Autotrophic growth activity of complete ammonia oxidizers in an upflow biological contact filter for drinking water treatment.

Biological filters effectively remove ammonium from drinking water via nitrification. In a pilot-scale upflow biological contact filter (U-BCF), complete ammonia oxidizers (comammox), which are capable of oxidizing ammonia to nitrate in one cell, were more abundant than ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). However, little information is available on the contribution of comammox to nitrification. In this study, we evaluated the autotrophic growth activity of comammox associated with biological activated carbon (BAC) in a U-BCF by DNA-stable isotope probing (DNA-SIP). BAC samples collected from the U-BCF were continuously fed mineral medium containing 0.14 mg N L-1 ammonium and 12C- or 13C-labeled bicarbonate for 20 days. DNA-SIP analysis revealed that comammox (clades A and B) as well as AOA assimilated bicarbonate after 10 days of incubation, proving that dominant comammox could contribute to nitrification. Contrarily, AOB remained inactive throughout the observation period. Amplicon sequencing of the 13C-labeled DNA fractions of comammox revealed that specific genotypes other than the most dominant genotype in the original sample were more enriched under the incubation condition for the DNA-SIP experiment. Thus, dominant genotypes of comammox in a U-BCF might utilize organic nitrogen to fuel nitrification in ammonia-limited environments.

Dynamics of the Microbial Community and Opportunistic Pathogens after Water Stagnation in the Premise Plumbing of a Building.

In premise plumbing, microbial water quality may deteriorate under certain conditions, such as stagnation. Stagnation results in a loss of disinfectant residual, which may lead to the regrowth of microorganisms, including opportunistic pathogens. In the present study, microbial regrowth was investigated at eight faucets in a building over four seasons in one year. Water samples were obtained before and after 24 h of stagnation. In the first 100| |mL after stagnation, total cell counts measured by flow cytometry increased 14- to 220-fold with a simultaneous decrease in free chlorine from 0.17-0.36| |mg L-1 to <0.02| |mg L-1. After stagnation, total cell counts were not significantly different among seasons; however, the composition of the microbial community varied seasonally. The relative abundance of Pseudomonas spp. was dominant in winter, whereas Sphingomonas spp. were dominant in most faucets after stagnation in other seasons. Opportunistic pathogens, such as Legionella pneumophila, Mycobacterium avium, Pseudomonas aeruginosa, and Acanthamoeba spp., were below the quantification limit for real-time quantitative PCR in all samples. However, sequences related to other opportunistic pathogens, including L. feeleii, L. maceachernii, L. micdadei, M. paragordonae, M. gordonae, and M. haemophilum, were detected. These results indicate that health risks may increase after stagnation due to the regrowth of opportunistic pathogens.

Optimized Cultivation and Syntrophic Relationship of Anaerobic Benzene-Degrading Enrichment Cultures under Methanogenic Conditions.

Current challenges in the anaerobic bioremediation of benzene are the lack of capable cultures and limited knowledge on the biodegradation pathway. Under methanogenic conditions, benzene may be mineralized by syntrophic interactions between microorganisms, which are poorly understood. The present study developed an optimized formula for anoxic medium to successfully promote the growth of the putative benzene degrader Deltaproteobacterium Hasda-A and enhance the benzene degradation activity of methanogenic enrichment cultures. Within 70| |d of incubation, the benzene degradation activity and relative abundance of Hasda-A in cultures in the new defined medium increased from 0.5 to >3| |mg L-1 d-1 and from 2.5% to >17%, respectively. Together with Hasda-A, we found a strong positive relationship between the abundances of superphylum OD1 bacteria, three methanogens (Methanoregula, Methanolinea, and Methanosaeta) and benzene degradation activity. The syntrophic relationship between these microbial taxa and Hasda-A was then demonstrated in a correlation analysis of longitudinal data. The involvement of methanogenesis in anaerobic benzene mineralization was confirmed by inhibition experiments. The high benzene degradation activity and growth of Hasda-A were quickly recovered in successive dilutions of enrichment cultures, proving the feasibility of using the medium developed in the present study to produce highly capable cultures. The present results will facilitate practical applications in bioremediation and research on the molecular mechanisms underlying benzene activation and syntrophic interactions in benzene mineralization.

