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Phenol is an important pollutant widely discharged as a component of hydrocarbon fuels, but its degradation in cold regions is challenging due to the harsh environmental conditions. To date, there is little information available concerning the capability for phenol biodegradation by indigenous Antarctic bacteria. In this study, enzyme activities and genes encoding phenol degradative enzymes identified using whole genome sequencing (WGS) were investigated to determine the pathway(s) of phenol degradation of Arthrobacter sp. strains AQ5-05 and AQ5-06, originally isolated from Antarctica. Complete phenol degradative genes involved only in the ortho-cleavage were detected in both strains. This was validated using assays of the enzymes catechol 1,2-dioxygenase and catechol 2,3-dioxygenase, which indicated the activity of only catechol 1,2-dioxygenase in both strains, in agreement with the results from the WGS. Both strains were psychrotolerant with the optimum temperature for phenol degradation, being between 10 and 15 °C. This study suggests the potential use of cold-adapted bacteria in the bioremediation of phenol pollution in cold environments.

Membrane vesicles (MVs) are released by various prokaryotes and play a role in the delivery of various cell-cell interaction factors. Recent studies have determined that these vesicles are capable of functioning as mediators of horizontal gene transfer. Outer membrane vesicles (OMVs) are a type of MV that is released by Gram-negative bacteria and primarily composed of outer membrane and periplasm components; however, it remains largely unknown why DNA is contained within OMVs. Our study aimed to understand the mechanism by which DNA that is localized in the cytoplasm is incorporated into OMVs in Gram-negative bacteria. We compared DNA associated with OMVs using Escherichia coli BW25113 cells harboring the non-conjugative, non-mobilized, and high-copy plasmid pUC19 and its hypervesiculating mutants that included ΔnlpI, ΔrseA, and ΔtolA. Plasmid copy per vesicle was increased in OMVs derived from ΔnlpI, in which peptidoglycan (PG) breakdown and synthesis are altered. When supplemented with 1% glycine to inhibit PG synthesis, both OMV formation and plasmid copy per vesicle were increased in the wild type. The bacterial membrane condition test indicated that membrane permeability was increased in the presence of glycine at the late exponential phase, in which cell lysis did not occur. Additionally, quick-freeze deep-etch and replica electron microscopy observations revealed that outer-inner membrane vesicles (O-IMVs) are formed in the presence of glycine. Thus, two proposed routes for DNA incorporation into OMVs under PG-damaged conditions are suggested. These routes include DNA leakage due to increased membrane permeation and O-IMV formation. Additionally, our findings contribute to a greater understanding of the vesicle-mediated horizontal gene transfer that occurs in nature and the utilization of MVs for DNA cargo.

Recycling fly ashes is a good alternative to disposal with the significant economic and environmental benefits. Characterization of fly ashes can be helpful to evaluate their use potentials. This study aimed to investigate the physical, chemical and mineralogical properties of fly ashes from five thermal power stations in Northern Vietnam in relation to use as sandy soil amendments. The results showed that the fly ashes were dominated by silt-sized and spherical particles and had low bulk densities. There was almost not significant difference in the surface charges among the fly ashes; however, their surface areas varied widely. The fly ashes were alkaline. The electrical conductivity and cation exchange capacity in the fly ashes were higher than those in the sandy soil. The concentrations of extractable K, P, Ca2+ and Mg2+ in the fly ashes were higher compared with the sandy soil. The major matrix elements in the fly ashes were Si, Al, and Fe together with significant percentages of K, Mg, Ca and Ti. Quartz was the most predominant mineral present in the fly ashes. Several radioactive elements were found in the fly ashes with very low concentrations. The potential to release trace elements from the fly ashes was below the regulatory guidelines. The amendment of fly ashes to the sandy soil led to the substantial decrease in the hydraulic conductivity but the increase in the plant-available water contents of the sandy soil. It is recommended to use the fly ashes as soil amendments for sandy soil amelioration.

Membrane vesicles (MVs) are lumen-containing spheres of lipid bilayers secreted by all prokaryotes into the extracellular milieu. They have multifunctional roles in stress response, virulence transfer, biofilm formation, and microbial interactions. Remarkably, MVs contain various components, including lytic enzymes, genetic materials, and hydrophobic signals, at high concentrations and transfer them effectively to the target microbial cells. Therefore, MVs act as carriers for bactericidal effects, horizontal gene transfer, and quorum sensing. Although the purpose of secreted MVs remains unclear, recent reports have provided evidence that MVs selectively interact with microbial cells in order to transfer their content to the target species. Herein, we review microbial interactions using MVs and discuss MV-mediated selective delivery of their content to target microbial cells.

We report here the complete genome sequence of Buttiauxella agrestis DSM 9389, which harbors eight 16S rRNA genes classified into three types. The genome sequence of this strain showed a higher average nucleotide identity value (97.3%) than that of the highly membrane vesicle-producing strain B. agrestis ATCC 33320T., We report here the complete genome sequence of Buttiauxella agrestis DSM 9389, which harbors eight 16S rRNA genes classified into three types. The genome sequence of this strain showed a high average nucleotide identity (97.3%) with that of the highly membrane vesicle-producing strain B. agrestis ATCC 33320T.

