Assessment of human adenovirus removal in a full-scale membrane bioreactor treating municipal wastewater

Human adenoviruses (HAdVs) in wastewater samples taken from four different treatment stages of a full-scale municipal wastewater treatment plant (i.e., incoming raw sewage, primary sedimentation effluent, membrane bioreactor (MBR) influent, and MBR effluent) were quantified by real-time PCR assays to further estimate removal efficiency of the HAdVs. Based on hexon gene sequence comparisons, HAdV species A, C, and F were consistently found in the wastewater samples. In general, all three identified HAdV species were detected in most of the wastewater samples using the real-time PCR assays. Overall HAdV concentrations were rather stable over the entire 8-month study period (January-August, 2008) (approximately 10⁶-10⁷ viral particles/L of wastewater for the raw sewage and primary effluent; 10⁸-10⁹ viral particles/L for the MBR influent; and, 10³-10⁴ viral particles/L for the MBR effluent). No significant seasonal differences were noticed for the HAdV abundances. Removal efficiencies of the viral particles in the full-scale MBR process were assessed and showed an average HAdV removal of 5.0 ± 0.6 logs over the study period. The removal efficiencies for F species (average log removal of 6.5 ± 1.3 logs) were typically higher (p-value <0.05) than those of the other two species (average of 4.1 ± 0.9 and 4.6 ± 0.5 logs for species A and C, respectively). These results demonstrate that the full-scale MBR system efficiently removed most HAdV from the wastewater leaving about 10³ viral particles/L in the MBR effluent.     

Removal of viruses, parasitic protozoa and microbial indicators in conventional and membrane processes in a wastewater pilot plant

The aim of this study was to investigate variations in the occurrence and removal of enterovirus and norovirus genomes, Giardia cysts, Cryptosporidium oocysts and the most commonly used faecal indicators in a Swedish wastewater pilot plant. Paired samples were taken from the inlet and outlet of each treatment line: tertiary filtration, membrane bioreactor (MBR) and upflow anaerobic sludge blankets (UASB). (Oo)cysts and indicators were enumerated using standard methods and viruses using RT-PCR. Giardia cysts and enteroviruses were constantly detected, mean numbers 10(3.11) cysts and 10(4.0) PCR units L(-1), respectively. Oocysts were found in 5/19 samples, mean number 5 L(-1). Noroviruses were found in 6/7 influent samples, with an average titre of 10(3.28)L(-1), during winter, but only in 2/15 in the rest of the year (mean 200 L(-1)). MBR treatment removed indicators more efficiently than did the other two lines, with 5log removal of E. coli. Human virus genome removal did not differ between the MBR and tertiary treatment line. Microorganism removal in UASB was significantly lower for all the organisms studied. E. coli, enterococci and Cl. perfringens removal was correlated (p<0.05) with enterovirus genome removal, with R-values around 0.4. However, values for removal of indicators were more strongly correlated to each other. Removal of viruses based on enumeration using RT-PCR probably underestimates infectious virion removal.     

Evaluation of human adenovirus and human polyomavirus as indicators of human sewage contamination in the aquatic environment

Discharge of inadequately treated human wastewater into surface waters used for recreation, drinking water, irrigation and shellfish cultivation may present a public health hazard due to the potential shedding of high concentrations of pathogenic viruses from the human gastrointestinal tract. Human adenovirus (HAdV) and human polyomavirus (HPyV) are ubiquitous in humans and have excellent survival characteristics in the environment, so are potential candidates for indicators of human sewage contamination. Using qPCR assays, the prevalence and quantity of HAdV and HPyV JC and BK were determined in influent and effluent wastewater and receiving waters (river, urban stream, estuarine), then compared with norovirus (NoV) presence, a significant human pathogen which is not necessarily ubiquitously excreted into the environment. HAdV and HPyV were frequently detected in high concentrations in wastewater and wastewater-contaminated waters confirming their use as potential indicators for the presence of human sewage. Overall, there was a correlation between the presence of HAdV and HPyV with NoV but there were some notable exceptions including the higher frequency of NoV compared to HAdV and HPyV in estuarine waters impacted by wastewater overflows. We found that HAdV and HPyV detection by qPCR was a suitable tool for evaluating water quality and that their detection can aid in determining pollution sources, thus providing useful information for health risk assessments.     

