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Satoshi Tsuneda

Publications and source records attributed to Satoshi Tsuneda.

At least 19 recordsLinked to original sources

Redox-stratification controlled biofilm (ReSCoBi) for completely autotrophic nitrogen removal: the effect of co- versus counter-diffusion on reactor performance.

A multi-population biofilm model for completely autotrophic nitrogen removal was developed and implemented in the simulation program AQUASIM to corroborate the concept of a redox-stratification controlled biofilm (ReSCoBi). The model considers both counter- and co-diffusion biofilm geometries. In the counter-diffusion biofilm, oxygen is supplied through a gas-permeable membrane that supports the biofilm while ammonia (NH(4)(+)) is supplied from the bulk liquid. On the contrary, in the co-diffusion biofilm, both oxygen and NH(4)(+) are supplied from the bulk liquid. Results of the model revealed a clear stratification of microbial activities in both of the biofilms, the resulting chemical profiles, and the obvious effect of the relative surface loadings of oxygen and NH(4)(+) (J(O(2))/J(NH(4)(+))) on the reactor performances. Steady-state biofilm thickness had a significant but different effect on T-N removal for co- and counter-diffusion biofilms: the removal efficiency in the counter-diffusion biofilm geometry was superior to that in the co-diffusion counterpart, within the range of 450-1,400 microm; however, the efficiency deteriorated with a further increase in biofilm thickness, probably because of diffusion limitation of NH(4)(+). Under conditions of oxygen excess (J(O(2))/J(NH(4)(+)) > 3.98), almost all NH(4)(+) was consumed by aerobic ammonia oxidation in the co-diffusion biofilm, leading to poor performance, while in the counter-diffusion biofilm, T-N removal efficiency was maintained because of the physical location of anaerobic ammonium oxidizers near the bulk liquid. These results clearly reveal that counter-diffusion biofilms have a wider application range for autotrophic T-N removal than co-diffusion biofilms.

Ammonia↗

Effects of acetate and nitrite addition on fraction of denitrifying phosphate-accumulating organisms and nutrient removal efficiency in anaerobic/aerobic/anoxic process.

The effects of acetate and nitrite on the performance of sequencing batch reactors (SBRs) employing an anaerobic/aerobic/anoxic (AOA) process were investigated. Three types of SBR operations were used: sodium acetate addition at the start of anoxic condition for heterotrophic denitrification (Type 1); sodium acetate addition at the start of aerobic condition for anoxic phosphate removal by denitrifying phosphate-accumulating organisms (DNPAOs) (Type 2: conventional AOA process); and nitrite addition at the start of aerobic condition for inhibition of phosphate-accumulating organisms (PAOs) (Type 3). A track experiment shows that Type 2 led to the best performance of SBRs among the three types. An analysis by fluorescence in situ hybridization (FISH) revealed that nitrite addition decreased the ratio of PAOs with a decrease in phosphorus removal efficiency. The fraction of DNPAOs in Type 2 was the highest at 13%, indicating that Type 2 is suitable for the simultaneous nitrogen and phosphorus removal in the AOA process.

Acetates↗

Identification of acetate- or methanol-assimilating bacteria under nitrate-reducing conditions by stable-isotope probing.

