Method evaluation for viruses in activated sludge: Concentration, sequencing, and identification.

Activated sludge (AS) in wastewater treatment plants is one of the largest artificial microbial ecosystems on earth and it makes enormous contributions to human societies. Viruses are an important component in AS with a high abundance. However, their communities and functionalities have not been as widely explored as those of other microorganisms, such as bacteria. This gap is mainly due to technical challenges in effective viral concentration, extraction, and sequencing. In this study, we compared four kinds of concentration methods, two sequencing approaches, and four identification bioinformatic tools to evaluate the whole analysis workflow for viruses in AS. Results showed flocculation, filtration, and resuspension (FFR) could get the longest DNA lengths and ultracentrifugation obtained the highest DNA yields for viruses in AS. Based on the results of present study, FFR and tangential flow filtration with the membrane pore size of 100 kDa were most recommended to concentrate viruses in AS samples with huge volumes. Besides, different concentration methods could get different viral catalogs and thus multiple methods should be combined to get the whole picture of viruses in the system. In addition, geNomad was the most recommended identification tool for viruses in the present study and the long-read sequencing could improve the assembly statistics of viruses when compared with the short-read sequencing. For the 8192 viral operational taxonomic units in this study, 95.1 % of them were phages and belonged to the same lineage at the order level of Caudovirales. Virulent phages dominated the AS system and Pseudomonadota were the main host. Taken together, this study provides new insights into methods selection for virus research of AS.

Melon: metagenomic long-read-based taxonomic identification and quantification using marker genes.

Long-read sequencing holds great potential for characterizing complex microbial communities, yet taxonomic profiling tools designed specifically for long reads remain lacking. We introduce Melon, a novel marker-based taxonomic profiler that capitalizes on the unique attributes of long reads. Melon employs a two-stage classification scheme to reduce computational time and is equipped with an expectation-maximization-based post-correction module to handle ambiguous reads. Melon achieves superior performance compared to existing tools in both mock and simulated samples. Using wastewater metagenomic samples, we demonstrate the applicability of Melon by showing it provides reliable estimates of overall genome copies, and species-level taxonomic profiles.

Metatranscriptomics-guided genome-scale metabolic reconstruction reveals the carbon flux and trophic interaction in methanogenic communities.

BACKGROUND: Despite rapid advances in genomic-resolved metagenomics and remarkable explosion of metagenome-assembled genomes (MAGs), the function of uncultivated anaerobic lineages and their interactions in carbon mineralization remain largely uncertain, which has profound implications in biotechnology and biogeochemistry. RESULTS: In this study, we combined long-read sequencing and metatranscriptomics-guided metabolic reconstruction to provide a genome-wide perspective of carbon mineralization flow from polymers to methane in an anaerobic bioreactor. Our results showed that incorporating long reads resulted in a substantial improvement in the quality of metagenomic assemblies, enabling the effective recovery of 132 high-quality genomes meeting stringent criteria of minimum information about a metagenome-assembled genome (MIMAG). In addition, hybrid assembly obtained 51% more prokaryotic genes in comparison to the short-read-only assembly. Metatranscriptomics-guided metabolic reconstruction unveiled the remarkable metabolic flexibility of several novel Bacteroidales-affiliated bacteria and populations from Mesotoga sp. in scavenging amino acids and sugars. In addition to recovering two circular genomes of previously known but fragmented syntrophic bacteria, two newly identified bacteria within Syntrophales were found to be highly engaged in fatty acid oxidation through syntrophic relationships with dominant methanogens Methanoregulaceae bin.74 and Methanothrix sp. bin.206. The activity of bin.206 preferring acetate as substrate exceeded that of bin.74 with increasing loading, reinforcing the substrate determinantal role. CONCLUSION: Overall, our study uncovered some key active anaerobic lineages and their metabolic functions in this complex anaerobic ecosystem, offering a framework for understanding carbon transformations in anaerobic digestion. These findings advance the understanding of metabolic activities and trophic interactions between anaerobic guilds, providing foundational insights into carbon flux within both engineered and natural ecosystems. Video Abstract.

