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Little is known about the cell physiology of anammox bacteria growing at extremely low growth rates. Here, “Candidatus Brocadia sinica” and “Candidatus Scalindua sp.” were grown in continuous anaerobic membrane bioreactors (MBRs) with complete biomass retention to determine maintenance energy (i.e., power) requirements at near-zero growth rates. After prolonged retentostat cultivations, the specific growth rates (μ) of “Ca. B. sinica” and “Ca. Scalindua sp.” decreased to 0.000023 h(−1) (doubling time of 1255 days) and 0.000157 h(−1) (184 days), respectively. Under these near-zero growth conditions, substrate was continuously utilized to meet maintenance energy demands (m(e)) of 6.7 ± 0.7 and 4.3 ± 0.7 kJ mole of biomass-C(−1) h(−1) for “Ca. B. sinica” and “Ca. Scalindua sp.”, which accorded with the theoretically predicted values of all anaerobic microorganisms (9.7 and 4.4 kJ mole of biomass-C(−1) h(−1)at 37 °C and 28 °C, respectively). These m(e) values correspond to 13.4 × 10(−15) and 8.6 × 10(−15) watts cell(−1) for “Ca. B. sinica” and “Ca. Scalindua sp.”, which were five orders of magnitude higher than the basal power limit for natural settings (1.9 × 10(−19) watts cells(−1)). Furthermore, the minimum substrate concentrations required for growth (S(min)) were calculated to be 3.69 ± 0.21 and 0.09 ± 0.05 μM NO(2)(−) for “Ca. B. sinica” and “Ca. Scalindua sp.”, respectively. These results match the evidence that “Ca. Scalindua sp.” with lower maintenance power requirement and S(min) are better adapted to energy-limited natural environments than “Ca. B. sinica”, suggesting the importance of these parameters on ecological niche differentiation in natural environments.

Anaerobic ammonium-oxidizing (anammox) bacteria are slow-growing and fastidious bacteria, and limited numbers of enrichment cultures have been established. A metagenomic ana-lysis of our 5 established anammox bacterial enrichment cultures was performed in the present study. Fourteen high-quality metagenome-assembled genomes (MAGs) were obtained, including those of 5 anammox Planctomycetota (Candidatus Brocadia, Ca. Kuenenia, Ca. Jettenia, and Ca. Scalindua), 4 Bacteroidota, and 3 Chloroflexota. Based on the gene sets of metabolic pathways involved in the degradation of polymeric substances found in Chloroflexota and Bacteroidota MAGs, they are expected to be scavengers of extracellular polymeric substances and cell debris.

Anammox bacteria produce N

Wastewater-based epidemiology has attracted attention as a COVID-19 surveillance tool. Here, we developed a practical method for detecting SARS-CoV-2 RNA in wastewater (the EPISENS-S method), which employs direct RNA extraction from wastewater pellets formed via low-speed centrifugation. The subsequent multiplex one-step RT-preamplification reaction with forward and reverse primers for SARS-CoV-2 and a reverse primer only for pepper mild mottle virus (PMMoV) allowed for qPCR quantification of the targets with different abundances in wastewater from the RT-preamplification product. The detection sensitivity of the method was evaluated using wastewater samples seeded with heat-inactivated SARS-CoV-2 in concentrations of 2.11 x 10(3) to 2.11 x 10(6) copies/L. The results demonstrated that the sensitivity of the EPISENS-S method was two orders of magnitude higher than that of the conventional method (PEG precipitation, followed by regular RT-qPCR; PEG-QVR-qPCR). A total of 37 untreated wastewater samples collected from two wastewater treatment plants in Sapporo, Japan when 1.6 to 18 new daily reported cases per 100,000 people were reported in the city (March 4 to July 8, 2021), were examined using the EPISENS-S method to confirm its applicability to municipal wastewater. SARS-CoV-2 RNA was quantified in 92 % (34/37) of the samples via the EPISENS-S method, whereas none of the samples (0/37) was quantifiable via the PEG-QVR-qPCR method. The PMMoV concentrations measured by the EPISENS-S method ranged from 2.60 x 10(6) to 1.90 x 10(8) copies/L, and the SARS-CoV-2 RNA concentrations normalized by PMMoV ranged from 5.71 x 10(-6 )to 9.51 x 10(-4) . The long-term trend of normalized SARS-CoV-2 RNA concentration in wastewater was consistent with that of confirmed COVID-19 cases in the city. These results demonstrate that the EPISENS-S method is highly sensitive and suitable for routine COVID-19 wastewater surveillance.