Stable-isotope probing (SIP) was used to identify acetate- or methanol-assimilating bacteria under nitrate-reducing conditions in activated sludge. A sludge sample obtained from wastewater treatment systems was incubated in a denitrifying batch reactor fed with synthetic wastewater containing [(13)C]acetate or [(13)C]methanol as the main carbon source and nitrate as the electron acceptor. We analyzed how growth of bacterial populations was stimulated by acetate or methanol as the external carbon source in nitrogen-removal systems. Most of the acetate- or methanol-assimilating bacteria identified by SIP have been known as denitrifiers in wastewater treatment systems. When acetate was used as the carbon source, 16S rRNA gene sequences retrieved from (13)C-labeled DNA were closely related to the 16S rRNA genes of Comamonadaceae (e.g., Comamonas and Acidovorax) and Rhodocyclaceae (e.g., Thauera and Dechloromonas) of the Betaproteobacteria, and Rhodobacteraceae (e.g., Paracoccus and Rhodobacter) of the Alphaproteobacteria. When methanol was used as the carbon source, 16S rRNA gene sequences retrieved from (13)C-DNA were affiliated with Methylophilaceae (e.g., Methylophilus, Methylobacillus, and Aminomonas) and Hyphomicrobiaceae. Rarefaction curves for clones retrieved from (13)C-DNA showed that the diversity levels for methanol-assimilating bacteria were considerably lower than those for acetate-assimilating bacteria. Furthermore, we characterized nitrite reductase genes (nirS and nirK) as functional marker genes for denitrifier communities in acetate- or methanol-assimilating populations and detected the nirS or nirK sequence related to that of some known pure cultures, such as Alcaligenes, Hyphomicrobium, and Thauera. However, most of the nirS or nirK sequences retrieved from (13)C-DNA were clustered in some unidentified groups. On the basis of 16S rRNA gene clone libraries retrieved from (13)C-DNA, these unidentified nir sequences might be identified by examining the nir gene in candidates for true denitrifiers (e.g., the families Comamonadaceae, Hyphomicrobiaceae, Methylophilaceae, and Rhodobacteraceae).

Acetates↗

Sequencing batch membrane biofilm reactor for simultaneous nitrogen and phosphorus removal: novel application of membrane-aerated biofilm.

A sequencing batch membrane biofilm reactor (SBMBfR) was developed for simultaneous carbon, nitrogen, and phosphorus removal from wastewater. This reactor was composed of two functional parts: (1) a gas-permeable membrane on which a nitrifying biofilm formed and (2) a bulk solution in which bacteria, mainly denitrifying polyphosphate-accumulating organisms (DNPAOs), were suspended. The reactor was operated sequentially under anaerobic condition and then under membrane aeration condition in one cycle. During the anaerobic period, organic carbon was consumed by DNPAOs; this was accompanied by phosphate release. During the subsequent membrane aeration period, nitrifying bacteria utilized oxygen supplied directly to them from the inside of the membrane. Consequently, the nitrite and nitrate products diffused into the bulk solution, where they were used by DNPAOs as electron acceptors for phosphate uptake. In a long-term sequencing batch operation, the mean removal efficiencies of total organic carbon (TOC), total nitrogen (T-N), and total phosphorus (T-P) under steady-state condition were 99%, 96%, and 90%, respectively. In addition, fluorescence in situ hybridization (FISH) clearly demonstrated the difference in bacterial community structure between the membrane biofilm and the suspended sludge: ammonia-oxidizing bacteria belonging to the Nitrosomonas group were dominant in the region adjacent to the membrane throughout the operation, and the occupation ratio of the well-known polyphosphate-accumulating organism (PAO) Candidatus "Accumulibacter phosphates" in the suspended sludge gradually increased to a maximum of 37%.

Bacteria↗

Real-time quantitative LAMP (loop-mediated isothermal amplification of DNA) as a simple method for monitoring ammonia-oxidizing bacteria.

Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique for the amplification of DNA under isothermal conditions. For the first time, we applied this method to develop a simple and quantitative monitoring method for environmental microorganisms targeting amoA gene in ammonia-oxidizing bacteria. Quantitative analysis was performed first by measuring fluorescence derived from an intercalation dye using a real-time thermal cycler, and then by measuring the turbidity of the reaction solution using a real-time turbidimeter. As a result, it was possible to quantify the initial amoA DNA concentration from an environment with a sensitivity down to 10(2) DNA copies of target DNA and a dynamic range of 7-9 orders in magnitude. Background DNA from nontargeted bacteria (Pseudomonas denitrificans) that does not encode amoA gene did not affect the quantitative capability of LAMP. Over results suggested that the real-time LAMP is effective for monitoring microorganisms and their gene expression in environments.

Ammonia↗

Affinity capillary electrophoresis with a DNA-nanoparticle conjugate as a new tool for genotyping.

