NTC and CTC treatments led to greater decreases in 5/10 ARGs and two integrons than ITC, and the abundances of ARGs (tetC, tetG, and tetQ) and int1 only declined in the NTC and CTC treatments. The abundances of HPB decreased by 82.8%, 76.9%, and 96.9% under ITC, NTC, CTC, respectively. Redundancy analysis showed that both bacterial succession and horizontal gene transfer play important roles in the variation of ARGs, and the changes in different ARGs were due to diverse mechanisms. CTC performed significantly better at reducing ARGs, integrons, and HPB, thus it may be used to manage the public health risks of ARGs in animal manure.201627598571
773870.9637The microbiome and its association with antibiotic resistance genes in the hadal biosphere at the Yap Trench. The hadal biosphere, the deepest part of the ocean, is known as the least-explored aquatic environment and hosts taxonomically diverse microbial communities. However, the microbiome and its association with antibiotic resistance genes (ARGs) in the hadal ecosystem remain unknown. Here, we profiled the microbiome diversity and ARG occurrence in seawater and sediments of the Yap Trench (YT) using metagenomic sequencing. Within the prokaryote (bacteria and archaea) lineages, the main components of bacteria were Gammaproteobacteria (77.76 %), Firmicutes (8.36 %), and Alphaproteobacteria (2.25 %), whereas the major components of archaea were Nitrososphaeria (6.51 %), Nanoarchaeia (0.42 %), and Thermoplasmata (0.25 %), respectively. Taxonomy of viral contigs showed that the classified viral communities in YT seawater and sediments were dominated by Podoviridae (45.96 %), Siphoviridae (29.41 %), and Myoviridae (24.63 %). A large majority of viral contigs remained uncharacterized and exhibited endemicity. A total of 48 ARGs encoding resistance to 12 antibiotic classes were identified and their hosts were bacteria and viruses. Novel ARG subtypes mexF(YTV-1), mexF(YTV-2), mexF(YTV-3), vanR(YTV-1), vanS(YTV-1) (carried by unclassified viruses), and bacA(YTB-1) (carried by phylum Firmicutes) were detected in seawater samples. Overall, our findings imply that the hadal environment of the YT is a repository of viral and ARG diversity.202235870206
40380.9637Nucleotide sequence and expression of the mercurial-resistance operon from Staphylococcus aureus plasmid pI258. The mercurial-resistance determinant from Staphylococcus aureus plasmid pI258 is located on a 6.4-kilobase-pair Bgl II fragment. The determinant was cloned into both Bacillus subtilis and Escherichia coli. Mercury resistance was found only in B. subtilis. The 6404-base-pair DNA sequence of the Bgl II fragment was determined. The mer DNA sequence includes seven open reading frames, two of which have been identified by homology with the merA (mercuric reductase) and merB (organomercurial lyase) genes from the mercurial-resistance determinants of Gram-negative bacteria. Whereas 40% of the amino acid residues overall were identical between the pI258 merA polypeptide product and mercuric reductases from Gram-negative bacteria, the percentage identity in the active-site positions and those thought to be involved in NADPH and FAD contacts was above 90%. The 216 amino acid organomercurial lyase sequence was 39% identical with that from a Serratia plasmid, with higher conservation in the middle of the sequences and lower homologies at the amino and carboxyl termini. The remaining five open reading frames in the pI258 mer sequence have no significant homologies with the genes from previously sequenced Gram-negative mer operons.19873037534
40590.9636Characterization of a small plasmid (pMBCP) from bovine Pseudomonas pickettii that confers cadmium resistance. This is the first report of isolation of Pseudomonas pickettii from a normal adult bovine duodenum. This organism was one of several bacteria isolated as part of a study to examine cadmium resistance genes (cad(r)) for use in generating transgenic plants to reclaim cadmium-contaminated soils in Kansas. P. pickettii containing a plasmid of 2.2kb (designated pMBCP) grew in Luria-Bertani broth and agar containing up to 800 microM of cadmium chloride and was resistant to 16 antibiotics. Curing the organism of plasmid revealed that antibiotic resistances were not plasmid-mediated. Low-level cadmium resistance was conferred by the plasmid because uncured organism grew significantly better (P<0.05) at 55 microM compared to cured organism. Both plasmid and chromosomal DNA were probed by DNA-DNA hybridization for the presence of known cadmium resistance genes (cadA, cadC, and cadD from Gram-positive (Staphylococcus aureus), but none were detected. The plasmid had one restriction site each for BamHI, PstI, SmaI, and XhoI; two sites each for HincII, SacI, and SphI; and multiple sites for AluI and XcmI. DNA sequence analyses of the cloned and original plasmids showed a GC content of greater than 60% and no homology to any published sequences in the GenBank, European Bioinformatics Institute, or Japanese Genome Net databases. The DNA sequence is contained in GenBank accession number AF144733. Thus, pMBCP offers low-level cadmium resistance to P. picketttii.200312651180