Release of antibiotic resistant bacteria and genes in the effluent and biosolids of five wastewater utilities in Michigan

The purpose of this study was to quantify the occurrence and release of antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB) into the environment through the effluent and biosolids of different wastewater treatment utilities including an MBR (Membrane Biological Reactor) utility, conventional utilities (Activated Sludge, Oxidative Ditch and Rotatory Biological Contactors-RBCs) and multiple sludge treatment processes (Dewatering, Gravity Thickening, Anaerobic Digestion and Lime Stabilization). Samples of raw wastewater, pre- and post-disinfected effluents, and biosolids were monitored for tetracycline resistant genes (tetW and tetO) and sulfonamide resistant gene (Sul-I) and tetracycline and sulfonamide resistant bacteria. ARGs and ARB concentrations in the final effluent were found to be in the range of ND(non-detectable)-2.33 × 10⁶ copies/100 mL and 5.00 × 10²-6.10 × 10⁵ CFU/100 mL respectively. Concentrations of ARGs (tetW and tetO) and 16s rRNA gene in the MBR effluent were observed to be 1-3 log less, compared to conventional treatment utilities. Significantly higher removals of ARGs and ARB were observed in the MBR facility (range of removal: 2.57-7.06 logs) compared to that in conventional treatment plants (range of removal: 2.37-4.56 logs) (p < 0.05). Disinfection (Chlorination and UV) processes did not contribute in significant reduction of ARGs and ARB (p > 0.05). In biosolids, ARGs and ARB concentrations were found to be in the range of 5.61 × 10⁶-4.32 × 10⁹ copies/g and 3.17 × 10⁴-1.85 × 10⁹ CFU/g, respectively. Significant differences (p < 0.05) were observed in concentrations of ARGs (except tetW) and ARB between the advanced biosolid treatment methods (i.e., anaerobic digestion and lime stabilization) and the conventional dewatering and gravity thickening methods.     

Coliphages as indicators of enteric viruses in activated sludge

The use of indigenous coliphage as indicators of enteric viruses during activated sludge treatment of domestic wastewater was examined. Enteric viruses, coliphage attacking three strains of Escherichia coli and a number of wastewater parameters were monitored in the primary effluent, secondary effluent and secondary sludge of a wastewater treatment plant.Removal of viruses during secondary treatment appeared to be the result of rapid adsorption of influent virions to mixed liquor suspended solids. Adsorption was followed by inactivation of the viruses during aeration of the mixed liquor. A larger proportion of influent enteric viruses than coliphage was recovered from the secondary sludge. This suggests that activated sludge treatment was less antagonistic towards enteric viruses than towards the coliphage.Coliphage plaques of less than or equal to 1 mm in diameter were composed of large viruses (approx. 100 nm in diameter). Plaques greater than 3 mm in diameter appeared to be the result of host infection by a much smaller virus (approx. 45 nm in diameter). Plaques between 1 and 3 mm in diameter were composed of both small and large coliphages.Coliphage concentrations could not be correlated with enteric virus concentrations in either primary or secondary effluent. However, it was found that coliphage giving rise to plaques greater than 3 mm in diameter were positively related with enteric viruses in the secondary effluent. This result suggests that this group of coliphage may serve as an indicator of the efficacy of activated sludge treatment of enteric viruses.     