15 N/ 14 N analysis for aqueous N species is a powerful tool for obtaining insights into the global N cycle, but the procedure is cumbersome and laborious. The combined use of colorimetric reagents and MALDI-TOF MS, designated color MALDI-TOF MS, enabled us to determine the 15 N atom% of common aqueous N species without laborious sample preparation and chromatographic separation steps; for instance, the 15 N atom% of NO 2 − can be determined from >1,000 liquid samples daily at

In the nitrogen (N) cycle, nitrogenous compounds are chemically and biologically converted to various aqueous and gaseous N species. The (15)N-labeling approach is a powerful culture-dependent technique to obtain insights into the complex nitrogen transformation reactions that occur in cultures. In the (15)N-labeling approach, the fates of supplemented (15)N- and/or unlabeled gaseous and aqueous compounds are tracked by mass spectrometry (MS) analysis, whereas MS analysis of aqueous N species requires laborious sample preparation steps and is performed using isotope-ratio mass spectrometry, which requires an expensive mass spectrometer. We developed a simple and high-throughput MS method for determining the (15)N atoms percent of NH(4)(+), NO(2)(−), NO(3)(−), NH(2)OH, and N(2)H(4), where liquid samples (1,000 liquid samples daily at

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is known to be present in sewage, and wastewater -based epidemiology has attracted much attention. However, the physical partitioning of SARS-CoV-2 in wastewater and the removal efficiency of treatment systems require further investigation. This study aimed to investigate the de-tectability and physical partitioning of SARS-CoV-2 in wastewater and assess its removal in a large-scale septic tank employing anaerobic, anoxic, and oxic processes in a sequential batch reactor, which was installed in a coronavirus disease 2019 (COVID-19) quarantine facility. The amount of SARS-CoV-2 RNA in wastewater was determined with polyethylene glycol (PEG) precipitation followed by quantitative polymerase chain reaction (qPCR), and the association of SARS-CoV-2 with wastewater solids was evaluated by the effect of filtration prior to PEG precipitation (pre-filtration). The amount of SARS-CoV-2 RNA detected from pre-filtered samples was substantially lower than that of samples without pre-filtration. These results suggest that most SARS-CoV-2 particles in wastewater are associ-ated with the suspended solids excluded by pre-filtration. The removal efficiency of SARS-CoV-2 in the septic tank was evaluated based on the SARS-CoV-2 RNA concentrations in untreated and treated wastewater, which was determined by the detection method optimized in this study. Escherichia coli and pepper mild mottle virus (PMMoV) were also quantified to validate the wastewater treatment system's performance. The mean log10 reduction values of SARS-CoV-2, E. coli, and PMMoV were 2.47 (range, 2.25-2.68), 2.81 (range, 2.45-3.18), and 0.66 (range, 0.61-0.70), respec-tively, demonstrating that SARS-CoV-2 removal by the wastewater treatment system was comparable to or better than the removal of fecal indicators. These results suggest that SARS-CoV-2 can be readily removed by the septic tank. This is the first study to determine the removal efficiency of SARS-CoV-2 in a facility-level sequencing batch activated sludge system.

RNA virus populations are not clonal; rather, they comprise a mutant swarm in which sequences are slightly different from the master sequence. Genetic diversity within a population (intrapopulation genetic diversity) is critical for RNA viruses to survive under environmental stresses. Disinfection has become an important practice in the control of pathogenic viruses; however, the impact of intrapopulation genetic diversity on the sensitivity of disinfection, defined as –log10 (postdisinfected infectious titer/predisinfected titer), has not been elucidated. In this study, we serially passaged populations of rhesus rotavirus. We demonstrated that populations with reduced chlorine sensitivity emerged at random and independently of chlorine exposure. Sequencing analysis revealed that compared with sensitive populations, less-sensitive ones had higher non-synonymous genetic diversity of the outer capsid protein gene, suggesting that changes in the amino acid sequences of the outer capsid protein were the main factors influencing chlorine sensitivity. No common mutations were found among less-sensitive populations, indicating that rather than specific mutations, the diversity of the outer capsid protein itself was associated with the disinfection sensitivity and that the disinfection sensitivity changed stochastically. Simulation results suggest that the disinfection sensitivity of a genetically diverse population is destabilized if cooperative viral clusters including multiple sequences are formed. These results advocate that any prevention measures leading to low intrapopulation genetic diversity are important to prevent the spread and evolution of pathogenic RNA viruses in society.

