Ecological linkages between top-down designed benzothiazole-degrading consortia and selection strength: From performance to community structure and functional genes.

The inefficient biodegradation and incomplete mineralization of nitrogenous heterocyclic compounds (NHCs) have emerged as a pressing environmental concern. The top-down design offers potential solutions to this issue by targeting improvements in community function, but the ecological linkages between selection strength and the structure and function of desired microbiomes remain elusive. Herein, the integration of metagenomics, culture-based approach, non-targeted metabolite screening and enzymatic verification experiments revealed the effect of enrichment concentration on the top-down designed benzothiazole (BTH, a typical NHC)-degrading consortia. Significant differences were observed for the degradation efficiency and community structure under varying BTH selections. Notably, the enriched consortia at high concentrations of BTH were dominated by genus Rhodococcus, possessing higher degradation rates. Moreover, the isolate Rhodococcus pyridinivorans Rho48 displayed excellent efficiencies in BTH removal (98 %) and mineralization (∼ 60 %) through the hydroxylation and cleavage of thiazole and benzene rings, where cytochrome P450 enzyme was firstly reported to participate in BTH conversion. The functional annotation of 460 recovered genomes from the enriched consortia revealed diverse interspecific cooperation patterns that accounted for the BTH mineralization, particularly Nakamurella and Micropruina under low selection strength, and Rhodococcus and Marmoricola under high selection strength. This study highlights the significance of selection strength in top-down design of synthetic microbiomes for degrading refractory organic pollutants, providing valuable guidance for designing functionally optimized microbiomes used in environmental engineering.

Application of earthworm can enhance biological power generation and accelerate sulfamethoxazole removal in agricultural soils.

Microbial degradation plays a crucial role in removing sulfonamides from soil, enhancing sulfamethoxazole (SMX) remediation. To further augment SMX removal efficiency and mitigate the transmission risk associated with antibiotic resistance genes (ARGs), this study proposes a novel approach that integrates micro-animals, microorganisms, and microbial fuel cell (MFC) technology. The results showed that earthworm-MFC synergy substantially reduces SMX content and ARGs abundance in soil. The introduction of earthworms enhances humus content, facilitating electron transfer within MFC and consequently improving current generation. Furthermore, electrical stimulation applied to earthworms led to increased protein secretion and enhanced antioxidant system activity, thereby accelerating SMX degradation. Earthworms also foster MFC-associated bacterial growth and SMX-degrading bacteria proliferation, augmenting MFC treatment efficacy. This synergistic effect significantly augmented the overall efficacy of MFC treatment for antibiotics. Overall, integrating earthworm activity with MFC technology effectively optimizes electricity generation and enhances pollutant removal.

HPB-Chip: An accurate high-throughput qPCR-based tool for rapidly profiling waterborne human pathogenic bacteria in the environment.

Waterborne pathogens are threatening public health globally, but profiling multiple human pathogenic bacteria (HPBs) in various polluted environments is still a challenge due to the absence of rapid, high-throughput and accurate quantification tools. This work developed a novel chip, termed the HPB-Chip, based on high-throughput quantitative polymerase chain reactions (HT-qPCR). The HPB-Chip with 33-nL reaction volume could simultaneously complete 10,752 amplification reactions, quantifying 27 HPBs in up to 192 samples with two technical replicates (including those for generating standard curves). Specific positive bands of target genes across different species and single peak melting curves demonstrated high specificity of the HPB-Chip. The mixed plasmid serial dilution test validated its high sensitivity with the limit of quantification (LoD) of averaged 82 copies per reaction for 25 target genes. PCR amplification efficiencies and R2 coefficients of standard curves of the HPB-Chip averaged 101 % and 0.996, respectively. Moreover, a strong positive correlation (Pearson' r: 0.961-0.994, P < 0.001) of HPB concentrations (log10 copies/L) between HPB-Chip and conventional qPCR demonstrated high accuracy of the HPB-Chip. Subsequently, the HPB-Chip has been successfully applied to absolutely quantify 27 HPBs in municipal and hospital wastewater treatment plants (WWTPs) after PMA treatment. A total of 17 HPBs were detected in the 6 full-scale WWTPs, with an additional 19 in the hospital WWTP. Remarkably, Acinetobacter baumannii, Legionella pneumophila, and Arcobacter butzler were present in the final effluent of each municipal WWTP. Overall, the HPB-Chip is an efficient and accurate high-throughput quantification tool to comprehensively and rapidly quantify 27 HPBs in the environment.

