Linking non-culturable (qPCR) and culturable enterococci densities with hydrometeorological conditions

Quantitative polymerase chain reaction (qPCR) measurement of enterococci has been proposed as a rapid technique for assessment of beach water quality, but the response of qPCR results to environmental conditions has not been fully explored. Culture-based E. coli and enterococci have been used in empirical predictive models to characterize their responses to environmental conditions and to increase monitoring frequency and efficiency. This approach has been attempted with qPCR results only in few studies. During the summer of 2006, water samples were collected from two southern Lake Michigan beaches and the nearby river outfall (Burns Ditch) and were analyzed for enterococci by culture-based and non-culture-based (i.e., qPCR) methods, as well as culture-based E. coli. Culturable enterococci densities (log CFU/100 ml) for the beaches were significantly correlated with enterococci qPCR cell equivalents (CE) (R = 0.650, P < 0.0001, N = 32). Enterococci CE and CFU densities were highest in Burns Ditch relative to the beach sites; however, only CFUs were significantly higher (P < 0.0001). Culturable enterococci densities at Burns Ditch and the beaches were significantly correlated (R = 0.565, P < 0.0001, N = 32). Culturable E. coli and enterococci densities were significantly correlated (R = 0.682, P < 0.0001, N = 32). Regression analyses suggested that enterococci CFU could be predicted by lake turbidity, Burns Ditch discharge, and wind direction (adjusted R² = 0.608); enterococci CE was best predicted by Burns Ditch discharge and log-transformed lake turbidity × wave height (adjusted R² = 0.40). In summary, our results show that analytically, the qPCR method compares well to the non-culture-based method for measuring enterococci densities in beach water and that both these approaches can be predicted by hydrometeorological conditions. Selected predictors and model results highlight the differences between the environmental responses of the two method endpoints and the potentially high variance in qPCR results.     

Pyrosequencing of the 16S rRNA gene to reveal bacterial pathogen diversity in biosolids

Given the potential for a variety of bacterial pathogens to occur in variably stabilized sewage sludge (biosolids), an understanding of pathogen diversity and abundance is necessary for accurate assessment of infective risk when these products are land applied. 16S rDNA was PCR amplified from genomic DNA extracted from municipal wastewater residuals (mesophilic- and thermophilic-phased anaerobic digestion (MAD and TPAD), composting (COM)), and agricultural soil (SOIL), and these amplicons were sequenced using massively parallel pyrosequencing technology. Resulting libraries contained an average of 30,893 16S rDNA sequences per sample with an average length of 392 bases. FASTUNIFRAC-based comparisons of population phylogenetic distance demonstrated similarities between the populations of different treatment plants performing the same stabilization method (e.g. different MAD samples), and population differences among samples from different biosolids stabilization methods (COM, MAD, and TPAD). Based on a 0.03 Jukes-Cantor distance to 80 potential bacterial pathogens, all samples contained pathogens and enrichment ranged from 0.02% to 0.1% of sequences. Most (61%) species identified were opportunistic pathogens of the genera Clostridium and Mycobacterium. As risk sciences continue to evolve to address scenarios that include multiple pathogen exposure, the analysis described here can be used to determine the diversity of pathogens in an environmental sample. This work provides guidance for prioritizing subsequent culturable and quantitative analysis, and for the first time, ensuring that potentially significant pathogens are not left out of risk estimations.     

Quantification of enterococci and bifidobacteria in Georgia estuaries using conventional and molecular methods