Migratory geese could influence the microbiological water quality; however, their impacts on pathogen dynamics remain largely unknown. In this study, we analyzed the population dynamics of Campylobacter and Arcobacter group bacteria (AGB) in a freshwater lake in Japan over two years. The bacteria were quantified by using both culture-dependent and -independent methods. The potential sources of these bacteria were examined by a high-throughput flaA sequencing approach. Campylobacter was abundantly detected both by culture-dependent and -independent methods in the lake, especially when migratory geese were present in the lake. High-throughput flaA sequencing suggests that geese were the likely source of Campylobacter in the lake. The viable population of Campylobacter exceeds the concentrations that can potentially cause 10

To harvest directly usable electrical energy from real domestic wastewater, a new power management system (PMS), transistor-based low voltage boosters followed by a voltage rectifier (LVBR), was developed and tested for its energy harvesting performance. Three air-cathode MFCs were individually linked with LVBs, which were electrically stacked in parallel and then connected with a single voltage rectifier (MFC-LVBR). The MFC-LVBR system could increase V

A simple analytical method was developed to determine the arsenite (As(III)) concentration using a DNA aptamer and gold nanoparticles (AuNPs). Prior to sample measurements, the method sensing mechanism was confirmed by analyzing the particle size of the AuNPs at each step of the analysis procedure, and the key operational parameters that affect the method performance were optimized. The optimal final NaCl concentration, incubation time with NaCl and pH of a 3-(N-morpholino) propanesulfonic acid buffer were 60 mM, 10 min and 7.3, respectively. A calibration curve was created under optimized operational conditions. The calibration curve was linear from a 1.0- to 10-μM As(III) concentration. The detection limit was 2.1 μM (161 μg/L). Using the calibration curve, we evaluated groundwater samples spiked with As(III). As(III) concentrations in groundwater pretreated with a 0.2-μm-pore-size membrane filter and cation-exchange resin were determined by using the method, which suggests that the proposed method can be used to determine the As(III) concentration in groundwater.

Natural abundance of stable nitrogen (N) and oxygen (O) isotopes are invaluable biogeochemical tracers for assessing the N transformations in the environment. To fully exploit these tracers, the N and O isotope effects ((15)ε and (18)ε) associated with the respective nitrogen transformation processes must be known. However, the N and O isotope effects of anaerobic ammonium oxidation (anammox), one of the major fixed N sinks and NO(3)(−) producers, are not well known. Here, we report the dual N and O isotope effects associated with anammox by three different anammox bacteria including “Ca. Scalindua japonica”, a putative marine species, which were measured in continuous enrichment culture experiments. All three anammox species yielded similar N isotope effects of NH(4)(+) oxidation to N(2) ((15)ε(NH4→N2)) ranging from 30.9‰ to 32.7‰ and inverse kinetic isotope effects of NO(2)(−) oxidation to NO(3)(−) ((15)ε(NO2→NO3) = −45.3‰ to −30.1‰). In contrast, (15)ε(NO2→N2) (NO(2)(−) reduction to N(2)) were significantly different among three species, which is probably because individual anammox bacteria species might possess different types of nitrite reductase. We also report the combined O isotope effects for NO(2)(−) oxidation ((18)E(NO2→NO3)) by anammox bacteria. These obtained dual N and O isotopic effects could provide significant insights into the contribution of anammox bacteria to the fixed N loss and NO(2)(−) reoxidation (N recycling) in various natural environments.

This study presents a novel methodology for estimating the concentration of environmental pollutants in water, such as pathogens, based on environmental parameters. The scientific uniqueness of this study is the prevention of excess conformity in the model fitting by applying domain knowledge, which is the accumulated scientific knowledge regarding the correlations between response and explanatory variables. Sign constraints were used to express domain knowledge, and the effect of the sign constraints on the prediction performance using censored datasets was investigated. As a result, we confirmed that sign constraints made prediction more accurate compared to conventional sign-free approaches. The most remarkable technical contribution of this study is the finding that the sign constraints can be incorporated in the estimation of the correlation coefficient in Tobit analysis. We developed effective and numerically stable algorithms for fitting a model to datasets under the sign constraints. This novel algorithm is applicable to a wide variety of the prediction of pollutant contamination level, including the pathogen concentrations in water. This article has been made Open Access thanks to the generous support of a global network of libraries as part of the Knowledge Unlatched Select initiative.

