Metin Duran

Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

ABSTRACT Quantitative differentiation of live cells in biosolids samples, without the use of culturing-based approaches, is highly critical from a public health risk perspective, as recent studies have shown significant regrowth and reactivation of indicator organisms. Persistence of DNA in the environment after cell death in the range of days to weeks limits the application of DNA-based approaches as a measure of live cell density. Using selective nucleic acid intercalating dyes like ethidium monoazide (EMA) and propidium monoazide (PMA) is one of the alternative approaches to detecting and quantifying viable cells by quantitative PCR. These compounds have the ability to penetrate only into dead cells with compromised membrane integrity and intercalate with DNA via their photoinducible azide groups and in turn inhibit DNA amplification during PCRs. PMA has been successfully used in different studies and microorganisms, but it has not been evaluated sufficiently for complex environmental samples such as biosolids. In this study, experiments were performed with Escherichia coli ATCC 25922 as the model organism and the uidA gene as the target sequence using real-time PCR via the absolute quantification method. Experiments with the known quantities of live and dead cell mixtures showed that PMA treatment inhibits PCR amplification from dead cells with over 99% efficiency. The results also indicated that PMA-modified quantitative PCR could be successfully applied to biosolids when the total suspended solids (TSS) concentration is at or below 2,000 mg·liter−1.

Anaerobic treatability and biogas production potential studies of different agro-industrial wastewaters in Turkey.

The anaerobic treatability and methane generation potential of the wastewaters of the three important agro-industries in Turkey, namely, cheese-making, poultry breeding and the olive-oil mill industries were studied. Biochemical methane potential (BMP) experiments were conducted for different initial chemical oxygen demand (COD) concentrations. The results indicate that anaerobic treatment was possible for all the wastewaters studied and the biogas produced had a high methane content.

Temperature-staged anaerobic processes

This study compares the mesophilic and thermophilic anaerobic biotreatment processes treating a common substrate in laboratory scale CSTRs in terms of their effluent quality. The improvement in the performance and the effluent quality of these systems by temperature-staged processes, i.e., mesophilic and thermophilic reactors in series, were also investigated. The data presented in this paper showed that thermophilic anaerobic microorganisms had a 26% higher initial substrate utilization rates (SUR0) for COD as compared to the mesophilic ones. Mesophilic bacteria, on the other hand, produced a better quality effluent as indicated by lower effluent soluble COD concentrations. The results also indicated that the poor effluent quality of thermophilic anaerobic processes can be improved by a temperature-staged process configuration with a mesophilic secondary unit. As high as 95% COD removal rates were achieved by the thermophilic-mesophilic staged process at an organic rate of 2 g.l−1.day−1. In a temperature...

Detection and Occurrence of Indicator Organisms and Pathogens.

This review summarizes the literature pertaining to the occurrence and detection of indicator organisms and pathogens published during 2014. It is organized into the following sections: i) detection and quantification of fecal indicators and waterborne pathogens, ii) microbial source tracking (MST) using genotypic and phenotypic methods, iii) antibiotic resistant bacteria (ARB), iv) live vs. dead cell differentiation methods, and v) next generation sequencing (NGS).

Use of RNA-based genotypic approaches for quantification of viable but non-culturable Salmonella sp. in biosolids

Recent research efforts demonstrated an increase in fecal coliform counts in anaerobically digested biosolids after dewatering. Variety of bacteria enters viable but nonculturable (VNC) state as a survival response when exposed to environmental stress. Increase in coliform concentration after digestion and dewatering processes have been attributed to cells going into a viable but non-culturable state implying that traditional coliform enumeration methods are not sufficient to determine number of viable cells. Therefore, this research has been undertaken to develop a method for rapid and accurate quantification of viable but non-culturable pathogens in biosolids via monitoring and quantifying stress-related genes in Salmonella sp. The proposed method has the potential to allow accurate detection of pathogens in biosolids even when the cells are non-culturable due to environmental stress. The research proposed identification of stress related genes in Salmonella when cells are exposed to heat for different durations by using available Salmonella microarrays. In the context of this study the identified stress genes can be quantified through reverse transcription, complementary DNA (cDNA) synthesis, and amplification of cDNA via quantitative reverse transcription polymerase chain reaction (qRT-PCR). Then quantity of mRNA can be correlated to cell viability and cells ability to grow, i.e., their culturability. Development of a novel approach to understand the pathogen behaviour in biosolids is key to ensure low public health risks from biosolids. Nevertheless, the initial results suggest that intact RNA isolation from biosolids is still challenging task.

Choice of indicator organism and library size considerations for phenotypic microbial source tracking by FAME profiling.

The primary objective of this study was to investigate the effects of choice of the indicator organisms on the accuracy of classifying the fatty acid methyl ester (FAME) profiles of the known-source library isolates. First, a known-source library containing the FAME profiles of Enterococcus isolates cultured from six different possible sources of microbial pollution was developed. A total of 511 Enterococcus isolates were profiled: 120 isolates from sewage samples representing humans; 69 from dairy and cattle cow; 74 from chicken; 76 from swine; 94 from deer; and 78 from waterfowl. Classification of known-source Enterococcus isolates into their respective host categories resulted with a 66% average rate of correct classification (ARCC) in a six-way discriminant analysis (DA). The ARCC increased to 75% when the individual hosts were pooled into larger categories of human, livestock, and wildlife. The accuracy was 80% when isolates of human origin were discriminated against those of non-human origins. Recently, several studies reported the ARCCs for various classification schemes associated with total coliform (TC), fecal coliform (FC), and Escherichia coli of the known-source isolates. When the accuracy of classification of Enterococcus isolates was compared to those reported for TC, FC, and E. coli isolates, the lowest ARCCs were associated with classification of E. coli isolates, the only species level indicator organism among the four compared. It was found that the degree of discrimination increases as the indicator becomes more inclusive of bacteria from different genus. In addition, random cluster formation analysis indicates that known-source libraries with isolate numbers between 300 and 500 might be sufficient for MST by FAME.