Fecal pollution is a serious threat to the estuarine environment along the Georgia coast. Culture-dependant and molecular methodologies were utilized to compare and evaluate the abundance of fecal indicator bacteria in four Georgia estuaries (Darien River, Frederica River, Gulley Hole Creek, and St. Marys River). The functionality of enterococci and bifidobacteria as indicator organisms in marine environments was assessed, as well as Bifidobacterium adolescentis densities. At each study site, enterococci were enumerated as colony forming units (CFU) on mEI agar. For quantitative polymerase chain reaction (qPCR), genus- and species-specific primer sets were used to quantify bifidobacteria and B. adolescentis as 16S rRNA gene copies and enterococci as tuf gene copies. A high correlation (r=0.925) was observed between CFU and qPCR enumeration of enterococci. Enterococci densities in the estuarine rivers ranged from 3-449CFU/100ml on mEI plates and 4.58-5.39Log(10) gene copies/100ml by qPCR. Bifidobacteria densities ranged from 3.62-4.14Log(10) gene copies/100ml and suggested the Frederica River as least affected by fecal bacteria and the Darien River as most affected by fecal pollution. A correlation of 0.46 was observed among qPCR densities of enterococci and bifidobacteria at all sample sites. Quantitative polymerase chain reaction detection of B. adolescentis was a rapid (i.e., less than 2h) indicator of presumptive human fecal pollution and suggested that Gulley Hole Creek, the Darien River, and the St. Marys River were affected by fecal bacteria derived from a human source. Gulley Hole Creek and the Darien River had the highest levels of fecal pollution detected in the studied estuaries. Molecular quantification of bifidobacteria may be a more accurate method of determining immediate health risks associated with fecal pollution in estuarine water than traditional and contemporary assessments of enterococci.     

Occurrence, molecular characterization and antibiogram of water quality indicator bacteria in river water serving a water treatment plant

Water pollution by microorganisms of fecal origin is a current world-wide public health concern. Total coliforms, fecal coliforms (Escherichia coli) and enterococci are indicators commonly used to assess the microbiological safety of water resources. In this study, influent water samples and treated water were collected seasonally from a water treatment plant and two major water wells in a Black Belt county of Alabama and evaluated for water quality indicator bacteria. Influent river water samples serving the treatment plant were positive for total coliforms, fecal coliforms (E. coli), and enterococci. The highest number of total coliform most probable number (MPN) was observed in the winter (847.5 MPN/100 mL) and the lowest number in the summer (385.6 MPN/100 mL). Similarly E. coli MPN was substantially higher in the winter (62.25 MPN/100 mL). Seasonal variation of E. coli MPN in influent river water samples was strongly correlated with color (R² = 0.998) and turbidity (R² = 0.992). Neither E. coli nor other coliform type bacteria were detected in effluent potable water from the treatment plant. The MPN of enterococci was the highest in the fall and the lowest in the winter. Approximately 99.7 and 51.5 enterococci MPN/100 mL were recorded in fall and winter seasons respectively. One-way ANOVA tests revealed significant differences in seasonal variation of total coliforms (P < 0.05), fecal coliforms (P < 0.01) and enterococci (P < 0.01). Treated effluent river water samples and well water samples revealed no enterococci contamination. Representative coliform bacteria selected by differential screening on Coliscan Easygel were identified by 16S ribosomal RNA gene sequence analysis. E. coli isolates were sensitive to gentamicin, trimethoprim/sulfamethazole, ciprofloxacin, vancomycin, tetracycline, ampicillin, cefixime, and nitrofurantoin. Nonetheless, isolate BO-54 displayed decreased sensitivity compared to other E. coli isolates. Antibiotic sensitivity pattern can be employed in microbial source tracking.     

Degradation of yew, ragwort and rhododendron toxins during composting

Recent concerns have been raised that plants such as ragwort (Senecio jacobaea), yew (Taxus baccata) and rhododendron (Rhododendron ponticum) that are toxic to livestock may be included in compost windrows but may not be fully detoxified by the composting process. This study investigates the decomposition during composting of toxic pyrrolizidine alkaloids present in ragwort, taxines (A and B) present in yew, and grayanotoxins (GTX I, II, and III) present in rhododendron during composting. Plant samples were contained within microporous bags either towards the edge or within the centre of a pilot-scale compost heap. They were destructively harvested at regular intervals over 1200 °C cumulative temperature (about three months). Samples were analysed for levels of toxins by liquid chromatography time of flight mass spectrometry (LC-TOF-MS). Pyrrolizidine alkaloids and taxines were shown to degrade completely during the composting process. While GTX I showed significant reductions, concentrations of GTX III remained unchanged after 1200 °C cumulative temperature. However, estimates of exposure to grazing livestock coming into contact with source-segregated green waste compost containing up to 7% rhododendron suggest that GTX III poses no appreciable risk.     