6117100.9636Isolation and characterization of the heavy metal resistant bacteria CCNWRS33-2 isolated from root nodule of Lespedeza cuneata in gold mine tailings in China. A total of 108 strains of bacteria were isolated from root nodules of wild legumes growing in gold mine tailings in northwest of China and were tested for heavy metal resistance. The results showed that the bacterial strain CCNWRS33-2 isolated from Lespedeza cuneata was highly resistant to copper, cadmium, lead and zinc. The strain had a relatively high mean specific growth rate under each heavy metal stress test and exhibited a high degree of bioaccumulation ability. The partial sequence of the copper resistance gene copA was amplified from the strain and a sequence comparison with our Cu-resistant PCR fragment showed a high homology with Cu-resistant genes from other bacteria. Phylogenetic analysis based on the 16S rRNA gene sequence showed that CCNWRS33-2 belongs to the Rhizobium-Agrobacterium branch and it had 98.9% similarity to Agrobactrium tumefaciens LMG196.200918562095
7160110.9635High-throughput profiling of antibiotic resistance genes in the Yellow River of Henan Province, China. Profiling antibiotic resistance genes (ARGs) in the Yellow River of China's Henan Province is essential for understanding the health risks of antibiotic resistance. The profiling of ARGs was investigated using high-throughput qPCR from water samples in seven representative regions of the Yellow River. The absolute and relative abundances of ARGs and moble genetic elements (MGEs) were higher in summer than in winter (ANOVA, p < 0.001). The diversity and abundance of ARGs were higher in the Yellow River samples from PY and KF than the other sites. Temperature (r = 0.470 ~ 0.805, p < 0.05) and precipitation (r = 0.492 ~ 0.815, p < 0.05) positively influenced the ARGs, while pH had a negative effect (r = - 0.462 ~ - 0.849, p < 0.05). Network analysis indicated that the pathogenic bacteria Rahnella, Bacillus, and Shewanella were the possible hub hosts of ARGs, and tnpA1 was the potential MGE hub. These findings provide insights into the factors influencing ARG dynamics and the complex interaction among the MGEs, pathogenic bacteria and environmental parameters in enriching ARGs in the Yellow River of Henan Province.202439080455
8016120.9635Transmission and retention of antibiotic resistance genes (ARGs) in chicken and sheep manure composting. Transmission of ARGs during composting with different feedstocks (i.e., sheep manure (SM), chicken manure (CM) and mixed manure (MM, SM:CM = 3:1 ratio) was studied by metagenomic sequencing. 53 subtypes of ARGs for 22 types of antibiotics were identified as commonly present in these compost mixes; among them, CM had higher abundance of ARGs, 1.69 times than that in SM, while the whole elimination rate of CM, MM and SM were 55.2%, 54.7% and 42.9%, respectively. More than 50 subtypes of ARGs (with 8.6%, 11.4% and 20.9% abundance in the initial stage in CM, MM and SM composting) were "diehard" ARGs, and their abundance grew significantly to 56.5%, 63.2% and 69.9% at the mature stage. These "diehard" ARGs were transferred from initial hosts of pathogenic and/or probiotic bacteria to final hosts of thermophilic bacteria, by horizontal gene transfer (HGT) via mobile gene elements (MGEs), and became rooted in composting products.202337196739
3567130.9635Cloning and sequence analysis of ermQ, the predominant macrolide-lincosamide-streptogramin B resistance gene in Clostridium perfringens. The erythromycin resistance determinant from Clostridium perfringens JIR100 has been cloned, sequenced, and shown to be expressed in Escherichia coli. An open reading frame with sequence similarity to erm genes from other bacteria was identified and designated the ermQ gene. On the basis of comparative sequence analysis, it was concluded that the ermQ gene represented a new Erm hybridization class, designated ErmQ. Genes belonging to the ErmQ class were found to be widespread in C. perfringens, since 30 of 38 macrolide-lincosamide-streptogramin B-resistant C. perfringens strains, from diverse sources, hybridized to an ermQ-specific gene probe. The ermQ gene therefore represents the most common erythromycin resistance determinant in C. perfringens.19948067735
