Endpoint Recombinase Polymerase Amplification (RPA) Assay for Enumeration of Thiocyanate-degrading Bacteria.

An endpoint recombination amplification reaction (RPA) assay for assessing the abundance of the gene encoding thiocyanate dehydrogenase (TcDH) in Thiohalobacter has been developed. The RPA reaction was performed at 37°C for 30| |min, terminated by the addition of sodium dodecyl sulfate (SDS) solution, and the DNA concentration of the RPA product was fluorometrically measured. The abundance of TcDH in 22 activated sludge samples and 7 thiocyanate-degrading enrichment cultures ranged between 2.5×103 and 1.5×106 copies µL-1, showing a linear relationship (R2=0.83) with those measured using a conventional quantitative PCR assay.

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.

Complete Genome Sequence of Thiohalobacter sp. Strain COW1, Isolated from Activated Sludge Treating Coke Oven Wastewater.

A thiocyanate-degrading bacterium, Thiohalobacter sp. strain COW1, was isolated from activated sludge treating coke oven wastewater, and the complete genome sequence was determined. COW1 contained a single circular chromosome (3.23 Mb; G+C content, 63.4%) in which 2,788 protein-coding genes, 39 tRNA genes, and 3 rRNA genes were identified.

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.

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.

Thiocyanate Degradation by a Highly Enriched Culture of the Neutrophilic Halophile Thiohalobacter sp. Strain FOKN1 from Activated Sludge and Genomic Insights into Thiocyanate Metabolism.

Thiocyanate (SCN-) is harmful to a wide range of organisms, and its removal is essential for environmental protection. A neutrophilic halophile capable of thiocyanate degradation, Thiohalobacter sp. strain FOKN1, was highly enriched (relative abundance; 98.4%) from activated sludge collected from a bioreactor receiving thiocyanate-rich wastewater. The enrichment culture degraded 3.38 mM thiocyanate within 140 h, with maximum activity at pH 8.8, 37°C, and 0.18 M sodium chloride. Thiocyanate degradation was inhibited by 30 mg L-1 phenol, but not by thiosulfate. Microbial thiocyanate degradation is catalyzed by thiocyanate dehydrogenase, while limited information is currently available on the molecular mechanisms underlying thiocyanate degradation by the thiocyanate dehydrogenase of neutrophilic halophiles. Therefore, (meta)genomic and proteomic analyses of enrichment cultures were performed to elucidate the whole genome sequence and proteome of Thiohalobacter sp. strain FOKN1. The 3.23-Mb circular Thiohalobacter sp. strain FOKN1 genome was elucidated using a PacBio RSII sequencer, and the expression of 914 proteins was identified by tandem mass spectrometry. The Thiohalobacter sp. strain FOKN1 genome had a gene encoding thiocyanate dehydrogenase, which was abundant in the proteome, suggesting that thiocyanate is degraded by thiocyanate dehydrogenase to sulfur and cyanate. The sulfur formed may be oxidized to sulfate by the sequential oxidation reactions of dissimilatory sulfite reductase, adenosine-5'-phosphosulfate reductase, and dissimilatory ATP sulfurylase. Although the Thiohalobacter sp. strain FOKN1 genome carried a gene encoding cyanate lyase, its protein expression was not detectable. The present study advances the understanding of the molecular mechanisms underlying thiocyanate degradation by the thiocyanate dehydrogenase of neutrophilic halophiles.

[A Case of Primary Lymphoma of the Breast with Hepatic Cirrhosis].

We report a case of primary lymphoma of the breast complicated by heart failure and alcoholic-decompensated hepatic cirrhosis. The patient was a woman in her 60s who noticed a right breast tumor growing 3 months previously. The size of the tumor was approximately 5 cm, and the tumor had infiltrated the skin. There was no metastasis to the axillary lymph node or other organs by CT. We performed right breast mastectomy. Pathology indicated diffuse large B cell lymphoma(DLBCL). We considered chemotherapy, but her general condition was not good because of hepatic cirrhosis, so we administered palliative care. Although chemotherapy is the first choice of treatment for DLBCL, it is necessary to individually consider each patient's circumstances.

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.

Draft Genome Sequence of Thiohalobacter thiocyanaticus Strain FOKN1, a Neutrophilic Halophile Capable of Thiocyanate Degradation.