Escherichia coli, enterococci, and Bacteroides thetaiotaomicron qPCR signals through wastewater and septage treatment

Fecal indicators such as Escherichia coli and enterococci are used as regulatory tools to monitor water with 24 h cultivation techniques for possible input of sewage or feces and presence of potential enteric pathogens yet their source (human or animal) cannot be determined with routine methods. This critical uncertainty has furthered water pollution science toward new molecular approaches. Members of Bacteroides genus, such as Bacteroides thetaiotaomicron are found to have features that allow their use as alternative fecal indicators and for Microbial Source Tracking (MST). The overall aim of this study was to evaluate the concentration and fate of B. thetaiotaomicron, throughout a wastewater treatment facility and septage treatment facility. A large number of samples were collected and tested for E. coli and enterococci by both cultivation and qPCR assays. B. thetaiotaomicron qPCR equivalent cells (mean: 1.8 × 10⁷/100 mL) were present in significantly higher concentrations than E. coli or enterococci in raw sewage and at the same levels in raw septage. The removal of B. thetaiotaomicron target qPCR signals was similar to E. coli and enterococci DNA during the treatment of these wastes and ranged from 3 to 5 log₁₀ for wastewater and was 7 log₁₀ for the septage. A significant correlation was found between B. thetaiotaomicron marker and each of the conventional indicators throughout the waste treatment process for both raw sewage and septage. A greater variability was found with enterococci when compared to E. coli, and CFU and equivalent cells could be contrasted by various treatment processes to examine removal and inactivation via septage and wastewater treatment. These results are compared and contrasted with other qPCR studies and other targets in wastewater samples providing a view of DNA targets in such environments.     

Persistence and partitioning of eight selected pharmaceuticals in the aquatic environment: Laboratory photolysis, biodegradation, and sorption experiments

We selected eight pharmaceuticals with relatively high potential ecological risk and high consumption—namely, acetaminophen, atenolol, carbamazepine, ibuprofen, ifenprodil, indomethacin, mefenamic acid, and propranolol—and conducted laboratory experiments to examine the persistence and partitioning of these compounds in the aquatic environment. In the results of batch sunlight photolysis experiments, three out of eight pharmaceuticals—propranolol, indomethacin, and ifenprodil—were relatively easily photodegraded (i.e., half-life < 24 h), whereas the other five pharmaceuticals were relatively stable against sunlight. The results of batch biodegradation experiments using river water suggested relatively slow biodegradation (i.e., half-life > 24 h) for all eight pharmaceuticals, but the rate constant was dependent on sampling site and time. Batch sorption experiments were also conducted to determine the sorption coefficients to river sediments and a model soil sample. The determined coefficients (K_d values) were much higher for three amines (atenolol, ifenprodil, and propranolol) than for neutral compounds or carboxylic acids; the K_d values of the amines were comparable to those of a four-ring polycyclic aromatic hydrocarbon (PAH) pyrene. The coefficients were also higher for sediment/soil with higher organic content, and the organic carbon-based sorption coefficient (log K_oc) showed a poor linear correlation with the octanol-water distribution coefficient (log D_ow) at neutral pH. These results suggest other sorption mechanisms—such as electrochemical affinity, in addition to hydrophobic interaction—play an important role in sorption to sediment/soil at neutral pH.     

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.     

Pre-treatment mechanisms during thermophilic-mesophilic temperature phased anaerobic digestion of primary sludge

Pre-treatment is used extensively to improve degradability and hydrolysis rate of material being fed into digesters. One emerging process is temperature phased anaerobic digestion (TPAD), which applies a short (2 day) 50-70 °C pre-treatment step prior to 35 °C digestion in the main stage (10-20 days). In this study, we evaluated a thermophilic-mesophilic TPAD against a mesophilic-mesophilic TPAD treating primary sludge. Thermophilic-mesophilic TPAD achieved 54% VS destruction compared to 44% in mesophilic-mesophilic TPAD, with a 25% parallel increase in methane production. Measurements of soluble COD and NH₄⁺-N showed increased hydrolysis extent during thermophilic pre-treatment. Model based analysis indicated the improved performance was due to an increased hydrolysis coefficient rather than an increased inherent degradability, suggesting while TPAD is suitable as an intensification process, a larger main digester could achieve similar impact.     