Metagenomic insights into ecological risk of antibiotic resistome and mobilome in riverine plastisphere under impact of urbanization.

Microplastics (MPs) are of increasing concern due to their role as reservoirs for antibiotic resistance genes (ARGs) and pathogens. To date, few studies have explored the influence of anthropogenic activities on ARGs and mobile genetic elements (MGEs) within various riverine MPs, in comparison to their natural counterparts. Here an in-situ incubation was conducted along heavily anthropogenically-impacted Houxi River to characterize the geographical pattern of antibiotic resistome, mobilome and pathogens inhabiting MPs- and leaf-biofilms. The metagenomics result showed a clear urbanization-driven profile in the distribution of ARGs, MGEs and pathogens, with their abundances sharply increasing 4.77 to 19.90 times from sparsely to densely populated regions. The significant correlation between human fecal marker crAssphage and ARG (R2 = 0.67, P=0.003) indicated the influence of anthropogenic activity on ARG proliferation in plastisphere and natural leaf surfaces. And mantel tests and random forest analysis revealed the impact of 17 socio-environmental factors, e.g., population density, antibiotic concentrations, and pore volume of materials, on the dissemination of ARGs. Partial least squares-path modeling further unveiled that intensifying human activities not only directly boosted ARGs abundance but also exerted a comparable indirect impact on ARGs propagation. Furthermore, the polyvinylchloride plastisphere created a pathogen-friendly habitat, harboring higher abundances of ARGs and MGEs, while polylactic acid are not likely to serve as vectors for pathogens in river, with a lower resistome risk score than that in leaf-biofilms. This study highlights the diverse ecological risks associated with the dissemination of ARGs and pathogens in varied MPs, offering insights for the policymaking of usage and control of plastics within urbanization.

Tracking diarrhea viruses and mpox virus using the wastewater surveillance network in Hong Kong.

The wastewater surveillance network successfully established for COVID-19 showed great potential to monitor other infectious viruses, such as norovirus, rotavirus and mpox virus. In this study, we established and validated detection methods for these viruses in wastewater. We developed a supernatant-based method to detect RNA viruses from wastewater samples and applied it to the monthly diarrhea viruses (norovirus genogroup I & II, and rotavirus) surveillance in wastewater treatment plants (WWTPs) at a city-wide level for 16 months. Significant correlations were observed between the diarrhea viruses concentrations in wastewater and detection rates in faecal specimens by clinical surveillance. The highest norovirus concentration in wastewater was obtained in winter, consistent with the seasonal pattern of norovirus outbreak in Hong Kong. Additionally, we established a pellet-based method to monitor DNA viruses in wastewater and detected weak signals for mpox virus in wastewater from a WWTP serving approximately 16,700 people, when the first mpox patient in Hong Kong was admitted to the hospital within the catchment area. Genomic sequencing provided confirmatory evidence for the validity of the results. Our findings emphasized the efficacy of the wastewater surveillance network in WWTPs as a cost-effective tool to track the transmission trend of diarrhea viruses and to provide sensitive detection of novel emerging viruses such as mpox virus in low-prevalence areas. The developed methods and surveillance results provide confidence for establishing robust wastewater surveillance programs to control infectious diseases in the post-pandemic era.

Treatment of fresh leachate by anaerobic membrane bioreactor: On-site investigation, long-term performance and response of microbial community.