The collapse of Comamonas testosteroni R2 under chemostat conditions and the aerobic growth of strain R2 under batch conditions with phenol as the sole carbon source were investigated using physiological and transcriptomic techniques. Phenol-/catechol-degrading activities under chemostat conditions gradually decreased, suggesting that metabolites produced from strain R2 accumulated in the culture, which caused negative feedback. The competitive inhibition of phenol hydroxylase and catechol dioxygenase was observed in a crude extract of the supernatant collected from the collapsed culture. Transcriptomic analyses showed that genes related to nitrogen transport were up-regulated; the ammonium transporter amtB was up-regulated approximately 190-fold in the collapsed status, suggesting an increase in the concentration of ammonium in cells. The transcriptional levels of most of the genes related to gluconeogenesis, glycolysis, the pentose phosphate pathway, and the TCA and urea cycles decreased by ~0.7-fold in the stable status, whereas the activities of glutamate synthase and glutamine synthetase increased by ~2-fold. These results suggest that ammonium was assimilated into glutamate and glutamine via 2-oxoglutarate under the limited supply of carbon skeletons, whereas the synthesis of other amino acids and nucleotides was repressed by 0.6-fold. Furthermore, negative feedback appeared to cause an imbalance between carbon and nitrogen metabolism, resulting in collapse. The effects of amino acids on negative feedback were investigated. L-arginine allowed strain R2 to grow normally, even under growth-inhibiting conditions, suggesting that the imbalance was corrected by the stimulation of the urea cycle, resulting in the rescue of strain R2.

Study of the potential of Antarctic microorganisms for use in bioremediation is of increasing interest due to their adaptations to harsh environmental conditions and their metabolic potential in removing a wide variety of organic pollutants at low temperature. In this study, the psychrotolerant bacterium Rhodococcus sp. strain AQ5-07, originally isolated from soil from King George Island (South Shetland Islands, maritime Antarctic), was found to be capable of utilizing phenol as sole carbon and energy source. The bacterium achieved 92.91% degradation of 0.5 g/L phenol under conditions predicted by response surface methodology (RSM) within 84 h at 14.8 &deg, C, pH 7.05, and 0.41 g/L ammonium sulphate. The assembled draft genome sequence (6.75 Mbp) of strain AQ5-07 was obtained through whole genome sequencing (WGS) using the Illumina Hiseq platform. The genome analysis identified a complete gene cluster containing catA, catB, catC, catR, pheR, pheA2, and pheA1. The genome harbours the complete enzyme systems required for phenol and catechol degradation while suggesting phenol degradation occurs via the &beta, ketoadipate pathway. Enzymatic assay using cell-free crude extract revealed catechol 1,2-dioxygenase activity while no catechol 2,3-dioxygenase activity was detected, supporting this suggestion. The genomic sequence data provide information on gene candidates responsible for phenol and catechol degradation by indigenous Antarctic bacteria and contribute to knowledge of microbial aromatic metabolism and genetic biodiversity in Antarctica.

A complete understanding of the plant microbiome has not yet been achieved due to its complexity and temporal shifts in the community structure. To overcome these issues, we created a synthetic bacterial community of the aquatic plant, duckweed. The synthetic community established with six bacterial strains showed a stable composition for 50 days, which may have been because duckweed maintains a similar physiological status through its clonal reproduction. Additionally, the synthetic community reflected the taxonomic structure of the natural duckweed microbiome at the family level. These results suggest the potential of a duckweed-based synthetic community as a useful model system for examining the community assembly mechanisms of the plant microbiome.

Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of tolB, which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming Buttiauxella agrestis type strain JCM 1090. The ΔtolB mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the ΔtolB mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of ΔtolB mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the B. agrestis ΔtolB mutant to form unique vesicles. IMPORTANCE Membrane vesicle (MV) formation has been recognized as a common mechanism in prokaryotes, and MVs play critical roles in intercellular interaction. However, a broad range of MV types and their multiple production processes make it difficult to gain a comprehensive understanding of MVs. In this work, using vesicle separation and electron microscopic analyses, we demonstrated that diverse types of outer membrane vesicles (OMVs) were released from an engineered strain, Buttiauxella agrestis JCM 1090(T) ΔtolB mutant. We also discovered a previously undiscovered type of vesicle, multilamellar/multivesicular outer membrane vesicles (M-OMVs), which were released by this mutant using unconventional processes. These findings have facilitated considerable progress in understanding MV diversity and expanding the utility of MVs in biotechnological applications.

We report the draft genome sequence of Variovorax boronicumulans strain c24, which was isolated from a soil-inoculated chemostat culture amended with phenol as a sole carbon and energy source. The genome data will provide insights into phenol and other xenobiotic compound degradation mechanisms for bioremediation applications.

The coexisting mechanism of a synthetic bacterial community (SBC) was investigated to better understand how to manage microbial communities. The SBC was constructed with three kinds of phenol-utilizing bacteria, Pseudomonas sp. LAB-08, Comamonas testosteroni R2, and Cupriavidus sp. P-10, under chemostat conditions supplied with phenol as a sole carbon and energy source. Population densities of all strains were monitored by real-time quantitative PCR (qPCR) targeting the gene encoding the large subunit of phenol hydroxylase. Although the supply of phenol was stopped to allow perturbation in the SBC, all of the strains coexisted and the degradation of phenol was maintained for more than 800 days. The qPCR analyses showed that strains LAB-08 and R2 became dominant simultaneously, whereas strain P-10 was a minor population. This phenomenon was observed before and after the phenol-supply stoppage. The kinetic parameters for phenol of the SBC changed before and after the phenol-supply stoppage, which suggests a change in functional roles of strains in the SBC. Transcriptional levels of phenol hydroxylase and catechol dioxygenases of three strains were monitored by reverse-transcription qPCR (RT-qPCR). The RT-qPCR analyses revealed that all strains shared phenol and survived independently before the phenol-supply stoppage. After the stoppage, strain P-10 would incur the cost for degradation of phenol and catechol, whereas strains LAB-08 and R2 seemed to be cheaters using metabolites, indicating the development of the metabolic network. These results indicated that it is important for the management and redesign of microbial communities to understand the metabolism of bacterial communities.