Quantification and molecular characterization of enteric viruses detected in effluents from two hospital wastewater treatment plants

Hospital wastewater has been described as an important source of spreading pathogenic microorganisms in the environment. However, there are few studies reporting the presence and concentrations of gastroenteric viruses and hepatitis A viruses in these environmental matrices. The aim of this study was to assess the contamination by viruses responsible for acute gastroenteritis and hepatitis derived from hospital wastewater treatment plants (WWTPs). Rotavirus A (RV-A), human adenoviruses (HAdV), norovirus genogroup I and II (NoV GI/GII) and hepatitis A viruses (HAV) were detected and quantified in sewage samples from two WWTPs located in Rio de Janeiro (Brazil) that operates different sewage treatments. WWTP-1 uses an Upflow Anaerobic Sludge Blanket (UASB reactor) and three serial anaerobic filters while WWTP-2 uses aerobic processes, activated sludge with extended aeration and final chlorination of the effluents. Viruses’ detection was investigated by using conventional PCR/RT-PCR, quantitative real-time PCR (qPCR) and partial sequencing of the genome of the viruses detected. Rate of viruses detection ranged from 7% (NoV GI in WWTP-1) to 95% (RV-A in WWTP-2) and genome from all viruses were detected. The most prevalent genotypes were RV-A SG I, HAdV species D and F, NoV GII/4 and HAV subgenotype IA. Mean values of viral loads (genome copies (GC)/ml) obtained in filtered effluents from anaerobic process was 1.9 × 10³ (RV-A), 2.8 × 10³ (HAdV) and 2.4 × 10³ (NoV GII). For chlorinated effluents from activated sludge process, the mean values of viral loads (GC/ml) was 1.2 × 10⁵ (RV-A), 1.4 × 10³ (HAdV), 8.1 × 10² (NoV GII) and 2.8 × 10⁴ (HAV). Data on viral detection in treated effluents of hospital WWTPs confirmed the potential for environmental contamination by viruses and could be useful to establish standards for policies on wastewater management.     

Peracetic acid (PAA) disinfection of primary, secondary and tertiary treated municipal wastewaters

The efficiency of peracetic acid (PAA) disinfection against enteric bacteria and viruses in municipal wastewaters was studied in pilot-scale. Disinfection pilot-plant was fed with the primary or secondary effluent of Kuopio municipal wastewater treatment plant or tertiary effluent from the pilot-scale dissolved air flotation (DAF) unit. Disinfectant doses ranged from 2 to 7 mg/l PAA in the secondary and tertiary effluents, and from 5 to 15 mg/l PAA in the primary effluents. Disinfection contact times were 4-27 min. Disinfection of secondary and tertiary effluents with 2-7 mg/l PAA and 27 min contact time achieved around 3 log reductions of total coliforms (TC) and enterococci (EC). PAA disinfection also significantly improved the hygienic quality of the primary effluents: 10-15 mg/l PAA achieved 3-4 log reductions of TC and EC, 5 mg/l PAA resulting in below 2 log reductions. F-RNA coliphages were more resistant against the PAA disinfection and around 1 log reductions of these enteric viruses were typically achieved in the disinfection treatments of the primary, secondary and tertiary effluents. Most of the microbial reductions occurred during the first 4-18 min of contact time, depending on the PAA dose and microorganism. The PAA disinfection efficiency remained relatively constant in the secondary and tertiary effluents, despite of small changes of wastewater quality (COD, SS, turbidity, 253.7 nm transmittance) or temperature. The disinfection efficiency clearly decreased in the primary effluents with substantially higher microbial, organic matter and suspended solids concentrations. The results demonstrated that PAA could be a good alternative disinfection method for elimination of enteric microbes from different wastewaters.     

Ecological impact of application of wastewater biosolids to agricultural soil

There has been little investigation of the ecological interactions between major organisms brought about by the application of wastewater biosolids to agricultural soil. This paper provides an overview of the possible effects of biosolids application on these ecosystems by using what is known about the ecology and diversity of microbial populations in soil and biosolids. Areas considered include interactions between indigenous and introduced bacterial populations, impacts of biosolids application on soil protozoa and the role of phages in bacterial population dynamics. These ecological processes are particularly relevant, for instance, to increase an understanding of the fundamental mechanisms responsible for inactivation of potentially infectious enteric microorganisms in biosolids‐amended agricultural soil.     

