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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 C-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 C-DCM and C-benzene, respectively, compared with the reference, but no shifts were induced by C-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. [ABSTRACT FROM AUTHOR]

Microbial regrowth causes problems during water reuse. Comprehensive understanding of the microorganisms that can regrow in reclaimed water and their substrate requirements are necessary. In this study, potential regrowth organisms were isolated from seven water reclamation plants in Japan. Based on 16S rDNA analysis, the isolates were grouped into 34 operational taxonomic units, belonging to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Substrate utilization profiling using Biolog microplate™ classified the isolates into four groups. Bacteria in Cluster 1 (e.g., Methylobacterium sp. and Acinetobacter sp.) mainly utilized polymers, esters, amides, and alcohol. Isolates in Cluster 2 (e.g., Flavobacterium sp. and Microbacterium sp.) preferred to utilize polymers, carbohydrates, and esters. Isolates in Cluster 3 (e.g., Pseudomonas sp. and Acidovorax sp.) mainly utilized esters, carboxylic acids, and amino acids. Isolates in Cluster 4 (e.g., Enterobacter sp. and Rhodococcus sp.) utilized carbohydrates, esters, and amino acids. All isolates grew in reclaimed water treated by sand filtration, whereas some isolates could not grow in reclaimed water treated by coagulation and ozonation. Most bacteria in the same Biolog clusters exhibited similar growth characteristics in water samples. The potential of bacteria to regrow in reclaimed water likely depended on substrate requirement. [ABSTRACT FROM AUTHOR]

The goal of this study was to develop and validate a novel fosmid-clone-based metagenome isotope array approach - termed the community isotope array ( CIArray) - for sensitive detection and identification of microorganisms assimilating a radiolabeled substrate within complex microbial communities. More specifically, a sample-specific CIArray was used to identify anoxic phenol-degrading microorganisms in activated sludge treating synthetic coke-oven wastewater in a single-sludge predenitrification-nitrification process. Hybridization of the CIArray with DNA from the 14C-phenol-amended sample indicated that bacteria assimilating 14C-atoms, presumably directly from phenol, under nitrate-reducing conditions were abundant in the reactor, and taxonomic assignment of the fosmid clone end sequences suggested that they belonged to the Gammaproteobacteria. The specificity of the CIArray was validated by quantification of fosmid-clone-specific DNA in density-resolved DNA fractions from samples incubated with 13C-phenol, which verified that all CIArray-positive probes stemmed from microorganisms that assimilated isotopically labeled carbon. This also demonstrated that the CIArray was more sensitive than DNA- SIP, as the former enabled positive detection at a phenol concentration that failed to yield a 'heavy' DNA fraction. Finally, two operational taxonomic units distantly related to marine Gammaproteobacteria were identified to account for more than half of 16S r RNA gene clones in the 'heavy' DNA library, corroborating the CIArray-based identification. [ABSTRACT FROM AUTHOR]

The use of randomly generated DNA fragment sequences as probes on DNA arrays offers a unique potential for exploring unsequenced microorganisms. In this study, the detection specificity was evaluated with respect to probe-target sequence similarity using genomic DNAs of four Pseudomonas strains. Genome fragments averaging 2000 bp were found to be specific enough to discriminate 85-90% similarity under highly stringent hybridization conditions. Such stringent conditions compromised signal intensities; however, specific signals remained detectable at the highest stringency (at 75 °C hybridization) with negligible false negatives. These results suggest that, without any probe design or selection, genomic fragments can provide a reasonable specificity for microbial diagnostics or species delineation by DNA- DNA similarities. [ABSTRACT FROM AUTHOR]

Abstract: Microbial degradation of benzene under anaerobic conditions plays an important role in remediation of contaminated sites but the microorganisms and metabolic pathways involved remain poorly understood. In this study, we evaluated degradation of benzene by a methanogenic enrichment culture obtained from non-contaminated lotus field soil, alone and in the presence of several putative metabolic intermediates, that is, toluene, benzoate and phenol. Using stable isotope (13C substrate, benzene was shown to be degraded almost completely to equimolar concentrations of methane and carbon dioxide, without detectable accumulation of extracellular metabolites. Concurrently, toluene, benzoate and phenol were also effectively mineralized, but probably by microorganisms other than the benzene degraders. The latter included Hasda-A, which is putative benzene-degrading deltaproteobacterium present in the culture. While toluene and benzoate did not affect benzene degradation, phenol had a moderate inhibitory effect although it was not a major metabolic intermediate of benzene in our culture. Finally, 4-hydroxycoumarin was detected as a compound formed from phenol but further experiments are required to elucidate its relationship to degradation of phenol. [Copyright &y& Elsevier]