We report the draft genome sequence of Variovorax boronicumulans strain HAB-30, which was isolated from a phenol-degrading chemostat culture. This strain contains genes encoding a multicomponent type of phenol hydroxylase, with degradation pathways for catechol and other aromatic compounds. The genome sequence will be useful for understanding the metabolic pathways involved in phenol degradation.

Pulp and paper mill sludge (PPMS) was found to be poorly colonised with thermophilic microorganisms. However, evidence to support the need for inoculation to facilitate PPMS composting has only been demonstrated in one instance. In this study, we aimed to: screen and identify PPMS digesting thermophilic bacterial strains; investigate effects of the mixture of selected thermophilic bacterial strains on PPMS digestion; and utilize this mixture as start inoculum in PPMS composting and assess the quality of compost product. The results showed that eleven thermophilic bacterial strains were isolated from Bai Bang PPMS by the enrichment culture method. Among these, three strains which reflected high growth rates on the plates of Minimal Media Agar supplemented with Bai Bang PPMS and showed hydrolytic and ligninolytic activities on the agar plates containing appropriate inductive substrates were selected. Based on the morphological, biochemical characteristics and 16S rRNA gene sequencing, they were identified as Bacillus subtilis. The inoculation with the mixture of selected strains enhanced remarkably Bai Bang PPMS digestion. The dry weight decrease, volatile suspended solids removal, dehydrogenase and protease activities in the inoculated sludge were 2.1-, 1.5-, 1.3- and 1.2- fold higher, respectively, compared to the non-inoculated sludge. The assessment of compost quality based on stability using the alkaline trap method and maturity using the germination and root elongation test showed that the inoculated compost was stable and mature while the non-inoculated compost was unstable and immature. These thermophilic bacterial strains therefore have great potential for Bai Bang PPMS composting.

A bacterium capable of complete ammonia oxidation (comammox) has been widely found in various environments, whereas its industrial application is limited due to the difficulty of cultivation and/or enrichment. We developed a biological system to produce a high-quality nitrate solution for use in hydroponic fertilizer. The system was composed of two separate reactors for ammonification and nitrification and was found to have a stable and efficient performance in the conversion of organic nitrogen to nitrate. To determine the key microbes involved and better understand the system, the microbial communities in the reactors were analyzed by 16S rRNA gene sequencing in combination with a shotgun metagenomic analysis. Canonical ammonia-oxidizing bacteria, which can only catalyze the oxidation of ammonia to nitrite, were detected with negligible relative abundances, while a comammox Nitrospira-related operational taxonomic unit (OTU) dominated the nitrification reactor. Furthermore, the comammox-type ammonia monooxygenase was found to be 500 times more highly expressed than the canonical one by quantitative PCR, indicating that comammox was the main driver of the stable and efficient ammonia oxidation in the system. A microbial co-occurrence analysis revealed a strong positive correlation between Nitrospira and several OTUs, some of which, such as Anaerolinea OTU, have been found to co-exist with comammox Nitrospira in the biofilms of water treatment systems. Given that these OTUs were abundant only on microbe-attached carriers in the system, their co-existence within the biofilm could be beneficial to stabilize the Nitrospira abundance, possibly by physically preventing oxygen exposure as well as cell spillage.

A conjugative F plasmid induces mature biofilm formation by Escherichia coli by promoting F-pili-mediated cell-cell interactions and increasing the expression of biofilm-related genes. We herein demonstrated another function for the F plasmid in E. coli biofilms; it contributes to the emergence of genetic and phenotypic variations by spontaneous mutations. Small colony variants (SCVs) were more frequently generated in a continuous flow-cell biofilm than in the planktonic state of E. coli harboring the F plasmid. E. coli SCVs represented typical phenotypic changes such as slower growth, less biofilm formation, and greater resistance to aminoglycoside antibiotics than the parent strain. Genomic and complementation analyses indicated that the small colony phenotype was caused by the insertion of Tn1000, which was originally localized in the F plasmid, into the hemB gene. Furthermore, the Tn1000 insertion was removed from hemB in the revertant, which showed a normal colony phenotype. This study revealed that the F plasmid has the potential to increase genetic variations not only by horizontal gene transfer via F pili, but also by site-specific recombination within a single cell.

Microbial fuel cells equipped with SPEEK-MEA (SPEEK-MFC) and Nafion-MEA (Nafion-MFC) were constructed with organic waste as electron donor and lake sediment as inoculum and were then evaluated comprehensively by electrochemical and microbial analyses. The proton conductivity of SPEEK was several hundreds-fold lower than that of Nafion 117, whereas the oxygen mass and diffusion transfer coefficients of SPEEK were 10-fold lower than those of Nafion 117. It was difficult to predict which was better membrane for MFC based on the feature of membrane. Analyses of polarization curves indicated that the potential of electricity production was similar in both MFCs, as the SPEEK-MFC produced 50–80% of the practical current density generated by the Nafion-MFC. Chronopotentiometry analyses indicated that the Nafion-MEA kept the performance longer than the SPEEK-MEA for long period, whereas performance of both anodes improved on time. Multidimensional scaling analyses based on DGGE profiles revealed the anolytic and biofilm communities of the SPEEK-MFC had developed differently from those of the Nafion-MFC. Clone library analyses indicated that Geobacter spp. represented 6.3% of the biofilm bacterial community in the Nafion-MFC but not detected in the SPEEK-MFC. Interestingly, the clone closely related to Acetobacterium malicum strain HAAP-1, belonging to the homoacetogens, became dominant in both anolytic and biofilm communities of the SPEEK-MFC. It was suggested that the lower proton conductivity of SPEEK-MEA allowed the bacteria closely related to strain HAAP-1 to be dominant specifically in SPEEK-MFC. These results indicated that Nafion-MFC ranked with SPEEK-MFC and that MEAs had strong selective pressure for electricity-producing bacterial community.