Evaluation of public health risks at recreational beaches in Lake Michigan via detection of enteric viruses and a human-specific bacteriological marker

Each year the National Resource Defense Council addresses the quality of US beaches by routine bacterial indicators. In the Great Lakes region the indicator used is Escherichia coli and for 2007 more beaches were closed and impacted than ever before. In this study, water quality was addressed at two Lake Michigan Beaches over the 2004 swimming season by monitoring infectious enteric viruses by cell culture and integrated PCR and for a human sewage marker based on the Enterococcal Surface Protein (esp). Our goals for this study were to 1) examine the occurrence and variety of human enteric viruses present during peak usage of the beaches 2) determine key variables for development of predictive models for viruses; and 3) use quantitative risk assessment to estimate the potential health impact. Our results demonstrate that for both beaches predictive models of virus pollution were best described utilizing physical parameters like wind speed, wind direction and water temperature. The esp marker was not predictive of human viruses. The daily risk of acquiring a viral infection at either of the beaches ranged from 0.2 to 2.4/1000 swimmers using a quantitative microbial risk assessment model, with three swims during a day at the beach for children and over the season, the risk was 9-15/1000 swimmers using adenovirus as the model.Conclusions: Lake Michigan recreational beaches are being adversely impacted by human fecal pollution. Monitoring for the traditional indicators of water quality does not address viral risks and models can be developed and potentially used as real-time water quality forecasting tools.     

水解酸化-膜生物反应器处理印染废水的试验研究

研究了水解酸化与膜生物反应器组合工艺对印染废水的处理效能。当进水CODCr为3500～4500mg/L、NH4+-N为55～60mg/L、色度为600倍时,经UASB-MBR系统处理后出水CODCr为120～150mg/L、NH4+-N为4～8mg/L、色度小于50倍,出水水质达到《纺织染整工业水污染物排放标准》的一级标准要求。试验结果表明,UASB-MBR系统运行良好,用于印染废水处理是可行的。     

Effects of chemical sludge disintegration on the performances of wastewater treatment by membrane bioreactor

A new wastewater treatment process combining a membrane bioreactor (MBR) with chemical sludge disintegration was tested in bench scale experiments. In particular, the effects of the disintegration treatment on the excess sludge production in MBR were investigated. Two MBRs were operated. In one reactor, a part of the mixed liquor was treated with NaOH and ozone gas consecutively and was returned to the bioreactor. The flow rate of the sludge disintegration stream was 1.5% of the influent flow rate. During the 200 days of operation, the MLSS level in the bioreactor with the disintegration treatment was maintained relatively constant at the range of 10,000-11,000 mg/L while it increased steadily up to 25,000 mg/L in the absence of the treatment. In the MBR with the sludge disintegration, relatively constant transmembrane pressures (TMPs) could be maintained for more than 6 months while the MBR without disintegration showed an abrupt increase of TMP in the later phase of the operation. In conclusion, a complete control of excess sludge production in the membrane-coupled bioreactor was possible without significant deterioration of the treated water quality and membrane performances.     

DNA extraction and Escherichia coli quantification of anaerobically digested biosolids using the competitive touchdown PCR method

Accurate enumeration of indicator organisms such as Escherichia coli is important for assessing the safety of water and wastewater samples. Recent research has shown that E. coli can enter a viable but non-culturable state; therefore, traditional cultivation methods could potentially underestimate the quantities of the organisms. The goals of the research were to develop and verify a DNA extraction protocol and a quantitative polymerase chained reaction (PCR) method for E. coli enumeration in digested biosolids. A solvent-based DNA extraction protocol with extensive cell lysis recovered approximately 78-84% of spiked DNA. In comparison, a commercial kit only recovered 28-42% of DNA, likely from inefficient cell lysis. The developed competitive touchdown PCR (cPCR) method for E. coli enumeration was comparable to both real-time PCR (rt-PCR) and cultivation methods with sensitivity of approximately 50,000-500,000 E. coli per gram dry solids (DS), which is suitable for Class B biosolids monitoring in the US and "conventional" biosolids in the European Union. The cPCR protocol provides a less expensive alternative than the rt-PCR as a culturing independent method for enumerating E. coli.     

