The paucity of proper sanitation facilities has contributed to the spread of waterborne diseases in many developing countries. The primary goal of this study was to demonstrate the feasibility of using a wastewater electrolysis cell (WEC) for toilet wastewater disinfection. The treated wastewater was designed to reuse for toilet flushing and agricultural irrigation. Laboratory-scale electrochemical (EC) disinfection experiments were performed to investigate the disinfection efficiency of the WEC with four seeded microorganisms (Escherichia coli, Enterococcus, recombinant adenovirus serotype 5, and bacteriophage MS2). In addition, the formation of organic disinfection byproducts (DBPs) trihalomethanes (THMs) and haloacetic acids (HAA5) at the end of the EC treatment was also investigated. The results showed that at an applied cell voltage of +4 V, the WEC achieved 5-log10 reductions of all four seeded microorganisms in real toilet wastewater within 60 min. In contrast, chemical chlorination (CC) disinfection using hypochlorite [NaClO] was only effective for the inactivation of bacteria. Due to the rapid formation of chloramines, less than 0.5-log10 reduction of MS2 was observed in toilet wastewater even at the highest [NaClO] dosage (36 mg/L, as Cl2) over a 1 h reaction. Experiments using laboratory model waters showed that free reactive chlorine generated in situ during EC disinfection process was the main disinfectant responsible for the inactivation of microorganisms. However, the production of hydroxyl radicals [OH], and other reactive oxygen species by the active bismuth-doped TiO2 anode were negligible under the same electrolytic conditions. The formation of THMs and HAA5 were found to increase with higher applied cell voltage. Based on the energy consumption estimates, the WEC system can be operated using solar energy stored in a DC battery as the sole power source.

In this work, we report digital loop-mediated isothermal amplification (LAMP) or reverse-transcription LAMP (RT-LAMP) on a commercial membrane, without the need for complex chip fabrication or use of specialized equipment. Due to the pore size distribution, the theoretical error for digital LAMP on these membranes was analyzed, using a combination of Random Distribution Model and Multi-volume Theory. A facile peel-off process was developed for effective droplets formation on the commercial track-etched polycarbonate (PCTE) membrane. Each pore functions as an individual nanoreactor for single DNA amplification. Absolute quantification of bacteria genomic DNA was realized with a dynamic range from 11 to 1.1  105 copies/µL. One-step digital RT-LAMP was also successfully performed on the membrane for the quantification of MS2 virus in wastewater. With the introduction of new probes, the positive pores can be easily distinguished from negative ones with 100 times difference in fluorescence intensities. Finally, the cost of a disposable membrane is less than $0.1/piece, which, to the best of our knowledge, is the most inexpensive way to perform digital LAMP. The membrane system offers opportunities for point-of-care users or common laboratories to perform digital quantification, single cell analysis, or other bioassays in an inexpensive, flexible and simplified way.

Model coliphages (e.g., ΦX174, MS2, and PRD1) have been widely used in place of pathogenic viruses to study their fate and transport in the environment and during wastewater treatment. Two groups of coliphages (F-specific and somatic) are being explored as indicators of viral fecal pollution in ambient water. However, the detection and quantification of coliphages still largely relies on time-consuming culture-based plaque assays. In this study, we developed an in-gel loop mediated isothermal amplification (gLAMP) system enabling coliphage MS2 quantification within 30 minutes using standard laboratory devices. Viral particles (MS2) were immobilized with LAMP reagents in polyethylene glycol hydrogel, and then viral RNAs were amplified through a LAMP reaction. Due to the restriction effect of the hydrogel matrix, one viral particle would only produce one amplicon dot. Therefore, the sample virus concentrations can be determined based on the number of fluorescent amplicon dots using a smartphone for imaging. The method was validated by using artificially spiked and naturally contaminated water samples. gLAMP results were shown to correlate well with plaque assay counts (R²=0.984, p<0.05) and achieved similar sensitivity to quantitative reverse transcription PCR (RT-qPCR) (1 PFU/reaction). Moreover, gLAMP demonstrated a high level of tolerance against inhibitors naturally present in wastewater, in which RT-qPCR was completely inhibited. Besides MS2, gLAMP can also be used for the quantification of other microbial targets (e.g., E. coli and Salmonella). Considering its simplicity, sensitivity, rapidity, and versatility, gLAMP holds a great potential for microbial water quality analysis, especially in resource-limited settings.

In this work, we introduce an asymmetric membrane as a simple and robust nanofluidic platform for digital detection of single pathogenic bacteria directly in 10 mL of unprocessed environmental water samples. The asymmetric membrane, consisting of uniform micropores on one side and a high density of vertically aligned nanochannels on the other side, was prepared within 1 min by a facile method. The single membrane covers all the processing steps from sample concentration, purification, and partition to final digital loop-mediated isothermal amplification (LAMP). By simple filtration, bacteria were enriched and partitioned inside the micropores, while inhibitors typically found in the environmental samples (i.e., proteins, heavy metals, and organics) were washed away through the nanochannels. Meanwhile, large particles, indigenous plankton, and positively charged pollutants in the samples were excluded by using a sacrificial membrane stacked on top. After initial filtration, modified LAMP reagents, including NaF and lysozyme, were loaded onto the membrane. Each pore in the asymmetric membrane functioned as an individual nanoreactor for selective, rapid, and efficient isothermal amplification of single bacteria, generating a bright fluorescence for direct counting. Even though high levels of inhibitors were present, absolute quantification of Escherichia coli and Salmonella directly in an unprocessed environmental sample (seawater and pond water) was achieved within 1 h, with sensitivity down to single cell and a dynamic range of 0.3–10000 cells/mL. The simple and low-cost analysis platform described herein has an enormous potential for the detection of pathogens, exosomes, stem cells, and viruses as well as single-cell heterogeneity analysis in environmental, food, and clinical research.