Contribution of specific interactions between human enteric viruses and wastewater suspended solids on human enteric virus removal by microfiltration was studied. A cross-flow microfiltration system was used with rotavirus HAL1166 and Enterobacter cloacae SENG-6 as the model virus and wastewater suspended solid. Cleavage of rotavirus HAL1166 protein VP4 by trypsin produces the VP8* subunit, which specifically interacts with histo-blood group antigen (HBGA). In the presence of Enterobacter cloacae SENG-6, the trypsin-treated rotavirus concentration reduced with time (R2 > 0.6) compared to the reduction of non-trypsin treated rotavirus. Calculation of the gel/cake layer deposited on the membrane, consisting of Enterobacter cloacae SENG-6 and either trypsin-treated or non-trypsin treated rotavirus HAL1166, revealed that the microflocs consisting of trypsin-treated rotavirus and Enterobacter cloacae SENG-6 have lower porosity and permeability, displaying higher resistance to virus passage through the membrane. The results provide evidence that specific wastewater suspended solids–human enteric virus interaction can contribute to increasing the removal of human enteric viruses by microfiltration.

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.

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.

Performances of anodic ammonia oxidation have been investigated for various bioelectrochemical systems at a wide range of poised anodic potentials in the literature. The effect of poised cathodic potential on ammonium nitrogen (NH

RATIONALE Oxygen isotope ratio measurements of NO2- and NO3- by the azide method and denitrifier method are sensitive to the δ18 O value of the sample water. However, the influence of δ18 OH2O on those measurements has not been quantitatively evaluated and documented so far. Therefore, we investigated the influence of δ18 OH2O of a sample on the δ18 O analysis of NO2- and NO3- . METHODS We prepared NO2- and NO3- standards (with known δ18 ONO2- and δ18 ONO3- values) dissolved in waters having different δ18 OH2O values (δ18 OH2O = -12.6, 25.9, 56.7, and 110.1‰). Nitrite and nitrate were converted into N2 O using the azide method and the denitrifier method, respectively. The isotope ratios of the generated N2 O were measured with a Sercon purge-and-trap gas chromatography/isotope ratio mass spectrometry (PT-GC/IRMS) system. The measured δ18 O values of the produced N2 O were plotted against known δ18 ONO2- and δ18 ONO3- values to evaluate the influence of exchange of an oxygen atom with H2 O during the conversion of NO2- into N2 O and NO3- into N2 O, respectively. RESULTS The degree of oxygen isotope exchange was 10.8 ± 0.3% in the azide method and 5.5 ± 1.0% in the denitrifier method, indicating that the azide method is more susceptible to artifacts arising from differences in the δ18 OH2O value of water than the denitrifier method. Thus, the intercept of the standard calibration curve must be corrected to account for differences in δ18 OH2O . Abiotic NO2 -H2 O equilibrium isotope effect experiments yielded a rate constant of (1.13 ± 007) × 10-2 (h-1 ) and an equilibrium isotope effect of 11.9 ± 0.1‰ under the condition of pH = 7.5, 30°C, and 2.5% salinity. CONCLUSIONS Oxygen isotope ratio measurements of NO2- by the azide method are highly sensitive to δ18 OH2O as a result of significant oxygen isotope exchange between NO2- and H2 O. Therefore, to obtain the most accurate measurements water with the same δ18 OH2O value as that of the sample must be used to make the NO2- and NO3- standards.

The activity of anaerobic ammonia-oxidizing (anammox) bacteria is considered to depend on cell density; however, this has not yet been confirmed due to the fastidious nature of anammox bacteria (e.g., slow growth, oxygen sensitivity, and rigid aggregate formation). In the present study, the cell density-dependent occurrence of anammox activity (14-15N2 gas production rate) was investigated using planktonic enrichment cultures of Candidatus Brocadia sinica. This activity was detectable when the density of cells was higher than 107‍ ‍cells‍ ‍mL–1 and became stronger with increases in cell density. At the cell densities, the transcription of the BROSI_A1042 and BROSI_A3652 genes, which are potentially involved in the biosynthesis and reception of N-acyl homoserine lactone (AHL), was detectable in Brocadia sinica cells. The presence of AHL molecules in the MBR culture of B. sinica was confirmed by an AHL reporter assay and gas chromatography mass spectrometry analysis. The exogenous addition of the MBR culture extract and AHL molecules (a cocktail of C6, C8, C10, and C12-homoserine lactones) increased the specific 14-15N2 production rate of B. sinica. These results suggest that the specific anammox activity of B. sinica is regulated by AHL-mediated quorum sensing.

