Hirofumi Tsutsui

Transfer of Antibiotic Multiresistant Plasmid RP4 from Escherichia coli to Activated Sludge Bacteria

In situ transfer of a self-transmissible, antibiotic-multiresistant plasmid RP4 from a laboratory Escherichia coli strain C600 to indigenous activated sludge bacteria was investigated using filter mating. The transfer frequency of RP4 from the donor E. coli to the bacteria that was sampled from two wastewater treatment plants was 5.1x10(-2) to 7.5x10(-1) and 4.6x10(-3) to 1.3x10(-2)/potential recipient. The isolated transconjugants showed resistance to Ap, Km, and Tc and the presence of a plasmid with a similar size to RP4. The traG gene on RP4 was also detected from all transconjugants. Reverse-transfer experiments from the transconjugants to E. coli HB101 indicated that RP4 maintained self-transmissibility in the transconjugants. The transconjugant strains were dominant bacteria in activated sludge including Pseudomonas fluorescens, P. putida, and Ochrobactrum anthropi and minor populations of enteric bacterial strains including Citrobacter freundii, E. coli, Enterobacter cloacae, E. asburiae, and Klebsiella pneumoniae ssp. pneumoniae. The transconjugant strains K. pneumoniae ssp. pneumonia, E. cloacae, and E. asburiae had several naturally occurring plasmids. These results suggest that in situ transfer of plasmids and the exchange of antibiotic-resistant genes can occur between released and indigenous bacteria in activated sludge.

Nitrous oxide emission mechanisms during intermittently aerated composting of cattle manure.

To investigate the mechanisms of nitrous oxide (N₂O) emission during intermittent aeration in the composting process, a laboratory scale experiment with continuous measurement of N₂O emission was conducted with cattle manure. A low oxygen mode (2.5% oxygen in the inlet for 1 day), anaerobic mode (0.13% oxygen for 0.25 day), and aerated mode (20.5% oxygen for 2 days) were sequentially set up three times after 22 days of continuous aeration to replicate intermittent aeration. The total N₂O emission was 0.26-0.35 mmol, 0.27-0.32 mmol, and 0.14-0.23 mmol during the low oxygen, anaerobic, and aerated modes, respectively. Denitrification was indicated as the main N₂O emission pathway in the anaerobic and low-oxygen modes, while nitrification was indicated as the main pathway in the aerated mode and under continuous aeration. Results from this study suggest that nitrification is an important pathway for N₂O emission as well as denitrification.

Application of real-time polymerase chain reaction (PCR) coupled with ethidium monoazide treatment for selective quantification of viable bacteria in aquatic environment

Ethidium monoazide (EMA) was used to quantify DNA selectively from viable cells with healthy membrane/cell wall system, but not from dead cells, of a target bacterium in the aquatic environment using real-time PCR. Spiking experiments to determine the EMA treatment conditions showed that EMA treatment with EMA at 10-25 microg/ml and subsequent halogen light exposure for 2 min was suitable for selective quantification of DNA from viable cells in an aquatic sample using real-time PCR coupled with EMA treatment (real-time EMA-PCR). Optimized real-time EMA-PCR was applied in combination with culture-based method and conventional real-time PCR without EMA treatment to elucidate the behavior of an Escherichia coli strain inoculated into a pond water microcosm. Quantification results obtained using real-time EMA-PCR were lower than those by conventional real-time PCR without EMA treatment and higher than those by culture-based method. The results suggest that quantification by real-time EMA-PCR seemed to represent the viable population, which would partly include viable but non-culturable state bacteria. Real-time EMA-PCR optimized here can be a useful tool for selective monitoring of the viable population of a target bacterium in the aquatic environment, and thereby contribute to assessment of potential microbial risks generated from waterborne pathogenic bacteria.

Comparative analysis of DNA-based microbial community composition and substrate utilisation patterns of activated sludge microorganisms from wastewater treatment plants operated under different conditions.

In this study, the microbial community structure and carbon source utilisation profile of activated sludge samples collected from full-scale municipal wastewater treatment plants (WWTPs) operated under different conditions were characterised and compared, respectively, using terminal-restriction fragment length polymorphism (T-RFLP) analysis and Biolog assay. Samples were collected from each biological treatment tank of six conventional activated sludge, two anaerobic-oxic, two anaerobic-anoxic-oxic, and one step-aeration processes in eight full-scale WWTPs in Osaka, Japan. Results of the T-RFLP analysis of eubacterial 16S rDNA showed that microbial communities of activated sludge differed greatly among samples, and that they were affected by process-based operational conditions. In contrast, the carbon source utilisation profiles of activated sludge samples were mutually similar, but appeared to be influenced slightly by aerated conditions at each reaction tank. Similar carbon source utilisation profiles among all samples suggest that the activated sludge community possesses functions that are necessary for wastewater treatment even if the phylogenetic composition is different. Different results from the T-RFLP analysis and Biolog assay suggest that the phylogenetic composition of microbial community might not necessarily reflect the wastewater treatment functions of the activated sludge.

