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.

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.

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.

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.

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.

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.

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.

Rational design of an on-site volume reduction system for source-separated urine.

Human urine contains nitrogen, phosphorus and potassium, which can be applied as fertilizer in agriculture, replacing commercial fertilizer. However, owing to the low nutrient content of the urine, huge quantities must be transported to farmland to meet the nutrient demand of crops. This highly increases the transportation cost for the farmers. To address the transportation issue, a new on-site volume reduction system was tested at the laboratory scale based on water evaporation from vertical gauze sheets. A mathematical water transport model was proposed to evaluate the performance of the system. The mass transfer coefficient and the resistance of water flow through the sheet in the water transport model were obtained from the experiments. The results agreed with the simulated data, thereby confirming the proposed model. The model was then applied to the dry climate of southern Pakistan, having an air temperature of 30-40 degrees C and air humidity of 20-40%, for an 80% volume reduction of 10 L urine per day, which corresponds to a family of 10 members (average for a household in Pakistan). The findings revealed that the estimated size of the vertical sheet is 440-2060 cm2, which is only a small area for setting up the system at a household level.

Survey on LPS endotoxin in rejected water from sludge treatment facility.

In this study, we applied the Limulus amebocyte lysate (LAL) test to rejected water samples from a sludge treatment facility in Sapporo, Japan. The endpoint LAL test was performed using a commercial kit and involved incubating a mixture of water samples and LAL reagent at 37 degrees C. We have observed that all of the rejected water showed endotoxic activity. Overflow from thickener and dewatering are the main contributor to this high concentration in the influent of treatment plants receiving return flow from sludge treatment facilities. The LAL test was also applied to sample batch tests, where sewage was mixed with rejected waters and aerated for 12 hours. It was found that the low biodegradability of endotoxic material in the mixture led to higher endotoxicity in comparison to the sewage and sludge aerated without rejected water. Several batch tests were also operated using sewage, sludge and different types of rejected water (overflow from thickener, dewatering, dehumidification, and desulfurization). The findings indicated that samples from reactors using water from thickener and dewatering had significant concentrations and these contributed to the high values of the effluent.

Assessment of endotoxin activity in wastewater treatment plants.

Endotoxic material, commonly associated to biological reactions, is thought to be one of the most important constituents in water. This has become a very important topic because of the common interest in microbial products governed by the possible shift to water reuse for drinking purposes. In this light, this study was conducted to provide an assessment of endotoxic activity in reclaimed wastewater. A bacterial endotoxin test (LAL test) was applied to water samples from several wastewater treatment plants (WWTP) in Sapporo, Japan keeping in view the seasonal variation. Samples were taken from several points in WWTP (influent, effluent, return sludge, advanced treatment effluent). The findings of this study indicated that wastewater shows high endotoxin activity. The value of Endotoxin (Endo) to COD ratio in the effluent is usually higher than that of the influent. Moreover, it is found that wastewater contains initially endotoxic active material. Some of those chemicals are biodegradable and but most of them are non-biodegradable. Batch scale activated sludge studies were undertaken to understand the origin of endotoxic active material in the effluent. This study showed that those chemicals are mainly produced during biological reactions, more precisely during decay process. Moreover, raw wastewater (RWW) contains high amounts of organic matter having endotoxicity which remains in the effluent.

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.

Evolution of ammonification potential in storage process of urine with fecal contamination.

The aim of this study was to obtain an evolution of the ammonification potentials in stored urine with fecal contamination. It was found that ammonification stopped after 20 days at 30 degrees C although more than half of urea still remained. The evolution of ammonification potentials showed three simple phases: the potential increased during the first 3.5 days, after which it decreased and reached a constant state significantly more than initial phase. It was concluded that the ammonification potentials remained more than the initial value after ammonia concentration was at the stable level. Thus inhibition process rather than decline process was exhibited in stored urine with fecal contamination as shown in constant phase.

Application of heat shock protein assay and proteome assay to water from wastewater treatment plant.

In this study we applied bioassay using Chinese hamster ovary (CHO) cells with a heat shock protein (HSP) 47 promoter to the effluent of the wastewater treatment plants in Sapporo and we observed the statistically significant HSP production. This implied the effluent contained some organic matter which can stress the CHO cells. To investigate the possible causes of the toxicity of the effluent, we applied the assay to the rejected water from the sludge treatment plant, the mixtures of sewage and rejected water. The evolution of HSP production during the aerobic decay process and thickening process of sludge was also examined. These assay results showed that dissolved microbial products generated and/or released from activated sludge during its decay process in the aeration tank and during thickening and dewatering process in the sludge treatment train contributed to develop HSP production. The proteomics analysis was also applied to the effluent and detected the production of elongation factor 1beta. This result implies that the effluent from wastewater treatment plants may cause changes in cell proteins involved in allergic reaction.

Biodegradability of fecal nitrogen in composting process.