Identification of new eligible indicator organisms for combined sewer overflow via 16S rRNA gene amplicon sequencing in Kanda River, Tokyo.

Fecal indicator bacteria (FIB) are commonly used to evaluate the pollution impact of combined sewer overflows (CSOs) in urban rivers. Although water quality assessment with FIB has a long tradition, recent studies demonstrated that FIB have a low correlation with pathogens and therefore are not accurate enough for the assessment of potential human hazards in water. Consequently, new eligible and more specific indicators have to be identified, which was done in this study via sequencing of genetic markers from total community DNA. To identify potential microbiome-based indicators, microbial communities in samples from an urban river in Tokyo under different climatic conditions (dry and rainy) were compared with the influent and effluent of three domestic wastewater treatment plants (WWTPs) by analyzing 16 S rRNA gene amplicon libraries. In the first part of this study, physicochemical parameters and FIB quantification with selective culture techniques facilitated the identification of samples contaminated with CSO, sewage, or both. This allowed the grouping of samples into CSO-contaminated and non-contaminated samples, an essential step prior to the microbiome comparison between samples. Increased turbidity, ammonia concentrations, and E. coli [up to (9.37 ± 0.95) × 102 CFU/mL after 11.5 mm of rainfall] were observed in CSO-contaminated river samples. Comparison of dry weather (including WWTP samples) and rainy weather samples showed a reduction in microbial diversity in CSO-contaminated samples. Furthermore, the results of this study suggest Bacteroides spp. as a novel indicator of sewage pollution in surface waters.

Non-target screening of dissolved organic matter in raw water, coagulated water, and chlorinated water by Orbitrap mass spectrometry.

This study aimed to classify the possible molecular formulas of precursors for disinfection by-products (DBPs) in raw, coagulated, and chlorinated water samples from the U-Tapao Canal, Songkhla, Thailand. The molecular formulas of DBPs in chlorinated water were investigated. Polyaluminum chloride (PACl) was employed as a coagulant. Orbitrap Fourier transform-mass spectrometry was able to estimate the composition of dissolved organic matter (DOM) with the carbon (C), hydrogen (H), oxygen (O), nitrogen (N), and sulfur (S) elements contained and DBPs at the molecular level. The molecular formulas of the DOM in the raw water primarily consisted of CHO and CHON when extracted by lichrolut EN. The CHO and CHON species were rich in lignin-, tannin-, and condensed aromatic-like substances. The DOM with high-molecular-weight from 300 to 500 Da were preferentially removed by coagulation. The PACl coagulation decreased the abundances of lignin-, tannin-, and condensed aromatic-like substances in the CHO formulas, while lignin- and condensed aromatic-like substances in the CHON formulas remained. The remaining precursors corresponded to CHON molecules in the coagulated water, which may result in the formation of some chlorine (Cl)-containing molecules. Several DBPs among the CHOCl and CHONCl species were produced in the chlorinated water through the addition reaction of chlorine. New chlorinated N-DBPs of 21 formulas were detected.

Changes in dissolved organic matter during water treatment by sequential solid-phase extraction and unknown screening analysis.