Changes in antibiotic resistance patterns of Escherichia coli during domestic wastewater treatment.

Changes in antibiotic resistance of Escherichia coli in the different stages of conventional domestic wastewater treatment were investigated. Over two years, more than 3500 E. coli isolates from four stages of the wastewater treatment process were tested for resistance to six different antibiotics. The percent resistance of bacteria from any of the stages was highly variable in different samples. Because of this variability, no statistically significant difference was found in the overall percent resistance of E. coli from influent to effluent. When comparing different stages within samples, however, there seemed to be an increase in resistance to ampicillin and amoxicillin between the raw influent and primary effluent. In addition, the percent of isolates with multiple antibiotic resistance, resistant to more than one and less than five antibiotics, and highly multiple antibiotic resistance, resistant to at least five antibiotics, increased through the treatment process.

Acetoclastic methanogens in an anaerobic digester could be susceptible to trace metal supplementation

The objective of this study was to investigate the effects of nutrient supplementation on anaerobic biomass. While many studies emphasized the importance of supplementing trace metals such as iron, cobalt, and nickel for maximum methanogenic activity, there is no evidence whether such supplements, even at relatively low concentration, could perturb anaerobic biomass. Effects of supplementing nutrients, including yeast extract, on anaerobic biomass from two full-scale mesophilic digesters, operating under different conditions, at the North East Water Pollution Control Plant in Philadelphia, Pennsylvania, USA, were assessed using biochemical methane potential tests. The results show that acetoclastic methanogens from a recently cleaned digester was not stimulated by nutrient supplementation at relatively low concentrations and a slight perturbation was observed when supplementation was at a relatively high concentration. Furthermore, greater degree of susceptibility to the trace metal supplementation was observed for biomass from another digester that had not been cleaned for over 10 years, thus it had reduced active volume due to grit accumulation. For instance, supplementation of 200 mg/L of iron as FeCl(2)·4H(2)O to the biomass from the reduced-active-volume digester caused 17% reduction in CH(4) production, as compared to a control which did not receive any supplements, while the same concentration had no effect on the biomass from full-active-volume digester. Results strongly suggest that acetoclastic methanogens stressed due to reduced hydraulic/solids retention time may be susceptible to trace metal addition. Therefore, trace metal supplementation for anaerobic digesters should be considered on a case by case basis.

Odor control during post-digestion processing of biosolids through bioaugmentation of anaerobic digestion

The effects of bioaugmenting anaerobic biosolids digestion with a commercial product that contained selected strains of bacteria from genera Bacillus, Pseudomonas, and Actinomycetes, along with ancillary organic compounds containing various micronutrients were evaluated. The main objective of the study was to investigate the effects of bioaugmentation specifically on the performance of methanogenesis during anaerobic digestion, as well as on the generation and fate of odor-causing compounds during the storage of the digested biosolids. The bench-scale digester with 5 g/L bioaugment generated 29% more net CH4 than a control during the eight weeks of operation. In addition, the average residual propionic acid concentration in the bioaugmented digester was 46% lower than that in the control. The biosolids digested in the bioaugmented digester generated a negligible amount of methyl mercaptan (CH3SH) during 10 days of post-digestion storage, while CH3SH concentration in the control reached nearly 300 ppmv during the same period. Similarly peak dimethyl sulfide (CH3SCH3) generated by stored biosolids from the bioaugmented digester was only 37% of that from the control. Similar results were obtained in a subsequent short term study designed to confirm the repeatability of the findings.

A comparison of complex electron donors for anaerobic dechlorination of PCE

The potential of sugar, flour, corn steep liquor, molasses, non-fat milk, and whey to serve as electron donors for anaerobic dechlorination of tetrachloroethene (PCE) was examined. The electron donors were compared based on acclimation time, the extent of PCE dechlorination achieved, the minimum electron donor dose necessary to achieve PCE removal, and unit cost. The time required to achieve routine dechlorination of PCE (to any daughter product) for each donor was (in days): corn steep liquor (10), milk (10), whey (10), methanol (12), molasses (14), sugar (26), flour (30). Ethene production was achieved by milk-, whey-, and methanol-fed cultures, whereas the other donors did not facilitate ethene production over a 135-day period. Corn steep liquor-, whey-, molasses-, and sugar-fed cultures needed five times the stoichiometric amount (e.g., donor per eq PCE to ethene) to facilitate PCE conversion to dichloroethene (DCE). Cultures fed milk and flour needed 20 times the stoichiometric amount, and methanol-fed cultures required 50 times the stoichiometric amount, perhaps due to competition from methanogenic organisms. Minimum laboratory-scale electron donor costs to achieve stoichiometric conversion of PCE to DCE are (