Occurrence and risk assessment of nonylphenol and nonylphenol ethoxylates in sewage sludge from different conventional treatment processes

In the present work, the concentrations of the organic pollutants nonylphenol (NP) and nonylphenol mono- and diethoxylates (NP1EO and NP2EO, respectively) in primary, secondary, mixed, aerobically-digested, anaerobically-digested, dehydrated, compost and lagoon sludge samples from different sludge treatments have been evaluated. Toxicological risk assessment of these compounds in sludge and sludge-amended soil has also been reported. NP, NP1EO and NP2EO were monitored in sludge samples obtained from treatment plants located in Andalusia (south of Spain) based on anaerobic treatments (11 anaerobic-digestion wastewater treatment plants and 3 anaerobic wastewater stabilization ponds) or on aerobic treatments (3 aerobic-digestion wastewater treatment plants, 1 dehydration treatment plant and 2 composting plants). The sum of NP, NP1EO and NP2EO (NPE) concentrations has been evaluated in relation to the limit value of 50 mg/kg set by the European Union Sludge Directive draft published in April 2000 (Working Document on Sludge). In most of the samples, NP was present at higher concentration levels (mean value 88.0 mg/kg dm) than NP1EO (mean value 33.8 mg/kg dm) and NP2EO (mean value 14.0 mg/kg dm). The most contaminated samples were compost, anaerobically-digested sludge, lagoon sludge and aerobically-digested sludge samples, which contained NPE concentrations in the ranges 44-962 mg/kg dm, 8-669 mg/kg dm, 27-319 mg/kg dm and 61-282 mg/kg dm, respectively. Risk quotients, expressed as the ratios between environmental concentrations and the predicted no-effect concentrations, were higher than 1 for NP, NP1EO and NP2EO in the 99%, 92% and 36% of the studied samples, respectively; and higher than 1 in the 86%, 6% and 2%, respectively, after sludge application to soil, leading to a significant ecotoxicological risk mainly due to the presence of NP.     

Antimicrobial resistance of fecal bacteria in waters of the Seine river watershed (France)

We studied the prevalences of antimicrobial resistance (AR) and multiple antimicrobial resistance (MAR) among the fecal bacteria found in the rivers of a large watershed under strong anthropogenic pressures, the Seine river watershed (France). Two groups of fecal indicator bacteria, Escherichia coli and intestinal enterococci, were tested for their susceptibility to 16 and 10 antimicrobials respectively, using the disk diffusion method. We found that 42% of the 214 E. coli river isolates were AR (resistant to at least one antimicrobial) and 35% were MAR (resistant to at least two antimicrobials). Among the 148 intestinal enterococci isolates from rivers, 83% were AR and 49% were MAR. We also investigated the sources of AR fecal bacteria found in the rivers of the watershed. A total of 715 E. coli isolates and 476 intestinal enterococci isolates were collected in point sources (municipal and hospital wastewaters) and non-point sources (surface runoff and soil leaching waters from agricultural or forest areas). For E. coli, the prevalence of AR differed widely from source to source and ranked in this order: hospital wastewaters (71%) > municipal wastewaters (44%) > agricultural non-point sources (16%) > forest non-point sources (2%). The prevalence of MAR ranked similarly, and the same trend was observed for intestinal enterococci. The AR level of fecal bacteria in the sources was related to their expected exposure level to antimicrobials before their release into the environment. A MAR index was calculated for every source and a good discrimination between them was thus obtained. At the global scale of the Seine river watershed, domestic wastewaters seemed more likely to be the predominant source of the AR fecal bacteria found in the rivers. This was corroborated by the similarity of the MAR indices from river and municipal wastewater isolates for both fecal indicators.     