We have developed a novel method for genotyping based on free solution affinity capillary electrophoresis. We prepared DNA-nanoparticle conjugates by mixing biotin-modified DNA and NeutrAvidin-modified polystyrene nanoparticles; this mixture was then injected into a capillary. Subsequently, we injected the fluorescent-labeled sample DNAs into the capillary, applied the voltage, increased its temperature after 7 min, and detected the fluorescence at its anodic end. This novel method was applied for genotyping human c-K-ras, and the three genotypes were definitely distinguishable with high reproducibility. This method can be easily automated, and it is useful for high-throughput gene mutation analysis.

Base Sequence↗

Anaerobic/oxic/anoxic granular sludge process as an effective nutrient removal process utilizing denitrifying polyphosphate-accumulating organisms.

In a biological nutrient removal (BNR) process, the utilization of denitrifying polyphosphate-accumulating organisms (DNPAOs) has many advantages such as effective use of organic carbon substrates and low sludge production. As a suitable process for the utilization of DNPAOs in BNR, an anaerobic/oxic/anoxic granular sludge (AOAGS) process was proposed in this study. In spite of performing aeration for nitrifying bacteria, the AOAGS process can create anaerobic/anoxic conditions suitable for the cultivation of DNPAOs because anoxic zones exist inside the granular sludge in the oxic phase. Thus, DNPAOs can coexist with nitrifying bacteria in a single reactor. In addition, the usability of DNPAOs in the reactor can be improved by adding the anoxic phase after the oxic phase. These characteristics enable the AOAGS process to attain effective removal of both nitrogen and phosphorus. When acetate-based synthetic wastewater (COD: 600 mg/L, NH4-N: 60 mg/L, PO(4)-P: 10 mg/L) was supplied to a laboratory-scale sequencing batch reactor under the operation of anaerobic/oxic/anoxic cycles, granular sludge with a diameter of 500 microm was successfully formed within 1 month. Although the removal of both nitrogen and phosphorus was almost complete at the end of the oxic phase, a short anoxic period subsequent to the oxic phase was necessary for further removal of nitrogen and phosphorus. As a result, effluent concentrations of NH(4)-N, NO(x)-N and PO(4)-P were always lower than 1 mg/L. It was found that penetration depth of oxygen inside the granular sludge was approximately 100 microm by microsensor measurements. In addition, from the microbiological analysis by fluorescence in situ hybridization, existence depth of polyphosphate-accumulating organisms was further than the maximum oxygen penetration depth. The water quality data, oxygen profiles and microbial community structure demonstrated that DNPAOs inside the granular sludge may be responsible for denitrification in the oxic phase, which enables effective nutrient removal in the AOAGS process.

Bacteria, Aerobic↗

Bacterial adhesion to and viability on positively charged polymer surfaces.

Secondary and tertiary amino groups were introduced into polymer chains grafted onto a polyethylene flat-sheet membrane to evaluate the effects of surface properties on the adhesion and viability of a strain of the Gram-negative bacterium Escherichia coli and a strain of the Gram-positive bacterium Bacillus subtilis. The characterization of the surfaces containing amino groups, i.e. ethylamino (EA) and diethylamino (DEA) groups, revealed that the membrane potentials are proportional to amino-group densities and contact angle hysteresis. A high bacterial adhesion rate constant k was observed at high membrane potential, which indicates that membrane potential could be used as an indicator for estimating bacterial adhesion to the EA and DEA sheets, especially in B. subtilis. The bacterial adhesion rate constant of E. coli markedly increased at a membrane potential higher than -7.8 mV, whereas that of B. subtilis increased at a membrane potential higher than -8.3 mV, at which the dominant effect on bacterial adhesion is expected to change. The viability experiments revealed that approximately 80% of E. coli cells adhering to the sheets with high membrane potential were inactivated after a contact time of 8 h, whereas 60% of B. subtilis cells were inactivated. Furthermore, E. coli viability significantly decreased at a membrane potential higher than -8 mV, whereas B. subtilis viability decreased as membrane potential increased, which reflects differences in cell wall structure between E. coli and B. subtilis.