This study proposed fresh leachate treatment with anaerobic membrane bioreactor (AnMBR) based on the on-site investigation of the characteristics of fresh leachate. Temperature-related profiles of fresh leachate properties, like chemical oxygen demand (COD), were observed. In addition, AnMBR achieved a high COD removal of 98% with a maximum organic loading rate (OLR) of 19.27 kg-COD/m3/d at the shortest hydraulic retention time (HRT) of 1.5 d. The microbial analysis implied that the abundant protein and carbohydrate degraders (e.g., Thermovirga and Petrimonas) as well as syntrophic bacteria, such as Syntrophomonas, ensured the effective adaptation of AnMBR to the reduced HRTs. However, an excessive OLR at 36.55 kg-COD/m3/d at HRT of 1 d resulted in a sharp decrease in key microbes, such as archaea (from 37% to 15%), finally leading to the deterioration of AnMBR. This study provides scientific guidance for treating fresh leachate by AnMBR and its full-scale application for high-strength wastewater.

Functional role of mixed-culture microbe in photocatalysis coupled with biodegradation: Total organic carbon removal of ciprofloxacin.

Ciprofloxacin is an extensively used fluoroquinolone antibiotic, which exists in aquatic environment, causing detrimental effects to the aquatic ecosystem and thus, indirectly to humans. Thus, an efficient and rapid removal method for ciprofloxacin is urgently needed. Intimately coupled photocatalysis and biodegradation has proven to be highly efficient, low-cost, and eco-friendly. In this study, cube polyurethane sponges modified with visible light-responsive g-C3N4 and mixed culture microbes were used to increase the ciprofloxacin removal efficiency. Subsequently, 94% of ciprofloxacin was removed by photocatalytic-biodegradation and 12 degradation products and possible degradation pathways were analyzed. Photocatalytic-biodegradation had a 1.57 times higher total organic carbon (TOC) removal rate than photocatalytic degradation. The microbial community structure after 72 h of photocatalytic biodegradation was examined. High microbial richness, evenness, and functional dominant species belong to Proteobacteria, which were closely associated with the utilization of antibiotics, may be majorly responsible for the highly efficient removal degradation process. Additionally, microbes retarded the interaction of photogenerated electrons and holes, which may contribute to the increasing mineralization. The findings demonstrated the potential ability of photocatalytic biodegradation in degrading bio-recalcitrant compounds and provide new insights into photocatalytic coupled with biodegradation for removal of ciprofloxacin.

Antibiotic resistance genes are increased by combined exposure to sulfamethoxazole and naproxen but relieved by low-salinity.

Combined pollution of antibiotic and non-antibiotic pharmaceutical residues is ubiquitous in realistic polluted environments, which is regarded as a complicated emerging pollution. Herein, high-throughput sequencing and high-throughput quantitative PCR were applied to profile the overall changes in microbial communities and antibiotic resistance genes (ARGs) of biofilms in response to a combination of naproxen and sulfamethoxazole pollution. After continuous operation for 120 days, naproxen or/and sulfamethoxazole were efficiently removed, and the salinity of 1.00% enhanced the removal rate of sulfamethoxazole. The high-throughput sequencing revealed that Eubacterium spp. with abundances of over 40.00% dominated in all samples, and combined pollution of naproxen and sulfamethoxazole more readily promoted the occurrence of multidrug-resistant microbes, including Pseudomonas and Methylophilus. The high-throughput quantitative PCR results showed that the combined pollution of naproxen and sulfamethoxazole increased the total abundance of ARGs to approximately 9 copies per cell. In contrast, increasing the salinity to 1.00% greatly reduced the overall abundance of ARGs to below 2 copies per bacterial cell. Mantel test and Procrustes analysis indicated that microbiomes from different treatments had tight links to their respective antibiotic resistomes. Furthermore, network analysis revealed that multidrug-resistant microbes were potential hosts for greatly enriched numbers of ARGs in the combined treatment. As increased salinity eliminated those multidrug-resistant but salt-sensitive microbes, the abundance of ARGs was significantly decreased. These results showed the high probability of the transmission of ARGs in biofilms exposed to combined pollution of naproxen and sulfamethoxazole, which could be relieved by increased salinity.

Rapid and efficient removal of naproxen from water by CuFe2O4 with peroxymonosulfate.