Selected personal care products and endocrine disruptors in biosolids: an Australia-wide survey

Personal care products (PCPs) and endocrine disrupting compounds (EDCs) are groups of organic contaminants that have been detected in biosolids around the world. There is a shortage of data on these types on compounds in Australian biosolids, making it difficult to gain an understanding of their potential risks in the environment following land application. In this study, 14 biosolids samples were collected from 13 Australian wastewater treatment plants (WWTPs) to determine concentrations of eight compounds that are PCPs and/or EDCs: 4-t-octylphenol (4tOP), 4-nonylphenol (4NP), triclosan (TCS), bisphenol A (BPA), estrone (E1), 17β-estradiol (E2), estriol (E3) and 17α-ethinylestradiol (EE2). Concentration data were evaluated to determine if there were any differences between samples that had undergone anaerobic or aerobic treatment. The concentration data were also compared to other Australian and international data. Only 4tOP, 4NP, TCS, and BPA were detected in all samples and E1 was detected in four of the 14 samples. Their concentrations ranged from 0.05 to 3.08 mg/kg, 0.35 to 513 mg/kg, < 0.01 to 11.2 mg/kg, < 0.01 to 1.47 mg/kg and < 45 to 370 μg/kg, respectively. The samples that were obtained from WWTPs that used predominantly anaerobic treatment showed significantly higher concentrations of the compounds than those obtained from WWTPs that used aerobic treatment. Overall, 4NP, TCS and BPA concentrations in Australian biosolids were lower than global averages (by 42%, 12% and 62%, respectively) and 4tOP concentrations were higher (by 25%), however, of these differences only that for BPA was statistically significant. The European Union limit value for NP in biosolids is 50 mg/kg, which 4 of the 14 samples in this study exceeded.     

Chelate-assisted phytoextraction of mercury in biosolids

Mercury contaminated stockpiles of biosolids (8.4 mg kg^− 1 Hg) from Melbourne Water's Western Treatment Plant (MW-WTP) were investigated to evaluate the possibility of their Hg chelate-assisted phytoextraction. The effects of ammonium thiosulphate (NH₄)₂S₂O₃, cysteine (Cys), nitrilotriacetic acid (NTA), and potassium iodide (KI) were studied to mobilize Hg and to increase its uptake in plant shoots. Three plant species were selected for this study, one herbaceous and two grasses: Atriplex codonocarpa, Austrodanthonia caespitosa and Vetiveria zizanioides. KI proved to be the best candidate for Hg phytostabilization in biosolids because it facilitated the concentration of this metal mainly in roots. (NH₄)₂S₂O₃ was shown to be the most effective chelating agent among those tested for Hg phytoextraction as it allowed the highest translocation of Hg into the above-ground tissues of the selected plant species. The phytoextraction conditions using A. caespitosa as the best performing plant species were optimized at an (NH₄)₂S₂O₃ concentration of 27 mmol kg^− 1 and contact time with biosolids of seven day. Monitoring of the Hg concentration in biosolids and in leachate water during a 9-day treatment revealed that the biosolids Hg concentration decreased significantly after the first day of treatment and then it decreased only slightly with time reaching a value of 5.6 mg kg^− 1 Hg at the end of the 9-day period. From the corresponding results obtained for the leachate water, it was suggested that a relatively large fraction of Hg (0.7 mg kg^− 1 Hg) was promptly mobilized and consequently the plants were able to take up the metal and translocate it into shoots.     

A water quality modeling study of non-point sources at recreational marine beaches

A model study was conducted to understand the influence of non-point sources including bather shedding, animal fecal sources, and near shore sand, as well as the impact of the environmental conditions, on the fate and transport of the indicator microbe, enterococci, at a subtropical recreational marine beach in South Florida. The model was based on an existing finite element hydrodynamic and transport model, with the addition of a first order microbe deactivation function due to solar radiation. Results showed that dog fecal events had a major transient impact (hundreds of Colony Forming Units/100 ml [CFU/100 ml]) on the enterococci concentration in a limited area within several hours, and could partially explain the high concentrations observed at the study beach. Enterococci released from beach sand during high tide caused mildly elevated concentration for a short period of time (ten to twenty of CFU/100 ml initially, reduced to 2 CFU/100 ml within 4 h during sunny weather) similar to the average baseline numbers observed at the beach. Bather shedding resulted in minimal impacts (less than 1 CFU/100 ml), even during crowded holiday weekends. In addition, weak current velocity near the beach shoreline was found to cause longer dwelling times for the elevated concentrations of enterococci, while solar deactivation was found to be a strong factor in reducing these microbial concentrations.     