A draft genome sequence of a neutrophilic halophile capable of thiocyanate degradation, Thiohalobacter thiocyanaticus FOKN1, was determined using a PacBio RSII sequencer. A 3.23-Mb circular genome sequence was assembled, in which 3,026 gene-coding sequences, 45 tRNAs, and 1 rrn operon were annotated.

Membrane fouling potentials and cellular properties of bacteria isolated from fouled membranes in a MBR treating municipal wastewater.

Membrane fouling remains a major challenge for wider application of membrane bioreactors (MBRs) to wastewater treatment. Membrane fouling is mainly caused by microorganisms and their excreted microbial products. For development of more effective control strategies, it is important to identify and characterize the microorganisms that are responsible for membrane fouling. In this study, 41 bacterial strains were isolated from fouled microfiltration membranes in a pilot-scale MBR treating real municipal wastewater, and their membrane fouling potentials were directly measured using bench-scale cross-flow membrane filtration systems (CFMFSs) and related to their cellular properties. It was found that the fouling potential was highly strain dependent, suggesting that bacterial identification at the strain level is essential to identify key fouling-causing bacteria (FCB). The FCB showed some common cellular properties. The most prominent feature of FCB was that they formed convex colonies having swollen podgy shape and smooth lustrous surfaces with high water, hydrophilic organic matter and carbohydrate content. However, general and rigid biofilm formation potential as determined by microtiter plates and cell surface properties (i.e., hydrophobicity and surface charge) did not correlate with the fouling potential in this study. These results suggest that the fouling potential should be directly evaluated under filtration conditions, and the colony water content could be a useful indicator to identify the FCB.

Metagenomic and metaproteomic analyses of Accumulibacter phosphatis-enriched floccular and granular biofilm.

Biofilms are ubiquitous in nature, forming diverse adherent microbial communities that perform a plethora of functions. Here we operated two laboratory-scale sequencing batch reactors enriched with Candidatus Accumulibacter phosphatis (Accumulibacter) performing enhanced biological phosphorus removal. Reactors formed two distinct biofilms, one floccular biofilm, consisting of small, loose, microbial aggregates, and one granular biofilm, forming larger, dense, spherical aggregates. Using metagenomic and metaproteomic methods, we investigated the proteomic differences between these two biofilm communities, identifying a total of 2022 unique proteins. To understand biofilm differences, we compared protein abundances that were statistically enriched in both biofilm states. Floccular biofilms were enriched with pathogenic secretion systems suggesting a highly competitive microbial community. Comparatively, granular biofilms revealed a high-stress environment with evidence of nutrient starvation, phage predation pressure, and increased extracellular polymeric substance and cell lysis. Granular biofilms were enriched in outer membrane transport proteins to scavenge the extracellular milieu for amino acids and other metabolites, likely released through cell lysis, to supplement metabolic pathways. This study provides the first detailed proteomic comparison between Accumulibacter-enriched floccular and granular biofilm communities, proposes a conceptual model for the granule biofilm, and offers novel insights into granule biofilm formation and stability.

Molecular analysis of methanogens involved in methanogenic degradation of tetramethylammonium hydroxide in full-scale bioreactors.

This study investigated methanogenic communities involved in degradation of tetramethylammonium hydroxide (TMAH) in three full-scale bioreactors treating TMAH-containing wastewater. Based on the results of terminal-restriction fragment-length polymorphism (T-RFLP) and quantitative PCR analyses targeting the methyl-coenzyme M reductase alpha subunit (mcrA) genes retrieved from three bioreactors, Methanomethylovorans and Methanosarcina were the dominant methanogens involved in the methanogenic degradation of TMAH in the bioreactors. Furthermore, batch experiments were conducted to evaluate mcrA messenger RNA (mRNA) expression during methanogenic TMAH degradation, and the results indicated that a higher level of TMAH favored mcrA mRNA expression by Methansarcina, while Methanomethylovorans could only express considerable amount of mcrA mRNA at a lower level of TMAH. These results suggest that Methansarcina is responsible for methanogenic TMAH degradation at higher TMAH concentrations, while Methanomethylovorans may be important at a lower TMAH condition.

High-temperature EBPR process: the performance, analysis of PAOs and GAOs and the fine-scale population study of Candidatus "Accumulibacter phosphatis".