7744140.9634Dynamics and removal mechanisms of antibiotic and antibiotic resistance genes during the fermentation process of spectinomycin mycelial dregs: An integrated meta-omics study. Antibiotic mycelial dregs (AMDs) have been listed as industrial hazardous wastes. With the aim of reducing the environmental risk, the integrated-omics and qPCR approaches were used to reveal the dynamics and removal mechanisms of antibiotic and antibiotic resistance genes (ARGs) during the fermentation of different spectinomycin mycelial dregs (SMDs). The results showed that the removal efficiency of antibiotic in the fermentation of high moisture SMDs reached up to 98%. The high abundance of aadA1 gene encoded by Streptomyces, Lactobacillus, and Pseudomonas was associated with the efficient degradation of spectinomycin, and the inactivating enzymes secreted by degradative bacteria were identified. Furthermore, the dominant microbiota was impacted by moisture content significantly under high temperature environments. In the fermentation of low moisture SMDs, Saccharopolyspora was the dominant microbiota which secreted S8 endopeptidase, M14, M15, S10, S13 carboxypeptidases, M1, M28, S15 aminopeptidases, and antioxidant enzymes, while in the fermentation of high moisture SMDs, Bacillus and Cerasibacillus were dominant genera which mainly secreted S8 endopeptidase and antioxidant enzymes. The abundance of ARGs and mobile genetic elements decreased significantly at thermophilic phase, with maximum drops of 93.7% and 99.9%, respectively. Maintaining moisture content below 30% at the end phase could prevent the transmission of ARGs effectively.202234396972
407150.9634Molecular cloning and characterization of two lincomycin-resistance genes, lmrA and lmrB, from Streptomyces lincolnensis 78-11. Two different lincomycin-resistance determinants (lmrA and lmrB) from Streptomyces lincolnensis 78-11 were cloned in Streptomyces lividans 66 TK23. The gene lmrA was localized on a 2.16 kb fragment, the determined nucleotide sequence of which encoded a single open reading frame 1446 bp long. Analysis of the deduced amino acid sequence suggested the presence of 12 membrane-spanning domains and showed significant similarities to the methylenomycin-resistance protein (Mmr) from Streptomyces coelicolor, the QacA protein from Staphylococcus aureus, and several tetracycline-resistance proteins from both Gram-positive and Gram-negative bacteria, as well as to some sugar-transport proteins from Escherichia coli. The lmrB gene was actively expressed from a 2.7 kb fragment. An open reading frame of 837 bp could be localized which encoded a protein that was significantly similar to 23S rRNA adenine(2058)-N-methyltransferases conferring macrolide-lincosamide-streptogramin resistance. LmrB also had putative rRNA methyltransferase activity since lincomycin resistance of ribosomes was induced in lmrB-containing strains. Surprisingly, both enzymes, LmrA and LmrB, had a substrate specificity restricted to lincomycin and did not cause resistance to other lincosamides such as celesticetin and clindamycin, or to macrolides.19921328813
6144160.9633Efficient arsenate reduction by As-resistant bacterium Bacillus sp. strain PVR-YHB1-1: Characterization and genome analysis. Arsenate (AsV) reduction in bacteria is essential to alleviate their arsenic (As) toxicity. We isolated a Bacillus strain PVR-YHB1-1 from the roots of As-hyperaccumulator Pteris vittata. The strain was efficient in reducing AsV to arsenite (AsIII), but the associated mechanisms were unclear. Here, we investigated its As resistance and reduction behaviors and associated genes at genome level. Results showed that the strain tolerated up to 20 mM AsV. When grown in 1 mM AsV, 96% AsV was reduced to AsIII in 48 h, with its AsV reduction ability being positively correlated to bacterial biomass. Two ars operons arsRacr3arsCDA and arsRKacr3arsC for As metabolisms were identified based on draft genome sequencing and gene annotations. Our data suggested that both operons might have attributed to efficient As resistance and AsV reduction in PVR-YHB1-1, providing clues to better understand As transformation in bacteria and their roles in As transformation in the environment.201930609485