Biochar for the mitigation of nitrate leaching from soil amended with biosolids

Countries with sewage treatment plants produce on average 27 kg of dried biosolids/person/yr. Concerns about nitrate leaching limit the rate at which biosolids are added to soil. We sought to determine whether biochar, a form of charcoal that is added to soil, could reduce nitrate leaching from biosolids amended soil. We set up 24 (0.5 m × 0.75 m) lysimeters, filled with two soil types (Templeton Silt Loam and Ashley Dene silt loam) and amended with combinations of biochar (102 t/ha equivalent) and biosolids (600 and 1200 kg N/ha equivalent). Pasture and leachates were sampled over 5 months. Nitrate leaching from biochar plus biosolids amended soils were reduced to levels at or below the control treatments. Pasture N concentrations were similarly affected by biochar addition. Future research should focus on unravelling the mechanism responsible for the change in the nitrogen cycle in soils amended with biosolids and biochar.     

Relationships between sand and water quality at recreational beaches

Enterococci are used to assess the risk of negative human health impacts from recreational waters. Studies have shown sustained populations of enterococci within sediments of beaches but comprehensive surveys of multiple tidal zones on beaches in a regional area and their relationship to beach management decisions are limited. We sampled three tidal zones on eight South Florida beaches in Miami-Dade and Broward counties and found that enterococci were ubiquitous within South Florida beach sands although their levels varied greatly both among the beaches and between the supratidal, intertidal and subtidal zones. The supratidal sands consistently had significantly higher (p < 0.003) levels of enterococci (average 40 CFU/g dry sand) than the other two zones. Levels of enterococci within the subtidal sand correlated with the average level of enterococci in the water (CFU/100mL) for the season during which samples were collected (r_s = 0.73). The average sand enterococci content over all the zones on each beach correlated with the average water enterococci levels of the year prior to sand samplings (r_s = 0.64) as well as the average water enterococci levels for the month after sand samplings (r_s = 0.54). Results indicate a connection between levels of enterococci in beach water and sands throughout South Florida’s beaches and suggest that the sands are one of the predominant reservoirs of enterococci impacting beach water quality. As a result, beaches with lower levels of enterococci in the sand had fewer exceedences relative to beaches with higher levels of sand enterococci. More research should focus on evaluating beach sand quality as a means to predict and regulate marine recreational water quality.     

Monitoring bacterial indicators and pathogens in cattle feedlot waste by real-time PCR

Quantitative microbial health risk assessment requires accurate enumeration of pathogens in hazard-containing matrices as part of the risk characterization process. As part of a risk management-oriented study of cattle feedlot waste contaminants, we investigated the utility of quantitative real-time PCR (qPCR) for surveying the microbial constituents of different faecal wastes. The abundance of Escherichia coli and enterococci were first estimated in five cattle feedlot waste types from five localities. Bacteria were quantified using two culture methods and compared to the number of genome copies detected by qPCR targeted at E. coli and Enterococcus faecalis. Bacterial numbers detected in the different wastes (fresh faeces, pen manure, aged manure, composted manure, carcass manure compost) ranged from 10−⁷ to 10² g⁻¹ (dry weight). Both indicator groups were detected by qPCR with a comparable sensitivity to culture methods across this range. qPCR measurements of E. coli and E. faecalis correlated well with MPN and spread plate data. As a second comparison, we inoculated green fluorescent protein (GFP) labeled reference bacteria into manure samples. GFP labeled E. coli and Listeria monocytogenes were detected by qPCR in concentrations corresponding to between 18% and 71% of the initial bacterial numbers, compared to only 2.5-16% by plating. Our results supported our selection of qPCR as a fast, accurate and reliable system for surveying the presence and abundance of pathogens in cattle waste.     

