Evaluating human fecal contamination sources in Kranji Reservoir Catchment, Singapore

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Civil and Environmental Engineering, 2010.Cataloged from PDF version of thesis.Includes bibliographical references (p. 120-132).Singapore government through its Public Utilities Board is interested in opening Kranji Reservoir to recreational use. However, water courses within the Kranji Reservoir catchment contain human fecal indicator bacteria above recreational water quality criteria; their sources and distribution under dry and wet weather are also unknown. The goal of this study was to evaluate the distribution of E. coli under dry and wet weather, to determine the sources of the human fecal contamination, and to validate the use of human-specific 16S rRNA Bacteroides marker for human fecal source tracking in Singapore and tropical regions. Environmental water and DNA water samples (332) collected in the Kranji catchment in January and July 2009, and January 2010 were analyzed for E. coli using Hach m-ColiBlue24@ and IDEXX Colilert Quanti-Tray*/2000. Touchdown PCR and Nested-PCR HF183F assays were used to assess the absence or presence of the HF marker in Kranji catchment. Selected positive HF marker samples were sequenced and mapped using a phylogenetic tree to confirm their similarity in base order to the human factor identified in the temperate climate. The indicator bacteria (E. coli) results showed consistently high E. coli concentrations (geometric mean 3240 CFU/100 ml) in dry and wet weather in residential, horticultural and animal farming areas. The DNA analysis results showed that 94% of the 34 environmental DNA water samples collected in residential, horticultural and animal farming areas were positive to the HF marker. Generally, 74% and 94% of DNA samples respectively collected in dry and wet weather in the Kranji catchment were positive. The sequence and phylogenetic tree analysis confirmed that the HF marker identified was similar to the HF marker identified in temperate climates. Based on the results we conclude that human fecal contamination sources are widespread in the animal farming, horticultural and residential areas of Kranji catchment. The HF marker analysis validated its applicability as 16S rRNA gene of human-specific Bacteroides for human fecal source tracking in Singapore and elsewhere in tropical climates.by Jean Pierre Nshimyimana.S.M

Variably improved microbial source tracking with digital droplet PCR

This study addressed whether digital droplet PCR (ddPCR) could improve sensitivity and specificity of human-associated Bacteroidales genetic markers, BacHum and B. theta, and their quantification in environmental and fecal composite samples. Human markers were quantified by qPCR and ddPCR platforms obtained from the same manufacturer. A total of 180 samples were evaluated by each platform including human and animal feces, sewage, and environmental water. The sensitivity of ddPCR and qPCR marker assays in sewage and human stool was 0.85–1.00 with marginal reduction in human stool by ddPCR relative to qPCR (<10%). The prevalence and distribution of markers across complex sample types was similar (74–100% agreement) by both platforms with qPCR showing higher sensitivity for markers in environmental and composite samples and ddPCR showing greater reproducibility for marker detection in fecal composites. Determination of BacHum prevalence in fecal samples by ddPCR increased specificity relative to qPCR (from 0.58 to 0.88) and accuracy (from 0.77 to 0.94), while the B. theta assay performed similarly on both platforms (specificity = 0.98). In silico analysis indicated higher specificity of ddPCR for BacHum was not solely attributed to reduced sensitivity relative to qPCR. Marker concentrations measured by ddPCR for all sample types were consistently lower than those measured by qPCR, by a factor of 2.6 ± 2.8 for B. theta and 18.7 ± 10.0 for BacHum. We suggest that differences in assay performance on ddPCR and qPCR platforms may be linked to the characteristics of the assay targets (that is, genes with multiple versus single copies and encoding proteins versus ribosomal RNA) however further work is needed to validate these ideas. We conclude that ddPCR is a suitable tool for microbial source tracking, however, other factors such as cost-effectiveness and assay-specific performance should be considered.Fil: Nshimyimana, Jean Pierre. Michigan State University; Estados Unidos. Massachusetts Institute of Technology; Estados Unidos. Nanyang Technological University; SingapurFil: Cruz, Mercedes Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones para la Industria Química. Universidad Nacional de Salta. Facultad de Ingeniería. Instituto de Investigaciones para la Industria Química; Argentina. Nanyang Technological University; SingapurFil: Wuertz, Stefan. Nanyang Technological University; SingapurFil: Thompson, Janelle R.. Massachusetts Institute of Technology; Estados Unido

Techno-Economic Analysis of Electrocoagulation on Water Reclamation and Bacterial/Viral Indicator Reductions of a High-Strength Organic Wastewater—Anaerobic Digestion Effluent

This study investigated the use of iron and aluminum and their combinations as electrodes to determine the technically sound and economically feasible electrochemical approach for the treatment of anaerobic digestion effluent. The results indicated that the use of iron as anode and cathode is the most suitable solution among different electrode combinations. The reduction of turbidity, total chemical oxygen demand, total phosphorus, total coliforms, Escherichia coli, Enterococci, and phages in the reclaimed water were 99%, 91%, 100%, 1.5 log, 1.7 log, 1.0 log, and 2.0 log, respectively. The economic assessment further concluded that the average treatment cost is $3 per 1000 L for a small-scale operation handling 3000 L wastewater/day. This study demonstrated that the electrocoagulation (EC) is a promising technique for the recovery and reclamation of water from anaerobic digestion effluent. Even though its energy consumption is higher and the nitrogen removal is insufficient compared to some conventional wastewater treatment technologies, there are several advantages of the EC treatment, such as short retention time, small footprint, no mixing, and gradual addition of coagulants. These features make EC technology applicable to be used alone or combined with other technologies for a wide range of wastewater treatment application

Microbial source tracking and dynamics of bacterial communities and pathogens in the urban tropical environment