Comparative evaluation of SPE methods for biotoxicity assessment of water and wastewater: Linkage between chemical extracting efficiency and biotoxicity outcome.

Biotoxicity assessment results of environmental waters largely depend on the sample extraction protocols that enrich pollutants to meet the effect-trigger thresholds of bioassays. However, more chemical mixture does not necessarily translate to higher combined biotoxicity. Thus, there is a need to establish the link between chemical extracting efficiency and biotoxicity outcome to standardize extraction methods for biotoxicity assessment of environmental waters. This study compares the performance of five different extraction phases in solid phase extraction (SPE), namely HLB, HLB+Coconut, C18 cartridge, C18 disk and Strata-X, and evaluated their chemical extracting efficiencies and biotoxicity outcomes. We quantitatively assessed cytotoxicity, acute toxicity, genotoxicity, estrogenic activity, and neurotoxicity of the extracts using in vitro bioassays and characterized the chemical extracting efficiencies of the SPE methods through chemical recoveries of 23 model compounds with different polarities and total organic carbon. Using Pareto ranking, we identified HLB+Coconut as the optimal SPE method, which exhibited the highest level of water sample biotoxicity and recovered the most chemicals in water samples. We found that the biotoxicity outcomes of the extracted water samples significantly and positively correlated with the chemical extracting efficiencies of the SPE methods. Moreover, we observed synchronous changing patterns in biotoxicity outcome and chemical extracting efficiencies in response to increasing sample volumes per cartridge (SVPC) during SPE. Our findings underscore that higher chemical extracting efficiency of SPE corresponds to higher biotoxicity outcome of environmental water samples, providing a scientific basis for standardization of SPE methods for adequate assessment of biotoxicities of environmental waters.

Chemical-toxicological insights and process comparison for estrogenic activity mitigation in municipal wastewater treatment plants.

Efforts in water ecosystem conservation require an understanding of causative factors and removal efficacies associated with mixture toxicity during wastewater treatment. This study conducts a comprehensive investigation into the interplay between wastewater estrogenic activity and 30 estrogen-like endocrine disrupting chemicals (EEDCs) across 12 municipal wastewater treatment plants (WWTPs) spanning four seasons in China. Results reveal substantial estrogenic activity in all WWTPs and potential endocrine-disrupting risks in over 37.5 % of final effluent samples, with heightened effects during colder seasons. While phthalates are the predominant EEDCs (concentrations ranging from 86.39 %) for both estrogenic activity and major EEDCs (phthalates and estrogens), with the secondary and tertiary treatment segments contributing 88.59 ± 8.12 % and 11.41 ± 8.12 %, respectively. Among various secondary treatment processes, the anaerobic/anoxic/oxic-membrane bioreactor (A/A/O-MBR) excels in removing both estrogenic activity and EEDCs. In tertiary treatment, removal efficiencies increase with the inclusion of components involving physical, chemical, and biological removal principles. Furthermore, correlation and multiple liner regression analysis establish a significant (p < 0.05) positive association between solid retention time (SRT) and removal efficiencies of estrogenic activity and EEDCs within WWTPs. This study provides valuable insights from the perspective of prioritizing key pollutants, the necessity of integrating more efficient secondary and tertiary treatment processes, along with adjustments to operational parameters like SRT, to mitigate estrogenic activity in municipal WWTPs. This contribution aids in managing endocrine-disrupting risks in wastewater as part of ecological conservation efforts.

Identification of key degraders for controlling toxicity risks of disguised toxic pollutants with division of labor mechanisms in activated sludge microbiomes: Using nonylphenol ethoxylate as an example.