Food waste conversion options in Singapore: Environmental impacts based on an LCA perspective

Proper management and recycling of huge volumes of food waste is one of the challenges faced by Singapore. Semakau island — the only offshore landfill of the nation — only accepts inert, inorganic solid waste and therefore a large bulk of food waste is directed to incinerators. A remaining small percent is sent for recycling via anaerobic digestion (AD), followed by composting of the digestate material. This article investigates the environmental performance of four food waste conversion scenarios — based on a life cycle assessment perspective — taking into account air emissions, useful energy from the incinerators and AD process, as well as carbon dioxide mitigation from the compost products derived from the digestate material and a proposed aerobic composting system. The life cycle impact results were generated for global warming, acidification, eutrophication, photochemical oxidation and energy use. The total normalized results showed that a small-scale proposed aerobic composting system is more environmentally favorable than incinerators, but less ideal compared to the AD process. By making full use of the AD's Recycling Phase II process alone, the Singapore Green Plan's 2012 aim to increase the recycling of food waste to 30% can easily be achieved, along with reduced global warming impacts.     

Rapid decay of host-specific fecal Bacteroidales cells in seawater as measured by quantitative PCR with propidium monoazide

We investigated the persistence of feces-derived Bacteroidales cells and their DNA in seawater under natural conditions using an optimized chemical method based on co-extraction of nucleic acids with propidium monoazide (PMA), which interferes with PCR amplification of molecular markers from extracellular DNA and dead bacterial cells. The previously validated Bacteroidales assays BacUni-UCD, BacHum-UCD, BacCow-UCD, and BacCan-UCD were utilized to determine concentrations of Bacteroidales genetic markers targeting all warm-blooded animals, humans, cows and dogs, specifically, over a period of 24 d. Microcosms containing mixed feces in dialysis tubing were exposed to seawater under flow-through conditions at ambient temperature in the presence and absence of sunlight. Using a two-stage plus linear decay model, the average T₉₉ (two-log reduction) of host-specific Bacteroidales cells was 28 h, whereas that of host-specific Bacteroidales DNA was 177 h. Natural sunlight did not affect the survival of uncultivable Bacteroidales cells and their DNA with the exception of the BacCow-UCD marker. Bacteroidales DNA, as measured by quantitative PCR (qPCR) without PMA, persisted for as long as 24 d at concentrations close to the limit of detection. Culturable Enterococcus cells were detected for only 70 h, whereas Enterococcus cells measured by qPCR with and without PMA persisted for 450 h. In conclusion, measuring Bacteroidales DNA without differentiating between intact and dead cells or extracellular DNA may misinform about the extent of recent fecal pollution events, particularly in the case of multiple sources of contamination with variable temporal and spatial scales due to the relatively long persistence of DNA in the environment. In contrast, applying qPCR with and without PMA can provide data on the fate and transport of fecal Bacteroidales in water, and help implement management practices to protect recreational water quality.     

Detection of the antimicrobials triclocarban and triclosan in agricultural soils following land application of municipal biosolids

The occurrence of the antimicrobials triclocarban (TCC) and triclosan (TCS) was investigated in agricultural soils following land application of biosolids using liquid chromatography-tandem mass spectrometry (LC-MS-MS) with negative ion multimode ionization. The method detection limits were 0.58 ng TCC/g soil, 3.08 ng TCC/g biosolids, 0.05 ng TCS/g soil and 0.11 ng TCS/g biosolids and the average recovery from all of the sample matrices was >95%. Antimicrobial concentrations in biosolids from three Michigan wastewater treatment plants (WWTPs) ranged from 4890 to 9280 ng/g, and from 90 to 7060 ng/g, for TCC and TCS respectively. Antimicrobial analysis of soil samples, collected over two years, from ten agricultural sites previously amended with biosolids, indicated TCC was present at higher concentrations (1.24-7.01 ng/g and 1.20-65.10 ng/g in 2007 and 2008) compared to TCS (0.16-1.02 ng/g and from the method detection limit, <0.05-0.28 ng/g in 2007 and 2008). Soil antimicrobial concentrations could not be correlated to any soil characteristic, or to the time of last biosolids application, which occurred in either 2003, 2004 or 2007. To our knowledge, our data represent the first report of TCC, and the first comparison of TCC and TCS concentrations, in biosolids-amended agricultural soils. Such information is important because approximately 50% of US biosolids are land applied, therefore, any downstream effects of either antimicrobial are likely to be widespread.