A batch culture was enriched on phenol with trichloroethene-contaminated aquifer soil as an inoculum. Cupriavidus sp., A batch culture was enriched on phenol with trichloroethene-contaminated aquifer soil as an inoculum. Cupriavidus sp. strain P-10 was isolated from the culture using a diluted plating method. Here, we report the draft genome sequence and annotation of strain P-10, which provides insights into the metabolic processes of phenol degradation.

Compost teas were proved to be used as effective biocontrol agent owing to their ability to inhibit plant diseases. In this experiment, aerated compost tea (ACT) and molasses aerated compost tea (MACT) sourced from oil palm compost were assessed to suppress against fungal pathogen namely, Grammothele lineata (KX532185) that causes leaf spot on Melicope ptelefolia leaf. Characterization of both aerated compost teas for physiochemical and microbiological analysis was carried out. All different concentrations (15%, 25%, 30%, and 40% v/v) of ACT and MACT significantly retarded the mycelial growth of Grammothele lineata in the range of 14.5–77.8% and 12.7–75.9%, respectively. The highest inhibition over Grammothele lineata was obtained by both compost teas at concentration of 40% (v/v) resulted with 12% and 14% growth only, respectively. Peroxidase (PO) and polyphenol oxidase (PPO) activities peaked 2–4 days after Grammothele lineata fungal suspension (5 × 105 conidia/mL) challenged on fortified leaf disks. Besides, in Melicope ptelefolia leaf fortified with both compost teas, peroxidase (PO) reached the highest (245.05 IU min−1 g−1 fresh weight) after 7 days, whereas polyphenol oxidase (PPO) recorded the highest at 91.3 IU min−1 g−1 fresh weight after 4 days upon challenged by Grammothele lineata suspension (5 × 105 conidia/mL). Our result showed that the defense enzyme properties play a significant role in contributing disease resistance against phytopathogen attack.

Many bacteria convert bicyclic compounds, such as indole and naphthalene, to oxidized compounds, including hydroxyindoles and naphthols. Pseudomonas aeruginosa , a ubiquitous bacterium that inhabits diverse environments, shows pathogenicity against animals, plants, and other microorganisms, and increasing evidence has shown that several bicyclic compounds alter the virulence-related phenotypes of P. aeruginosa . Here, we revealed that hydroxyindoles (4- and 5-hydroxyindoles) and naphthalene derivatives bearing hydroxyl groups specifically inhibit swarming motility but have minor effects on other motilities, including swimming and twitching, in P. aeruginosa . Further analyses using 1-naphthol showed that this effect is also associated with clinically isolated hyperswarming P. aeruginosa cells. Swarming motility is associated with the dispersion of cells from biofilms, and the addition of 1-naphthol maintained biofilm biomass without cell dispersion. We showed that this 1-naphthol-dependent swarming inhibition is independent of changes of rhamnolipid production and the intracellular level of signaling molecule cyclic-di-GMP (c-di-GMP). Transcriptome analyses revealed that 1-naphthol increases gene expression associated with multidrug efflux and represses gene expression associated with aerotaxis and with pyochelin, flagellar, and pilus synthesis. In the present study, we showed that several bicyclic compounds bearing hydroxyl groups inhibit the swarming motility of P. aeruginosa , and these results provide new insight into the chemical structures that inhibit the specific phenotypes of P. aeruginosa .

Comamonas testosteroni strain R2 was isolated from a continuous culture enriched by a low concentration of phenol-oxygenating activities with low K s values (below 1 μM). The draft genome sequence of C. testosteroni strain R2 reported here may contribute to determining the phenol degradation gene cluster.

We investigated how bacterial communities adapted to external resistances and exhibited the performance of electricity production in microbial fuel cells (MFCs) with external resistance of 10 Ω (LR-MFC) and 1000 Ω (HR-MFC). The HR-MFC exhibited better performance than the LR-MFC. The power densities of the LR-MFC and the HR-MFC were 5.2 ± 1.6 mW m-2 and 28 ± 9.6 mW m-2 after day 197, respectively. Low-scan cyclic voltammetry analyses indicated that the onset potential of the HR-MFC was more negative than that of the LR-MFC, suggesting that the higher external resistance led to enrichment of the highly current producing bacteria on the anode surface. All clones of Geobacter retrieved from the LR-MFC and the HR-MFC were members of the Geobacter metallireducens clade. Although the population density of Geobacter decreased from days 366-427 in the HR-MFC, the current density was almost maintained. Multidimensional scaling analyses based on denaturing gradient gel electrophoresis profiles indicated that the dynamics of the biofilm and anolytic communities changed synchronously in the two MFCs, but the dynamics of the bacterial communities in the LR-MFC and the HR-MFC were different from each other, reflecting different processes in adaptation to the different external resistances. The results suggest that the microbial community structure was formed by adapting to higher external resistance, exhibiting more negative onset potential and higher performance of the HR-MFC through collaborating with anode-respiring bacteria and fermenters.

