Quantitative detection and reduction of potentially pathogenic bacterial groups of Aeromonas, Arcobacter, Klebsiella pneumoniae species complex, and Mycobacterium in wastewater treatment facilities.

Water quality parameters influence the abundance of pathogenic bacteria. The genera Aeromonas, Arcobacter, Klebsiella, and Mycobacterium are among the representative pathogenic bacteria identified in wastewater. However, information on the correlations between water quality and the abundance of these bacteria, as well as their reduction rate in existing wastewater treatment facilities (WTFs), is lacking. Hence, this study aimed to determine the abundance and reduction rates of these bacterial groups in WTFs. Sixty-eight samples (34 influent and 34 non-disinfected, treated, effluent samples) were collected from nine WTFs in Japan and Thailand. 16S rRNA gene amplicon sequencing analysis revealed the presence of Aeromonas, Arcobacter, and Mycobacterium in all influent wastewater and treated effluent samples. Quantitative real-time polymerase chain reaction (qPCR) was used to quantify the abundance of Aeromonas, Arcobacter, Klebsiella pneumoniae species complex (KpSC), and Mycobacterium. The geometric mean abundances of Aeromonas, Arcobacter, KpSC, and Mycobacterium in the influent wastewater were 1.2 × 104-2.4 × 105, 1.0 × 105-4.5 × 106, 3.6 × 102-4.3 × 104, and 6.9 × 103-5.5 × 104 cells mL-1, respectively, and their average log reduction values were 0.77-2.57, 1.00-3.06, 1.35-3.11, and -0.67-1.57, respectively. Spearman's rank correlation coefficients indicated significant positive or negative correlations between the abundances of the potentially pathogenic bacterial groups and Escherichia coli as well as water quality parameters, namely, chemical/biochemical oxygen demand, total nitrogen, nitrate-nitrogen, nitrite-nitrogen, ammonium-nitrogen, suspended solids, volatile suspended solids, and oxidation-reduction potential. This study provides valuable information on the development and appropriate management of WTFs to produce safe, hygienic water.

A novel approach for toluene gas treatment using a downflow hanging sponge reactor.

A novel gas-scrubbing bioreactor based on a downflow hanging sponge (DHS) reactor was developed as a new volatile organic compound (VOC) treatment system. In this study, the effects of varying the space velocity and gas/liquid ratio were investigated to assess the effectiveness of using toluene gas as a model VOC. Under optimal conditions, the toluene removal rate was greater than 80%, and the maximum elimination capacity was observed at approximately 13 g-C m-3 h-1. The DHS reactor demonstrated slight pressure loss (20 Pa) and a high concentration of suspended solids (up to 30,000 mg/L-sponge). Cloning analysis of the 16S rRNA and functional genes of toluene degradation pathways (tmoA, todC, tbmD, xylA, and bssA) revealed that the clones belonging to the toluene-degrading bacterium Pseudomonas putida constituted the predominant species detected at the bottom of the DHS reactor. The toluene-degrading bacteria Pseudoxanthomonas spadix and Pseudomonas sp. were also detected by tmoA- and todC-targeted cloning analyses, respectively. These results demonstrate the potential for the industrial application of this novel DHS reactor for toluene gas treatment.

Rapid and sensitive identification of marine bacteria by an improved in situ DNA hybridization chain reaction (quickHCR-FISH).

Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with rRNA-targeted oligonucleotide probes has significantly improved the identification of microorganisms in various environmental samples. However, one of the major constraints of CARD-FISH is the low probe penetration due to the high molecular weight of the horseradish peroxidase (HRP) label. Recently, this limitation has been overcome by a novel signal amplification approach termed in situ DNA-hybridization chain reaction (in situ DNA-HCR). In this study, we present an improved and accelerated in situ DNA-HCR protocol (quickHCR-FISH) with increased signal intensity, which was approximately 2 times higher than that of standard in situ DNA-HCR. In addition, the amplification time was only 15 min for the extension of amplifier probes from the initiator probe compared to 2h in the original protocol. The quickHCR-FISH was successfully tested for the quantification of marine bacteria with low rRNA contents in both seawater and sediment samples. It was possible to detect the same number of marine bacteria with quickHCR-FISH compared to CARD-FISH within only 3h. Thus, this newly developed protocol could be an attractive alternative to CARD-FISH for the detection and visualization of microorganisms in their environmental context.

