Inactivation kinetics modeling of Escherichia coli in concentrated urine for implementing predictive environmental microbiology in sanitation safety planning.

Urine concentration (condensation) leads to the inactivation of pathogens in urine owing to a hyperosmotic environment. This study proposed an inactivation kinetic model of Escherichia coli (E. coli), a surrogate of human bacterial pathogens, in concentrated synthetic urine. The model parameters were obtained under an assumption that the inactivation rate of E. coli followed a binomial distribution, which made it possible to accurately simulate the time-course decay of E. coli in synthetic urine. The inactivation rate constant values obtained in concentrated urine samples, ammonium buffer solutions and carbonate buffer solutions indicated that the osmotic pressure was a relatively predominant cause for the inactivation of E. coli. The appropriate storage time was estimated using the approach of quantitative microbial risk assessment, which indicated that the 5-fold concentrated urine could be safely collected after 1-day storage when urea was hydrolyzed, whereas 91-hour storage was required for non-concentrated urine. The occupational risk was not negligible even with 6-month storage at 20 °C when urea was not hydrolyzed, which suggested that the urine storage styles should be clarified more minutely. The present study highlights the importance of "predictive environmental microbiology," which deals with inactivation kinetic models of microorganisms under varied environmental conditions to fully implement the hazard analysis and critical control point (HACCP) approach for the safe use of human excreta in agriculture.

Fecal Source Tracking in A Wastewater Treatment and Reclamation System Using Multiple Waterborne Gastroenteritis Viruses.

Gastroenteritis viruses in wastewater reclamation systems can pose a major threat to public health. In this study, multiple gastroenteritis viruses were detected from wastewater to estimate the viral contamination sources in a wastewater treatment and reclamation system installed in a suburb of Xi'an city, China. Reverse transcription plus nested or semi-nested PCR, followed by sequencing and phylogenetic analysis, were used for detection and genotyping of noroviruses and rotaviruses. As a result, 91.7% (22/24) of raw sewage samples, 70.8% (17/24) of the wastewater samples treated by anaerobic/anoxic/oxic (A2O) process and 62.5% (15/24) of lake water samples were positive for at least one of target gastroenteritis viruses while all samples collected from membrane bioreactor effluent after free chlorine disinfection were negative. Sequence analyses of the PCR products revealed that epidemiologically minor strains of norovirus GI (GI/14) and GII (GII/13) were frequently detected in the system. Considering virus concentration in the disinfected MBR effluent which is used as the source of lake water is below the detection limit, these results indicate that artificial lake may be contaminated from sources other than the wastewater reclamation system, which may include aerosols, and there is a possible norovirus infection risk by exposure through reclaimed water usage and by onshore winds transporting aerosols containing norovirus.

Identification of the inactivating factors and mechanisms exerted on MS2 coliphage in concentrated synthetic urine.

Volume reduction (condensation) is a key for the practical usage of human urine as a fertilizer because it enables the saving of storage space and the reduction of transportation cost. However, concentrated urine may carry infectious disease risks resulting from human pathogens frequently present in excreta, though the survival of pathogens in concentrated urine is not well understood. In this study, the inactivation of MS2 coliphage, a surrogate for single-stranded RNA human enteric viruses, in concentrated synthetic urine was investigated. The infectious titer reduction of MS2 coliphage in synthetic urine samples was measured by plaque assay, and the reduction of genome copy number was monitored by reverse transcription-quantitative PCR (RTqPCR). Among chemical-physical conditions such as pH and osmotic pressure, uncharged ammonia was shown to be the predominant factor responsible for MS2 inactivation, independently of urine concentration level. The reduction rate of the viral genome number varied among genome regions, but the comprehensive reduction rate of six genome regions was well correlated with that of the infectious titer of MS2 coliphage. This indicates that genome degradation is the main mechanism driving loss of infectivity, and that RT-qPCR targeting the six genome regions can be used as a culture-independent assay for monitoring infectivity loss of the coliphage in urine. MS2 inactivation rate constants were well predicted by a model using ion composition and speciation in synthetic urine samples, which suggests that MS2 infectivity loss can be estimated solely based on the solution composition, temperature and pH, without explicitly accounting for effects of osmotic pressure.