Oxygen isotope ratio measurements of NOWe prepared NOThe degree of oxygen isotope exchange was 10.8 ± 0.3% in the azide method and 5.5 ± 1.0% in the denitrifier method, indicating that the azide method is more susceptible to artifacts arising from differences in the δOxygen isotope ratio measurements of NO

In this study, we investigated the impact of GD1a-expressing bacterial strains on the infectivity of murine norovirus (MNV). Eligible bacterial strains were screened from a sewage sample using flow cytometry, and their genetic sequences of 16S rRNA were determined. The enzyme-linked immunosorbent assay (ELISA) was employed to analyze the binding between bacteria and MNV particles, and the plaque assay was used to assess the effects of GD1a-positive and negative strains on MNV infectivity. The result from ELISA shows that MNV particles are able to bind to both GD1a-positive and negative bacterial strains, but the binding to the GD1a-positive strain is more significant. The infectivity assay result further shows that the MNV infectious titer declined with an increasing concentration of GD1a-positive bacteria. The addition of anti-GD1a antibody in the infectivity assay led to the recovery of the MNV infectious titer, further confirming that the binding between MNV particles and bacterial GD1a ganglioside compromises MNV infectivity. Our findings highlight the role indigenous bacteria may play in the lifecycle of waterborne enteric viruses as well as the potential of exploiting them for virus transmission intervention and water safety improvement.

Presence of glycogen granules in anaerobic ammonium-oxidizing (anammox) bacteria has been reported so far. However, very little is known about their glycogen metabolism and the exact roles. Here, we studied the glycogen metabolism in “Ca. Brocadia sinica” growing in continuous retentostat cultures with bicarbonate as a carbon source. The effect of the culture growth phase was investigated. During the growing phase, intracellular glycogen content increased up to 32.6 mg-glucose (g-biomass dry wt)(−1) while the specific growth rate and ATP/ADP ratio decreased. The accumulated glycogen begun to decrease at the onset of entering the near-zero growth phase and was consumed rapidly when substrates were depleted. This clearly indicates that glycogen was synthesized and utilized as an energy storage. The proteomic analysis revealed that “Ca. B. sinica” synthesized glycogen via three known glycogen biosynthesis pathways and simultaneously degraded during the progress of active anammox, implying that glycogen is being continuously recycled. When cells were starved, a part of stored glycogen was converted to trehalose, a potential stress protectant. This suggests that glycogen serves at least as a primary carbon source of trehalose synthesis for survival. This study provides the first physiological evidence of glycogen metabolism in anammox bacteria and its significance in survival under natural substrate-limited habitat.

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.

Currently, we are researching a single-carrier transmission with frequency domain equalization (SC-FDE), which is robust to power-amplifier distortion and superior in power efficiency for 4K and 8K ultrahigh-definition television (UHDTV) wireless cameras using 42-GHz band of millimeter wave. In this paper, we describe a prototype of a transmitter with the SC-FDE and evaluate its transmission performance. A compact transmitter that can transmit 200-Mbps-class UHDTV signals was obtained by doubling the channel bandwidth of our previous 80-Mbps-class high-definition television wireless camera through orthogonal frequency division multiplexing. The transmission power was doubled to compensate the noise generated by the doubled bandwidth.

The activity of anaerobic ammonia-oxidizing (anammox) bacteria is considered to depend on cell density; however, this has not yet been confirmed due to the fastidious nature of anammox bacteria (e.g., slow growth, oxygen sensitivity, and rigid aggregate formation). In the present study, the cell density-dependent occurrence of anammox activity (14-15N2 gas production rate) was investigated using planktonic enrichment cultures of Candidatus Brocadia sinica. This activity was detectable when the density of cells was higher than 107‍ ‍cells‍ ‍mL-1 and became stronger with increases in cell density. At the cell densities, the transcription of the BROSI_A1042 and BROSI_A3652 genes, which are potentially involved in the biosynthesis and reception of N-acyl homoserine lactone (AHL), was detectable in Brocadia sinica cells. The presence of AHL molecules in the MBR culture of B. sinica was confirmed by an AHL reporter assay and gas chromatography mass spectrometry analysis. The exogenous addition of the MBR culture extract and AHL molecules (a cocktail of C6, C8, C10, and C12-homoserine lactones) increased the specific 14-15N2 production rate of B. sinica. These results suggest that the specific anammox activity of B. sinica is regulated by AHL-mediated quorum sensing.