Isolation of complex dissolved organic matter (DOM) from environmental water is a major challenge for unknown screening analysis by high-resolution mass spectrometry. In this study, DOM in process water during advanced drinking water treatment was fractionated sequentially by three solid-phase extraction (SPE) cartridges based on the polarity and charge of DOM molecules. By sequential SPE with unknown screening analysis, over 3000 DOM features were found in raw water, whereas around 2000 were obtained by a single SPE. The hydrophobic neutral (HPON) fraction contained CHO features with highest averaged molecular weight followed by hydrophobic acid (HPOA) and then hydrophilic acid (HPIA). The average degree of carbon double bond equivalents and carbon oxidation states indicated that the HPON fraction contained molecules that were more unsaturated and less oxidized than those of the HPOA and HPIA fractions. Ozone selectively decomposed (1) more unsaturated and less oxidized HPON features, (2) more unsaturated HPOA compounds, and (3) less oxidized HPIA molecules. Oxidation by-products were mostly HPON and HPIA compounds that were more oxidized than the decomposed molecules. During biological activated carbon (BAC) filtration, less oxidized HPON were preferentially removed, whereas HPOA were removed without selectivity. HPON and HPIA molecules with more oxidized character were found to be refractory to BAC treatment. HPON with more unsaturated and HPIA with more oxidized characters were decomposed by chlorine. Many types of HPIA decomposed during chlorination were the oxidation by-products of ozonation that were refractory to BAC treatment. Sequential SPE with unknown screening analysis provided previously unknown details of the molecular characteristics of DOM and its changes during advanced water treatment.

Orbitrap mass spectrometry for the molecular characterization of water resource recovery from polluted surface water using membrane bioreactor.

The increasing organic contamination of surface water hinders the conventional tap water treatment process. Membrane bioreactors (MBRs) are a promising alternative technology for recovering water from polluted surface water. In this study, the composition changes of dissolved organic matters (DOMs) in MBR and ultraviolet/ozone (UV/O3)-MBR systems for polluted surface water treatment were investigated using Orbitrap mass spectrometry analysis with unknown screening. The intense DOM ions within a mass-to-charge ratio range of 100-500 was detected, and 2340 molecular formulae from 5743 peaks were assigned to the two systems. The most abundant components were formulae with C, H, O, N, and CHO only classes. The highest formulae decrease including CHO, CHON, CHOS, and CHONS were attributed to the bio-carrier used in both systems. Results showed that bioprocess was the main contributor in the DOM reduction, and the integration of UV/O3 into the MBR improved the DOM composition changes. Biodegradable components with low O/C ratio in the CHO and CHON classes remarkably increased in the UV/O3-MBR system. The integration of UV/O3 as a polishing step in the recirculation stream of MBR system was effective in improve the DOM removal.

Characterization of lower Phong river dissolved organic matters and formations of unknown chlorine dioxide and chlorine disinfection by-products by Orbitrap mass spectrometry.

Dissolved organic matter (DOM) have been reported as precursors of disinfection byproducts (DBPs) and its molecular characteristics are rarely investigated due to its complexity. In this study, changes in the characteristics of DOM were investigated in the lower Phong River in Thailand in dry season and after the first rain in rainy season, using a non-targeted analysis with Orbitrap mass spectrometry. The river was rich with CHO features dominated by lignin-like molecules, while lipid-like molecules increase after domestic wastewater discharges. Wastewater discharge released DOM with higher molecular weight (MW) that was less oxygenated (low O/C) and less oxidized (low carbon oxidation state [Cos]). A lake affected by anthropogenic activities contributed more oxidized DOM into the river, while surface runoff carried DOM that is more oxygenated (high O/C), less hydrogenated (low H/C), and more oxidized (high Cos) to the stream. Water treatment further modified DOM to be lower MW. Approximately three hundred Cl-containing features (CHOCl) detected upstream were also found downstream. Disinfection by chlorine (Cl2) or chlorine dioxide (ClO2) formed both CHO and CHOCl DBPs. Low chlorine dosage applied to upstream and downstream samples resulted in many common unknown DBPs while increasing chlorine dosage resulted in more unique DBPs. At the same dosage, Cl2 reacted with DOM more than ClO2, including more oxidized molecules that are refractory to ClO2. Both Cl2 and ClO2 produced chlorinated and non-chlorinated DBPs, and some DBPs were commonly found by both disinfections. Cl2-produced DBPs were more unsaturated (higher [DBE-O]/C) and oxidized (higher Cos) than ClO2-DBPs.