Bactericidal and virucidal mechanisms in the alkaline disinfection of compost using calcium lime and ash.

In the present study, the bactericidal and virucidal mechanisms in the alkaline disinfection of compost with calcium lime and ash were investigated. Two indicator microorganisms, Escherichia coli and MS2 coliphage, were used as surrogates for enteric pathogens. The alkaline-treated compost with calcium oxide (CaO) or ash resulted primarily in damage to the outer membrane and enzyme activities of E. coli. The alkaline treatment of compost also led to the infectivity loss of the coliphage because of the partial capsid damage and RNA exteriorization due to a raised pH, which is proportional to the amount of alkaline agents added. These results indicate that the alkaline treatment of compost using calcium oxide and ash is effective and can contribute to the safe usage of compost from a mixing type dry toilet.

Effect of post-treatment conditions on the inactivation of helminth eggs (Ascaris suum) after the composting process.

Safe and appropriate disposal of human waste is a basic requirement for sanitation and protection of public health. For proper sanitation and nutrient recovery, it is necessary to ensure effective treatment methods to complete pathogen destruction in excreta prior to reuse. Composting toilets convert faeces to a reusable resource such as fertilizer or humus for organic agriculture. A composting toilet for rural Burkina Faso was created by modifying a commercial model available in Japan to improve hygiene and increase food production. The toilet has shown to result in a degraded final product, but its effectiveness for pathogen destruction was unclear due to low temperatures generated from the toilet. This study aimed to sanitize compost withdrawn from the composting toilet for food production by setting post-treatment conditions. The inactivation kinetics of Ascaris suum eggs, selected as an indicator for helminth eggs, was determined during post-treatment at different temperatures (30°C, 40°C, 50°C and 60°C) with varying moisture contents (MC) (50%, 60% and 70%). The treatment of compost in a possible additional post-treatment after the composting process was tried in the laboratory test. Inactivation of A. suum eggs was fast with greater than two log reductions achieved within 2 h for temperature 50°C and 50% MC and greater than three log reductions for temperature 60°C and 50% MC within 3 h. Statistical analysis showed the significant impact of temperature and moisture on the inactivation rates of A. suum eggs. The post-treatment can efficiently increase helminth eggs destruction prior to reuse.

Effect of linear alkylbenzene sulfonate (LAS) on human intestinal Caco-2 cells at non cytotoxic concentrations.

Linear alkylbenzene sulfonate (LAS) is a cytotoxic synthetic anionic surfactant widely present in the environment due to its large-scale production and intensive use in the detergency field. In this study, we investigated the effect of LAS (CAS No. 25155-30-0) at non cytotoxic concentrations on human intestinal Caco-2 cells using different in vitro bioassays. As results, LAS increased Caco-2 cell proliferation at concentrations ranging from 1 to 15 ppm, more significantly for shorter exposure time (24 h), confirmed using flow cytometry and trypan blue exclusion methods. Moreover, proteomics analysis revealed that this effect was associated with an over-expression of elongation factor 2 and dipeptidyl peptidase 3, and a down-regulation of 14-3-3 protein theta, confirmed at mRNA level using real-time PCR. These findings suggest that LAS at non cytotoxic concentrations, similar to those observed at wastewater treatment plants outlets, increases the growth rate of colon cancer cells, raising thereby its tumor promotion effect potential.

Relationship between respiratory quotient, nitrification, and nitrous oxide emissions in a forced aerated composting process.

We assessed the relationship between respiratory quotient (RQ) and nitrification and nitrous oxide (N2O) emission in forced aerated composting using lab-scale reactors. Relatively high RQ values from degradation of readily degradable organics initially occurred. RQ then stabilized at slightly lower values, then decreased. Continuous emission of N2O was observed during the RQ decrease. Correlation between nitrification and N2O emission shows that the latter was triggered by nitrification. Mass balances demonstrated that the O2 consumption of nitrification (∼24.8mmol) was slightly higher than that of CO2 emission (∼20.0mmol), indicating that the RQ decrease was caused by the occurrence of nitrification. Results indicate that RQ is a useful index, which not only reflects the bioavailability of organics but also predicts the occurrence of nitrification and N2O emission in forced aerated composting.