Efficient management of disguised toxic pollutants (DTPs), which can undergo microbial degradation and convert into more toxic substances, necessitates the collaboration of diverse microbial populations in wastewater treatment plants. However, the identification of key bacterial degraders capable of controlling the toxicity risks of DTPs through division of labor mechanisms in activated sludge microbiomes has received limited attention. In this study, we investigated the key degraders capable of controlling the risk of estrogenicity associated with nonylphenol ethoxylate (NPEO), a representative DTP, in textile activated sludge microbiomes. The results of our batch experiments revealed that the transformation of NPEO into NP and subsequent NP degradation were the rate-limiting processes for controlling the risk of estrogenicity, resulting in an inverted V-shaped curve of estrogenicity in water samples during the biodegradation of NPEO by textile activated sludge. By utilizing enrichment sludge microbiomes treated with NPEO or NP as the sole carbon and energy source, a total of 15 bacterial degraders, including Sphingbium, Pseudomonas, Dokdonella, Comamonas, and Hyphomicrobium, were identified as capable of participating in these processes, Among them, Sphingobium and Pseudomonas were the two key degraders that could cooperatively interact in the degradation of NPEO with division of labor mechanisms. Co-culturing Sphingobium and Pseudomonas isolates exhibited a synergistic effect in degrading NPEO and reducing estrogenicity. Our study underscores the potential of the identified functional bacteria for controlling estrogenicity associated with NPEO and provides a methodological framework for identifying key cooperators engaged in labor division, contributing to the management of risks associated with DTPs by leveraging intrinsic microbial metabolic interactions.

Environmental exposure to low-dose perfluorohexanesulfonate promotes obesity and non-alcoholic fatty liver disease in mice fed a high-fat diet.

Perfluorohexanesulfonate (PFHxS) is one of the most prevalent perfluoroalkyls. It is widely distributed in both abiotic and biotic environments because of its prevalence and bioaccumulative properties. Exposure to PFHxS has been associated with the higher serum liver functions associated with steatosis in obese people. This study explores the impact of chronic exposure to low-dose PFHxS on predisposition to non-alcoholic fatty liver disease (NAFLD) as well as on metabolic functions in diet-induced obese mice. Results showed that 12-week exposure to PFHxS at a dose of 450 µg/L through drinking water significantly promoted obesity and metabolic syndrome in male C57 mice fed a high-fat diet. The PFHxS exposure markedly aggravated hepatic symptoms resembling NAFLD and caused systematic metabolic disorders as well as gut dysbiosis in the obese mice. Key genes of hepatic lipid metabolism, inflammation, and fibrosis were strongly altered, while gut microflora that have been associated with obesity and pathogenesis of NAFLD, including the Bacteroides/Firmicutes ratio, Desulfovibrio, Mucispirillum, and Akkermansia, were significantly affected by the PFHxS exposure. The findings of this study suggest that environmental PFHxS exposure is a tangible risk factor for metabolic diseases such as NAFLD, especially among obese individuals.

Varied interspecies interactions between anammox and denitrifying bacteria enhanced nitrogen removal in a single-stage simultaneous anammox and denitrification system.

The simultaneous anammox and denitrification (SAD) system has received growing interest for the enhanced nitrogen removal, while the ecological traits of microbial community including spatial distribution characteristics, assembly processes and interspecies interactions have not been fully unraveled. The present study applied metagenomics and ecological analysis methods to gain the ecological traits of microbial communities in the SAD system across different organic substrate loadings. Results showed that organic matter significantly affected the bioreactor performance, and the optimal total nitrogen removal efficiency reached 93.4 ± 0.7% under the COD concentrations of 180 ± 18.2 mg/L. Functional organisms including Candidatus Brocadia (3.9%), Denitratisoma (1.6%), Dokdonella (4.4%) and Thauera (4.6%) obviously enriched under the optimal organic loading conditions. Moreover, microbial communities were significantly governed by deterministic process under high organic concentrations, and the denitrifying organisms displayed important ecological roles in the communities. Although anammox bacteria obviously enriched at the middle of bioreactor, it possessed the highest expression activities at both bottom and middle sites. Denitrifying bacteria that enriched at the bottom sites strongly achieved nitrate reduction and provided nitrite for anammox bacteria, while these organisms trended to compete nitrite with anammox bacteria at the middle site. These findings highlight the importance of microbial ecology in the SAD systems, which may expand our understanding of the synergistic patterns between anammox and denitrifying bacteria.