This study was aimed at comparing the Zn and Cu binding parameters with dissolved organic matter (DOM) in road runoff, wastewater treatment plant (WWTP) effluent and influent. Conditional stability constant (K') and binding site concentration ([L]) are important to predict free or labile metal concentration and toxicity in the water environment. The values of K' and [L] of three DOMs were determined by metal titration and Scatchard linearization. The Zn and Cu titration data for DOMs in WWTP effluent and influent fitted to a two-ligand model, while DOM in road runoff fitted to a single-ligand model. The order of the values of K' was WWTP influent > WWTP effluent > road runoff both for Zn and Cu. Total ambient binding site concentration ([LT]) was lower in DOM of road runoff (1.4-1.5μM) than those in DOM of WWTP effluent (1.6-2.3μM) and influent (17-18μM), suggesting lower metal complexation capacity in DOM of road runoff. DOM in WWTP influent was expected to bind to both Zn and Cu more strongly than that of the effluent DOM. [ABSTRACT FROM AUTHOR]

Selective sequential dissolution (SSD) and isotopic dilution are two most commonly applied techniques for the measurement of mobile fraction of heavy metal present in the urban environment. This work examined the compliance between SSD proposed by the Community Bureau of Reference (BCR) and isotopic dilution technique (IDT) for determination of mobile pool of heavy metal contained in soakaway sediment, road dust, and soil sample. Heavy metals (Cu, Zn, Cd and Pb) were fractionated using the three-stage BCR protocol, while isotopically exchangeable metal concentrations (E-value) were investigated through isotopic tracers (111Cd, 65Cu, 207Pb and 66Zn). In general, total contamination level, E-value and BCR exchangeable fractions of different samples followed the similar order of road dust . soakaway sediment . soil. Results revealed that the E-value exceeded the BCR exchangeable fraction in all samples. In addition, the first three fractions of BCR which have potential to become mobile under certain environmental conditions were collectively termed as "potential mobile pool" and compared with E-value. It was concluded that metal extracted by weak acid underestimates the exchangeable fraction while the potential mobile pool extracted by three reagents overestimates the real mobile forms of heavy metals. However, better mobility characteristics of heavy metals can be assessed by coupling information obtained through sequential extraction and isotopic exchange. [ABSTRACT FROM AUTHOR]

Abstract: Five bacterial strains capable of utilizing 17β-estradiol (E2) and estrone (E1) were isolated from soil samples. Using their morphological and physiological features and 16S rDNA sequences, we classified these isolates into two groups: Group A (Rhodococcus sp. strains ED6, ED7, and ED10) and Group B (Sphingomonas sp. strains ED8 and ED9). All isolates used E2 and E1 as the sole carbon sources and showed high E1 and E2 degradation activities. In all strains, more than 50% of 0.8 mg of E1 or E2 was degraded in 4 mL of inorganic medium over 24 h, and 90% was degraded over 120 h. By incubating the resting ED8 cells with E2 and the meta-cleavage inhibitor 3-chlorocatechol, we identified two metabolites, 4-hydroxyestrone (4-OH-E1) and 4-hydroxyestradiol (4-OH-E2), and confirmed their identity using authentic chemicals. The 4-OH-E1 and 4-OH-E2 compounds were assumed to be intermediate metabolites formed before meta-cleavage, as they were not identified in culture without 3-chlorocatechol. Degradation of E2 by strain ED8 can be initiated by hydroxylation of the C-4 position, followed by meta-cleavage of the benzene ring. When strains ED8 degraded E2, we further identified hydroxy-E2, keto-E1 and -E2, and an additional degradation product via mass spectrometry. The presence of these compounds implied degradation through a second pathway initiated through an attack of the saturated ring. [Copyright &y& Elsevier]