Impacts of gene bioaugmentation with pJP4-harboring bacteria of 2,4-D-contaminated soil slurry on the indigenous microbial community

Gene bioaugmentation is a bioremediation strategy that enhances biodegradative potential via dissemination of degradative genes from introduced microorganisms to indigenous microorganisms. Bioremediation experiments using 2,4-dichlorophenoxyacetic acid (2,4-D)-contaminated soil slurry and strains of Pseudomonas putida or Escherichia coli harboring a self-transmissible 2,4-D degradative plasmid pJP4 were conducted in microcosms to assess possible effects of gene bioaugmentation on the overall microbial community structure and ecological functions (carbon source utilization and nitrogen transformation potentials). Although exogenous bacteria decreased rapidly, 2,4-D degradation was stimulated in bioaugmented microcosms, possibly because of the occurrence of transconjugants by the transfer of pJP4. Terminal restriction fragment length polymorphism analysis revealed that, although the bacterial community structure was disturbed immediately after introducing exogenous bacteria to the inoculated microcosms, it gradually approached that of the uninoculated microcosms. Biolog assay, nitrate reduction assay, and monitoring of the amoA gene of ammonia-oxidizing bacteria and nirK and nirS genes of denitrifying bacteria showed no irretrievable depressive effects of gene bioaugmentation on the carbon source utilization and nitrogen transformation potentials. These results may suggest that gene bioaugmentation with P. putida and E. coli strains harboring pJP4 is effective for the degradation of 2,4-D in soil without large impacts on the indigenous microbial community.

Transfer of plasmid pJP4 from Escherichia coli and Pseudomonas putida to bacteria in activated sludge developed under different sludge retention times.

To add the knowledge on the plasmid-mediated bioaugmentation in the activated sludge process, conjugative transfer of a self-transmissible, broad host range, mercury resistance, and partial 2,4-dichlorophenoxyactic acid (2,4-D)-degrading plasmid pJP4 from Escherichia coli HB101 or Pseudomonas putida KT2440 to activated sludge bacteria were examined by filter mating. Activated sludge samples were collected from an actual wastewater treatment plant and laboratory reactors operated under different sludge retention times (SRTs) of 5, 10, and 15 days. Transfer of pJP4 was observed in all combinations of donor and activated sludge (recipient) with the transfer frequency from 6.5 x 10⁻⁸ to 2.5 x 10⁻⁴ transconjugants per recipient, except from E. coli HB101 to the activated sludge sample of SRT 5 days. Although plasmid pJP4 was transferred mainly to dominant bacteria belonging to Pseudomonas, the host range of pJP4 in the activated sludge varied depending on the donor strain. Phenotypically various transconjugants were isolated from activated sludge sampled from the reactors with longer SRT operations. All transconjugants exhibited high mercury resistance equivalent to the donor strains; some were capable of degrading 2,4-D. These results suggest that the behavior of plasmids can be controlled partially through selection of appropriate donor strains and operational conditions of the activated sludge process.

Changes in bacterial community structure in a full-scale membrane bioreactor for municipal wastewater treatment.

This study investigated changes in the structure and metabolic capabilities of the bacterial community in a full-scale membrane bioreactor (MBR) treating municipal wastewater. Microbial monitoring was also conducted for a parallel-running conventional activated sludge (CAS) process treating the same influent. The mixed-liquor suspended solid concentration in the MBR reached a steady-state on day 73 after the start-up. Then the MBR maintained higher rates of removal of organic compounds and nitrogen than the CAS process did. Terminal restriction fragment length polymorphism analysis revealed that the bacterial community structure in the MBR was similar to that in the CAS process at the start-up, but it became very different from that in the CAS process in the steady state. The bacterial community structure of the MBR continued to change dynamically even after 20 months of the steady-state operation, while that of the CAS process was maintained in a stable condition. By contrast, Biolog assay revealed that the carbon source utilization potential of the MBR resembled that of the CAS process as a whole, although it declined transiently. Overall, the results indicate that the bacterial community of the MBR has flexibility in terms of its phylogenetic structure and metabolic activity to maintain the high wastewater treatment capability.

Relationship between respiratory quotient, nitrification, and nitrous oxide emissions in a forced aerated composting process.

We assessed the relationship between respiratory quotient (RQ) and nitrification and nitrous oxide (N2O) emission in forced aerated composting using lab-scale reactors. Relatively high RQ values from degradation of readily degradable organics initially occurred. RQ then stabilized at slightly lower values, then decreased. Continuous emission of N2O was observed during the RQ decrease. Correlation between nitrification and N2O emission shows that the latter was triggered by nitrification. Mass balances demonstrated that the O2 consumption of nitrification (∼24.8mmol) was slightly higher than that of CO2 emission (∼20.0mmol), indicating that the RQ decrease was caused by the occurrence of nitrification. Results indicate that RQ is a useful index, which not only reflects the bioavailability of organics but also predicts the occurrence of nitrification and N2O emission in forced aerated composting.

Occurrence and persistence of indigenous transconjugants carrying conjugative plasmids in soil

The transfer of the self-transmissible plasmids, RP4 and pJP4, from introduced bacteria to indigenous bacteria was examined in soil and slurry microcosms. The introduced plasmids persisted in indigenous transconjugants despite the low survival of introduced donors. The potential of the transconjugants for growth and conjugation affects the persistence of introduced plasmids in soil.