Shigeaki Harayama

Advances in the field of high‐molecular‐weight polycyclic aromatic hydrocarbon biodegradation by bacteria

Interest in understanding prokaryotic biotransformation of high‐molecular‐weight polycyclic aromatic hydrocarbons (HMW PAHs) has continued to grow and the scientific literature shows that studies in this field are originating from research groups from many different locations throughout the world. In the last 10 years, research in regard to HMW PAH biodegradation by bacteria has been further advanced through the documentation of new isolates that represent diverse bacterial types that have been isolated from different environments and that possess different metabolic capabilities. This has occurred in addition to the continuation of in‐depth comprehensive characterizations of previously isolated organisms, such as Mycobacterium vanbaalenii PYR‐1. New metabolites derived from prokaryotic biodegradation of four‐ and five‐ring PAHs have been characterized, our knowledge of the enzymes involved in these transformations has been advanced and HMW PAH biodegradation pathways have been further developed, expanded upon and refined. At the same time, investigation of prokaryotic consortia has furthered our understanding of the capabilities of microorganisms functioning as communities during HMW PAH biodegradation.

Iron corrosion activity of anaerobic hydrogen-consuming microorganisms isolated from oil facilities

The purpose of the present study was to test the hypothesis that anaerobic hydrogen-consuming microorganisms generally promote iron corrosion. We isolated 26 hydrogen-consuming microorganisms (acetogens, sulfate-reducing bacteria, and methanogens) from oil facilities in Japan using hydrogen as an electron donor. The iron corrosion activities of these microorganisms were examined using iron (Fe0) granules as the sole electron donor. Almost all the isolates consumed hydrogen that was chemically generated from iron granules but did not induce significant iron corrosion. The amount of corroded iron in the cultures of these organisms was less than 2-fold that in an abiotic chemical corrosion reaction. These results indicated that hydrogen consumption did not strongly stimulate iron corrosion. On the other hand, one isolate, namely, Methanococcus maripaludis Mic1c10, considerably corroded iron: this phenomenon was not accompanied by hydrogen consumption, methane formation, or cell growth. This finding also provided strong evidence that M. maripaludis Mic1c10 produced some material that caused iron to corrode.

Methanobacterium petrolearium sp. nov. and Methanobacterium ferruginis sp. nov., mesophilic methanogens isolated from salty environments.

Two methane-producing archaea, designated Mic5c12(T) and Mic6c05(T), were isolated from sludge deposited in a crude oil storage tank and a tubercle on the interior of a pipe transporting natural gas-containing brine, respectively. The isolates were Gram-staining-variable, non-motile rods and grew only on H(2)/CO(2). Strain Mic6c05(T) produced methane from some alcohols without showing any growth; strain Mic5c12(T) did not utilize alcohols. The optimum growth conditions for strain Mic5c12(T) were 35 °C, pH 6.5 and 0-0.68 M NaCl and for strain Mic6c05(T) were 40 °C, pH 6.0-7.5 and 0.34 M NaCl. Strain Mic5c12(T) was halotolerant and strain Mic6c05(T) was halophilic. Comparative 16S rRNA gene sequence analysis revealed that strains Mic5c12(T) and Mic6c05(T) belonged to the genus Methanobacterium and their closest relative was Methanobacterium subterraneum A8p(T) (97.3 and 97.9u200a% 16S rRNA gene sequence similarity, respectively). The findings from the 16S rRNA gene sequence analyses were supported by analysis of McrA, the alpha subunit of methyl-coenzyme M reductase. On the basis of phylogenetic analyses and phenotypic characteristics, two novel species are proposed, Methanobacterium petrolearium sp. nov. and Methanobacterium ferruginis sp. nov., with type strains Mic5c12(T) (=NBRC 105198(T) =DSM 22353(T)) and Mic6c05(T) (=NBRC 105197(T) =DSM 21974(T)), respectively.

Iron Corrosion Induced by Nonhydrogenotrophic Nitrate-Reducing Prolixibacter sp. Strain MIC1-1