Abstract: Anaerobic benzene-degrading enrichment cultures performing methanogenesis were obtained from non-contaminated lotus field soil. Stable isotope probing with 13C benzene was used to detect the bacteria that were involved in benzene degradation. Denaturing gradient gel electrophoresis (DGGE) of fractionated samples exhibited an obvious shift of some DGGE bands to a heavier DNA fraction. An almost full-length 16S rRNA gene sequence corresponding to the DGGE band, namely Hasda-A, was obtained by constructing a clone library of the heavier fraction. The Hasda-A sequence showed only 85.1% identity with the closest identified bacterium, Syntrophus gentianae. Hasda-A may be an important bacterium involved in the initial steps of benzene degradation under methanogenic conditions, as it was the most prominent bacterium that assimilated labeled benzene early in the process of benzene degradation. A primer set was designed to quantify the gene copies of Hasda-A by quantitative PCR. Hasda-A was present at a concentration of (3.5±0.8)×106 copies/mL and represented 8.4% of gene copies among bacteria in the enrichment culture. The enrichment culture consisted of three dominant bacterial groups: Hasda-A and both aceticlastic and hydrogenotrophic methanogens. Methane is believed to be produced from benzene by the sequential degradation of benzene by fermenting bacteria, hydrogen-producing acetogens, and methanogens. [Copyright &y& Elsevier]

Lead (Pb) isotopic data were used in this study to first distinguish the partitioning of anthropogenic and natural lead in different fractions, obtained by BCR sequential extraction, and then to anticipate their mixing process in the soakaway sediment of artificial infiltration facilities (AIF). Total metal content was found higher in soakaway sediment samples than that of soil. The lowest 206Pb/207Pb ratios were mostly observed in exchangeable fractions of soil and sediment samples, while residual fractions mostly showed the highest 206Pb/207Pb and 208Pb/207Pb ratios than those of other fractions. In general, both ratios were higher in the soil than those of sediments. Further among soil samples, residual fraction of bottom soil exhibited higher ratios than surface soil indicating higher contribution of natural lead with depth. In addition, the difference in Pb content, partitioning and its isotope signature among four sediment samples were also investigated considering their sampling locations. The plot of 206Pb/207Pb versus 208Pb/207Pb showed two well demarcated cluster formations by soil and sediments samples that describe the partitioning between anthropogenic and natural lead; and some points falling in between soil and sediment samples pertinently illustrated the mixing processes between these two different pools of lead. [ABSTRACT FROM AUTHOR]

Diverse environmental genes have been identified recently. To characterize their functions, it is necessary to understand which genes and what combinations of those genes are responsible for the biodegradation of soil contaminants. In this article, a 60-mer oligonucleotide microarray was constructed to simultaneously detect di- and monooxygenase genes for benzene and related compounds. In total, 148 probes were designed and validated by pure-culture hybridizations using the following criteria to discriminate between highly homologous genes: ≤53-bp identities and ≤25-bp continuous stretch to nontarget sequences. Microarray hybridizations were performed using PCR products amplified from five benzene-amended soils and two oil-contaminated soils. Six of the probes gave a positive signal for more than six soils; thus, they may represent key sequences for benzene degradation in the environment. The microarray developed in this study will be a powerful tool for the screening of key genes involved in benzene degradation and for the rapid profiling of benzene oxygenase gene diversity in contaminated soils. [ABSTRACT FROM AUTHOR]

Abstract: In situ uptake of [2,4,6,7-3H(N)]estrone ([3H]E1) by the major phylogenetic groups present in activated sludge samples from two different municipal wastewater treatment plants was investigated using microautoradiography–fluorescence in situ hybridization (MAR–FISH). Approximately 1–2% of the total cells confined in the samples by an EUB probe mix contributed to E1 assimilation. Almost all the detected E1-assimilating cells involved in the early phase of E1 degradation were affiliated with the Beta- and Gammaproteobacteria. In the early phase of E1 degradation, no E1-assimilating cells affiliated with the Alphaproteobacteria, Actinobacteria, the Cytophaga–Flavobacterium cluster of phylum Bacteroidetes, or the phyla Chloroflexi, Nitrospira and Planctomycetes were detected. Bacteria affiliated with the Betaproteobacteria in the shape of long rods or chains of rods were found to contribute most to in situ E1 degradation. They contributed 61% and 82% of total E1-assimilating cells in cultures from two sources of activated sludge spiked with [3H]E1. The E1-degrading bacteria related to the Betaproteobacteria differed phylogenetically from the aerobic E1-degrading bacterial isolates reported in previous studies. In addition, MAR–FISH revealed the significant contribution of E1-degrading bacteria affiliated with the Gammaproteobacteria in the degradation of E1 in activated sludge. [Copyright &y& Elsevier]