Naproxen, a widely used nonsteroidal anti-inflammatory drug, has been detected in many environmental matrixes and is regarded as an emerging pollutant. Sulfate radical (SO4·-) -based advanced oxidation processes have attracted wide attention due to their high efficiency and applicability in the removal of emerging contaminants. In this study, CuFe2O4 was used as an efficient catalyst to activate peroxymonosulfate to oxidize naproxen. The results suggested that 92.3% of naproxen was degraded and 50.3% total organic carbon was removed in 60 min in the presence of 0.3 g·L-1 CuFe2O4 and 2 mM peroxymonosulfate. This degradation system showed strong adaptability in a wide pH range from 4.0 to 10.0. Free radical scavenger experiments and electron spin resonance analysis indicated that 1O2, ·OH, and SO4·- are the main active species. Finally, the potential degradation pathways of naproxen were proposed by detecting and analyzing the degradation products with ultra-high-performance liquid chromatography combined with mass spectrometry. The results of this study suggest that the CuFe2O4-activated peroxymonosulfate system is a promising technology for the removal of naproxen from natural water.

Long-term operation of electroactive biofilms for enhanced ciprofloxacin removal capacity and anti-shock capabilities.

Few studies have focused on the feasibility of microbial fuel cells (MFCs) for removing quinolones antibiotics and their anti-shock capabilities. After 1.5 years of operation, the removal efficiency of 10 mg/L ciprofloxacin in MFCs increased to 99.00% in 88 h. These results are in accordance with the enhanced activity of biofilms and voltage output of MFCs. Additionally, the anti-shock capacities of the biofilms in MFCs were evaluated by treating ofloxacin and enrofloxacin and operating at different temperature and salinity. These MFCs can remove 87.31% and 40.81% of ofloxacin and enrofloxacin in 72 h, respectively. Even exposed to a low temperature of 10 °C or a salinity of 3%, the MFCs can achieve greater than 50% and nearly 80% of ciprofloxacin removal efficiency, respectively. The enrichment of Alcaligenes and Chryseobacterium contributed mostly to the removal of quinolones antibiotics. This study provides scientific evidences for treating wastewater containing quinolones antibiotics using MFCs.

Proteomic analysis of the reduction and resistance mechanisms of Shewanella oneidensis MR-1 under long-term hexavalent chromium stress.

Hexavalent chromium [Cr(VI)] is a priority heavy metal pollutant causing a series of environmental issues, and bio-reduction of Cr(VI) to trivalent chromium can remarkably decrease the environmental risk of Cr(VI). The reduction and resistance abilities of microorganisms to Cr(VI) can be dramatically improved by acclimatization. In the present study, we collected Shewanella oneidensis MR-1 from a 120-day acclimatization by increasing Cr(VI) concentration in the culture media to investigate its adaptation mechanisms under long-term Cr(VI) stress at the proteome level. Tandem mass tag-based quantitative proteomic analysis was performed to study the differences between 9 collected samples. A total of 2500 proteins were quantified from 2723 identified protein groups. Bioinformatics analysis showed that the differentially expressed proteins after the 120-day Cr(VI) acclimatization were mostly related to flagellar assembly, ribosomes, transport, sulfur metabolism, and energy metabolism. The findings of this study present novel insights into the molecular mechanisms for the reduction and resistance of S. oneidensis MR-1 responding to long-term Cr(VI) stress at the proteome level.

Leaching of vanadium from waste V2O5-WO3/TiO2 catalyst catalyzed by functional microorganisms.