Membrane vesicles (MVs) are secreted from a wide range of microbial species and transfer their content to other cells. Although MVs play critical roles in bacterial communication, whether MVs selectively interact with bacterial cells in microbial communities is unclear. In this study, we investigated the specificity of the MV-cell interactions and evaluated the potential of MVs to target bacterial cells for delivery. MV association with bacterial cells was examined using a fluorescent membrane dye to label MVs. MVs derived from the enterobacterium Buttiauxella agrestis specifically interacted with cells of the parent strain but interacted less specifically with those of other genera tested in this study. Electron microscopic analyses showed that MVs were not only attached on B. agrestis cells but also fused to them. The interaction energy, which was characterized by hydrodynamic diameter and zeta potential based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, was significant low between MVs and cells in B. agrestis, compared to those between B. agrestis MVs and cells of other genera. Similar specific interaction was also occurred between B. agrestis MVs and cells of six other species belonging to Buttiauxella spp. B. agrestis harboring plasmid pBBR1MCS-1 secreted plasmid-containing MVs (p-MVs), and plasmid DNA in p-MVs was transferred to the same species. Moreover, antibiotic-associated MVs enabled effective killing of target species; the survival rate of B. agrestis was lower than those of Escherichia coli and Pseudomonas aeruginosa in the presence of gentamicin-associated MVs derived from B. agrestis. Altogether, we provide the evidence that MVs selectively interact with target bacterial cells and offer a new avenue for controlling specific bacterial species using bacterial MVs in microbial communities.

The relationship between the bacterial communities in anolyte and anode biofilms and the electrochemical properties of microbial fuel cells (MFCs) was investigated when a complex organic waste-decomposing solution was continuously supplied to MFCs as an electron donor. The current density increased gradually and was maintained at approximately 100 to 150 mA m(-2). Polarization curve analyses revealed that the maximum power density was 7.4 W m(-3) with an internal resistance of 110 Ω. Bacterial community structures in the organic waste-decomposing solution and MFCs differed from each other. Clonal analyses targeting 16S rRNA genes indicated that bacterial communities in the biofilms on MFCs developed to specific communities dominated by novel Geobacter. Multidimensional scaling analyses based on DGGE profiles revealed that bacterial communities in the organic waste-decomposing solution fluctuated and had no dynamic equilibrium. Bacterial communities on the anolyte in MFCs had a dynamic equilibrium with fluctuations, while those of the biofilm converged to the Geobacter-dominated structure. These bacterial community dynamics of MFCs differed from those of control-MFCs under open circuit conditions. These results suggested that bacterial communities in the anolyte and biofilm have a gentle symbiotic system through electron flow, which resulted in the advance of current density from complex organic waste.

We initiated this research topic based on the question “how can we develop microbial ecological theory?” We incorporated three important aspects, stability, plasticity, and evolution, which are key areas for progress in microbial ecological theory. Advances in microbial ecological techniques have allowed us to meticulously consider the dynamics of community structures, interspecies networks, and metabolic flux of microbial ecosystems. Studies using these techniques have demonstrated the stability (i.e., resistance, resilience, and persistence), dynamic equilibrium, and self-organizing ability of micro-ecosystems. Here, the fine contributors in this volume aimed to determine the central and general tenets of the ecological theory that describes the features of microbial ecosystems.

In the past couple of decades, molecular ecological techniques have been developed to elucidate microbial diversity and distribution in microbial ecosystems. Currently, modern techniques, represented by meta-omics and single cell observations, are revealing the incredible complexity of microbial ecosystems and the large degree of phenotypic variation. These studies propound that microbiological techniques are insufficient to untangle the complex microbial network. This minireview introduces the application of advanced mathematical approaches in combination with microbiological experiments to microbial ecological studies. These combinational approaches have successfully elucidated novel microbial behaviors that had not been recognized previously. Furthermore, the theoretical perspective also provides an understanding of the plasticity, robustness and stability of complex microbial ecosystems in nature.

Pseudomonas sp. LAB-08 was isolated from a phenol-fed bioreactor constructed with contaminated aquifer soil as the inoculum. Strain LAB-08 utilized phenol as a sole carbon and energy source. Here, we report the genome sequence and annotation of Pseudomonas sp. LAB-08.

Aerobic aromatic-hydrocarbon-degrading bacteria from a semi-anaerobic microbial microcosm that exhibited apparent complete dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were isolated through enrichment and plating culture procedures with dibenzofuran as the model substrate. By 16S rRNA gene sequence comparisons, these dibenzofuran-degrading isolates were identified as being members of the phyla Actinobacteria, Firmicutes, and Proteobacteria, among which those of the genera Paenibacillus and Rhizobium were most abundant. All of the isolates utilized naphthalene as the sole carbon and energy source and degraded dibenzofuran metabolically or co-metabolically; however, they hardly attacked monochlorinated dibenzofuran and dibenzo-p-dioxin. By PCR cloning and sequencing, genes predicted to encode aromatic-ring-hydroxylating dioxygenase (AhDO) were detected in all test isolates. Real-time quantitative PCR assays with specific primer sets detected approximately 10⁵ copies of the AhDO large subunit genes g⁻¹ wet wt in the microcosm from which the isolates were obtained. This order of the copy number corresponded to approximately 1% of the 16S rRNA gene copies from "Dehalococcoides" and its relatives present as potent dechlorinators. These results suggest that aerobic AhDO-containing bacteria co-exist and play a role in the oxidative degradation of less chlorinated and completely dechlorinated products in the PCDD/F-dechlorinating process, thereby achieving the apparent complete dechlorination of PCDD/Fs.