Nonylphenol ethoxylates biodegradation increases estrogenicity of textile wastewater in biological treatment systems.

The formation of estrogenic intermediates, i.e. nonylphenol diethoxylate (NP2EO), nonylphenol monoethoxylate (NP1EO), and nonylphenol (NP), following nonylphenol ethoxylates (NPEOs) biodegradation in textile wastewater raises concerns about its endocrine disruptive activity, but the estrogenicity changes of textile wastewater throughout biological treatment processes remain unknown. In the present study, the estrogenicity of textile wastewater sampled from 10 wastewater treatment plants (WWTPs) were investigated using the reporter gene-based T47D-KBluc bioassay. Results showed that the estrogenicity of the textile wastewater significantly increased after either anaerobic or aerobic treatment in all WWTPs, with an average fold change of 3.21, although traditional pollutants were effectively removed. The estradiol equivalents of the effluent (ranging from 1.50 to 4.12 ng-E2/L) were generally higher than published effect based trigger values, indicating an increased risk for the receiving waters. Removal efficiency was high (84.46%) for NPEOs, but was low for NP2EO and NP1EO in the biological treatment processes. Nevertheless, NP had increased concentrations after the treatment. Bioanalytical equivalent concentration of the textile wastewater and that of NP2EO, NP1EO, and NP showed a good linear correlation, of which NP alone contributed more than 70% to the observed estrogenicity. Extending hydraulic retention time was found effective in reducing the estrogenicity as it allows relatively complete degradation of NP, which was further confirmed by running lab-scale A/O reactors fed with NP10EO. The results may extend our knowledge regarding the estrogenicity of textile wastewater and its reduction technologies used in WWTPs.

Structural and functional alterations of gut microbiome in mice induced by chronic cadmium exposure.

Mammalian gut microbiome is readily affected by acute or subchronic cadmium (Cd) intoxication, but it susceptibility following chronic Cd exposure at environmentally-relevant levels remains unknown. This study comprehensively assessed the effects of Cd exposure at doses of 10 and 50 ppm in drinking water for 20 weeks on gut microbiome in mice. Results showed that the Cd exposure induced alterations in gut morphology with potentially increased gut permeability and inflammation. These changes were accompanied by marked perturbation of gut microbiota characterized by significantly decreased gut microbial richness and lowered abundance of short chain fatty acid (SCFA)-producing bacteria, resulting in reduced SCFAs production in the gut. Moreover, the Cd exposure caused substantial metabolic functional changes of the gut microbiome, with significant inhibitions on gene pathways associated with metabolism of amino acid, carbohydrate, and energy, as well as promotions on metabolic pathways such as glutathione metabolism and aminobenzoate degradation. Our findings provide new insights into the hazards assessment of environmental Cd exposure towards gut microbiome.

Effects of chronic cadmium exposure at food limitation-relevant levels on energy metabolism in mice.

Cadmium (Cd) exposure has been implicated in the perturbation of energy metabolism and the development of cardiometabolic disease, but disease predisposition from chronic low-dose Cd exposure remains unclear. This study employed a mouse model to investigate the toxic effects of chronic Cd exposure at food limitation-relevant levels on energy metabolism and the associated liver and gut microbiome functions. Results showed that the Cd exposure induced the perturbation of energy metabolism in mice, evidenced by the alteration of various metabolites associated with the phosphorogen (adenosine triphosphate-creatine phosphate) system, tricarboxylic acid cycle, and lipid metabolism, as well as the increase of the cardiometabolic risk factor, triglyceride. Moreover, both liver and gut microbiome underwent marked structural/histological and functional alterations, prone to the onset of cardiometabolic disease following the Cd exposure. Certain hepatic transcription factors and gut microbes, specifically PPARα, SREBP1c, HNF4A and the Clostridiales_vadinBB60_group, were identified to be highly correlated with altered urinary metabolites, revealing potential toxicological interactions between the liver and gut microbiome, and energy metabolism. Our findings provide new insights into the progression of metabolic diseases induced by Cd exposure. We also propose a stricter Cd limitation in future food safety standards.