电解/膜生物反应器组合工艺处理造纸废水

利用电解/膜生物反应器(MBR)组合工艺处理造纸废水,并与MBR工艺的单独处理效果进行对比。结果表明,电解/MBR组合工艺对造纸废水具有良好的处理效果,在原水的COD为1 100～2 000 mg/L、色度为160～220倍的条件下,组合工艺的出水COD可降至80 mg/L左右、色度在40倍左右,达到了《山东省半岛流域水污染物综合排放标准》(DB 37/676—2007)中的一级标准要求。而采用MBR单独处理时,其出水COD在200 mg/L左右、色度为140倍左右,不能满足DB 37/676—2007的要求。     

One-year monthly quantitative survey of noroviruses, enteroviruses, and adenoviruses in wastewater collected from six plants in Japan

Sewerage systems are important nodes to monitor human enteric pathogens transmitted via water. A quantitative virus survey was performed once a month for a year to understand the seasonal profiles of noroviruses genotype 1 and genotype 2, enteroviruses, and adenoviruses in sewerage systems. A total of 72 samples of influent, secondary-treated wastewater before chlorination and effluent were collected from six wastewater treatment plants in Japan. Viruses were successfully recovered from 100ml of influent and 1000ml of the secondary-treated wastewater and effluent using the acid rinse method. Viruses were determined by the RT-PCR or PCR method to obtain the most probable number for each sample. All the samples were also assayed for fecal coliforms (FCs) by a double-layer method. The seasonal profiles of noroviruses genotype 1 and genotype 2 in influent were very similar, i.e. they were abundant in winter (from November to March) at a geometric mean value of 190 and 200 RT-PCR units/ml, respectively, and less frequent in summer (from June to September), at 4.9 and 9.1 RT-PCR units/ml, respectively. The concentrations of enteroviruses and adenoviruses were mostly constant all the year round, 17 RT-PCR units/ml and 320 PCR units/ml in influent, and 0.044 RT-PCR units/ml and 7.0 PCR units/ml in effluent, respectively.     

Quantitative real-time PCR analysis of total and propidium monoazide-resistant fecal indicator bacteria in wastewater

A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. These methods were used in the analyses of wastewater samples to investigate their feasibility as alternatives to current fecal indicator bacteria culture methods for predicting the efficiency of viral pathogen removal by standard treatment processes. PMA treatment was effective in preventing qPCR detection of target sequences from non-viable cells. Concentrates of small volume, secondary-treated wastewater samples, collected from a publicly owned treatment works (POTW) under normal operating conditions, had little influence on this effectiveness. Higher levels of total suspended solids, such as those associated with normal primary treatment and all treatment stages during storm flow events, appeared to interfere with PMA effectiveness under the sample preparation conditions employed. During normal operating conditions at three different POTWs, greater reductions were observed in PMA-qPCR detectable target sequences of both Enterococcus and Bacteroidales than in total qPCR detectable sequences. These reductions were not as great as those observed for cultivable fecal indicator bacteria in response to wastewater disinfection. Reductions of PMA-qPCR as well as total qPCR detectable target sequences from enterococci and, to a lesser extent, Bacteroidales correlated well with reductions in infectious viruses during both normal and storm flow operating conditions and therefore may have predictive value in determining the efficiency at which these pathogens are removed.     

水解酸化/MBR处理偶氮染料废水的研究

采用水解酸化/MBR工艺处理活性艳红X-3B偶氮染料废水.结果表明,水解酸化/MBR工艺对活性艳红X-3B偶氮染料废水的处理效果较好,平均脱色率为81.58%,对COD和氨氮的平均去除率分别为83.53%、80.39%;对各工艺单元进、出水的紫外-可见吸收光谱分析表明,兼氧微生物通过水解酸化作用可将活性艳红X-3B染料分子中的偶氮双键、苯环、蔡环和二氯均三嗪活性基等降解,形成易被生物降解的小分子有机物,提高了染料废水的可生化性,为后续的MBR处理创造了条件.