Bradyrhizobium sp. S23321 is an oligotrophic bacterium isolated from paddy field soil. Although S23321 is phylogenetically close to Bradyrhizobium japonicum USDA110, a legume symbiont, it is unable to induce root nodules in siratro, a legume often used for testing Nod factor-dependent nodulation. The genome of S23321 is a single circular chromosome, 7,231,841 bp in length, with an average GC content of 64.3%. The genome contains 6,898 potential protein-encoding genes, one set of rRNA genes, and 45 tRNA genes. Comparison of the genome structure between S23321 and USDA110 showed strong colinearity; however, the symbiosis islands present in USDA110 were absent in S23321, whose genome lacked a chaperonin gene cluster (groELS3) for symbiosis regulation found in USDA110. A comparison of sequences around the tRNA-Val gene strongly suggested that S23321 contains an ancestral-type genome that precedes the acquisition of a symbiosis island by horizontal gene transfer. Although S23321 contains a nif (nitrogen fixation) gene cluster, the organization, homology, and phylogeny of the genes in this cluster were more similar to those of photosynthetic bradyrhizobia ORS278 and BTAi1 than to those on the symbiosis island of USDA110. In addition, we found genes encoding a complete photosynthetic system, many ABC transporters for amino acids and oligopeptides, two types (polar and lateral) of flagella, multiple respiratory chains, and a system for lignin monomer catabolism in the S23321 genome. These features suggest that S23321 is able to adapt to a wide range of environments, probably including low-nutrient conditions, with multiple survival strategies in soil and rhizosphere.

A dechlorinating microbial enrichment culture designated TUT2264 was cultured with tetrachloroethene and then characterized for tetrachloroethene-dechlorination by culture-dependent and -independent methods. The fourth-transferred TUT2264 culture completely dechlorinated tetrachloroethene and trichloroethene, and accumulated more trans-1,2-dichloroethene than cis-1,2-dichloroethene. A real-time PCR analysis revealed that "Dehalococcoides" cells made up only 0.3% of the total. Eight distinct reductive-dehalogenase-homologous genes (rdh) were detected with degenerate primers. Phylogenetic analyses revealed 5 of the 8 RdhAs to be very similar to RdhAs reported previously but not to share 100% identity. Transcriptional levels were quantified as the number of transcripts per rdhA by combining the reverse transcription real-time PCR and exogenous internal reference mRNA methods. TUT2264 responded to all the chloroethenes tested. rdhA4 was transcribed with all chloroethenes except vinyl chloride, whereas rdhA8 was only transcribed on tetrachloroethene. Furthermore, multiple rdhAs were induced to express by a single chloroethene as a growth-supporting or non-supporting substrate. These results suggested that Rdhs are multi-functional and rdhAs are a powerful tool to evaluate the potential of contaminated sites and isolates to dechlorinate chloroethenes.

The reductive dechlorinating abilities for chloroethenes of seven enrichment cultures from polychlorinated-dioxin-dechlorinating microcosm were investigated using culture-independent and -dependent methods. These cultures were constructed and maintained with 1,2,3-trichlorobenzene (1,2,3-TCB) or fthalide as an electron acceptor and hydrogen as an electron donor. Denaturing gradient gel electrophoresis (DGGE) analysis of the amplified fragments targeting the 16S rRNA gene showed one or two major bands, whose nucleotide sequences were then analyzed and were found to suggest that Dehalococcoides was one of the dominant bacteria in all enrichment cultures. The nucleotide sequence data revealed that the identity of the major band was 100% identical to the 16S rRNA gene sequence of the Pinellas subgroup of the Dehalococcoides clusters, that is, strains CBDB1 and FL2. Genetic diagnosis targeting the pceA, tceA, bvcA, vcrA and reductive dehalogenase homologous (rdh) gene was performed to investigate the potential for reductive chloroethene dechlorination of cultures. The required length of PCR-amplified fragments was not observed, suggesting that these cultures are not capable of reductively dechlorinating chloroethenes. However, a culture-dependent test indicated that two cultures, TUT1903 and TUT1952, reductively dechlorinated tetrachloroethene (PCE) to trichloroethene (TCE), although not completely. While, TUT2260 and TUT2264 completely converted PCE to TCE and dichloroethenes, but not further. These results suggest that these TUT cultures might include a novel type of bacteria belonging to the Dehalococcoides group and that currently available information on both the 16S rRNA gene and rdh gene sequences is insufficient to definitively evaluate the potential abilities for reductive dechlorination.

This study investigated the factors that determine the dynamics of bacterial communities in a complex system using multidisciplinary methods. Since natural and engineered microbial ecosystems are too complex to study, six types of synthetic microbial ecosystems (SMEs) were constructed under chemostat conditions with phenol as the sole carbon and energy source. Two to four phenol-degrading, phylogenetically and physiologically different bacterial strains were used in each SME. Phylogeny was based on the nucleotide sequence of 16S rRNA genes, while physiologic traits were based on kinetic and growth parameters on phenol. Two indices, J parameter and "interspecies interaction," were compared to predict which strain would become dominant in an SME. The J parameter was calculated from kinetic and growth parameters. On the other hand, "interspecies interaction," a new index proposed in this study, was evaluated by measuring the specific growth activity, which was determined on the basis of relative growth of a strain with or without the supernatant prepared from other bacterial cultures. Population densities of strains used in SMEs were enumerated by real-time quantitative PCR (qPCR) targeting the gene encoding the large subunit of phenol hydroxylase and were compared to predictions made from J parameter and interspecies interaction calculations. In 4 of 6 SEMs tested the final dominant strain shown by real-time qPCR analyses coincided with the strain predicted by both the J parameter and the interspecies interaction. However, in SMEII-2 and SMEII-3 the final dominant Variovorax strains coincided with prediction of the interspecies interaction but not the J parameter. These results demonstrate that the effects of interspecies interactions within microbial communities contribute to determining the dynamics of the microbial ecosystem.