Bacillus subtilis↗

Long-term monitoring of the succession of a microbial community in activated sludge from a circulation flush toilet as a closed system.

The microbial diversity and community succession of a circulation flush toilet were investigated by terminal restriction fragment length polymorphism and cloning analyses. Clonal libraries of 16S rRNA gene on day 3 and day 127 were constructed. On day 3, 102 clones were sequenced; Proteobacteria and Bacteroidetes accounted for 27% and 45%, respectively. On day 127, Proteobacteria had increased to 43% and Bacteroidetes had decreased to 26% of a total of 100 clones. Terminal restriction fragment length polymorphism peaks were identified by in silico analysis of clone libraries. The relative abundances of Nitrosomonas increased from 1% to 6% with commencement of nitrification and denitrification. Similarly, the relative abundance of terminal restriction fragments generated from Xanthomonas increased from 3% to 10%. Therefore, these bacteria could play a prominent role in this process. To reveal the relationship between stability of the microbial community and performance of the system, microbial community succession was visualized by multidimensional scaling analysis. The microbial community structure changed markedly, particularly during the start-up period of the system. The plots then became stable after the start of nitrification and denitrification. This result suggests that the succession of microbial community structure had a correlation with the performance of the system.

Bacteroidetes↗

Highly sensitive real-time PCR assay for quantification of toxic cyanobacteria based on microcystin synthetase A gene.

The presence of cyanobacterial bloom in water supply reservoirs can cause potential health hazards. In this study, we aimed at the quantification of microcystin-producing cyanobacteria based on the microcystin synthetase A (mcyA) gene using real-time PCR. To perform a highly sensitive real-time PCR assay, the novel primer MSR-2R was designed and a coprecipitation DNA extraction method was used in this study. Cyanobacterial cells could be collected efficiently by coprecipitation with other bacteria suspended in solution even in the case of low concentrations of cyanobacteria. The detection limit of the method was found to be 8.8 cells per reaction. When cyanobacterial growth was monitored in pure culture, the cell concentration determined by real-time PCR positively correlated with the cell concentration determined from direct microscopic count. Furthermore, we could detect and quantify the mcyA gene in lake water samples using real-time PCR. It was concluded that the quantification of the mcyA gene based on real-time PCR is a powerful tool for the rapid quantification of microcystin-producing cyanobacteria in environmental samples.

Bacterial Proteins↗

Molecular analysis of halophilic bacterial community for high-rate denitrification of saline industrial wastewater.

A denitrification system for saline wastewater utilizing halophilic denitrifying bacteria has not been developed so far. In this study, denitrification performance and microbial community under various saline conditions were investigated using denitrifying sludge acclimated under low-salinity condition for a few years as seed sludge. A continuous denitrification experiment showed that denitrification performance and microbial community at 10% salinity was higher than that at 1% salinity. The microbial community in the denitrification sludge that was acclimated under low salinity was monitored by terminal-restriction fragment length polymorphism (T-RFLP) analysis during acclimation to high-salinity condition. T-RFLP profiles and clone analysis based on 16S rRNA-encoding genes in the sludge of the denitrification system with 10% salinity indicated that the gamma-Proteobacteria, particularly Halomonas spp., were predominant species, suggesting that these bacterial members were possibly responsible for a high denitrification activity under high-salinity conditions. Furthermore, the investigation of denitrification performance under various saline conditions revealed that 4-10% salinity results in the highest denitrification rate, indicating that this salinity was optimal for predominant bacterial species to exhibit denitrification activity. These results indicate the possibility that an appropriate denitrification system for saline wastewater can be designed using acclimated sludge with a halophilic community.

Bacteria↗

Growth characteristic of anaerobic ammonium-oxidizing bacteria in an anaerobic biological filtrated reactor.