In situ DNA-hybridization chain reaction (HCR): a facilitated in situ HCR system for the detection of environmental microorganisms.

In situ detection of microorganisms by fluorescence in situ hybridization (FISH) is a powerful tool for environmental microbiology, but analyses can be hampered by low rRNA content in target organisms, especially in oligotrophic environments. Here, we present a non-enzymatic, hybridization chain reaction (HCR)-based signal amplified in situ whole-cell detection technique (in situ DNA-HCR). The components of the amplification buffer were optimized to polymerize DNA amplifier probes for in situ DNA-HCR. In situ hybridization of initiator probes followed by signal amplification via HCR produced bright signals with high specificity and probe permeation into cells. The detection rates for Bacteria in a seawater sample and Archaea in anaerobic sludge samples were comparable with or greater than those obtained by catalyzed reporter deposition (CARD)-FISH or standard FISH. Detection of multiple organisms (Bacteria, Archaea and Methanosaetaceae) in an anaerobic sludge sample was achieved by simultaneous in situ DNA-HCR. In summary, in situ DNA-HCR is a simple and easy technique for detecting single microbial cells and enhancing understanding of the ecology and behaviour of environmental microorganisms in situ.

Enrichment of denitrifying methane-oxidizing microorganisms using up-flow continuous reactors and batch cultures.

Denitrifying anaerobic methane oxidizing (DAMO) microorganisms were enriched from paddy field soils using continuous-flow and batch cultures fed with nitrate or nitrite as a sole electron acceptor. After several months of cultivation, the continuous-flow cultures using nitrite showed remarkable simultaneous methane oxidation and nitrite reduction and DAMO bacteria belonging to phylum NC10 were enriched. A maximum volumetric nitrite consumption rate of 70.4±3.4 mg-N·L(-1)·day(-1) was achieved with very short hydraulic retention time of 2.1 hour. In the culture, about 68% of total microbial cells were bacteria and no archaeal cells were detected by fluorescence in situ hybridization. In the nitrate-fed continuous-flow cultures, 58% of total microbial cells were bacteria while archaeal cells accounted for 7% of total cell numbers. Phylogenetic analysis of pmoA gene sequence showed that enriched DAMO bacteria in the continuous-flow cultivation had over 98% sequence similarity to DAMO bacteria in the inoculum. In contrast, for batch culture, the enriched pmoA gene sequences had 89-91% sequence similarity to DAMO bacteria in the inoculum. These results indicate that electron acceptor and cultivation method strongly affect the microbial community structures of DAMO consortia.

A long-term cultivation of an anaerobic methane-oxidizing microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor.

Anaerobic oxidation of methane (AOM) in marine sediments is an important global methane sink, but the physiological characteristics of AOM-associated microorganisms remain poorly understood. Here we report the cultivation of an AOM microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor with polyurethane sponges, called the down-flow hanging sponge (DHS) bioreactor. We anaerobically incubated deep-sea methane-seep sediment collected from the Nankai Trough, Japan, for 2,013 days in the bioreactor at 10°C. Following incubation, an active AOM activity was confirmed by a tracer experiment using 13C-labeled methane. Phylogenetic analyses demonstrated that phylogenetically diverse Archaea and Bacteria grew in the bioreactor. After 2,013 days of incubation, the predominant archaeal components were anaerobic methanotroph (ANME)-2a, Deep-Sea Archaeal Group, and Marine Benthic Group-D, and Gammaproteobacteria was the dominant bacterial lineage. Fluorescence in situ hybridization analysis showed that ANME-1 and -2a, and most ANME-2c cells occurred without close physical interaction with potential bacterial partners. Our data demonstrate that the DHS bioreactor system is a useful system for cultivating fastidious methane-seep-associated sedimentary microorganisms.

Laser absorption spectrometer using frequency chirped intensity modulation at 1.57 µm wavelength for CO2 measurement.