Grey water treatment by the slanted soil system with unsorted soil media.

This study evaluated the performance of unsorted soil media in the slanted soil treatment system, in terms of removal efficiency in suspended solids (SS), chemical oxygen demand (COD), linear alkylbenzene sulphonate (LAS) and Escherichia coli, and lifetime until clogging occurs. Unsorted soil performed longer lifetime until clogging than sorted fine soil. Removal of SS, COD, and LAS also performed same or better level in unsorted soil than fine soil. As reaction coefficients of COD and LAS were described as a function of the hydraulic loading rate, we can design a slanted soil system according to the expected hydraulic loading rate and the targeted level of COD or LAS in effluent. Regarding bacteria removal, unsorted soil performed sufficient reduction of E. coli for 5 weeks; however, the removal process occurred throughout all four chambers, while that of fine soil occurred in one to two chambers.

Suitability of biochar as a matrix for improving the performance of composting toilets.

To evaluate the suitability of biochar (rice husk charcoal) as a matrix in composting toilets that can decompose human faeces and recover fertiliser components, the composting process during toilet operation and the agricultural value of the resulting compost were characterised by performing a comparison with sawdust, rice husks, and corn stalks. The faecal decomposition ratio in biochar was 42%, similar to the values for rice husks (46%) and corn stalks (41%), but higher than the value for sawdust (25%). Heterotroph micro-organism acidity is qualitatively higher in biochar than in sawdust. However, nitrogen loss in biochar was 19%, lower than that in rice husks (36%) and corn stalks (25%), but similar to that in sawdust (16%). Although the biochar compost had no significant impact on the cation exchange capacity and water retention of sandy soil, the ratio of nitrogen transportation into plants was 12.8%, higher than that for the other materials. These results suggest that biochar is effective for achieving high faecal decomposition, low nitrogen loss, and high nutrient supply.

Cytotoxic effect of linear alkylbenzene sulfonate on human intestinal Caco-2 cells: associated biomarkers for risk assessment.

Linear alkylbenzene sulfonate (LAS) is a synthetic anionic surfactant widely present in the environment due to its intensive production and use in the detergency field. Admitting that current procedure of risk assessment has limits in providing realistic risk assessment data and predicting the cumulative effect of the toxicant mixtures, the incorporation of information regarding the mode of action and cell response mechanism seems to be a potential solution to overcome these limits. In this regard, we investigated in this study the LAS cytotoxicity on human intestinal Caco-2 cells, trying to unveil the protein actors implicated in the cell response using proteomics approach in order to give a better understanding of the toxicological effect and allow the identification of appropriate biomarkers reflecting the mode of action associated with LAS. As results, we demonstrated that LAS induces a time- and dose-dependent cytotoxicity in Caco-2 cells accompanied by an induction of oxidative stress followed by an excessive increase of intracellular calcium level. Proteomics approach helped in discovering three informative biomarkers of effect associated with LAS cytotoxic effect, reported for the first time: calreticulin, thioredoxin, and heat shock cognate 71 (HSP7C), confirmed by real-time PCR and western blot analysis. These biomarkers could serve for more reliable future risk assessment studies that consider the toxicants mode of action in order to help in the prediction of potential cumulative effects of environmentally coexisting contaminants.

Estimation of contamination sources of human enteroviruses in a wastewater treatment and reclamation system by PCR-DGGE.

A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method was employed to estimate the contamination sources of human enteroviruses and understand how their dominant strains vary in a wastewater treatment and reclamation system consisting of sewage collection, wastewater treatment with membrane bioreactor and open lakes for reclaimed water storage and reuse. After PCR-DGGE using a selected primer set targeting enteroviruses, phylogenetic analysis of acquired enterovirus gene sequences was performed. Enteroviruses identified from the septic tank were much more diverse than those from grey water and kitchen wastewater. Several unique types of enterovirus different from those in wastewater samples were dominant in a biological wastewater treatment unit. Membrane filtration followed by chlorination was proved effective for physically eliminating enteroviruses; however, secondary contamination likely occurred as the reclaimed water was stored in artificial lakes. Enterovirus 71 (EV71), a hand-foot-and-mouth disease (HFMD) viral pathogen, was detected mainly from the artificial lakes, implying that wastewater effluent was not the contamination source of EV71 and that there were unidentified non-point sources of the contamination with the HFMD viral pathogen in the reclaimed water stored in the artificial lakes. The PCR-DGGE targeting enteroviruses provided robust evidence about viral contamination sources in the wastewater treatment and reclamation system.