8049170.9633Microalgae simultaneously promote antibiotic removal and antibiotic resistance genes/bacteria attenuation in algal-bacterial granular sludge system. This study investigated the effects of microalgae growth on antibiotic removal and the attenuation of antibiotic resistance genes (ARGs)/ARGs host bacteria in algal-bacterial granular sludge (ABGS) system. In the presence of tetracycline (TC) and sulfadiazine (SDZ) mixture (2-4 mg/L), microalgae could grow on bacterial granular sludge (BGS) to form ABGS, with a chlorophyll-a content of 7.68-8.13 mg/g-VSS being achieved. The removal efficiencies of TC and SDZ by ABGS were as high as 79.0 % and 94.0 %, which were 4.3-5.0 % higher than those by BGS. Metagenomic analysis indicated that the relative abundances of TC/SDZ- related ARGs and mobile genetic elements (MGEs) in BGS were 56.1 % and 22.1 % higher than those in ABGS. A total of 26 ARGs were detected from the granules, and they were identified to associate with 46 host bacteria. 13 out of 26 ARGs and 13 out of 46 hosts were shared ARGs and hosts, respectively. The total relative abundance of host bacteria in BGS was 30.8 % higher than that in ABGS. Scenedesmus and Chlorella were the dominant microalgae that may reduce the diversity of ARGs hosts. Overall, ABGS is a promising biotechnology for antibiotic-containing wastewater treatment.202235777142
8015180.9632Distribution, horizontal transfer and influencing factors of antibiotic resistance genes and antimicrobial mechanism of compost tea. Compost tea was alternatives of chemical pesticide for green agriculture, but there were no reports about antibiotic resistance genes (ARGs) in compost tea. This study investigated the effect of livestock manures, sewage sludge, their composting products and liquid fermentation on ARGs, mobile genetic elements (MGEs), metal resistance genes (MRGs) and antimicrobial properties of various compost tea. The results showed aerobic liquid fermentation reduced ARGs by 65.93 % and 45.20 % in the compost tea of chicken manure and sludge, enriched ARGs by 8.57 % and 37.41 % in the compost tea of pig manure and bovine manure, and increased MGEs and MRGs by 1.25 × 10(-5)-5.53 × 10(-3) and 2.03 × 10(-5)-2.03 × 10(-3) in the four compost tea. The correlation coefficient of tetracycline and sulfonamide resistance genes between compost product and compost tea were 0.98 and 0.91. aadA2-02, sul2 and tetX abundant in the compost tea were positively correlated with MGEs and MRGs. Furthermore, liquid fermentation enriched the potential host of tetracycline and vancomycin resistance genes. Tetracycline resistance genes occupied 62.7 % of total ARGs in the compost tea. Alcaligenes and Bacillus enriched by 0.78-39.31 % in the four compost tea, which metabolites had high antimicrobial activity. The potential host of ARGs accounted for 42.1 % bacteria abundance in the four compost tea.202235803190
8046190.9632Responses of aerobic granular sludge to fluoroquinolones: Microbial community variations, and antibiotic resistance genes. In this study, aerobic granular sludge (AGS) was operated under high levels of ammonium for removing three fluoroquinolones (FQs), i.e., ciprofloxacin (CFX), ofloxacin (OFX), and norfloxacin (NFX) at 3, 300, and 900 µg/L, respectively. Two key objectives were to investigate the differential distribution of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in sludge fractions and to evaluate correlations between ARGs and MGEs to nitrifying and denitrifying bacteria. AGS showed excellent stability under the exposure of FQs, with nitrite-oxidizing bacteria (NOB) more sensitive to FQs than ammonium-oxidizing bacteria (AOB). Specific oxygen utilization rates (SOUR) showed a reduction of 26.9% for NOB but only 4.0% of the reduced activity of AOB by 3 μg/L FQs. AGS performed better removal efficiencies for CFX and NFX than OFX, and the efficiencies increased with their elevated concentrations, except at 900 μg/L FQs. The elevated FQ concentrations led to a significant enrichment of intI1 and genus Thauera, while qnrD and qnrS showed no accumulation. Compared to nitrifiers, FQs relevant ARGs and the intI1 gene preferred to exist in denitrifiers, and the abundance of denitrifiers behaved a decreasing trend with the sludge size. Two quinoline-degrading bacteria were found in the AGS system, i.e., Alicycliphilus and Brevundimonas, possibly carrying qnrS and qnrD, respectively. Their relative abundance increased with the sludge size, which was 2.18% in sludge <0.5 mm and increased to 3.70% in sludge >2.0 mm, suggesting that the AGS may be a good choice in treating FQs-containing wastewater.202133676249