We have demonstrated the laser-absorption spectrometer system using frequency chirped intensity modulation at 1.57 µm wavelength for measurement of CO(2) concentration. Using this technique, backscattered laser radiation from different ranges can be discriminated in the frequency domain of the electrical signal. We have reported the discrimination of two signals from the targets with different ranges. It is shown that stable measurements with short time fluctuation corresponding to 4 ppm (rms) were obtained with 32 s measurement intervals. Furthermore, there is qualitative good agreement on, at least, the diurnal changes between the results of the laser absorption spectrometer system and the in-situCO(2) sensor.

Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes.

In situ detection of functional genes with single-cell resolution is currently of interest to microbiologists. Here, we developed a two-pass tyramide signal amplification (TSA)-fluorescence in situ hybridization (FISH) protocol with PCR-derived polynucleotide probes for the detection of single-copy genes in prokaryotic cells. The mcrA gene and the apsA gene in methanogens and sulfate-reducing bacteria, respectively, were targeted. The protocol showed bright fluorescence with a good signal-to-noise ratio and achieved a high efficiency of detection (>98%). The discrimination threshold was approximately 82-89% sequence identity. Microorganisms possessing the mcrA or apsA gene in anaerobic sludge samples were successfully detected by two-pass TSA-FISH with polynucleotide probes. The developed protocol is useful for identifying single microbial cells based on functional gene sequences.

Feasibility study on 1.6 µm continuous-wave modulation laser absorption spectrometer system for measurement of global CO2 concentration from a satellite.

A feasibility study is carried out on a 1.6 µm continuous-wave modulation laser absorption spectrometer system for measurement of global CO(2)concentration from a satellite. The studies are performed for wavelength selection and both systematic and random error analyses. The systematic error in the differential absorption optical depth (DAOD) is mainly caused by the temperature estimation error, surface pressure estimation error, altitude estimation error, and ON wavelength instability. The systematic errors caused by unwanted backscattering from background aerosols and dust aerosols can be reduced to less than 0.26% by using a modulation frequency of around 200 kHz, when backscatter coefficients of these unwanted backscattering have a simple profile on altitude. The influence of backscattering from cirrus clouds is much larger than that of dust aerosols. The transmission power required to reduce the random error in the DAOD to 0.26% is determined by the signal-to-noise ratio and the carrier-to-noise ratio calculations. For a satellite altitude of 400 km and receiving aperture diameter of 1 m, the required transmission power is approximately 18 W and 70 W when albedo is 0.31 and 0.08, respectively; the total measurement time in this case is 4 s, which corresponds to a horizontal resolution of 28 km.

Performance improvement and analysis of a 1.6 µm continuous-wave modulation laser absorption spectrometer system for CO2 sensing.

In a previous study, we developed a 1.6 µm continuous-wave (cw) modulation laser absorption spectrometer system for CO(2) sensing and demonstrated the measurement of small fluctuations in CO(2) corresponding to a precision of 4 parts per million (ppm) with a measurement interval of 32 s. In this paper, we present the process to achieve this highly specific measurement by introducing important points, which have not been shown in the previous study. Following the results of preliminary experiments, we added a function for speckle averaging on the optical antenna unit. We additionally came up with some ideas to avoid the influences of etalon effects and polarization dependence in optical components. Because of the new functions, we realized a calibration precision of 0.006 dB (rms), which corresponds to a CO(2) concentration precision of less than 1 ppm for a 2 km path. We also analyzed the CO(2) sensing performance after the improvements described above. The measured short time fluctuation of the differential absorption optical depth was reasonably close to that calculated using the carrier-to-noise ratio of the received signal.

Performance of a pilot-scale sewage treatment: an up-flow anaerobic sludge blanket (UASB) and a down-flow hanging sponge (DHS) reactors combined system by sulfur-redox reaction process under low-temperature conditions.