The community structure of pink-colored microbial mats naturally occurring in a swine wastewater ditch was studied by culture-independent biomarker and molecular methods as well as by conventional cultivation methods. The wastewater in the ditch contained acetate and propionate as the major carbon nutrients. Thin-section electron microscopy revealed that the microbial mats were dominated by rod-shaped cells containing intracytoplasmic membranes of the lamellar type. Smaller numbers of oval cells with vesicular internal membranes were also found. Spectroscopic analyses of the cell extract from the biomats showed the presence of bacteriochlorophyll a and carotenoids of the spirilloxanthin series. Ubiquinone-10 was detected as the major quinone. A clone library of the photosynthetic gene, pufM , constructed from the bulk DNA of the biomats showed that all of the clones were derived from members of the genera Rhodobacter and Rhodopseudomonas . The dominant phototrophic bacteria from the microbial mats were isolated by cultivation methods and identified as being of the genera Rhodobacter and Rhodopseudomonas by studying 16S rRNA and pufM gene sequence information. Experiments of oxygen uptake with lower fatty acids revealed that the freshly collected microbial mats and the Rhodopseudomonas isolates had a wider spectrum of carbon utilization and a higher affinity for acetate than did the Rhodobacter isolates. These results demonstrate that the microbial mats were dominated by the purple nonsulfur bacteria of the genera Rhodobacter and Rhodopseudomonas , and the bioavailability of lower fatty acids in wastewater is a key factor allowing the formation of visible microbial mats with these phototrophs.

Vertical profiles of polychlorinated dioxins and microbial biomass including "Dehalococcoides" populations in cores of sediment of Lake Suwa, Japan, were investigated. The core samples were analyzed in 3-cm intervals at 0-15 cm and 50 cm, where a sharp gradient of Eh from 5 to −110 mV with depth occurred. The concentration of polychlorinated dioxins was relatively constant at 0-15 cm, ranging from 7.6 to 8.3 ng (7.0-9.2 pg-TEQ [toxic equivalent]) g-1 dry wt, but decreased sharply at 50 cm. The total bacterial count was in the order of 108 to 109 g-1 dry wt, being highest at 3-6 cm and decreasing in the deeper sediment. A similar vertical profile was found for respiratory quinones with larger amounts of ubiquinones than menaquinones at 0-15 cm. Quantitative real-time PCR with a specific primer set showed that "Dehalococcoides" and its phylogenetic relatives occurred in the order of 104 g -1 (dry wt) at 0-12 cm but were absent at 50 cm. The amplified clones showed 91-100% similarity (mostly

Semi-anaerobic microcosms containing different levels of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were constructed by seeding with different mass ratios of lake sediment and dioxin-contaminated soil and incubating with organic medium for 1 year. In all microcosms, PCDD/Fs were reduced as a first-order reaction with similar removal rate coefficients, and only trace amounts of less chlorinated congeners were produced as the intermediate and end products. This apparent complete dechlorination of PCDD/Fs seemed to be due to a combination of reductive dechlorination of PCDD/Fs and oxidative degradation of the dechlorinated products. Total cell counting, 16S rRNA gene clone library analyses and quinone profiling showed that the microcosms contained relatively constant total populations with members of the phyla Bacteroidetes, Firmicutes and Proteobacteria (especially “Deltaproteobacteria”) as the major constituents, independent of pollution levels. Quantitative real-time PCR with a specific primer set showed that the population density of “Dehalococcoides” and its phylogenetic relatives was highly correlated with the concentration of PCDD/Fs present. Some “Dehalococcoides” strains were isolated from the microcosms by repeated enrichment with chloroaromatics as the terminal electron acceptor. However, these isolates did not match with the major “Dehalococcoides”-related clones directly PCR-amplified. The results of this study suggest that PCDD/Fs in natural environments under given conditions are transformed with similar half-reduction rates independent of their concentrations, and a wide variety of “Dehalococcoides”-related bacteria play the primary role in this process.

Aerobic chemoorganotrophic bacteria in soils polluted with different levels of polychlorinated dioxins (6.8 to 4,600 pg-TEQ g-1 dry weight) were isolated by the quantitative agar-plating method and tested for their ability to degrade dibenzofuran (DF) using DF-overlaid agar and DF-containing liquid media. For comparison, bacteria isolated from river sediments were also tested. Out of the 5,069 strains thus isolated, 23 strains were found to be able to degrade DF, and the majority produced soluble yellow metabolites during the degradation. A higher isolation frequency for DF degraders was obtained with samples containing higher concentrations of polychlorinated dioxins. Most of the DF-degrading isolates were identified as members of the class Actinobacteria, particularly of the genera Nocardioides and Rhodococcus. These results suggest that particular actinobacterial species constitute the major populations of culturable DF-degrading bacteria in dioxin-polluted environments.

We did tests using swarm plates, to examine the effects of various salts and their concentrations on the chemotaxis of fluorescent Pseudomonas strains. As a result, we found that the swarming ability of the Pseudomonas strains was inhibited by high concentrations of Ca2+. The growth of the strains was not affected at the high concentration of Ca2+, but the cells grown in swarm agar under the condition were extended in the filaments. Most of the cells had reached 10 microm to 40 microm in length. Such cell elongation was not observed with salts other than calcium salts. A significant correlation between the cell elongation and the decrease of swarming ability by the high concentration of Ca2+ was observed.

