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The current water quality monitoring and regulation approaches use fecal indicator bacteria (FIB) to indirectly assess health risks from fecal pathogens. Direct detection of waterborne pathogens is expected to provide more accurate and comprehensive risk assessment, which however has been hindered by the lack of methods for simultaneous detection of the numerous waterborne pathogens. This study aimed to develop a mPCR-NGS approach that uses the high sequencing depth of NGS and sequence-based detection to significantly increase the multiplex level of mPCR for direct pathogen detection in water. Individual PCR primers were designed for 16 target marker genes of nine different bacterial pathogens, and an optimal combination of primers with least primer complementarities was identified for the multiplex setting. Using an artificial tester sample, the mPCR system was optimized for annealing temperature and primer concentration, and bioinformatic procedures were developed to directly detect the target marker gene amplicons in NGS sequence reads, which showed simultaneous detection of 14 different target genes in one reaction. The effectiveness of the developed mPCR-NGS approach was subsequently demonstrated on DNA extracts from stream water samples and their counterparts that were spiked with various target pathogen DNA, and all target genes spiked into the environmental water samples were successfully detected. Several key issues for further improving the mPCR-NGS approach were also identified and discussed.

Recently, ozone injection technique was developed as a novel biomass control method to reduce bed clogging in biofilters treating volatile organic compounds (VOCs). However, the effects of ozone on the characteristics of biofilms are still unknown. In this study, two identical lab-scale biofilters treating gaseous toluene were operated in parallel except that one was continuously injected with 200 mg/m(3) ozone. Four glass slides were placed inside each biofilter on day 57 and then were taken out sequentially after 1, 2, 4, and 6 weeks of cultivation. The biofilms grown on the glass slides were stained by the ViaGram™ Red + Bacterial Gram Stain and Viability Kit and observed through the confocal laser scanning microscopy (CLSM). According to the CLSM images of 1, 2, and 4 weeks, the ozonated biofilm was significantly thinner than the control biofilm, which demonstrated that ozone could effectively control the biomass in the biofilter. For the biofilter without ozone injection, the ratios of viable cells (0.51~0.89) and the ratios of Gram-positive bacteria (0.22~0.57) both decreased within 4 weeks of cultivation. The CLSM image analysis results also demonstrated that a continuous injection of 200 mg/m(3) ozone was able to significantly enhance the ratio of viable cells to 0.77~0.97 and allow the dominance of Gram-positive bacteria in the biofilms with the ratio 0.46~0.88 instead of Gram-negative bacteria. For the 6-week samples, the biofilm thickness of the control system was reduced significantly which indicated the detachment of accumulated biofilms might occur in the samples without ozone.

Biofilters treating high-concentration gaseous volatile organic compounds (VOC) can be subject to bed clogging induced by excess biomass accumulation. In this study, O3 was continuously injected into biofilters to control biomass. Its effects on the performance of the biofilters and on biomass accumulation were investigated. Four identical biofilters designed to treat gaseous toluene were operated for 70 days, and three of them were continuously injected with O3 at different concentrations (from 80 to 320 mg/m(3)). The results showed that continuous O3 injection could effectively keep the bed pressure drop stable and had no adverse effect on toluene removal when O3 concentrations were 180-220 mg/m(3). The maximum toluene elimination capacity of the four biofilters was 140 g-toluene/m(3)/h, and the bed pressure drop of the biofilter fed with 180-220 mg/m(3) O3 remained below 3 mmH2O/m throughout the operation period. The biomass accumulation rates of the three biofilters with O3 at 80-320 mg/m(3) were lowered by 0.15-0.25 g/L/day compared with the biofilter without O3. The decreases in biomass accumulation resulted in higher void fractions of the filter beds with O3 injection. Carbon balance analysis indicated that CO2 production had increased while biomass accumulation and leachate waste production decreased in response to O3 injection. Based on the experimental results, it was concluded here that continuous O3 injection can reduce increases in bed pressure effectively, preserve VOC removal capacity, and prevent production of extra leachate waste.

Biofilters treating high-concentration gaseous volatile organic compounds (VOC) can be subject to bed clogging induced by excess biomass accumulation. In this study, O3 was continuously injected into biofilters to control biomass. Its effects on the performance of the biofilters and on biomass accumulation were investigated. Four identical biofilters designed to treat gaseous toluene were operated for 70 days, and three of them were continuously injected with O3 at different concentrations (from 80 to 320 mg/m(3)). The results showed that continuous O3 injection could effectively keep the bed pressure drop stable and had no adverse effect on toluene removal when O3 concentrations were 180-220 mg/m(3). The maximum toluene elimination capacity of the four biofilters was 140 g-toluene/m(3)/h, and the bed pressure drop of the biofilter fed with 180-220 mg/m(3) O3 remained below 3 mmH2O/m throughout the operation period. The biomass accumulation rates of the three biofilters with O3 at 80-320 mg/m(3) were lowered by 0.15-0.25 g/L/day compared with the biofilter without O3. The decreases in biomass accumulation resulted in higher void fractions of the filter beds with O3 injection. Carbon balance analysis indicated that CO2 production had increased while biomass accumulation and leachate waste production decreased in response to O3 injection. Based on the experimental results, it was concluded here that continuous O3 injection can reduce increases in bed pressure effectively, preserve VOC removal capacity, and prevent production of extra leachate waste.