Risk assessment of heavy metal toxicity of soil irrigated with treated wastewater using heat shock proteins stress responses: case of El Hajeb, Sfax, Tunisia.

Heavy metal contamination of soil resulting from treated wastewater irrigation can cause serious concerns resulting from consuming contaminated crops. Therefore, it is crucial to assess hazard related to wastewater reuse. In the present investigation, we suggest the use of biomarker approach as a new tool for risk assessment of wastewater reuse in irrigation as an improvement to the conventional detection of physicochemical accumulation in irrigated sites. A field study was conducted at two major sites irrigated with treated wastewater and comparisons were made with a control site. Different soil depths were considered to investigate the extent of heavy metal leaching, the estrogenic activity, and the biomarker response. Results have shown that a longer irrigation period (20 years) caused a slight decrease in soil metal levels when compared to the soil irrigated for 12 years. The highest levels of Cr, Co, Ni, Pb, and Zn were detected at 20 and 40 cm horizons in plots irrigated with wastewater for 12 years. The latter finding could be attributed to chemical leaching to deeper plots for longer irrigation period. Furthermore, the treated wastewater sample showed a high estrogenic activity while none of the soil samples could induce any estrogenic activity. Regarding the stress response, it was observed that the highest stress shown by the HSP47 promoter transfected cells was induced by a longer irrigation period. Finally, the treated wastewater and the irrigated soils exhibited an overexpression of HSP60 in comparison with reference soil following 1 h exposure. In conclusion, in vitro techniques can be efficiently used to assess potential hazard related to wastewater reuse.

Nitrous oxide emission mechanisms during intermittently aerated composting of cattle manure.

To investigate the mechanisms of nitrous oxide (N2O) emission during intermittent aeration in the composting process, a laboratory scale experiment with continuous measurement of N2O emission was conducted with cattle manure. A low oxygen mode (2.5% oxygen in the inlet for 1 day), anaerobic mode (0.13% oxygen for 0.25 day), and aerated mode (20.5% oxygen for 2 days) were sequentially set up three times after 22 days of continuous aeration to replicate intermittent aeration. The total N2O emission was 0.26-0.35 mmol, 0.27-0.32 mmol, and 0.14-0.23 mmol during the low oxygen, anaerobic, and aerated modes, respectively. Denitrification was indicated as the main N2O emission pathway in the anaerobic and low-oxygen modes, while nitrification was indicated as the main pathway in the aerated mode and under continuous aeration. Results from this study suggest that nitrification is an important pathway for N2O emission as well as denitrification.

Production of slow-released nitrogen fertilizer from urine.

Human excreta, especially urine is rich in nitrogen that can be utilized for agricultural purposes, while the slow-release fertilizer allows effective utilization of nutrients in agricultural production. The direct formation of slow-release fertilizer--methylene urea--from urine was being proposed in this study. The experiments were tried to prove formation of methylene urea from human urine, and to investigate the effect of pH and salt concentration on the reaction rate. The synthetic urine and real urine were used for the urea source of the reaction. As a result, the precipitates were prepared from synthetic urine, while the small molecule fractions generated then they grew into precipitate. The nuclear magnetic resonance, infrared spectroscopy, element analyses showed the precipitates in synthetic urine were the same compound found in the urea solution, which was methylene urea. The reaction rate was high at low pH value. The reaction rate in the buffer solution was lower than the synthetic urine at the same pH, because some salts may work as a catalyst. The urea concentration reduction rate in real urine showed the same trend with synthetic urine at the same pH, while the precipitation was quite similar to methylene urea.

Photocatalytic decomposition of crotamiton over aqueous TiO(2) suspensions: determination of intermediates and the reaction pathway.