Research aims were (1) to examine general bacterial community composition and specific sequences characteristic of sewage associated taxa and pathogen-like sequences as a function of water quality, (2) to validate host-associated Bacteroidales genetic markers for microbial sources tracking in Singapore, (3) to compare the performance of digital droplet PCR and quantitative PCR in detecting and quantifying markers, and (4) to evaluate sediment-associated decay rates of the best-performing human-associated markers B. thetaiotaomicron α-1-6, mannanase (B. theta) and BacHum and those of fecal indicator bacteria in the presence and absence of grazing protozoa. Four Bacteroidales markers were validated for microbial source tracking in Singapore and Southeast Asia (Human: B. theta and BacHum; Dog: BacCan, and total Bacteroidales: BacUni). Bacteroidales viable cells and DNA had similar decay rate constants in sediments, with two-log reduction times between five and six days, and rates were not affected by the presence of protozoa.Doctor of Philosophy (CEE

Bacteroidales markers for microbial source tracking in Southeast Asia

The island city country of Singapore served as a model to validate the use of host-associated Bacteroidales 16S rRNA gene marker assays for identifying sources of fecal pollution in the urban tropical environment of Southeast Asia. A total of 295 samples were collected from sewage, humans, domesticated animals (cats, dogs, rabbits and chicken), and wild animals (birds, monkeys and wild boars). Samples were analyzed by real time PCR using five human-associated assays (HF183-SYBR Green, HF183, BacHum, BacH and B. thetaiotaomicron α-1-6, mannanase (B. theta), one canine-associated assay (BacCan), and a total Bacteroidales assay (BacUni). The best performing human-associated assay was B. theta with a diagnostic sensitivity of 69% and 100% in human stool and sewage, respectively, and a specificity of 98%. BacHum achieved the second highest sensitivity and specificity for human stool at 65% and 91%, respectively. The canine-associated Bacteroidales assay (BacCan) had a sensitivity and specificity above 80% and was validated for tracking fecal pollution from dogs. BacUni demonstrated a sensitivity and specificity of 100% for mammals, thus BacUni was confirmed for total Bacteroidales detection in the region. We showed for the first time that rabbit fecal samples cross-react with human-associated assays (HF183-SYBR Green, HF183, BacHum and BacH) and with BacCan. Our findings regarding the best performing human-associated assays differ from those reported in Bangladesh and India, which are geographically close to Southeast Asia, and where HF183 and BacHum were the preferred assays, respectively.Fil: Nshimyimana, Jean Pierre. Nanyang Technological University; Singapur. Singapore Centre for Environmental Life Sciences Engineering; Singapur. Massachusetts Institute of Technology; Estados Unidos. Singapore-MIT Alliance for Research and Technology; SingapurFil: Cruz, Mercedes Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Singapore Centre for Environmental Life Sciences Engineering; SingapurFil: Thompson, R. Janelle. Massachusetts Institute of Technology; Estados Unidos. Singapore-MIT Alliance for Research and Technology; SingapurFil: Wuertz, Stefan. Nanyang Technological University; Singapur. Singapore Centre for Environmental Life Sciences Engineering; Singapu

Next-generation sequencing (NGS) for assessment of microbial water quality: current progress, challenges, and future opportunities

Water quality is an emergent property of a complex system comprised of interacting microbial populations and introduced microbial and chemical contaminants. Studies leveraging next-generation sequencing (NGS) technologies are providing new insights into the ecology of microbially mediated processes that influence fresh water quality such as algal blooms, contaminant biodegradation, and pathogen dissemination. In addition, sequencing methods targeting small subunit (SSU) rRNA hypervariable regions have allowed identification of signature microbial species that serve as bioindicators for sewage contamination in these environments. Beyond amplicon sequencing, metagenomic and metatranscriptomic analyses of microbial communities in fresh water environments reveal the genetic capabilities and interplay of waterborne microorganisms, shedding light on the mechanisms for production and biodegradation of toxins and other contaminants. This review discusses the challenges and benefits of applying NGS-based methods to water quality research and assessment. We will consider the suitability and biases inherent in the application of NGS as a screening tool for assessment of biological risks and discuss the potential and limitations for direct quantitative interpretation of NGS data. Secondly, we will examine case studies from recent literature where NGS based methods have been applied to topics in water quality assessment, including development of bioindicators for sewage pollution and microbial source tracking, characterizing the distribution of toxin and antibiotic resistance genes in water samples, and investigating mechanisms of biodegradation of harmful pollutants that threaten water quality. Finally, we provide a short review of emerging NGS platforms and their potential applications to the next generation of water quality assessment tools

Effects of a Changing Earth on Microbial Dynamics and Human Health Risks in the Water/Sand Continuum

Humans may be exposed to microbial pathogens at recreational beaches via environmental sources such as water and sand. Although infectious disease risk from exposure to waterborne pathogens, and the fecal indicator bacteria (FIB) used to monitor water quality are active areas of research, sand is a relatively unexplored reservoir of pathogens and FIB. Sand and water at beaches experience continuous exchange of microorganisms, and these habitats provide unique advantages and challenges to pathogen introduction, growth, and persistence. Models of FIB and pathogen fate and transport in beach habitats can aid prediction of the risk of infectious disease from recreational water use, but filling knowledge gaps is necessary for accurate modeling. Climate change predictions estimate an increase in global temperatures of 2.5 – 10° F, sea level rise, and intensification of storms and precipitation in some regions. Other global change factors like population growth and urbanization may exacerbate predicted impacts. These changes can alter microbial population dynamics in beach habitats, and may consequently affect the assumptions and relationships used in numerical models. We discuss literature on microbial population and transport dynamics in sand/beach habitats, with an emphasis on how climate change and other anthropogenic influences (e.g., land use, urbanization) should be considered when using and developing models.N/