The doubling time of anaerobic ammonium-oxidizing (anammox) bacteria in an anaerobic biological filtrated (ABF) reactor was determined. Fluorescence in situ hybridization analysis was used to detect and count anammox bacteria cells in anammox sludge. As a result, the populations of anammox bacteria at 14th and 21st days were 1.1 x 10(6) and 1.7 x 10(7) cells/ml reactor, respectively. From these results, the doubling time of anammox bacteria was calculated as 1.8 days, and the specific growth rate (mu) was 0.39 day(-1). This result indicated that the anammox bacteria have higher growth rate than the reported value (doubling time, 11 days). Furthermore, it was clearly demonstrated that nitrogen conversion rate was proportional to the population of anammox bacteria. Maintaining the ideal environment for the growth of anammox bacteria in the ABF reactor might lead to faster growth. This is the first report of the growth rate of anammox bacteria based on the direct counting of anammox bacteria.

Anaerobiosis↗

Elucidation of dominant effect on initial bacterial adhesion onto polymer surfaces prepared by radiation-induced graft polymerization.

Surface-modified polyethylene (PE) membrane sheets were prepared by the radiation-induced graft polymerization (RIGP) of an epoxy-group-containing monomer, glycidyl methacrylate (GMA). The epoxy ring of GMA was opened by introducing diethylamine (DEA) or sodium sulfite (SS). We examined the properties of these sheets by measuring the amount of grafting polymer, surface roughness and membrane potential, and also investigated the adhesion of five Gram-negative bacteria, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens and Paracoccus denitrificans, onto the prepared sheet surfaces. A linear relationship between the degree of grafting (dg) and surface roughness was observed. Moreover, membrane potential was dependent on the amount of DEA or SS as the ionizable group. These results indicate that RIGP enables the control of the physicochemical properties of such a sheet surface by adjusting dg and the subsequent conversion of functional groups. A batch test on bacterial adhesion onto the sheets clarified that the DEA-containing sheet (DEA sheet) exhibited an adhesion rate constant, k, significantly greater than those of other types of sheet. Clearly, the adhesion rate constant of the DEA sheet increased with dg, indicating that electrostatic interaction is the most decisive factor for bacterial adhesion when it works as an attractive force. Furthermore, the densities of bacteria adhering onto the GMA-containing sheet (GMA sheet) and the SS-containing sheet (SS sheet) were almost the same as that onto a PE sheet, whereas that onto a DEA sheet significantly increased. Thus, the introduction of the GMA- and SS-containing graft chain did not have much influence on bacterial adhesion onto the surfaces, supporting the conclusion that the promotion of bacterial adhesion onto the GMA and SS sheets was due to an increase in surface area resulting from RIGP. Moreover, the scanning electron microscopy images of the sheet surfaces indicate that the conditions and morphologies of initial bacterial adhesion are dependent on surface properties, particularly membrane potential.

Bacterial Adhesion↗

Quantification of genetically modified soybean by quenching probe polymerase chain reaction.

Quenching probe (QProbe) polymerase chain reaction (PCR) is a simple and cost-effective real-time PCR assay in comparison with other real-time PCR assays such as the TaqMan assay. We used QProbe-PCR to quantify genetically modified (GM) soybean (Roundup Ready soybean). We designed event-specific QProbes for Le1 (soy endogenous gene) and RRS (recombinant gene), and we quantified certified reference materials containing 0.1, 0.5, 1, 2, and 5% GM soybean. The TaqMan assay was also applied to the same samples, and the results were compared. The accuracy of QProbe-PCR was similar to that of TaqMan assay. When GM soybean content was 0.5% or more, the relative standard deviations of QProbe-PCR were less than 20%. QProbe-PCR is sensitive enough to monitor labeling systems and has acceptable levels of accuracy and precision.

DNA Probes↗

Effect of salinity on nitrous oxide emission in the biological nitrogen removal process for industrial wastewater.