This study investigated the effects of ammonium and nitrite on ammonia-oxidizing bacteria (AOB) from an activated sludge process in laboratory-scale continuous-flow reactors. AOB communities were analyzed using specific PCR followed by denaturing gel gradient electrophoresis, cloning and sequencing of the 16S rRNA gene, and AOB populations were quantified using real-time PCR. To study the effect of ammonium, activated sludge from a sewage treatment system was enriched in four reactors receiving inorganic medium containing four different ammonium concentrations (2, 5, 10 and 30 mM NH4+-N). One of several sequence types of the Nitrosomonas oligotropha cluster predominated in the reactors with lower ammonium loads (2, 5 and 10 mM NH4+-N), whereas Nitrosomonas europaea was the dominant AOB in the reactor with the highest ammonium load (30 mM NH4+-N). The effect of nitrite was studied by enriching the enriched culture possessing both N. oligotropha and N. europaea in four reactors receiving 10-mM-ammonium inorganic medium containing four different nitrite concentrations (0, 2, 12 and 22 mM NO2−-N). Nitrosomonas oligotropha comprised the majority of AOB populations in the reactors without nitrite accumulation (0 and 2 mM NO2−-N), whereas N. europaea was in the majority in the 12- and 22-mM NO2−-N reactors, in which nitrite concentrations were 2.1–5.7 mM (30–80 mg N L−1). [ABSTRACT FROM AUTHOR]

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. • Sequential solid-phase extraction effectively recovers dissolved organic matter (DOM). • Ozone preferentially decomposes more unsaturated and less oxidized DOM. • Ozone oxidation by-products are found as highly oxidized hydrophobic neutral (HPON) and hydrophilic acid (HPIA). • Biological activated carbon filtration removes less oxidized HPON and hydrophobic acid (HPOA). • Chlorine selectively reacts with HPON and HPIA species. [ABSTRACT FROM AUTHOR]

The shotgun isotope array method has been proposed to be an effective new tool for use in substrate-specific microbe exploration without any prior knowledge of the community composition. Proof of concept was demonstrated by detection of acetate-degrading microorganisms in activated sludge and further verified by independent stable isotope probing (SIP). [ABSTRACT FROM AUTHOR]

Granular activated carbon (GAC) has been widely introduced to advanced drinking water purification plants to remove organic matter and ammonium. Backwashing, which is the routine practice for GAC maintenance, is an important operational factor influencing the performance of GAC and its microbial biomass. In this study, the effects of backwashing on the ammonium removal potential of GAC were evaluated. In addition, abundances of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) on GAC were analyzed. GAC samples before and after backwashing were collected from a full-scale drinking water purification plant. Samplings were conducted before and after implementation of prechlorination of raw water. The results showed that the ammonium removal potential of the GAC increased by 12% after backwashing before prechlorination (p < 0.01). After implementing the prechlorination, the ammonium removal potential of the GAC decreased by 12% even after backwashing (p < 0.01). The AOA was predominant on the GAC in the two samplings. Regardless of prechlorination, the amounts of the AOA and the AOB remained at the same level before and after backwashing. Analysis of the backwashing water indicated that the amounts of the AOA and AOB washed out from the GAC were negligible (0.08%–0.26%) compared with their original amounts on the GAC. These results revealed the marginal role of backwashing on the biomass of ammonia oxidizers on GAC. However, the results also revealed that backwashing could have a negative impact on the ammonium removal potential of GAC during prechlorination. [ABSTRACT FROM AUTHOR]

Abstract: Artificial infiltration facilities (AIFs) that enhance groundwater recharge and regulate urban runoff are going to be an integral element of the urban infrastructure. However, AIFs provide a sink which trap pollutants that are likely to cause groundwater contamination. The current study aimed first to examine the mobility characteristics of Cu, Zn, and Pb through soil and soakaway sediment using an integrated analytical approach for column leaching with artificial road runoff (ARR) and then to differentiate the sorption patterns among different samples, (i.e., surface soil, underlying soil, and soakaway sediments) using mass balances. In addition, the study compares metal retention and release under continuous and intermittent flow conditions. Column leaching experiments were conducted using batches for 10 and 30d under continuous flow condition and for 20d of intermittent leaching. Heavy metal content and partitioning in soil and sediment used in columns were well characterized before and after leaching experiments. The results showed that a gradual increase in pH and decrease in dissolved organic carbon had pronounced effect on the mobilization of heavy metals. Pb showed the highest retention compared to Cu and Zn which implies that metal complexes play a pivotal role in metal transport. Labile fractions were found to be trapped by the solid materials for retention and their high concentration in ARR is a major concern from the pollution point of view through infiltration facilities. Results obtained in this study predict the risk associated with the release of retained heavy metal under changing environmental conditions in AIFs. [Copyright &y& Elsevier]