The photocatalytic degradation of crotamiton in aqueous solution using TiO(2) was investigated. To investigate the effect of initial pH, the photodegradation behaviors of three types of pharmaceuticals were compared (crotamiton, clofibric acid, sulfamethoxazole). The degradation rates of crotamiton in the pH range 3-9 were nearly equal, but those of clofibric acid and sulfamethoxazole were affected by pH. At pH>6.5, TiO(2) particles, clofibric acid and sulfamethoxazole had negative charge, therefore, the repulsive force between TiO(2) particles and anionic pharmaceuticals occurred and a low reaction rate at high pH was observed. The effect of UV intensity and TiO(2) concentration on photodegradation efficiency was also investigated. Linear and logarithmical relationships between UV intensity, TiO(2) concentration and the reaction rate constant were confirmed. Furthermore, the structures of photodegradation intermediates formed concomitantly with the disappearance of crotamiton were estimated. Seven intermediates were characterized by LC/MS/MS analyses, and it was assumed that the photocatalytic degradation of crotamiton was initiated by the attack of electrophilic hydroxyl radicals on aromatic rings and alkyl chains.

Occurrence of hand-foot-and-mouth disease pathogens in domestic sewage and secondary effluent in Xi'an, China.

Hand, foot and mouth disease (HFMD), caused by a group of enteric viruses such as Enterovirus 71 (EV71), Coxsackievirus A16 (CVA16) and Coxsackievirus A10 (CVA10), is heavily epidemic in East Asia. This research focused on investigating the occurrence of HFMD pathogens in domestic sewage and secondary effluent before disinfection in a wastewater treatment plant (WWTP) in Xi'an, the largest megacity in northwest China. In order to simultaneously detect all three HFMD pathogens, a semi-nested RT-PCR assay was constructed with a newly designed primer set targeting conservative gene regions from the 5' untranslated region (UTR) to VP2. As a result, 86% of raw sewage samples and 29% of the secondary effluent samples were positive for the HFMD viral gene, indicating that HFMD pathogens were highly prevalent in domestic wastewater and that they could also persist, even with lower probability, in the secondary effluent before disinfection. Of the three HFMD pathogens, CVA10 was positive in 48% of the total samples, while the occurrences of CVA16 and EV71 were 12% and 2%, respectively. It could thus be stated that CVA10 is the main HFMD pathogen prevailing in the study area, at least during the investigation period. High genetic diversity in the conservative gene region among the same serotype of the HFMD pathogen was identified by phylogenetic analysis, implying that this HFMD pathogen replicates frequently among the population excreting the domestic sewage.

Heat shock protein 47 stress responses in Chinese hamster ovary cells exposed to raw and reclaimed wastewater.

As wastewater reclamation and reuse becomes more widespread, risks of exposure to treated wastewater increase. Moreover, an unlimited number of pollutants can be identified in wastewater. Therefore, comprehensive toxicity assessment of treated wastewater is imperative. The objective of this study was to perform a comprehensive toxicity assessment of wastewater treatment systems using stress response bioassays. This powerful tool can comprehensively assess the toxicity of contaminants. In this study, samples from conventional activated sludge treatment, membrane bioreactors (MBRs) with different pore sizes and sludge retention times (SRTs), rapid sand filtration, coagulation, nano-filtration (NF) and reverse osmosis (RO) were investigated. The results of stress response bioassays confirmed that the secondary effluent showed higher stress response than influent indicating that biological treatment generates toxic compounds. The results obtained from molecular weight fractionation of water samples demonstrated that organic matter with a higher molecular weight fraction (>0.1 µm) causes toxicity in secondary effluent. Furthermore, supernatant from MBR reactors showed toxicity regardless of SRT. On the other hand, stress response was not detected in MBR permeates except for an MBR equipped with a larger pore size membrane (0.4 µm) and with a short SRT (12 days). While rapid sand filtration could not remove the toxic compounds found in secondary effluent, coagulation tests, operated at an appropriate pH, were effective for reducing stress response in the secondary effluent. Experimental findings also showed that stress response was not detected in cases of NF and RO permeate subsequent to MBR treatment.

Performance evaluation of an on-site volume reduction system with synthetic urine using a water transport model.

