Transcriptomic response of HepG2 cells exposed to three common anti-inflammatory drugs: Ketoprofen, mefenamic acid, and diclofenac in domestic wastewater effluents.

The biological impacts of residual pharmaceuticals in the complex wastewater effluents have not been fully understood. Here, we investigated changes in the transcriptomic responses of hepatobrastoma (HepG2) cells exposed to a single or partially combined three common non-steroidal anti-inflammatory drugs (NSAIDs); ketoprofen (KPF), mefenamic acid (MFA) and diclofenac (DCF), in domestic wastewater effluents. After 48 h sub-lethal exposure to single compounds, the DNA microarray analysis identified 57-184 differently expressed genes (DEGs). The hierarchical clustering analysis and GO enrichment of the DEGs showed that gene expression profiles of the NSAIDs were distinct from each other although they are classified into the same therapeutic category. Four maker genes (i.e., EGR1, AQP3, SQSTM1, and NAG1) were further selected from the common DEGs, and their expressions were quantified by qPCR assay in a dose-dependent manner (ranging from µg/L to mg/L). The results revealed the insignificant induction of the marker genes at 1 µg/L of KPF, MFA, and DCF, suggesting negligible biological impacts of the NSAIDs on gene expression (early cellular responses) of HepG2 at typical concentration levels found in the actual wastewater effluents. Based on the quantitative expression analysis of the selected marker genes, the present study indicated that the presence of wastewater effluent matrix may mitigate the potentially adverse cellular impacts of the NSAIDs.

Viral Interference as a Factor of False-Negative in the Infectious Adenovirus Detection Using Integrated Cell Culture-PCR with a BGM Cell Line.

This study investigated the influence of viral interference on the detection of enteric viruses using the integrated cell culture (ICC)-PCR with a BGM cell line. It was possible to detect 102 plaque-forming units (PFU)/flask of enterovirus 71 (EV71) in spite of the presence of 104 PFU/flask of adenovirus 40 (AdV40). Meanwhile, 104 PFU/flask of AdV40 was not detected in the presence of 102 PFU/flask of EV71. This inhibition of AdV40 detection using ICC-PCR was attributable to the growth of EV71, because the addition of a growth inhibitor of EV71 (rupintrivir) neutralized the detection inhibition of AdV40. The growth inhibition of AdV40 under co-infection with EV71 is probably caused by the immune responses of EV71-infected cells. AdV is frequently used as a fecal contamination indicator of environmental water, but this study demonstrated that false-negative detection of infectious AdV using ICC-PCR could be caused by the co-existence of infectious EV in a water sample. The addition of rupintrivir could prevent false-negative detection of AdV using ICC-PCR. This study, therefore, emphasizes the importance of confirming the presence of multiple enteric viruses in a sample derived from environmental water prior to the application of ICC-PCR because the viral interference phenomenon may lead to the false-negative detection of target viruses.

Simultaneous removal of nitrate and heavy metals in a continuous flow nitrate-dependent ferrous iron oxidation (NDFO) bioreactor.

Nitrogen and heavy metals can co-occur in various industrial wastewaters such as coke-oven wastewater. Removal of these contaminants is important, but cost-efficient removal technology is limited. In this study, we examined the usefulness of nitrate-dependent ferrous iron oxidation (NDFO) for the simultaneous removal of nitrate and heavy metals (iron and zinc), by using an NDFO strain Pseudogulbenkiania sp. NH8B. Based on the batch culture assays, nitrate, Fe, and Zn were successfully removed from a basal medium as well as coke-oven wastewater containing 5 mM nitrate, 10 mM Fe(II), and 10 mg/L Zn. Zinc in the water was most likely co-precipitated with Fe(III) oxides produced during the NDFO reaction. Simultaneous removal of nitrate, Fe, and Zn was also achieved in a continuous-flow reactor fed with a basal medium containing 10 mM nitrate, 5 mM Fe(II), 4 mM acetate, and 10 mg/L Zn. However, when the reactor is fed with coke-oven wastewater supplemented with 10 mM nitrate, 5 mM Fe(II), 4 mM acetate, and 10 mg/L ZnCl2, the reactor performance significantly decreased, most likely due to the inhibition of bacterial growth by thiocyanate or organic contaminants present in the coke-oven wastewater. Use of mixed culture of NDFO bacteria and thiocyanate/organic-degrading denitrifiers should help improve the reactor performance.