The applicability of the enhanced biological phosphorus removal (EBPR) process for the removal of phosphorus in warm climates is uncertain due to frequent reports of EBPR deterioration at temperature higher than 25 °C. Nevertheless, a recent report on a stable and efficient EBPR process at 28 °C has inspired the present study to examine the performance of EBPR at 24 °C-32 °C, as well as the PAOs and GAOs involved, in greater detail. Two sequencing batch reactors (SBRs) were operated for EBPR in parallel at different temperatures, i.e., SBR-1 at 28 °C and SBR-2 first at 24 °C and subsequently at 32 °C. Both SBRs exhibited high phosphorus removal efficiencies at all three temperatures and produced effluents with phosphorus concentrations less than 1.0 mg/L during the steady state of reactor operation. Real-time quantitative polymerase chain reaction (qPCR) revealed Accumulibacter-PAOs comprised 64% of the total bacterial population at 24 °C, 43% at 28 °C and 19% at 32 °C. Based on fluorescent in situ hybridisation (FISH), the abundance of Competibacter-GAOs at both 24 °C and 28 °C was rather low (<10%), while it accounted for 40% of the total bacterial population at 32 °C. However, the smaller Accumulibacter population and larger population of Competibacter at 32 °C did not deteriorate the phosphorus removal performance. A polyphosphate kinase 1 (ppk1)-based qPCR analysis on all studied EBPR processes detected only Accumulibacter clade IIF. The Accumulibacter population shown by 16S rRNA and ppk1 was not significantly different. This finding confirmed the existence of single clade IIF in the processes and the specificity of the clade IIF primer sets designed in this study. Habitat filtering related to temperature could have contributed to the presence of a unique clade. The clade IIF was hypothesised to be able to perform the EBPR activity at high temperatures. The clade's robustness most likely helps it to fit the high-temperature EBPR sludge best and allows it not only to outcompete other Accumulibacter clades but coexist with GAOs without compromising EBPR activity.

Transcriptional responses of bacterial amoA gene to dimethyl sulfide inhibition in complex microbial communities.

This study presented an approach by combining the real-time reverse transcription polymerase chain reaction with the terminal restriction fragment length polymorphism (T-RFLP) to investigate transcriptional responses of ammonia-oxidizing bacteria (AOB) to dimethyl sulfide (DMS) inhibition. Batch experiments with added ammonium and DMS were conducted using three activated sludges and Nitrosomonas europaea, and the transcriptional responses of the amo subunit A (amoA) mRNA were evaluated. It was found that DMS inhibited ammonium oxidation and amoA mRNA expression in all batch experiments but the inhibition degree observed was different for different sludges examined. It is likely that the different inhibitory effects of DMS on ammonium oxidation and amoA mRNA expression stemmed from different dominant AOB populations in the sludges. The T-RFLP results for amoA mRNA suggested that inhibition of ammonium oxidation by DMS to Nm. europaea-like AOB with T-RF 219/270 is relatively minor compared to other AOB populations in the examined sludges, such as Nm. europaea-like AOB with T-RF 491/491.

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.

Linking TFT-LCD wastewater treatment performance to microbial population abundance of Hyphomicrobium and Thiobacillus spp.

This study investigated the linkage between performance of two full-scale membrane bioreactor (MBR) systems treating thin-film transistor liquid crystal display (TFT-LCD) wastewater and the population dynamics of dimethylsulfoxide (DMSO)/dimethylsulfide (DMS) degrading bacteria. High DMSO degradation efficiencies were achieved in both MBRs, while the levels of nitrification inhibition due to DMS production from DMSO degradation were different in the two MBRs. The results of real-time PCR targeting on DMSO/DMS degrading populations, including Hyphomicrobium and Thiobacillus spp., indicated that a higher DMSO oxidation efficiency occurred at a higher Hyphomicrobium spp. abundance in the systems, suggesting that Hyphomicrobium spp. may be more important for complete DMSO oxidation to sulfate compared with Thiobacillus spp. Furthermore, Thiobacillus spp. was more abundant during poor nitrification, while Hyphomicrobium spp. was more abundant during good nitrification. It is suggested that microbial population of DMSO/DMS degrading bacteria is closely linking to both DMSO/DMS degradation efficiency and nitrification performance.

Impact of food to microorganism (F/M) ratio and colloidal chemical oxygen demand on nitrification performance of a full-scale membrane bioreactor treating thin film transistor liquid crystal display wastewater.