Inhibition of Mitochondrial Fatty Acid Oxidation Contributes to Development of Nonalcoholic Fatty Liver Disease Induced by Environmental Cadmium Exposure.

Cadmium (Cd) is one of the most prevalent toxic metal pollutants widely distributed in water and soil environments. Epidemiological studies have shown that exposure to Cd is implicated in the prevalence of nonalcoholic fatty liver disease (NAFLD) in middle-aged human population, but biological evidence is lacking and its toxicological mechanism remains unclear for the disease predisposition from environmental Cd exposure. In this study, we established a chronic Cd-exposure mouse model mimicking the liver Cd deposition in middle-aged human population to determine whether the environmental Cd exposure can induce NAFLD. Results showed that hepatic Cd burden at levels of 0.95 and 6.04 µg/g wet weight resulting from 20-week Cd exposure at different doses induced NAFLD and nonalcoholic steatohepatitis-like phenotypes in mice, respectively. The Cd exposure caused marked hepatic mitochondrial dysfunction and fatty acid oxidation deficiency, along with significant suppression of sirtuin 1 (SIRT1) signaling pathway in the liver. In vitro study confirmed that Cd evidently inhibited the mitochondrial fatty acid oxidation in hepatocytes and that SIRT1 signaling was potentially involved in the process. Our findings suggest that exposure to environmental Cd is a tangible risk factor for NAFLD, and the induced public health risks deserve greater attention.

Roles and correlations of functional bacteria and genes in the start-up of simultaneous anammox and denitrification system for enhanced nitrogen removal.

Simultaneous anammox and denitrification (SAD) is a newly developed wastewater treatment process efficient in nitrogen removal, but its underlying microbiological mechanisms during start-up remains unknown. This study investigated the changing patterns of functional bacteria and genes, as well as their correlation during the start-up (260 d) of the SAD systems in two lab-scale up-flow anaerobic sludge blanket bioreactors separately inoculated with anaerobic granular sludge (R1) and aerobic floccular sludge (R2). Results showed that high total nitrogen removal was achieved in the SAD systems of both R1 (88.25%) and R2 (89.42%). High-throughput sequencing of 16S rRNA gene amplicons revealed that Armatimonadetes phylum had a high abundance (44.34%) in R2, while was not detectable in R1 during the anammox stage. However, the SAD bioreactors retained inherent microbial community and the inoculation with different sludge showed less notable effects on their microbial composition. In the SAD systems, Candidatus Brocadia had high abundance in R1 (2.93%) and R2 (4.64%) and played important role in anammox. Network analysis indicated that Denitratisoma and Dokdonella were positively correlated with nitrite reductase genes nirS and nirK (p < 0.05), while Thermomonas and Pseudomonas showing a positive correlation with nitrate reductase gene narG (p < 0.05) were mainly responsible for the nitrate reduction in the SAD systems. Moreover, the overwhelming dominance of narG v.s. napA revealed the crucial roles of respiratory nitrate reduction in the bioreactors. The results extend our knowledge regarding the microbial ecology of the SAD system, which might be practically helpful for application of the process in wastewater treatment.

Altered gut microbiome promotes proteinuria in mice induced by Adriamycin.

Inflammation has recently been attributed to dysbiosis of the gut microbiome, which has been linked to proteinuria in chronic kidney disease. Since Adriamycin® (ADR) is widely used to induce proteinuria in mouse models, the aim of this study was to explore the potential effect of gut microbiome on this process. Both ADR resistant (C57BL/6) and susceptible (BALB/C) strains were part of the induced nephropathy with ADR injection. BALB/C mice significantly presented increased urinary albumin/creatinine ratio (UACR) with renal lesions in pathology, but C57BL/6 mice were absent from kidney damage. Species and genus level resolution analysis showed a shift in gut microbial profile between BALB/C and C57BL/6 mice. ADR further altered the stool microbiome in BALB/C mice, particularly with enrichment of Odoribacter and depletion of Turicibacter, Marvinbryantia and Rikenella. Moreover, the level of UACR in BALB/C mice was marked related to the abundance of Marvinbryantia, Odoribacter and Turicibacter in stool. Meanwhile, ADR remarkably increased the serum levels of interleukin (IL)-2 in BALB/C mice, but not in C57BL/6 mice. It is suggested that the favorably altered stools as shown in the microbiome might promote the inflammation and proteinuria in ADR-sensitive mice, which provides a new insight on the pathogenicity of chronic kidney disease.