Performance of a wastewater treatment system utilizing a sulfur-redox reaction of microbes was investigated using a pilot-scale reactor that was fed with actual sewage. The system consisted of an up-flow anaerobic sludge blanket (UASB) reactor and a down-flow hanging sponge (DHS) reactor with a recirculation line. Consequently, the total CODCr (465±147 mg L(-1); total BOD of 207±68 mg L(-1)) at the influent was reduced (70±14 mg L(-1); total BOD of 9±2 mg L(-1)) at the DHS effluent under the conditions of an overall hydraulic retention time of 12 h, a recirculation ratio of 2, and a low-sewage temperature of 7.0±2.8 °C. A microbial analysis revealed that sulfate-reducing bacteria contributed to the degradation of organic matter in the UASB reactor even in low temperatures. The utilized sulfur-redox reaction is applicable for low-strength wastewater treatment under low-temperature conditions.

Detection of single copy genes by two-pass tyramide signal amplification fluorescence in situ hybridization (Two-Pass TSA-FISH) with single oligonucleotide probes.

In situ detection of functional genes is informative for understanding microbial physiology. Most methods of detecting functional genes employ multiple oligonucleotides or polynucleotide probes. However, single oligonucleotide probes are superior in terms of specificity and flexibility in probe design. Here we describe the detection of a single copy functional gene, the methyl coenzyme M reductase gene, in a methanogen by two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with a single oligonucleotide probe without pre-amplification of target nucleic acids. Locked-nucleic-acid-incorporated DNA probes were employed to achieve high specificity and affinity. Although problems associated with non-removable nonspecific binding of the antibody could not be overcome completely, single copy gene detection was carried out with single mismatch descriminatable specificity; however, only around 15% of cells were detected. The detection rate increased when a multiple copy gene like rrn in Escherichia coli was targeted, indicating that a certain number of target molecules are necessary to achieve a high detection rate. Although possible applications of this technique to environmental samples remain restricted, the results presented the potential of gene detection by FISH with single oligonucleotide probes.

Development of 1.6 microm continuous-wave modulation hard-target differential absorption lidar system for CO2 sensing.

We have demonstrated the 1.6 mum cw modulation hard-target differential absorption lidar system for CO(2) sensing. In this system, ON and OFF wavelength laser lights are intensity modulated with cw signals. Received lights of the two wavelengths from the hard target are discriminated by modulation frequencies in the electrical signal domain. The optical circuit is fiber based, and this makes the system compact and reliable. It is shown that a stable CO(2) concentration measurement corresponding to a fluctuation of 4 ppm (rms) (ppm is parts per million) has been achieved in 32 s measurement intervals and the 1 km path.

Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens.

Several enzymatic permeabilization protocols (utilizing lysozyme, proteinase K, achromopeptidase, or recombinant pseudomurein endopeptidase [PeiW]) were evaluated for application of in situ hybridization with horseradish peroxidase-labeled oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) to methanogens. In this study, twelve methanogens were selected that have typical cell surface structures: pseudomurein, surface layer, methanochondroitin and sheath. Among the treatments tested, PeiW treatment was observed to be the most effective one, although methanogens having a sheath were stained heterogeneously and methanogens having methanochondroitin were not permeabilized. On the other hand, lysozyme, proteinase K, and achromopeptidase treatments were ineffective or caused cell-lysis, resulting in weak or no signals. Applicability of PeiW treatment was further evaluated using an anaerobic granular sludge sample. The detection rate of Archaea by CARD-FISH increased remarkably after the treatment. Based on the results obtained in this study, we propose PeiW treatment as a novel permeabilization method for CARD-FISH application to methanogens.

Microbial community that catalyzes partial nitrification at low oxygen atmosphere as revealed by 16S rRNA and amoA genes.

We previously reported that partial nitrification in the down-flow hanging sponge (DHS) system was satisfactorily accomplished under oxygen-limited conditions [Chuang et al., Water Res., 41, 295-302 (2007)]. In this study, we investigated the microbes that are responsible for the partial nitrification in this unique system by 16S rRNA- and amoA-based cloning analyses and fluorescence in situ hybridization. Microbes related to Nitrosomonas species were found to be chiefly responsible for catalyzing the partial nitrification. Microbes affiliated with the uncultivated phyla OP10 and Bacteroidetes were also numerous in the DHS, but their ecological niches are still unknown.