Candidal Prosthetic Hip Infection in a Patient with Previous Candidal Sepsis - A Case Report.

A 60 year-old woman with hip dysplasia battled with duodenal cancer that was complicated with Candida tropicalis sepsis. Two years later, the patient underwent a total hip arthroplasty (THA). She complained of a persisting low-grade fever and local heat on the THA scar. Arthrocentesis of the hip was performed and the Candida tropicalis was detected. Debridement and polyethylene liner/modular head exchange were performed 28 days after the primary THA. Fluconazole was administrated for one year. The patient reported no symptoms five years later. It was found that periprosthetic infection could be prevented by implant preservation surgery.

Required Chlorination Doses to Fulfill the Credit Value for Disinfection of Enteric Viruses in Water: A Critical Review.

A credit value of virus inactivation has been assigned to the disinfection step in international and domestic guidelines for wastewater reclamation and reuse. To fulfill the credit value for water disinfection, water engineers need to apply an appropriate disinfection strength, expressed as a CT value (mg × min/L), which is a product of disinfectant concentration and contact time, against enteric viruses in wastewater. In the present study, we extracted published experimental data on enteric virus inactivation using free chlorine and monochloramine and applied the Tobit analysis and simple linear regression analysis to calculate the range of CT values (mg × min/L) needed for 4-log10 inactivation. Data were selected from peer-reviewed papers containing kinetics data of virus infectivity and chlorine residual in water. Coxsackie B virus and echovirus require higher CT values (lower susceptibility) for 4-log10 inactivation than adenovirus and a human norovirus surrogate (murine norovirus) with free chlorine. On the other hand, adenovirus has lower susceptibility to monochloramine compared to murine norovirus, coxsackievirus, and echovirus. The factors that influence the required CT value are virus type, pH, water temperature, and water matrix. This systematic review demonstrates that enteroviruses and adenovirus are appropriate representative enteric viruses to evaluate water disinfection using free chlorine and monochloramine, respectively.

Mixture toxicity of the combinations of silver nanoparticles and environmental pollutants.

Although toxicity of silver nanoparticles (AgNPs) has been well studied, the mixture toxicity of the combination of AgNPs and other environmental pollutants is still largely unknown. Here, we investigated the mixture toxicity of the combinations of AgNPs and common environmental pollutants such as arsenic (As), cadmium (Cd), and chromium (Cr) on human hepatoma cell line (HepG2) at noncytotoxic concentrations based on analyses of cytotoxicity, genotoxicity, reactive oxygen species (ROS) generation, and modes of cell death. In addition, DNA microarray analysis was performed to understand the cellular responses at a molecular level. AgNPs-As and AgNPs-Cd combinations exhibited synergistic effect on cytotoxicity while AgNPs-Cr showed additive effect. The AgNPs-Cd combination caused much stronger synergism than AgNPs-As combination. Based on cellular and molecular level analyses, the synergistic effect could be explained by overproduction of reactive oxygen species (ROS), which induced DNA damage and consequently apoptotic cell death. On the other hand, the additive effect caused by AgNPs-Cr could be attributed to reduction of the mixture toxicity by precipitation of Cr ions. Taken together, our results clearly demonstrated that the mixture toxicity of AgNPs with As, Cd, or Cr at noncytotoxic concentrations had different toxicity effects. Particularly, toxicogenomic approach using DNA microarray was useful to assess the mechanisms of the mixture toxicity.

Transcriptomic analysis of HepG2 cells exposed to fractionated wastewater effluents suggested humic substances as potential inducer of whole effluent toxicity.

We performed a transcriptome-based bioassay (TSB assay) using human hepatoma HepG2 cells to evaluate the potential toxicity of whole wastewater effluents from two membrane bioreactors (MBRs) and a conventional activated sludge process (AS). The biologically active agent(s) in the wastewater effluents were characterized based on expression of the marker genes (i.e., CYP1A1, AKR1B10, GCLM and GPX2) selected by DNA microarray analysis, after the wastewater effluent samples were concentrated by a reverse osmosis (RO) membrane and further fractionated by various manipulations. The qPCR assay of marker genes demonstrated that the induction of CYP1A1 and GPX2 was mitigated after passing through C18 and chelate columns. In addition, clear induction of CYP1A1 was observed in the smallest size fraction with 1 k Da or smaller organic molecules in all the tested effluents. These results together with the water quality data of the fractionated samples suggested that responsible constituents for potentially adverse and abnormal transcriptomic responses in HepG2 could have hydrophobic nature and act with metal-dissolved organic matter (DOM) complexes in 1 k Da or smaller size fraction. Although DOM is known to play two contradictory roles as a protector and an inducer of toxicants, our present study indicated the DOM in wastewater effluent, particularly humic substances with acidic nature, functioned as a toxicity inducer of residual chemicals in the effluents. This study provided a new insight into the nature of "toxic unknowns" in the wastewater effluents, which should be monitored whole through the reclamation process and prioritized for removal.