Risk assessment of silica nanoparticles on liver injury in metabolic syndrome mice induced by fructose.

This study aims to assess the effects and the mechanisms of silica nanoparticles (SiNPs) on hepatotoxicity in both normal and metabolic syndrome mouse models induced by fructose. Here, we found that SiNPs exposure lead to improved insulin resistance in metabolic syndrome mice, but markedly worsened hepatic ballooning, inflammation infiltration, and fibrosis. Moreover, SiNPs exposure aggravated liver injury in metabolic syndrome mice by causing serious DNA damage. Following SiNPs exposure, liver superoxide dismutase and catalase activities in metabolic syndrome mice were stimulated, which is accompanied by significantly increased malondialdehyde and 8-hydroxy-2-deoxyguanosine levels as compared to normal mice. Scanning electron microscope (SEM) revealed that SiNPs were more readily deposited in the liver mitochondria of metabolic syndrome mice, resulting in more severe mitochondrial injury as compared to normal mice. We speculated that SiNPs-induced mitochondrial injury might be the cause of hepatic oxidative stress, which further lead to a series of liver lesions as observed in mice following SiNPs exposure. Based on these results, it is likely that SiNPs will increase the risk and severity of liver disease in individuals with metabolic syndrome. Therefore, SiNPs should be used cautiously in food additives and clinical settings.

Different impacts of manure and chemical fertilizers on bacterial community structure and antibiotic resistance genes in arable soils.

Both manure and chemical fertilizers are widely used in modern agriculture. However, the impacts of different fertilizers on bacterial community structure and antibiotic resistance genes (ARGs) in arable soils still remain unclear. In this study, high-throughput sequencing and quantitative PCR were employed to investigate the bacterial community structure, ARGs and mobile genetic elements (MGEs) influenced by the application of different fertilizers, including chemical fertilizers, piggery manure and straw ash. The results showed that the application of fertilizers could significantly change the soil bacterial community and the abundance of Gaiella under phylum Actinobacteria was significantly reduced from 12.9% in unfertilized soil to 4.1%-7.4% in fertilized soil (P < 0.05). It was also found that the application of manure could cause a transient effect on soil resistome composition and the relative abundance of ARGs increased from 7.37 ppm to 32.10 ppm. The abundance of aminoglycoside, sulfonamide and tetracycline resistance genes greatly increased after manure fertilization and then gradually returned to normal levels with the decay of some intestinal bacteria carrying ARGs. In contrast, the application of chemical fertilizers and straw ash significantly changed the bacterial community structure but exerted little effect on soil resistome. Overall, the results of this study illustrated the different effects of different fertilizers on the soil resistome and revealed that the changes of soil resistome induced by manure application mainly resulted from alteration of bacteria community rather than the horizontal gene transfer.

Diversity, abundance, and possible sources of fecal bacteria in the Yangtze River.

The fecal bacteria in natural waters may pose serious risks on human health. Although many source tracking methods have been developed and used to determine the possible sources of the fecal pollution, little is known about the overall diversity and abundance of fecal bacterial community in natural waters. In this study, a method based on fecal bacterial sequence library was introduced to evaluate the fecal bacterial profile in the Yangtze River (Nanjing section). Our results suggested that the Yangtze River water harbors diverse fecal bacteria. Fifty-eight fecal operational taxonomic units (97% identity level) were detected in the Yangtze River water samples and the relative abundance of fecal bacteria in these samples ranged from 0.1 to 8%. It was also found that the relative abundances of the fecal bacteria in locations near to the downstream of wastewater treatment plants were obviously higher than those in other locations. However, the high abundance of fecal bacteria could decrease to the normal level in 2~4 km in the river due to degradation or dilution, and the overall fecal bacteria level changed little when the Yangtze River flew through the Nanjing City. Moreover, the fecal bacteria in the Yangtze River water were found to be highly associated (Spearman rho = 0.804, P < 0.001) with the potential pathogenic bacteria. Collectively, the findings in this study reveal the diversity, abundance, and possible sources of fecal bacteria in the Yangtze River and advance our understandings of the fecal bacteria community in the natural waters.