Monitoring of Escherichia coli concentrations at wastewater treatment plants (WWTPs) is important to ensure process performance and protect public health. However, conventional E. coli enumeration methods are complicated and time- and labor-consuming. Here, we report a novel simple and reliable method based on β- d -glucuronidase (GUS) activity assay to enumerate E. coli concentrations in wastewater (WW) samples. An aliquot (20 μL) of the medium with fluorogenic enzyme substrate for E. coli and 180 μL of a WW sample were added to one well of a 96-well microplate. The microplate was placed in a microplate reader at 37 °C. To this end, the fluorescence intensity of a fluorogenic enzyme substrate for E. coli was measured every 10 min over 3 h to determine GUS activity. The linear increase in the fluorescence intensity representing the GUS activities showed a positive correlation with E. coli concentrations in wastewater samples. However, the correlation equations were specific to WWTPs, which could be due to the difference in the E. coli population structures among WWTPs. We observed that the wastewater matrix is not a limitation to measure the GUS activity, and a WWTP-specific correlation equation can be used as a calibration curve to estimate the E. coli concentrations in the samples collected from that site. A comparison of the results with those of culture-dependent Colilert method proved that the current method is simple and useful for the enumeration of E. coli concentrations in wastewater samples reliably.

In this study, we investigated a bottleneck effect on an RRV population that may drastically affect the viral population structure. RRV populations were serially passaged under two levels of a bottleneck effect, which exemplified human-to-human transmission. As a result, the genetic diversity and specific growth rate of RRV populations increased under the stronger bottleneck effect, which implied that a bottleneck created a new space in a population for minor mutants originally existing in a hidden layer, which includes minor mutations that cannot be distinguished from a sequencing error. The results of this study suggest that the genetic drift caused by a bottleneck in human-to-human transmission explains the random appearance of new genetic lineages causing viral outbreaks, which can be expected according to molecular epidemiology using next-generation sequencing in which the viral genetic diversity within a viral population is investigated., RNA viruses form a dynamic distribution of mutant swarms (termed “quasispecies”) due to the accumulation of mutations in the viral genome. The genetic diversity of a viral population is affected by several factors, including a bottleneck effect. Human-to-human transmission exemplifies a bottleneck effect, in that only part of a viral population can reach the next susceptible hosts. In the present study, two lineages of the rhesus rotavirus (RRV) strain of rotavirus A were serially passaged five times at a multiplicity of infection (MOI) of 0.1 or 0.001, and three phenotypes (infectious titer, cell binding ability, and specific growth rate) were used to evaluate the impact of a bottleneck effect on the RRV population. The specific growth rate values of lineages passaged under the stronger bottleneck (MOI of 0.001) were higher after five passages. The nucleotide diversity also increased, which indicated that the mutant swarms of the lineages under the stronger bottleneck effect were expanded through the serial passages. The random distribution of synonymous and nonsynonymous substitutions on rotavirus genome segments indicated that almost all mutations were selectively neutral. Simple simulations revealed that the presence of minor mutants could influence the specific growth rate of a population in a mutant frequency-dependent manner. These results indicate a stronger bottleneck effect can create more sequence spaces for minor sequences. IMPORTANCE In this study, we investigated a bottleneck effect on an RRV population that may drastically affect the viral population structure. RRV populations were serially passaged under two levels of a bottleneck effect, which exemplified human-to-human transmission. As a result, the genetic diversity and specific growth rate of RRV populations increased under the stronger bottleneck effect, which implied that a bottleneck created a new space in a population for minor mutants originally existing in a hidden layer, which includes minor mutations that cannot be distinguished from a sequencing error. The results of this study suggest that the genetic drift caused by a bottleneck in human-to-human transmission explains the random appearance of new genetic lineages causing viral outbreaks, which can be expected according to molecular epidemiology using next-generation sequencing in which the viral genetic diversity within a viral population is investigated.