Determination of Cadmium in Brown Rice Samples by Fluorescence Spectroscopy Using a Fluoroionophore after Purification of Cadmium by Anion Exchange Resin.

Simple analytical methods are needed for determining the cadmium (Cd) content of brown rice samples. In the present study, we developed a new analytical procedure consisting of the digestion of rice using HCl, Cd purification using anion exchange resin, and then determining the Cd content using fluorescence spectroscopy. Digestion with 0.1 M HCl for 10 min at room temperature was sufficient to extract Cd from the ground rice samples. The Cd in the extract was successfully purified in preference to other metals using Dowex 1X8 chloride form resin. Low concentrations of Cd in the eluate could be determined using fluorescence spectroscopy with a fluoroionophore. Overall, the actual limit of quantification value for the Cd content in rice was about 0.1 mg-Cd/kg-rice, which was sufficiently low compared with the regulatory value (0.4 mg-Cd/kg-rice) given by the Codex Alimentarius Commission. We analyzed authentic brown rice samples using our new analytical procedure and the results agreed well with those determined using inductively coupled plasma optical emission spectrometry (ICP-OES). Since the fluoroionophore recognized Zn2+ and Hg2+ as well as Cd2+, a sample containing high concentration of Zn2+ or Hg2+ might cause a false positive result.

Multiple-endpoints gene alteration-based (MEGA) assay: A toxicogenomics approach for water quality assessment of wastewater effluents.

Wastewater effluents contain a significant number of toxic contaminants, which, even at low concentrations, display a wide variety of toxic actions. In this study, we developed a multiple-endpoints gene alteration-based (MEGA) assay, a real-time PCR-based transcriptomic analysis, to assess the water quality of wastewater effluents for human health risk assessment and management. Twenty-one genes from the human hepatoblastoma cell line (HepG2), covering the basic health-relevant stress responses such as response to xenobiotics, genotoxicity, and cytotoxicity, were selected and incorporated into the MEGA assay. The genes related to the p53-mediated DNA damage response and cytochrome P450 were selected as markers for genotoxicity and response to xenobiotics, respectively. Additionally, the genes that were dose-dependently regulated by exposure to the wastewater effluents were chosen as markers for cytotoxicity. The alterations in the expression of an individual gene, induced by exposure to the wastewater effluents, were evaluated by real-time PCR and the results were validated by genotoxicity (e.g., comet assay) and cell-based cytotoxicity tests. In summary, the MEGA assay is a real-time PCR-based assay that targets cellular responses to contaminants present in wastewater effluents at the transcriptional level; it is rapid, cost-effective, and high-throughput and can thus complement any chemical analysis for water quality assessment and management.

Enhancement of organic matter degradation and methane gas production of anaerobic granular sludge by degasification of dissolved hydrogen gas.

A hollow fiber degassing membrane (DM) was applied to enhance organic matter degradation and methane gas production of anaerobic granular sludge process by reducing the dissolved hydrogen gas (D-H2) concentration in the liquid phase. DM was installed in the bench-scale anaerobic granular sludge reactors and D-H2 was removed through DM using a vacuum pump. Degasification improved the organic matter degradation efficiency to 79% while the efficiency was 62% without degasification at 12,000mgL-1 of the influent T-COD concentration. Measurement of D-H2 concentrations in the liquid phase confirmed that D-H2 was removed by degasification. Furthermore, the effect of acetate concentrations on the organic matter degradation efficiency was investigated. At acetate concentrations above 3gL-1, organic matter degradation deteriorated. Degasification enhanced the propionate and acetate degradation. These results suggest that degasification reduced D-H2 concentration and volatile fatty acids concentrations, prevented pH drop, and subsequent enhanced organic matter degradation.