Solid wastes are currently produced in large amounts. Although bioleaching of metals from solid wastes is an economical and sustainable technology, it has seldom been used to recycle metals from abandoned catalyst. In this study, the bioleaching of vanadium from V2O5-WO3/TiO2 catalyst were comprehensively investigated through five methods: Oligotrophic way, Eutrophic way, S-mediated way, Fe-mediated way and Mixed way of S-mediated and Fe-mediated. The observed vanadium bioleaching effectiveness of the assayed methods was follows: S-mediated > Mixed > Oligotrophic > Eutrophic > Fe-mediated, which yielded the maximum bioleaching efficiencies of approximately 90%, 35%, 33%, 20% and 7%, respectively. The microbial community analysis suggested that the predominant genera Acidithiobacillus and Sulfobacillus from the S-mediated bioleaching way effectively catalyzed the vanadium leaching, which could have occurred through the indirect mechanism from the microbial oxidation of S0. In addition, the direct mechanism, involving direct electron transfer between the catalyst and the microorganisms that attached to the catalyst surface, should also help the vanadium to be leached more effectively. Therefore, this work provides guidance for future research and practical application on the treatment of waste V2O5-WO3/TiO2 catalyst.

[Oxytetracycline Wastewater Treatment in Microbial Fuel Cells and the Analysis of Microbial Communities].

Oxytetracycline (OTC) as an important broad spectrum antibiotic has been widely used in animal husbandry. However, the abuse of OTC not only has a direct adverse impact on ecosystems, but also exacerbates the appearance of antibiotic resistance bacteria and antibiotic resistance genes. OTC-containing wastewater was treated using microbial fuel cells (MFCs), and the removal efficiency of OTC in MFCs in different operation periods was investigated. Result showed that the removal efficiency of 10 mg·L-1 OTC by MFCs within 132 h was up to 99.0% after 150 d of operation. The bacterial communities in raw pig mature and anodic biofilms were studied by high-throughput sequencing. This showed that Firmicutes were both dominant on phylum a level; However, compared to the raw pig mature, the abundance of Proteobacteria greatly increased from 2.84% to 8.92%-22.75% in the anodic biofilm. In addition, the abundance of Eubacterium spp. in the anodic biofilm increased obviously from nearly 0.00% to 20.49%-49.00%. It has been reported that some Eubacterium spp. were able to biodegrade oxygen heterocyclic aromatic compounds contained in OTC. Therefore, Eubacterium spp. is suggested as potential functional species in the biodegradation of OTC and/or its metabolites. This work proves the feasibility and effectiveness of removing OTC-containing wastewater by using MFCs.

Light-excited photoelectrons coupled with bio-photocatalysis enhanced the degradation efficiency of oxytetracycline.

Intimately coupled photocatalysis and biodegradation (ICPB) is a novel wastewater treatment technique that has potential applications in refractory degradation. This paper reports a synergistic degradation protocol that allowing the transfer of photoelectrons between photocatalysts and microbes without supplementary electron donors or improving the loading rate of the photocatalysts. As a result, a degradation rate of ∼94% was sustained for 400 h in a perturbation setup with a hydraulic retention time of 4.0 h. We achieved the degradation of ß-apo-oxytetracycline, a stable antimicrobial intermediate compound (half-life of 270 d in soil interstitial water), within 10 min, and no accumulation was observed. Moreover, the required loading rate of the photocatalyst was dramatically reduced to 18.3% compared to previous reports which mentioned much higher rates. The results of our study provided a new strategy to improve the degradation efficiency of oxytetracycline and give new insight into the degradation mechanism of the bio-photocatalytic degradation system.

The changes of bacterial communities and antibiotic resistance genes in microbial fuel cells during long-term oxytetracycline processing.