The effects of wastewater salinity on both nitrogen removal efficiency and N2O emission rate were investigated in a single nitrification process, a single denitrification process and an anoxic-oxic activated sludge process. In the single nitrification process, by increasing the salt concentration from 1.0 to 2.0 wt%, the N2O conversion ratio in the steady state increased by 2.2 times, from 0.22 to 0.48%. In the single denitrification process, a minimal change in the N2O conversion ratio was observed in the steady state even when the salt concentration was increased from 3.0 to 5.0 wt%. From the results of the anoxic-oxic activated sludge process, it was found that a salt concentration increase from 1.6 to 3.0 wt% significantly increases the N2O conversion ratio from 0.7 to 13%. It is suggested that an increase in salt concentration markedly influences N2O emission both directly and indirectly via the inhibition of N2O reductase activity. The indirect inhibition is due to the high concentration of dissolved oxygen which is transported from the oxic tank to the anoxic tank through the circulated liquid. Thus, the salt concentration should be maintained below 3.0% to suppress N2O emission in an anoxic-oxic activated sludge process.

Anesthetics, Inhalation↗

Characterization of denitrifying polyphosphate-accumulating organisms in activated sludge based on nitrite reductase gene.

Nitrite reductase gene (nirS) fragments in the activated sludge obtained from a sequencing batch reactor (SBR) under anaerobic-aerobic condition were cloned and classified by restriction fragment length polymorphism (RFLP) analysis, and representative fragments were sequenced. One of the nirS clones was approximately 70% of all nirS clones in anaerobic/aerobic (existing oxygen and nitrate) cycle operation in which a large amount of anoxic phosphate uptake was observed. Although the activated sludge samples analyzed might contain bacteria that did not accumulate polyphosphate, it was likely that this nirS fragment sequence was that from denitrifying polyphosphate-accumulating organisms (DNPAOs) which can utilize both oxygen and nitrate as electron acceptors. The sequence was similar to the nirS sequences of Thauera mechernichensis (83% similarity) and Azoarcus tolulyticus (83% similarity) both of which belong to the Rhodocyclus group.

Genes, Bacterial↗

Significance of cell electrokinetic properties determined by soft-particle analysis in bacterial adhesion onto a solid surface.

The influence of extracellular polymeric substances (EPSs) on bacterial cell electrokinetic properties and on cell adhesion onto glass beads in connection with bacterial cell electrokinetic properties was investigated using 12 heterotrophic bacterial strains. Bacterial cell surface properties such as the softness 1/lambda and charge density ZN were determined by Ohshima's soft-particle analysis using the measured electrophoretic mobility as a function of ionic strength. In 10 of 12 strains, when EPSs covering the cell surface were removed, the softness of the cell decreased, indicating that EPS adsorption enhanced the ease of liquid fluid in the ion-penetrable layer on the cell surface. On the other hand, the negative charge density of the cell surface increased for 9 of 12 strains, suggesting that EPSs covering the cell surface decreased the negative charge density of the cell surface layer. In addition, the characteristics of bacterial cell adhesion onto glass beads were evaluated by the packed-bed method and the data were interpreted to indicate cell adhesiveness. As a result, the efficiency of cell adhesion onto glass beads increased as negative cell surface potential psi0 decreased, whereas there seemed to be no correlation between zeta potential and cell adhesiveness. Cell surface potential psi0, which was derived by taking the bacterial polymer layer with EPSs into consideration, provided a more detailed understanding of the electrokinetic properties of bacterial cells.

Bacteria↗

Expression of amoA mRNA in wastewater treatment processes examined by competitive RT-PCR.

The expression of ammonia monooxygenase encoding mRNA (amoA mRNA) in a wastewater treatment process was analyzed in an attempt to propose an effective target for the monitoring of nitrifying bacteria in engineered systems or natural environments. The quick response (1-2 h) of amoA mRNA transcription to the recovery of ammonia oxidation activity induced by the sudden exposure to ammonia was observed in a short-time batch-mode incubation whereas the amount of amoA DNA did not markedly change during the incubation under any conditions. In the continuous feeding-operation, amoA mRNA level dynamically changed in response to the change in the surrounding environmental conditions and increase in ammonia oxidation rate. Although, amoA mRNA level did not quickly respond to the decrease in ammonia oxidation activity, it decreases over long time scales. These results suggest that the profiles of amoA mRNA expression can be used as an indicator of the ammonia oxidation activity.

Ammonia↗