Temperature-Dependent Ammonium Removal Capacity of Biological Activated Carbon Used in a Full-Scale Drinking Water Treatment Plant.

Nitrification is a key function of biological activated carbon (BAC) filters for drinking water treatment. It is empirically known that the nitrification activity of BAC filters depends on water temperature, potentially resulting in the leakage of ammonium from BAC filters when the water temperature decreases. However, the ammonium removal capacity of BAC filters and factors governing the capacity remain unknown. This study employed a bench-scale column assay to determine the volumetric ammonium removal rate (VARR) of BAC collected from a full-scale drinking water treatment plant. VARR was determined at a fixed loading rate under different conditions. Seasonal variations of the VARR as well as impacts of the water matrix and water temperature on ammonium removal were quantitatively analyzed. While the VARR in an inorganic medium at 25 °C was maintained even during low water temperature periods and during breakpoint chlorination periods, the water matrix factor reduced the VARR in ozonated water at 25 °C by 33% on average. The VARR in ozonated water was dependent on water temperature, indicating that the microbial activity of BAC did not adapt to low water temperature. The Arrhenius equation was applied to reveal the relationship between VARR and water temperature. The actual ammonium removal performance of a full-scale BAC filter was predicted. VARR is useful for water engineers to reexamine the loading and filter depth of BAC filters.

Metal partitioning and leaching vulnerability in soil, soakaway sediments, and road dust in the urban area of Japan.

Isotope dilution techniques (IDT) and sequential extraction procedures (SEPs) were compared to apprehend the differences between two techniques in determining metal exchangeability and vulnerability to pollute the urban groundwater. For this purpose, soil (n = 2), "soakaway" sediment deposited in the artificial infiltration facilities (AIF) (n = 4), and road dust (n = 2) were sampled from Tokyo metropolitan. Sorption coefficients of four metals (Cu, Zn, Cd and Pb) were assessed through isotopic exchangeability (E-value) and potential mobile pool i.e. addition of exchangeable, reducible and oxidizable fraction obtained by Community Bureau of Reference (BCR)-procedures. The E-value for the three samples were found smaller than the potential mobile pool but were higher than BCR-exchangeable fractions. The use of strong extractants are likely to play an active role in the disagreement between SEPs and IDT. IDT accounts for the isotopic exchangeability while BCR provides information of vulnerability of metals associated with different fractions that can leach under different environmental conditions. Sorption coefficients measured in soakaway sediment was found comparable to soil thus likely to retain metals. However, as variability in environmental conditions is likely to affect Kd, the soakaway sediment may become an active metal source in future rather than acting as the permanent sink. The study concludes that there is the possibility of errors while predicting metal vulnerability to groundwater with both techniques and thus a model compliance integrating the virtue of both techniques will be a way forward.

Exploring the operating factors controlling Kouleothrix (type 1851), the dominant filamentous bacterial population, in a full-scale A2O plant.

This study reveals that the abundance of the filament Kouleothrix (Eikelboom type 1851) correlated positively with poor settleability of activated sludge biomass in a Japanese full-scale nutrient removal wastewater treatment plant sampled over a one-year period. 16S rRNA amplicon sequence data confirmed that Kouleothrix was the dominant filament in the plant, with a relative abundance of 3.06% positively correlated with sludge volume index (SVI) (R = 0.691). Moreover, Kouleothrix (type 1851) appeared to form interfloc bridges, typical of bulking sludge, regardless of season. Together with earlier studies that indicated the responsibility of Kouleothrix (type 1851) on bulking events, these data suggest that their high relative abundances alone may be responsible for sludge bulking. 16S rRNA qPCR data for this filament showed changes in its relative abundance correlated with changes in several operational parameters, including mixed liquor temperature, sludge retention time, and suspended solids concentration, and it may be that manipulating these may help control Kouleothrix bulking.