Microbial fuel cell (MFC) is regarded as a promising alternative for enhancing the removal of antibiotic pollutants. In this study, oxytetracycline served as an electron donor in the anode chamber of MFCs, and after continuous operation for 330 days, the efficiency of removal of 10 mg/L oxytetracycline in MFCs increased to 99.00% in 78 h, whereas removal efficiency of only 58.26% was achieved in microbial controls. Compared to microbial controls, higher ATP concentration and persistent electrical stimulation mainly contributed to bioelectrochemical reactions more rapidly to enhance oxytetracycline removal in MFCs. In addition, the analysis of bacterial communities revealed that Eubacterium spp.-as the main functional bacterial genus responsible for oxytetracycline biodegradation-flourished starting from merely 0.00%-91.69% ±â€¯0.27% (mean ±â€¯SD) in MFCs. High-throughput quantitative PCR showed that the normalized copy numbers of total antibiotic resistance genes (ARGs) and mobile genetic elements in MFCs were 1.7364 and 0.0065 copies/cell respectively, which were markedly lower than those in the microbial controls. Furthermore, there was no significant correlation between oxytetracycline concentration in the influent and abundance of ARGs in effluent from MFCs. Nevertheless, Tp614, a transposase gene, was found to be enriched in both MFCs and microbial reactors, suggesting that it may be a common challenge for different biological processes for wastewater treatment. This study therefore showed a lower probability of upregulation and transmission of ARGs in MFCs when compared to a traditional anaerobic microbial treatment.

Enhanced bioleaching efficiency of metals from E-wastes driven by biochar.

Electronic wastes (E-wastes) contain a huge amount of valuable metals that are worth recovering. Bioleaching has attracted widespread attention as an environment-friendly and low-cost technology for the recycling of E-wastes. To avoid the disadvantages of being time-consuming or having a relatively low efficiency, biochar with redox activity was used to enhance bioleaching efficiency of metals from a basic E-waste (i.e., printed circuit boards in this study). The role of biochar was examined through three basic processes: Carbon-mediated, Sulfur-mediated and Iron-mediated bioleaching pathways. Although no obvious enhancement of bioleaching performance was observed in the C-mediated and S-mediated systems, Fe-mediated bioleaching was significantly promoted by the participation of biochar, and its leaching time was decreased by one-third compared with that of a biochar-free system. By mapping the dynamic concentration of Fe(II) and Cu(II), biochar was proved to facilitate the redox action between Fe(II) to Fe(III), which resulted in effective leaching of Cu. Two dominant functional species consisting of Alicyclobacillus spp. and Sulfobacillus spp. may cooperate in the Fe-mediated bioleaching system, and the ratio of these two species was regulated by biochar for enhancing the efficiency of bioleaching. Hence, this work provides a method to improve bioleaching efficiency with low-cost solid redox media.

High-throughput single-cell sequencing of activated sludge microbiome.

Wastewater treatment plants (WWTPs) represent one of biotechnology's largest and most critical applications, playing a pivotal role in environmental protection and public health. In WWTPs, activated sludge (AS) plays a major role in removing contaminants and pathogens from wastewater. While metagenomics has advanced our understanding of microbial communities, it still faces challenges in revealing the genomic heterogeneity of cells, uncovering the microbial dark matter, and establishing precise links between genetic elements and their host cells as a bulk method. These issues could be largely resolved by single-cell sequencing, which can offer unprecedented resolution to show the unique genetic information. Here we show the high-throughput single-cell sequencing to the AS microbiome. The single-amplified genomes (SAGs) of 15,110 individual cells were clustered into 2,454 SAG bins. We find that 27.5% of the genomes in the AS microbial community represent potential novel species, highlighting the presence of microbial dark matter. Furthermore, we identified 1,137 antibiotic resistance genes (ARGs), 10,450 plasmid fragments, and 1,343 phage contigs, with shared plasmid and phage groups broadly distributed among hosts, indicating a high frequency of horizontal gene transfer (HGT) within the AS microbiome. Complementary analysis using 1,529 metagenome-assembled genomes from the AS samples allowed for the taxonomic classification of 98 SAG bins, which were previously unclassified. Our study establishes the feasibility of single-cell sequencing in characterizing the AS microbiome, providing novel insights into its ecological dynamics, and deepening our understanding of HGT processes, particularly those involving ARGs. Additionally, this valuable tool could monitor the distribution, spread, and pathogenic hosts of ARGs both within AS environments and between AS and other environments, which will ultimately contribute to developing a health risk evaluation system for diverse environments within a One Health framework.