Evaluation of virus reduction efficiency in wastewater treatment unit processes as a credit value in the multiple-barrier system for wastewater reclamation and reuse.

The virus reduction efficiency of each unit process is commonly determined based on the ratio of virus concentration in influent to that in effluent of a unit, but the virus concentration in wastewater has often fallen below the analytical quantification limit, which does not allow us to calculate the concentration ratio at each sampling event. In this study, left-censored datasets of norovirus (genogroup I and II), and adenovirus were used to calculate the virus reduction efficiency in unit processes of secondary biological treatment and chlorine disinfection. Virus concentration in influent, effluent from the secondary treatment, and chlorine-disinfected effluent of four municipal wastewater treatment plants were analyzed by a quantitative polymerase chain reaction (PCR) approach, and the probabilistic distributions of log reduction (LR) were estimated by a Bayesian estimation algorithm. The mean values of LR in the secondary treatment units ranged from 0.9 and 2.2, whereas those in the free chlorine disinfection units were from -0.1 and 0.5. The LR value in the secondary treatment was virus type and unit process dependent, which raised the importance for accumulating the data of virus LR values applicable to the multiple-barrier system, which is a global concept of microbial risk management in wastewater reclamation and reuse.

A case of ANCA-negative renal small-vessel vasculitis with tubulointerstitial infiltration of IgG4-positive plasma cells.

A 62-year-old male patient presented with progressive renal dysfunction for 2 months. He had elevated serum C-reactive protein and IgG4 levels with absence of anti-neutrophil cytoplasmic antibodies. A renal biopsy showed severe tubulointerstitial nephritis (TIN) with extensive infiltration of IgG4-positive plasma cells, suggesting a diagnosis of IgG4-related kidney disease (IgG4-RKD). However, the identification of a few crescentic glomeruli and necrotizing vasculitis of an interlobular artery lead to a diagnosis of renal small-vessel vasculitis. This case indicates that a careful examination is required to distinguish between IgG4-RKD and TIN caused by renal small-vessel vasculitis.

Microfluidic quantitative PCR for simultaneous quantification of multiple viruses in environmental water samples.

To secure food and water safety, quantitative information on multiple pathogens is important. In this study, we developed a microfluidic quantitative PCR (MFQPCR) system to simultaneously quantify 11 major human viral pathogens, including adenovirus, Aichi virus, astrovirus, enterovirus, human norovirus, rotavirus, sapovirus, and hepatitis A and E viruses. Murine norovirus and mengovirus were also quantified in our MFQPCR system as a sample processing control and an internal amplification control, respectively. River water contaminated with effluents from a wastewater treatment plant in Sapporo, Japan, was collected and used to validate our MFQPCR system for multiple viruses. High-throughput quantitative information was obtained with a quantification limit of 2 copies/µl of cDNA/DNA. Using this MFQPCR system, we could simultaneously quantify multiple viral pathogens in environmental water samples. The viral quantities obtained using MFQPCR were similar to those determined by conventional quantitative PCR. Thus, the MFQPCR system developed in this study can provide direct and quantitative information for viral pathogens, which is essential for risk assessments.

Toxicity assessment of chlorinated wastewater effluents by using transcriptome-based bioassays and Fourier transform mass spectrometry (FT-MS) analysis.

Effects of chlorination on the toxicity of wastewater effluents treated by activated sludge (AS) and submerged membrane bioreactor (S-MBRB) systems to HepG2 human hepatoblastoma cells were investigated. In addition to the cytotoxicity and genotoxicity assays, the DNA microarray-based transcriptome analysis was performed to evaluate the change in types of biological impacts on HepG2 cells of the effluents by chlorination. Effluent organic matter (EfOM) and disinfection by-products (DBPs) were also characterized by using Fourier transform mass spectrometry (FT-MS). Although no significant induction of genotoxicity was observed by chlorination for both effluents, the chlorination elevated the cytotoxicity of AS effluent but reduced that of S-MBRB effluent. The FT-MS analyses revealed that more DBPs including nitrogenated DBPs (N-DBPs) were formed in the AS effluent than in the S-MBRB effluent by chlorination, supporting the increased cytotoxicity of AS effluent. The lower O/C ratio of S-MBRB EfOM suggests that a large number of organic molecules were detoxified by chlorination, which consequently decreased the cytotoxicity of S-MBRB effluent. Integration of all the results highlights that both cytotoxicity and biological impacts of chlorinated wastewater effluents were clearly dependent on the EfOM characteristics such as DBPs and O/C ratio, namely, on types of treatment systems.