Evaluation of five microbial and four mitochondrial DNA markers for tracking human and pig fecal pollution in freshwater.

This study systematically evaluated five microbial and four mitochondrial DNA (mtDNA) markers, including sensitivities and specificities under PCR method, and fecal concentrations and decay rates in water under qPCR method. The microbial DNA markers were the three human-associated (BacH, HF183 and B.adolescentis) and two pig-associated (Pig-2-Bac and L.amylovorus), while the mtDNA ones were two human- (H-ND6 and H-ND5) and two pig-associated (P-CytB and P-ND5). All the mtDNA markers showed higher sensitivity (100%) than the microbial ones (84.0-88.8%) except Pig-2-Bac (100%). Specificities of the human mtDNA markers (99.1 and 98.1%) were higher than those of the human-associated microbial ones (57.0-88.8%). But this pattern was not observed in the pig-associated markers where Pig-2-Bac had 100% specificity. The reliability of H-ND6 and H-ND5 was further evidenced to identify locations of the most polluted within the Taihu Lake watershed of China. In general, the microbial DNA markers demonstrated a higher fecal concentration than the mtDNA ones; increasing temperature and sunlight exposure accelerated significantly the decay of all the DNA markers. Results of this study suggest that DNA markers H-ND6, H-ND5, and Pig-2-Bac may be among the best for fecal source tracking in water.

Metagenomic analysis of bacterial community composition and antibiotic resistance genes in a wastewater treatment plant and its receiving surface water.

The presence of pathogenic bacteria and the dissemination of antibiotic resistance genes (ARGs) may pose big risks to the rivers that receive the effluent from municipal wastewater treatment plants (WWTPs). In this study, we investigated the changes of bacterial community and ARGs along treatment processes of one WWTP, and examined the effects of the effluent discharge on the bacterial community and ARGs in the receiving river. Pyrosequencing was applied to reveal bacterial community composition including potential bacterial pathogen, and Illumina high-throughput sequencing was used for profiling ARGs. The results showed that the WWTP had good removal efficiency on potential pathogenic bacteria (especially Arcobacter butzleri) and ARGs. Moreover, the bacterial communities of downstream and upstream of the river showed no significant difference. However, the increase in the abundance of potential pathogens and ARGs at effluent outfall was observed, indicating that WWTP effluent might contribute to the dissemination of potential pathogenic bacteria and ARGs in the receiving river.

Persistence of mitochondrial DNA markers as fecal indicators in water environments.

Mitochondrial DNA (mtDNA) polymerase chain reaction (PCR) technology has recently been developed to identify sources of fecal contamination, but information regarding environmental fate of mtDNA is limited. In this study, quantitative real-time PCR was used to determine the persistence of three species-specific mtDNA markers (human, pig and chicken) in river microcosms under different laboratory conditions and in dialysis tubes incubated in river environments during different seasons. Human feces had a higher abundance of mtDNA marker than pig and chicken feces. A biphasic decay pattern was observed for the mtDNA markers in microcosms incubated in darkness, and T90 (time needed for 90% reduction) ranged from 2.03 to 13.83 d. Each species-specific mtDNA marker persisted for relatively longer time at lower temperatures, and light exposure and predation increased the decay rates. Field experiments showed that the mtDNA markers could survive for longer time in winter (T90: 1.79-4.37 d) than in summer (T90: 0.60-0.75 d). Field application of mtDNA technology indicated that the markers were mainly distributed on the sites near animal breeding plants and had lower abundance in downstream water of the receiving river. This study expands our knowledge of the environmental fate of mtDNA markers and the results may be useful for practical application of the technology in fecal source tracking.