The parameters of a model of the transport of water from a wet cloth sheet to the air, developed for deionized water, to establish design procedures of an on-site volume reduction system, were identified for high salt concentrations present in synthetic urine. The results showed that the water penetration was affected neither by the salts, urea or creatinine present in the synthetic urine nor by the salts accumulated on the surface of the vertical gauze sheet. However, the saturated vapour pressure decreased, leading to reduction in the evaporation rate, which occurred as a result of the salts accumulating on the surface of the vertical gauze sheet. Furthermore, a steady-state evaporation condition was established, illustrating salts falling back to the tank from the vertical gauze sheet. Accordingly, the existing design procedure was amended by incorporating the calculation procedure for the saturated vapour pressure using Raoult's law. Subsequently, the effective evaporation area of the vertical gauze sheet was estimated using the amended deign procedures to assess feasibility. This estimation showed that the arid, tropical, temperate and cold climates are suitable for the operation of this system, which require requires a small place at household level for 80% volume reduction of 10 L of urine per day for 12 hours' operation in the daytime.

Assessing the removal potential of soil-aquifer treatment system (soil column) for endotoxin.

Soil-aquifer treatment (SAT) of wastewater is an increasingly valued practice for replenishing aquifers due to ease of operation and low maintenance needs and therefore low cost. In this study, we investigated the fate of endotoxins through laboratory-scale SAT soil columns over a four month period. The effluent of rapid sand filtration was run through the columns under gravity flow conditions. Four SAT columns were packed with four different filter materials (fine sand, medium sand, coarse sand and very coarse sand). The effluent of rapid sand filtration (average dissolved organic carbon (DOC) = 4 mg l(-1) and average endotoxin concentration = 4 EU ml(-1)) was collected from a domestic wastewater treatment plant in Sapporo, Japan. DOC removal ranged from 12.5% to greater than 22.5% during the study, with DOC levels averaging 3.1 and 3.5 mg l(-1) for the SAT columns packed with different soils. Endotoxin transformation exhibited different profiles, depending on the time and soil type. Reduction in endotoxin concentration averaged 64.3% and was as high as 86.7% across the soil columns 1, 2, 3 and 4, respectively. While DOC removal was gradual, the reductions in endotoxin levels were rather rapid and most of the removal was achieved in the top layers. Soil with a larger grain size had lower efficiency in removing endotoxin. Tests were performed to evaluate the transformation of organic matter showing endotoxicity and to determine the mechanisms responsible for changes in the structural and size properties of dissolved organic matter (OM) during SAT. Dissolved OM was fractionated using Sep-Pack C18 Cartridges into hydrophobic and hydrophilic fractions. Dialysis tubes with different molecular weight cut-offs were used to perform size fractions of OM showing endotoxicity. Evaluation of the transformation of organic matter showing endotoxicity during SAT indicated that both hydrophobic and large molecules were reduced. Moreover, experimental findings showed that adsorption test data fit to the Freundlich isotherm and were affected by the particle grain size with higher adsorption capacity for fine and medium sand.

Characterization of endotoxic indicative organic matter (2-keto-3deoxyoctulosonic acid) in raw and biologically treated domestic wastewater.

The aim of this research is to characterize the organic matter showing endotoxicity in domestic wastewater. It is assumed that endotoxicity is caused by lipo-polysaccharide (LPS), particularly large and hydrophobic molecules. In this study, a batch experiment (decay test for 12 h) was conducted to confirm whether LPS is the cause of endotoxicity or not. 2-keto-3deoxyoctulosonic acid (KDO) was used as an indicator of presence of LPS.A size and structural characterization of several samples from raw and domestic wastewater was also carried out in order know which fractions are causing endotoxicity. Endotoxin and KDO patterns were found to be similar, peaking at the same time. Thus, organic matter showing endotoxicity, such as LPS was released in the decay test. Moreover, the organic matter released from bacteria during decay test was partly biodegradable. Results from size characterization (Molecular Weight Distribution) showed that the majority of endotoxin (up to 82%), in domestic sewage and secondary effluents,is composed of molecules larger than 100 kDa and less than 0.1 µm. Similarly, structural characterization (hydrophobic and hydrophilic) showed that the majority of endotoxin, ranging from 59% to 83% of the total endotoxicity, is hydrophobic fractions. Therefore, removing large and hydrophobic molecules from wastewater can be an effective way to achieve a significant decrease in its endotoxicity.