Hana Yi

INTRODUCING EZTAXON-E: A PROKARYOTIC 16S RRNA GENE SEQUENCE DATABASE WITH PHYLOTYPES THAT REPRESENT UNCULTURED SPECIES

Despite recent advances in commercially optimized identification systems, bacterial identification remains a challenging task in many routine microbiological laboratories, especially in situations where taxonomically novel isolates are involved. The 16S rRNA gene has been used extensively for this task when coupled with a well-curated database, such as EzTaxon, containing sequences of type strains of prokaryotic species with validly published names. Although the EzTaxon database has been widely used for routine identification of prokaryotic isolates, sequences from uncultured prokaryotes have not been considered. Here, the next generation database, named EzTaxon-e, is formally introduced. This new database covers not only species within the formal nomenclatural system but also phylotypes that may represent species in nature. In addition to an identification function based on Basic Local Alignment Search Tool (blast) searches and pairwise global sequence alignments, a new objective method of assessing the degree of completeness in sequencing is proposed. All sequences that are held in the EzTaxon-e database have been subjected to phylogenetic analysis and this has resulted in a complete hierarchical classification system. It is concluded that the EzTaxon-e database provides a useful taxonomic backbone for the identification of cultured and uncultured prokaryotes and offers a valuable means of communication among microbiologists who routinely encounter taxonomically novel isolates. The database and its analytical functions can be found at http://eztaxon-e.ezbiocloud.net/.

A novel electrochemically active and Fe(III)-reducing bacterium phylogenetically related to Aeromonas hydrophila, isolated from a microbial fuel cell

A facultative anaerobic bacterium was isolated from a mediator-less microbial fuel cell fed with artificial wastewater containing acetate and designated as PA3. The isolate was identified as a strain of Aeromonas hydrophila based on its biochemical, physiological and morphological characteristics as well as 16S rDNA sequence analysis and DNA-DNA hybridization. PA3 used glucose, glycerol, pyruvate and hydrogen to reduce Fe(III), nitrate and sulfate. Cyclic voltammetry showed that PA3 was electrochemically active and was the culture collection strain A. hydrophila KCTC 2358. Electricity was generated from a fuel cell-type reactor, the anode compartment of which was inoculated with cell suspensions of the isolate or A. hydrophila KCTC 2358. The electrochemical activities are novel characteristics of A. hydrophila.

Selection of a variant of Geobacter sulfurreducens with enhanced capacity for current production in microbial fuel cells

Geobacter sulfurreducens produces current densities in microbial fuel cells that are among the highest known for pure cultures. The possibility of adapting this organism to produce even higher current densities was evaluated. A system in which a graphite anode was poised at -400 mV (versus Ag/AgCl) was inoculated with the wild-type strain of G. sulfurreducens, strain DL-1. An isolate, designated strain KN400, was recovered from the biofilm after 5 months of growth on the electrode. KN400 was much more effective in current production than strain DL-1. This was apparent with anodes poised at -400 mV, as well as in systems run in true fuel cell mode. KN400 had current (7.6A/m(2)) and power (3.9 W/m(2)) densities that respectively were substantially higher than those of DL1 (1.4A/m(2) and 0.5 W/m(2)). On a per cell basis KN400 was more effective in current production than DL1, requiring thinner biofilms to make equivalent current. The enhanced capacity for current production in KN400 was associated with a greater abundance of electrically conductive microbial nanowires than DL1 and lower internal resistance (0.015 versus 0.130 Omega/m(2)) and mass transfer limitation in KN400 fuel cells. KN400 produced flagella, whereas DL1 does not. Surprisingly, KN400 had much less outer-surface c-type cytochromes than DL1. KN400 also had a greater propensity to form biofilms on glass or graphite than DL1, even when growing with the soluble electron acceptor, fumarate. These results demonstrate that it is possible to enhance the ability of microorganisms to electrochemically interact with electrodes with the appropriate selective pressure and that improved current production is associated with clear differences in the properties of the outer surface of the cell that may provide insights into the mechanisms for microbe-electrode interactions.

Application of cyclic voltammetry to investigate enhanced catalytic current generation by biofilm-modified anodes of Geobacter sulfurreducens strain DL1 vs. variant strain KN400

A biofilm of Geobacter sulfurreducens will grow on an anode surface and catalyze the generation of an electrical current by oxidizing acetate and utilizing the anode as its metabolic terminal electron acceptor. Here we report qualitative analysis of cyclic voltammetry of anodes modified with biofilms of G. sulfurreducens strains DL1 and KN400 to predict possible rate-limiting steps in current generation. Strain KN400 generates approximately 2 to 8-fold greater current than strain DL1 depending upon the electrode material, enabling comparative electrochemical analysis to study the mechanism of current generation. This analysis is based on our recently reported electrochemical model for biofilm-catalyzed current generation expanded here to a five step model; Step 1 is mass transport of acetate, carbon dioxide and protons into and out of the biofilm, Step 2 is microbial turnover of acetate to carbon dioxide and protons, Step 3 is the non-concerted, 1-electron reduction of 8 equivalents of electron transfer (ET) mediator, Step 4 is extracellular electron transfer (EET) through the biofilm to the electrode surface, and Step 5 is the reversible oxidation of reduced mediator by the electrode. Five idealized voltammetric current vs. potential dependencies (voltammograms) are derived, one for when each step in the model is assumed to limit catalytic current. Comparison to experimental voltammetry of DL1 and KN400 biofilm-modified anodes suggests that for both strains, the microbial oxidation of acetate (Step 2) is fast compared to microbial reduction of ET mediator (Step 3), and either Step 3 or EET through the biofilm (Step 4) limits catalytic current generation. The possible limitation of catalytic current by Step 4 is consistent with proton concentration gradients observed within these biofilms and finite thicknesses achieved by these biofilms. The model presented here has been universally designed for application to biofilms other than G. sulfurreducens and could serve as a platform for future quantitative voltammetric analysis of non-corrosive anode and cathode reactions catalyzed by microorganisms.