Isolation and characterization of Pb-resistant plant growth promoting endophytic bacteria and their role in Pb accumulation by fast-growing trees.

Lead (Pb) contamination is one of the major environmental problems on a global scale. Bacterial endophytes have been accepted as a promising technique to assist phytoremediation. In this study, three Pb-tolerant endophytic bacteria were isolated from the roots of Pityrogramma calomelanos. Based on partial 16S rRNA gene sequencing analysis, all isolates were similar to Pseudomonas and tolerated Pb concentration up to 1850mg/L, producing siderophores and solubilized phosphate. Among them, Pc isolate closely related to Pseudomonas psychrophila showed the highest water-soluble Pb in solution (Pb solubilization) and in contaminated soil. This isolate was chosen to study the effects on Pb accumulation in the roots of Acacia mangium and Eucalyptus camaldulensis by a hydroponic experiment. The results showed that, in the Hoagland nutrient solution with no Pb spiking, the roots showed no significant difference (p > 0.05), and the concentration of Pb ranged from 10 to 89 mg/kg. In the nutrient solution in the presence of 30 mg/L Pb, there were no significant changes in Pb contents in roots. However, A. mangium showed an increase in Pb concentration in the roots (6829 ± 697 mg/kg), compared to non-inoculation (6242 ± 272 mg/kg). E. camaldulensis inoculation showed a decrease in Pb content (3763 ± 592 mg/kg), compared to non-inoculation (4233 ± 264 mg/kg). These results suggest that the Pc isolate closely related to P. psychrophila was effective in promoting the phytoremediation potential of A. mangium, but it was not useful for E. camaldulensis.

Simultaneous screening for chemically diverse micropollutants in public water bodies in Japan by high-performance liquid chromatography-Orbitrap mass spectrometry.

An improved assessment of environmental risks to public water bodies requires screening a large number of micropollutants. This study reports the development of a novel target screening method based on solid-phase extraction (SPE), HPLC, and high-resolution Orbitrap MS for the analysis of micropollutants with diverse chemical properties. First, target compounds were screened for their detectability by Orbitrap MS. An optimized SPE cartridge and HPLC column maximized recovery and separated most target compounds. The sensitivity and repeatability of the method was validated by determining the detection limits and relative standard deviation (RSD). Eighty-four compounds with highly diverse properties were simultaneously detected with detection limits of 0.1-100 ng/L. Of these compounds, 52 were quantitated, with R2 ≥ 0.99 by linearity analysis and SPE recovery ratios of ≥50%. The remaining 32 compounds were qualitatively detected, with R2 < 0.99 or SPE recovery ratio of <50%. Satisfactory repeatability was obtained (RSD < 13.5%). This method was applied to the surveillance of the Arakawa River in Japan in 2019. Thirty-two compounds, including pesticides, surfactants, plasticizers, adhesives, and industrial solvents, were detected in the river. The measured concentrations of 13 compounds were compared with their predicted no effect concentrations (PNECs). Decanoic acid showed a higher concentration than the corresponding PNEC value, suggesting that its risk to the Arakawa water environment required further evaluation. The concentrations of dicyclohexylamine, 1,3-diphenylguanidine, and 2,4-dichlorophenoxyacetic acid were higher than their corresponding PNEC/10 values, demonstrating that these compounds were of higher priority than other compounds.

Molecular characteristics of dissolved organic matter transformed by O3 and O3/H2O2 treatments and the effects on formation of unknown disinfection by-products.