This study investigated impact of food to microorganism (F/M) ratio and colloidal chemical oxygen demand (COD) on nitrification performance in one full-scale membrane bioreactor (MBR) treating monoethanolamine (MEA)/dimethyl sulfoxide (DMSO)-containing thin film transistor liquid crystal display (TFT-LCD) wastewater. Poor nitrification was observed under high organic loading and high colloidal COD conditions, suggesting that high F/M ratio and colloidal COD situations should be avoided to minimize their negative impacts on nitrification. According to the nonmetric multidimensional scaling (NMS) statistical analyses on terminal restriction fragment length polymorphism (T-RFLP) results of ammonia monooxygenase (amoA) gene, the occurrence of Nitrosomonas oligotropha-like ammonia oxidizing bacteria (AOB) was positively related to successful nitrification in the MBR systems, while Nitrosomonas europaea-like AOB was positively linked to nitrification rate, which can be attributed to the high influent total nitrogen condition. Furthermore, Nitrobacter- and Nitrospira-like nitrite oxidizing bacteria (NOB) were both abundant in the MBR systems, but the continuously low nitrite environment is likely to promote the growth of Nitrospira-like NOB.

Nitrifying bacterial community structures and their nitrification performance under sufficient and limited inorganic carbon conditions.

This study examined the hypothesis that different inorganic carbon (IC) conditions enrich different ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) populations by operating two laboratory-scale continuous-flow bioreactors fed with 15 and 100 mg IC/L, respectively. During this study, both bioreactors maintained satisfactory nitrification performance and stably oxidized 250 mg N/L of influent ammonium without nitrite accumulation. Based on results of cloning/sequencing and terminal restriction fragment length polymorphism targeting on the ammonia monooxygenase subunit A (amoA) gene, Nitrosomonas nitrosa lineage was identified as the dominant AOB population in the high-IC bioreactor, while Nitrosomonas europaea and Nitrosomonas nitrosa lineage AOB were dominant in the low-IC bioreactor. Results of real-time polymerase chain reactions for Nitrobacter and Nitrospira 16S rRNA genes indicated that Nitrospira was the predominant NOB population in the high-IC bioreactor, while Nitrobacter was the dominant NOB in the low-IC bioreactor. Furthermore, batch experiment results suggest that N. europaea and Nitrobacter populations are proliferated in the low-IC bioreactor due to their higher rates under low IC conditions despite the fact that these two populations have been identified as weak competitors, compared with N. nitrosa and Nitrospira, under low ammonium/nitrite environments. This study revealed that in addition to ammonium/nitrite concentrations, limited IC conditions may also be important in selecting dominant AOB/NOB communities of nitrifying bioreactors.

Responses of ammonia-oxidizing archaeal and betaproteobacterial populations to wastewater salinity in a full-scale municipal wastewater treatment plant.

The diversity and abundance of ammonia-oxidizing Betaproteobacteria and archaea were investigated in a full-scale municipal wastewater treatment plant where the wastewater conductivity level varied considerably (due to seawater salinity intrusion) during this study between 2004 and 2007. Based on the quantitative polymerase chain reaction of ammonia monooxygenase subunit A (amoA) genes, an increase in the ammonia oxidizing bacteria amoA gene copies occurred with a decrease in the wastewater salinity level. A corresponding decrease in the average ammonia-oxidizing archaea to bacteria ratio, from 1.22 (2004 and 2005), 0.17 (2006), and then to 0.07 (2007), was observed. Phylogenetic analyses on amoA gene sequences indicated that Nitrosomonas marina-like ammonia oxidizing bacteria and Thaumarcheota Ⅰ.1a (marina group) ammonia-oxidizing archaea were dominant when the wastewater salinity level fluctuated at high values with an average of 4.83 practical salinity unit (psu), while Nitrosomonas urea-like ammonia oxidizing bacteria and Thaumarcheota Ⅰ.1b (soil group) ammonia-oxidizing archaea became dominant when the wastewater salinity decreased to a more stable lower level with an average of 1.93 psu. Based on the amoA gene-based terminal restriction fragment length polymorphism analyses, results from this study demonstrated that the observed shift in ammonia oxidizing bacteria and archaea populations is likely caused by a change of the wastewater salinity level.