Hydrogenophaga electricum sp. nov., isolated from anodic biofilms of an acetate-fed microbial fuel cell.

A Gram-negative, non-spore-forming, rod-shaped bacterial strain, AR20(T), was isolated from anodic biofilms of an acetate-fed microbial fuel cell in Japan and subjected to a polyphasic taxonomic study. Strain AR20(T) grew optimally at pH 7.0-8.0 and 25°C. It contained Q-8 as the predominant ubiquinone and C16:0, summed feature 3 (C16:1ω7c and/or iso-C15:02OH), and C18:1ω7c as the major fatty acids. The DNA G+C content was 67.1 mol%. A neighbor-joining phylogenetic tree revealed that strain AR20(T) clustered with three type strains of the genus Hydrogenophaga (H. flava, H. bisanensis and H. pseudoflava). Strain AR20(T) exhibited 16S rRNA gene sequence similarity values of 95.8-97.7% to the type strains of the genus Hydrogenophaga. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain AR20(T) is considered a novel species of the genus Hydrogenophaga, for which the name Hydrogenophaga electricum sp. nov. is proposed. The type strain is AR20(T) (= KCTC 32195(T) = NBRC 109341(T)).

Evaluation of whole wastewater effluent impacts on HepG2 using DNA microarray-based transcriptome analysis.

DNA microarray-based transcriptome analysis with human hepatoma HepG2 cells was applied to evaluate the impacts of whole wastewater effluents from the membrane bioreactors (MBRs) and the activated sludge process (AS). In addition, the conventional bioassays (i.e., cytotoxicity tests and bioluminescence inhibition test), which were well-established for the evaluation of the overall effluent toxicity, were also performed for the same samples. Transcriptome analysis revealed that 2 to 926 genes, which were categorized to 0 to 225 biological processes, were differentially expressed after exposure to the effluents and the raw wastewater. Among the tested effluents, the effluent from a MBR operated at a relatively long solid retention time (i.e., 40 days) and small membrane pore size (i.e., 0.03 µm) showed the least impacts on the HepG2 even at the level comparable to tap water. The observed gene expression responses were in good agreement with the results of cytotoxicity tests, and provided additional molecular mechanistic information on adverse effects occurred in the sublethal region. Furthermore, the genes related to "lipid metabolism", "response to endogenous stimulus", and "response to inorganic substance" were selected as potential genetic markers, and their expression levels were quantified to evaluate the cellular impacts and treatability of wastewater effluents. Although the harmful impacts and innocuous impacts could not be distinguished at present, the results demonstrated that the DNA microarray-based transcriptome analysis with human HepG2 cells was a powerful tool to rapidly and comprehensively evaluate impacts of whole wastewater effluents.

15-year comparison of cementless total hip arthroplasty with anatomical or high cup placement for Crowe I to III hip dysplasia.

This study compared radiological and clinical results of Mallory-Head (Biomet, Warsaw, Indiana) cementless total hip arthroplasty (THA) by anatomical (AP group) or high cup placement (HP group) for Crowe I to III developmental dysplasia of the hip. Of the 68 hips studied, 43 hips were available for 15.3-year follow-up. Ten cups were placed at anatomical center with bulk bone grafting, and 33 cups were at high hip center without bulk bone grafting. No acetabular or femoral components showed loosening in either group. One standard polyethylene liner in a highly placed cup was revised due to excessive wear after 11 years. The average rate of polyethylene wear was 0.128 mm/year in the AP group and 0.148 mm/year in the HP group (except for the revision case). The extent of grafted bone coverage was 34.6% in the AP group. Hip center height was 24.5 mm from the inter-teardrop line in the HP group. The center of the hip horizontal location in the AP group (24.5 mm) and HP group (26.4 mm) was significantly shorter than in normal hips (35.6 mm). Postoperative center-edge angle was 11° (except grafted bone) in the AP group and 25° in the HP group. Mean Harris Hip Score in the AP group improved from 38 points preoperatively to 82 points postoperatively and in the HP group improved from 40 points preoperatively to 88 points postoperatively. Survivorship was 100% in the AP group and 97% in the HP group. Our results indicate that moderate high cup placement without bulk bone grafting at a horizontal locus more medial than that of a normal hip is an alternative durable solution.