The effects of different phenol-feeding conditions on trichloroethylene (TCE) biodegradation and bacterial population structure in an aquifer soil community were studied. The soil sample, minerals, phenol, and TCE were mixed in glass bottles, which were then incubated under three different phenol-feeding conditions. First, phenol was supplied only once at 0.2 mM (condition 0.2P); second, it was added at 2.0 mM (condition 2.0P); and third, it was periodically supplied ten times at 0.2 mM (condition 0.2PS). TCE concentrations remained stable under conditions 0.2P and 2.0P. In contrast, TCE was completely degraded under condition 0.2PS. TCE/phenol-degrading bacteria were enumerated indirectly and functionally by quantitative PCR. The low- K(s) (half saturation constant) group of phenol-degrading bacteria, exhibiting high TCE-degrading activity, yielded a 50-fold higher population under condition 0.2PS than under condition 2.0P. The bacterial community structure under condition 0.2PS was studied by denaturing gradient gel electrophoresis targeting the genes encoding 16S rRNA and the largest subunit of multicomponent phenol hydroxylase. Sequence analysis of the major bands detected indicated the predominance of the low- K(s) group of TCE/phenol-degrading bacteria belonging to beta-Proteobacteria. These results suggest that continuous supplementation with phenol at a low concentration increases the population of the low- K(s) group of TCE/phenol-degrading bacteria.

Molecular ecological approaches have detected diverse microorganisms that occur in response to pollution and bioremediation; however, most of these organisms have not been isolated, and their physiological traits are poorly understood. One important objective in current bioremediation studies would therefore be an assessment of the physiology and functions of the diverse microbial population at a polluted site. Among the parameters relating to the diversity of the microbial catabolic potential, e.g., substrate specificity, inducer specificity, number of catabolic routes and kinetics of catabolic enzymes, our studies have focused on the kinetic diversity of phenol-degrading bacteria. In one example, a kinetic analysis allowed functionally important phenol-degrading bacteria to be identified in activated sludge; this information could be used to improve the performance of phenol-degrading activated sludge. In an analysis of phenol-degrading bacteria present in trichloroethylene (TCE)-contaminated aquifer soil, the kinetic data could be linked to group-specific monitoring of their phenol-hydroxylase genes. The results have suggested that one group of phenol-degrading bacteria can effectively contribute to TCE bioremediation, while other groups work poorly. Based on this information, we have succeeded in developing a high-performance TCE-degrading bioreactor. We suggest that a careful analysis of the diversity of microbial catabolic potential, particularly of the kinetic traits, may facilitate the development of new bioremediation strategies.

The particulate methane monooxygenase genes (pMMO) were amplified from methane-biostimulated aquifer samples using two sets of primers, A189 plus A682 and pmof2 plus pmor, and the products were cloned and sequenced. The analysis of the sequences revealed a high diversity of pMMO genes in the aquifers. Some of the pMMO gene sequences (LP20 and LP21) isolated in this study were unique and unrelated to methanotrophs previously isolated from this site. Throughout all the samples collected from the different methane biostimulation trials, competitive PCR amplification using specific pMMO primers revealed that type I methanotrophs predominated over type II methanotrophs. The partial soluble methane monooxygenase genes were also amplified from the aquifer samples using specially designed primers but sequencing revealed a lower diversity.

Whole-cell kinetics of phenol- and trichloroethylene (TCE)-degrading activities expressed by 13 phenol-degrading bacteria were analyzed. The Ks (apparent affinity constant in Haldane's equation) values for TCE were unexpectedly diverse, ranging from 11 microM to over 800 microM. The Vmax/Ks values for phenol were three orders of magnitude higher than the values for TCE in all bacteria analyzed, suggesting that these bacteria preferentially degrade phenol rather than TCE. A positive correlation between Ks for phenol and Ks for TCE was found, i.e., bacteria exhibiting high Ks values for phenol showed high Ks values for TCE, and vice versa. A comparison of the Ks values allowed grouping of these bacteria into three types, i.e., low-, moderate- and high-Ks types. Pseudo-first-order degradation-rate constants for TCE at 3.8 microM were found to be adequate to rapidly discriminate among the three types of bacteria. When bacteria were grown on phenol at the initial concentration of 2 mM, Comamonas testosteroni strain R5, a representative of low-Ks bacteria, completely degraded TCE at 3.8 microM, while strain P-8, a representative of high-Ks bacteria, did not. A mixed culture of these two bacteria poorly degraded TCE under the same conditions, where P-8 outgrew R5. These results suggest that low-Ks bacteria should be selectively grown for effective bioremediation of TCE-contaminated groundwater.

Comamonas testosteroni strain R5 is a phenol-degrading bacterium which expresses a phenol-oxygenating activity that is characterized by low Ks (the apparent half-saturation constant in Haldane's equation) and low K(SI) (the apparent inhibition constant) values. We have now cloned the gene cluster encoding a phenol hydroxylase (phcKLMNOP) and its cognate regulator (phcR) from strain R5. Transformation of Pseudomonas aeruginosa PAO1c (Phenol Catechol+) with pROR502, a derivative of pRO1614 containing the cloned genes, confers the ability to grow on phenol as the sole carbon source. The Ks and K(SI) values for the phenol-oxygenating activity of PAO1c(pROR502) are almost identical to those of strain R5, suggesting that the phcKLMNOP genes encode the major phenol hydroxylase in strain R5. A phylogenetic analysis shows the phenol hydroxylase from strain R5 to be more closely related to toluene/benzene-2-monooxygenase (Tb2m) from Pseudomonas sp. JS150 than to the phenol hydroxylases from P. putida CF600 and BH, or to the phenol hydroxylase from Ralstonia eutropha E2. Analysis of the substrate specificity of PAO1c(pROR502) and PAO1c derivatives expressing phenol hydroxylase from P. putida BH or from R. eutropha E2 indicates that these phenol hydroxylases catalyze the oxidation not only of phenol and cresols but also of toluene and benzene.