Biodegradability of fecal nitrogen was studied in composting process. Fecal nitrogen was subdivided into two fractions: a type originally inert in biological activity (N(XI)), and a slowly biodegradable one (N(XS)). During the composting process, an inert type of organic matter (N(XIB)) was reproduced by endogenous respiration of heterotrophic microorganism. Evaluations for fecal nitrogen formed a conclusion of 75% (N(XS)) and 25% (N(XI)), respectively. It was estimated that the N(XIB) could be 9% of initial fecal nitrogen. Thus, approximately 34% (N(XI)+N(XIB)) of initial fecal nitrogen remained in the composting material (mixture of sawdust and feces) as biologically inert type of organic nitrogen.

Antibiotic effect of amoxicillin on the feces composting process and reactivation of bacteria by intermittent feeding of feces.

The aim of this work was to investigate the single exposure effect of amoxicillin on the feces composting process and the possibility of its bacterial reactivation by intermittent feeding of feces. The respiratory activity of the bacteria during the process after receiving exposure to several dosages of amoxicillin indicated a decrement of treatment performance, which was caused by the reduction of the initial viable bacterial count and activity brought about by the amoxicillin dosage. The amount of remaining feces was proportional to the initial concentration of amoxicillin, and even though no amoxicillin was detected, no automatic reactivation was observed, either. An intermittent feces feeding test was conducted to reactivate the bacteria. For the 10 and 100microg-amoxicillin/g dry systems, reactivation was observed, but for the 1000microg/g dry, no reactivation was seen. Finally, an intermittent feces feeding test was conducted with sterilized sawdust and the result indicated that the feces acted as a substrate rather than as a bacterial carrier.

Factors affecting the degradation of amoxicillin in composting toilet.

The biological and non-biological factors that affect the degradation of amoxicillin in the composting process of feces have been investigated. The effect of living bacteria and the enzyme (beta-lactamase) on amoxicillin decay was examined, and our results indicated that the biological effects are likely to be negligible. Consequently, the effect of phosphate, ammonia and pH level as non-biological factors was investigated by monitoring the reduction rate of amoxicillin in phosphate and ammonia buffer solutions with several pH levels. Each reduction rate constant was integrated by a simulation model, and the each calculated amoxicillin reduction profile was compared to the reduction profiles of amoxicillin in the composting process of feces. The calculated results corresponded almost exactly to the experimental profiles. We therefore concluded that the degradation of amoxicillin in a toilet matrix was dependent on the concentration of ammonia, phosphate and hydroxyl ion.

Human intestinal cells incubated with activated sludge and lipopolysaccharide express Hsp90b.

Intestinal cells are the first line of defense against toxic substances that may enter the body orally. Some of these substances may emanate from wastewater treatment plants and thus may eventually enter the food chain. In this study, human intestinal Caco-2 cells were incubated with biologically treated activated sludge and lipopolysaccharide (LPS) to determine changes in protein expression and cell monolayer integrity. Caco-2 cells incubated for 3 h with 24-h-aerated activated sludge or 220 microg/ml LPS showed enhanced expression of the heat shock protein 90b (Hsp90b). The results further show that 220 microg/ml LPS is sufficient to irreversibly disrupt tight-junction permeability in 30 min. The Hsp90b expression by the Caco-2 cells incubated with biologically treated activated sludge may be a cellular protective mechanism against LPS-induced stress.

Toxicity assessment of wastewater by proteomics analysis.

The effects of effluent from a wastewater treatment plant (EWWTP) on intestinal epithelial Caco-2 cells, a human intestinal epithelial cell line derived from a human colon carcinoma, were investigated. Previous studies have shown that the wastewater constituents nonylphenol and lipopolysaccharide (LPS) induce the overexpression of specific proteins (galectin-3, glutathione S-transferase A2 subunit, peroxiredoxin-1, and heat shock protein 90, beta (HSP90b)). In this study, the first screening of EWWTP was carried out using the HSP47-transformed cell assay, which is a highly sensitive toxicity assay. From the results of proteomics analysis of human intestinal Caco-2 cells treated with EWWTP, we found the overexpression of specific proteins, namely, elongation factor 1beta and enolase 1. These results suggest that specific proteins can be used as biomarkers for the risk assessment of water and wastewater.

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.

Design and operation of the bio-toilet system.

Criteria for the proper design and operation of the bio-toilet have not been established either by the manufacturers or by the authorities. Based on the results of experimental research already reported by us, criteria for the proper design and operation of the bio-toilet system were established. The establishment of operation criteria led to the formulation of an operation scheme where three main zones are distinguished: i) green zone, where the best composting performance is expected; ii) yellow zone, where biodegradation can be conducted but performance is not the most efficient; iii) other zones where operation of the bio-toilet is not recommended because odour problems and human health risks will develop, or in the worst case, biodegradation of faeces will not occur. In the design criteria, the surface area required for evaporating the water loads due to contributions of urine and faeces was determined by applying concepts of drying theory. This theory was also applied to estimate the mass and volume of dried sawdust required for dealing with the critical water loading. However, the accumulation of stabilised solids transpired to be more critical and, finally, mass and volume of dried sawdust were estimated based on this factor.