Removal of residual dissolved methane gas in an upflow anaerobic sludge blanket reactor treating low-strength wastewater at low temperature with degassing membrane.

In this study, we investigated the efficiency of dissolved methane (D-CH(4)) collection by degasification from the effluent of a bench-scale upflow anaerobic sludge blanket (UASB) reactor treating synthetic wastewater. A hollow-fiber degassing membrane module was used for degasification. This module was connected to the liquid outlet of the UASB reactor. After chemical oxygen demand (COD) removal efficiency of the UASB reactor became stable, D-CH(4) discharged from the UASB reactor was collected. Under 35 °C and a hydraulic retention time (HRT) of 10 h, average D-CH(4) concentration could be reduced from 63 mg COD L(-1) to 15 mg COD L(-1); this, in turn, resulted in an increase in total methane (CH(4)) recovery efficiency from 89% to 97%. Furthermore, we investigated the effects of temperature and HRT of the UASB reactor on degasification efficiency. Average D-CH(4) concentration was as high as 104 mg COD L(-1) at 15 °C because of the higher solubility of CH(4) gas in liquid; the average D-CH(4) concentration was reduced to 14 mg COD L(-1) by degasification. Accordingly, total CH(4) recovery efficiency increased from 71% to 97% at 15 °C as a result of degasification. Moreover, degasification tended to cause an increase in particulate COD removal efficiency. The UASB reactor was operated at the same COD loading rate, but different wastewater feed rates and HRTs. Although average D-CH(4) concentration in the UASB reactor was almost unchanged (ca. 70 mg COD L(-1)) regardless of the HRT value, the CH(4) discharge rate from the UASB reactor increased because of an increase in the wastewater feed rate. Because the D-CH(4) concentration could be reduced down to 12 ± 1 mg COD L(-1) by degasification at an HRT of 6.7 h, the CH(4) recovery rate was 1.5 times higher under degasification than under normal operation.

A polyphasic approach to study ecophysiology of complex multispecies nitrifying biofilms.

This chapter aims to highlight the great potential of the combined use of microautoradiography (MAR) combined with fluorescent in situ hybridization (FISH) and microsensor technology in studies of complex multispecies nitrifying biofilms. The combination of FISH and microsensor technology is a powerful and reliable tool to link the spatial organization of microbial communities and their in situ function at community levels. MAR-FISH can be used to simultaneously examine the 16S rRNA-based phylogenetic identity and specific metabolic activity of cultivable or uncultivable microorganisms within complex microbial communities at a single-cell level. Information obtained at both resolution levels must be combined to draw a clear picture of a complex multispecies biofilm ecosystem. In addition, ecophysiological interactions among community members in complex multispecies biofilms can be investigated by tracing the fate of radiolabeled [(14)C] atom incorporated in nitrifying bacteria with MAR-FISH. The structure, function, and ecophysiological interactions among community members in complex multispecies nitrifying biofilms will be illustrated as an example of the combined use of MAR-FISH and microsensor technology.

Ecophysiology of uncultured filamentous anaerobes belonging to the phylum KSB3 that cause bulking in methanogenic granular sludge.

A filamentous bulking of a methanogenic granular sludge caused by uncultured filamentous bacteria of the candidate phylum KSB3 in an upflow anaerobic sludge blanket (UASB) system has been reported. To characterize the physiological traits of the filaments, a polyphasic approach consisting of rRNA-based activity monitoring of the KSB3 filaments using the RNase H method and substrate uptake profiling using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was conducted. On the basis of rRNA-based activity, the monitoring of a full-scale UASB reactor operated continuously revealed that KSB3 cells became active and predominant (up to 54% of the total 16S rRNA) in the sludge when the carbohydrate loading to the system increased. Batch experiments with a short incubation of the sludge with maltose, glucose, fructose, and maltotriose at relatively low concentrations (approximately 0.1 mM) in the presence of yeast extract also showed an increase in KSB3 rRNA levels under anaerobic conditions. MAR-FISH confirmed that the KSB3 cells took up radioisotopic carbons from [(14)C]maltose and [(14)C]glucose under the same incubation conditions in the batch experiments. These results suggest that one of the important ecophysiological characteristics of KSB3 cells in the sludge is carbohydrate degradation in wastewater and that high carbohydrate loadings may trigger an outbreak of KSB3 bacteria, causing sludge bulking in UASB systems.