We investigated semiquantitative changes in almost 1000 dissolved organic matter (DOM) features during oxidation with 1 mg of O3 per liter (mg O3/L), 4 mg O3/L, or 4 mg O3/L + 2.5 mg of H2O2 per liter (advanced oxidation process, AOP) by unknown screening analysis with Orbitrap mass spectrometry. The consequential effects on formation of unknown disinfection by-products (DBPs) by chlorination were evaluated in laboratory-scale experiments. Several hundred unsaturated DOM features with positive oxygen-subtracted double bond equivalents per carbon ((DBE-O)/C) were decomposed by the ozone-only treatment and AOP. The AOP decomposed some saturated (negative (DBE-O)/C)) and reduced molecules, which had negative carbon oxidation states (Cos). Several hundred saturated oxidation by-products were detected after ozonation and the AOP. After chlorination, the samples pre-treated with ozone alone resulted in higher formation of unknown DBPs than the AOP pre-treated sample or the sample without oxidation. Over half of the DBP precursors, estimated by electrophilic substitution, were not totally decomposed by any oxidation process, but they were increased after the ozone-only process and AOP. DBP precursors produced by the ozone-only process or AOP formed unique unknown DBPs. Therefore, post-treatment processes after oxidation and before chlorination are important to minimize formation of unknown DBPs.

Incorporation of 13C-HCO3- by ammonia-oxidizing archaea and bacteria during ammonia oxidation of sludge from a municipal wastewater treatment plant.

Ammonia-oxidizing archaea (AOA) have recently been proposed as potential players for ammonia removal in wastewater treatment plants (WWTPs). However, there is little evidence directly showing the contribution of AOA to ammonia oxidation in these engineered systems. In this study, DNA-stable isotope probing (DNA-SIP) with labeled 13C-HCO3- was introduced to sludge from a municipal WWTP. Quantitative PCR demonstrated that AOA amoA genes outnumbered AOB amoA genes in this WWTP sludge. AOA amoA gene sequence analysis revealed that AOA present in this WWTP were specific to one subcluster within the group 1.1b Thaumarchaeota. When ammonia was supplied to DNA-SIP incubation, the DNA-SIP profiles demonstrated the incorporation of the 13C into AOA and AOB. However, the 13C was not found to be assimilated into both microorganisms in the incubation without ammonia. Specific primers were designed to target amoA genes of AOA belonging to the subcluster found in this WWTP. Applying the primers to DNA-SIP experiment revealed that AOA of this subcluter most likely utilized inorganic carbon during ammonia oxidation under the studied conditions.

Distinct Microbial Community Performing Dissimilatory Nitrate Reduction to Ammonium (DNRA) in a High C/NO3- Reactor.

A dissimilatory nitrate reduction to ammonium (DNRA) microbial community was developed under a high organic carbon to nitrate (C/NO3-) ratio in an anoxic semi-continuous sequencing batch reactor (SBR) fed with glucose as the source of carbon and NO3- as the electron acceptor. Activated sludge collected from a municipal wastewater treatment plant with good denitrification efficiency was used as the inoculum to start the system. The aim of this study was to examine the microbial populations in a high C/NO3- ecosystem for potential DNRA microorganisms, which are the microbial group with the ability to reduce NO3- to ammonium (NH4+). A low C/NO3- reactor was operated in parallel for direct comparisons of the microbial communities that developed under different C/NO3- values. The occurrence of DNRA in the high C/NO3- SBR was evidenced by stable isotope-labeled nitrate and nitrite (15NO3- and 15NO2-), which proved the formation of NH4+ from dissimilatory NO3-/NO2- reduction, in which both nitrogen oxides induced DNRA activity in a similar manner. An analysis of sludge samples with Illumina MiSeq 16S rRNA sequencing showed that the predominant microorganisms in the high C/NO3- SBR were related to Sulfurospirillum and the family Lachnospiraceae, which were barely present in the low C/NO3- system. A comparison of the populations and activities of the two reactors indicated that these major taxa play important roles as DNRA microorganisms under the high C/NO3- condition. Additionally, a beta-diversity analysis revealed distinct microbial compositions between the low and high C/NO3- SBRs, which reflected the activities observed in the two systems.

Changes in Dissolved Organic Matter Composition and Disinfection Byproduct Precursors in Advanced Drinking Water Treatment Processes.