Combustion characteristics of intake port injection type hydrogen fueled engine

Abstract This paper describes the experimental results on a hydrogen fueled single cylinder engine to study the characteristics of a solenoid-driven intake port injection type hydrogen injection valve. In experiments, the fuel-air equivalence ratio was varied from the lean limit at which stable operation was guaranteed to the rich limit at which flash-back occurred and spark timing was also changed. As a consequence, a hydrogen intake port injection system can be easily installed on a spark ignition engine only with simple modification and the flow rate of hydrogen supplied can also be controlled conveniently. In this case, the most serious problem is flash-back and it can be suppressed by accurate control of injection timing and elimination of hot spots on the surface of the combustion chamber.

Effect of Genetically Modified Poplars on Soil Microbial Communities during the Phytoremediation of Waste Mine Tailings

ABSTRACT The application of transgenic plants to clean up environmental pollution caused by the wastes of heavy metal mining is a promising method for removing metal pollutants from soils. However, the effect of using genetically modified organisms for phytoremediation is a poorly researched topic in terms of microbial community structures, despite the important role of microorganisms in the health of soil. In this study, a comparative analysis of the bacterial and archaeal communities found in the rhizosphere of genetically modified (GM) versus wild-type (WT) poplar was conducted on trees at different growth stages (i.e., the rhizospheres of 1.5-, 2.5-, and 3-year-old poplars) that were cultivated on contaminated soils together with nonplanted control soil. Based on the results of DNA pyrosequencing, poplar type and growth stages were associated with directional changes in the structure of the microbial community. The rate of change was faster in GM poplars than in WT poplars, but the microbial communities were identical in the 3-year-old poplars. This phenomenon may arise because of a higher rate and greater extent of metal accumulation in GM poplars than in naturally occurring plants, which resulted in greater changes in soil environments and hence the microbial habitat.

Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes

Advancement of DNA sequencing technology allows the routine use of genome sequences in the various fields of microbiology. The information held in genome sequences proved to provide objective and reliable means in the taxonomy of prokaryotes. Here, we describe the minimal standards for the quality of genome sequences and how they can be applied for taxonomic purposes.

Duplex-specific nuclease efficiently removes rRNA for prokaryotic RNA-seq

Next-generation sequencing has great potential for application in bacterial transcriptomics. However, unlike eukaryotes, bacteria have no clear mechanism to select mRNAs over rRNAs; therefore, rRNA removal is a critical step in sequencing-based transcriptomics. Duplex-specific nuclease (DSN) is an enzyme that, at high temperatures, degrades duplex DNA in preference to single-stranded DNA. DSN treatment has been successfully used to normalize the relative transcript abundance in mRNA-enriched cDNA libraries from eukaryotic organisms. In this study, we demonstrate the utility of this method to remove rRNA from prokaryotic total RNA. We evaluated the efficacy of DSN to remove rRNA by comparing it with the conventional subtractive hybridization (Hyb) method. Illumina deep sequencing was performed to obtain transcriptomes from Escherichia coli grown under four growth conditions. The results clearly showed that our DSN treatment was more efficient at removing rRNA than the Hyb method was, while preserving the original relative abundance of mRNA species in bacterial cells. Therefore, we propose that, for bacterial mRNA-seq experiments, DSN treatment should be preferred to Hyb-based methods.

Analytical Tools and Databases for Metagenomics in the Next-Generation Sequencing Era

Metagenomics has become one of the indispensable tools in microbial ecology for the last few decades, and a new revolution in metagenomic studies is now about to begin, with the help of recent advances of sequencing techniques. The massive data production and substantial cost reduction in next-generation sequencing have led to the rapid growth of metagenomic research both quantitatively and qualitatively. It is evident that metagenomics will be a standard tool for studying the diversity and function of microbes in the near future, as fingerprinting methods did previously. As the speed of data accumulation is accelerating, bioinformatic tools and associated databases for handling those datasets have become more urgent and necessary. To facilitate the bioinformatics analysis of metagenomic data, we review some recent tools and databases that are used widely in this field and give insights into the current challenges and future of metagenomics from a bioinformatics perspective.

The optimised mixture formation for hydrogen fuelled engines

Abstract Both intake port injection type and in-cylinder injection type hydrogen fuel supply systems are designed for a single-cylinder research engine to investigate the effect of mixture formation on the performance of the hydrogen-fuelled engines. The intake port injection is superior in thermal efficiency and engine operation stability at low load conditions. However, the in-cylinder injection is superior for high load condition. In an engine experiment with throttling condition, the engine with intake port injection operates more stably and efficiently when fuel–air equivalence ratio is maintained above a certain level despite the pumping loss due to stable combustion. Thus, the hydrogen-fuelled engines can be operated more stably with the in-cylinder injection at high load and more efficiently with the intake port injection at low load. Therefore, the optimised operation of the hydrogen-fuelled engines can be achieved with a dual injection system and throttle valve control.