Molecular changes in dissolved organic matter (DOM) from treatment processes at two drinking water treatment plants in Japan were investigated using unknown screening analysis by Orbitrap mass spectrometry. DOM formulas with carbon, hydrogen and oxygen (CHO-DOM) were the most abundant class in water samples, and over half of them were commonly found at both plants. Among the treatment processes, ozonation induced the most drastic changes to DOM. Mass peak intensities of less saturated CHO-DOM (positive (oxygen subtracted double bond equivalent per carbon (DBE-O)/C)) decreased by ozonation, while more saturated oxidation byproducts (negative (DBE-O)/C) increased and new oxidation byproducts (OBPs) were detected. By Kendrick mass analysis, ozone reactions preferred less saturated CHO-DOM in the same alkylation families and produced more saturated alkylation families of OBPs. Following ozonation, biological activated carbon filtration effectively removed <300 Da CHO-DOM, including OBPs. Following chlorination, over 50 chlorinated formulas of disinfection byproducts (DBPs) were found in chlorinated water samples where at least half were unknown. Putative precursors of these DBPs were determined based on electrophilic substitutions and addition reactions. Ozonation demonstrated better decomposition of addition reaction-type precursors than electrophilic substitution-type precursors; over half of both precursor types decreased during biological activated carbon filtration.

Bacterial Degraders of Coexisting Dichloromethane, Benzene, and Toluene, Identified by Stable-Isotope Probing.

Most bioremediation studies on volatile organic compounds (VOCs) have focused on a single contaminant or its derived compounds and degraders have been identified under single contaminant conditions. Bioremediation of multiple contaminants remains a challenging issue. To identify a bacterial consortium that degrades multiple VOCs (dichloromethane (DCM), benzene, and toluene), we applied DNA-stable isotope probing. For individual tests, we combined a 13C-labeled VOC with other two unlabeled VOCs, and prepared three unlabeled VOCs as a reference. Over 11 days, DNA was periodically extracted from the consortia, and the bacterial community was evaluated by next-generation sequencing of bacterial 16S rRNA gene amplicons. Density gradient fractions of the DNA extracts were amplified by universal bacterial primers for the 16S rRNA gene sequences, and the amplicons were analyzed by terminal restriction fragment length polymorphism (T-RFLP) using restriction enzymes: HhaI and MspI. The T-RFLP fragments were identified by 16S rRNA gene cloning and sequencing. Under all test conditions, the consortia were dominated by Rhodanobacter, Bradyrhizobium/Afipia, Rhizobium, and Hyphomicrobium. DNA derived from Hyphomicrobium and Propioniferax shifted toward heavier fractions under the condition added with 13C-DCM and 13C-benzene, respectively, compared with the reference, but no shifts were induced by 13C-toluene addition. This implies that Hyphomicrobium and Propioniferax were the main DCM and benzene degraders, respectively, under the coexisting condition. The known benzene degrader Pseudomonas sp. was present but not actively involved in the degradation.

Quantification of Chloroflexi Eikelboom morphotype 1851 for prediction and control of bulking events in municipal activated sludge plants in Japan.

The dominant filamentous bacteria associated with bulking incidents in Japanese activated sludge plants with nutrient removal were identified and their quantitative correlations with sludge settleability were assessed, with the aim of controlling bulking incidents by specifically suppressing bacterial growth. Fluorescence in situ hybridization (FISH) analyses using existing oligonucleotide FISH probes indicated that the presence of Eikelboom type 1851 filamentous bacteria belonging to the phylum Chloroflexi is correlated with biomass settleability in the municipal wastewater treatment plants examined. Real-time quantitative PCR (qPCR) assays developed in this study also showed a linear correlation between type 1851 filament members and sludge settleability, with the exception of some winter samples. The real-time qPCR assays and 16S ribosomal RNA gene amplicon sequencing to reveal the microbial community of activated sludge showed that the abundance of type 1851 at 200 mL g-1 of sludge volume index was estimated to be about 1.9% of the total microbial cells. The abundance of type 1851 served as a bulking indicator in plants where type 1851 was dominant.