ABSTRACT Microbiologically influenced corrosion (MIC) of metallic materials imposes a heavy economic burden. The mechanism of MIC of metallic iron (Fe0) under anaerobic conditions is usually explained as the consumption of cathodic hydrogen by hydrogenotrophic microorganisms that accelerates anodic Fe0 oxidation. In this study, we describe Fe0 corrosion induced by a nonhydrogenotrophic nitrate-reducing bacterium called MIC1-1, which was isolated from a crude-oil sample collected at an oil well in Akita, Japan. This strain requires specific electron donor-acceptor combinations and an organic carbon source to grow. For example, the strain grew anaerobically on nitrate as a sole electron acceptor with pyruvate as a carbon source and Fe0 as the sole electron donor. In addition, ferrous ion and l-cysteine served as electron donors, whereas molecular hydrogen did not. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MIC1-1 was a member of the genus Prolixibacter in the order Bacteroidales. Thus, Prolixibacter sp. strain MIC1-1 is the first Fe0-corroding representative belonging to the phylum Bacteroidetes. Under anaerobic conditions, Prolixibacter sp. MIC1-1 corroded Fe0 concomitantly with nitrate reduction, and the amount of iron dissolved by the strain was six times higher than that in an aseptic control. Scanning electron microscopy analyses revealed that microscopic crystals of FePO4 developed on the surface of the Fe0 foils, and a layer of FeCO3 covered the FePO4 crystals. We propose that cells of Prolixibacter sp. MIC1-1 accept electrons directly from Fe0 to reduce nitrate.

A simple and rapid GC/MS method for the simultaneous determination of gaseous metabolites

We modified and tuned a commercial model of a gas chromatography/mass spectrometry (GC/MS) instrument to develop a simple and rapid method for the simultaneous quantification of a variety of gas species. Using the developed method with the newly modified instrument, gas species such as H(2), N(2), O(2), CO, NO, CH(4), CO(2), and N(2)O, which are common components of microbial metabolism, were accurately identified based on their retention times and/or mass-to-charge ratios (m/z) in less than 2.5 min. By examining the sensitivities and dynamic ranges for the detection of H(2), N(2), O(2), CH(4), CO(2), and N(2)O, it was demonstrated that the method developed in this study was sufficient for accurately monitoring the production and the consumption of these gaseous species during microbial metabolism. The utility of the new method was demonstrated by a denitrification study with Pseudomonas aureofaciens ATCC 13985(T). This method will be suitable for a variety of applications requiring the identification of gaseous metabolites in microorganisms, microbial communities, and natural ecosystems.

Corrosion of iron by iodide-oxidizing bacteria isolated from brine in an iodine production facility.

Elemental iodine is produced in Japan from underground brine (fossil salt water). Carbon steel pipes in an iodine production facility at Chiba, Japan, for brine conveyance were found to corrode more rapidly than those in other facilities. The corroding activity of iodide-containing brine from the facility was examined by immersing carbon steel coupons in “native” and “filter-sterilized” brine samples. The dissolution of iron from the coupons immersed in native brine was threefold to fourfold higher than that in the filter-sterilized brine. Denaturing gradient gel electrophoresis analyses revealed that iodide-oxidizing bacteria (IOBs) were predominant in the coupon-containing native brine samples. IOBs were also detected in a corrosion deposit on the inner surface of a corroded pipe. These results strongly suggested the involvement of IOBs in the corrosion of the carbon steel pipes. Of the six bacterial strains isolated from a brine sample, four were capable of oxidizing iodide ion (I−) into molecular iodine (I2), and these strains were further phylogenetically classified into two groups. The iron-corroding activity of each of the isolates from the two groups was examined. Both strains corroded iron in the presence of potassium iodide in a concentration-dependent manner. This is the first report providing direct evidence that IOBs are involved in iron corrosion. Further, possible mechanisms by which IOBs corrode iron are discussed.

Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea

BackgroundMicroalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea.ResultsIn this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura+) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator.ConclusionsA self-cloning-based positive selection system for the genetic transformation of P. ellipsoidea was developed. Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.

Influence of extracellular polysaccharides (EPS) produced by two different green unicellular algae on membrane filtration in an algae-based biofuel production process

In the present study, two strains of green algae named S1 and S2, categorized as the same species of Pseudo-coccomyxa ellipsoidea but showing 99% homology, were cultivated under the same conditions and filtrated with a microfiltration membrane. On the basis of the results of the extracellular polysaccharides (EPS) characteristics of these two green algae and the degree of fouling, the influence of these characteristics on the performance of membrane filtration was investigated. There was no difference in the specific growth rate between the S1 and S2 strains; however, large differences were seen in the amount and quality of EPS between S1 and S2. When the S1 and S2 strains were filtered with a membrane, the trend in the increase in transmembrane pressure (TMP) was quite different. The filtration of the S1 strain showed a rapid increase in TMP, whereas the TMP of the filtration of the S2 strain did not increase at all during the operation. This clearly demonstrated that the characteristics of each strain affect the development of membrane fouling. On the basis of the detailed characterization of solved-EPS (s-EPS) and bound-EPS (b-EPS), it was clarified that s-EPS mainly contributed to irreversible fouling for both operations and the biopolymer-like organic matter contained in b-EPS mainly contributed to reversible fouling.

Biofilm-based photobioreactor absorbing water and nutrients by capillary action

Cells of the unicellular green alga, Pseudochoricystis ellipsoidea, were uniformly spread on a cellulosic sheet or on a polytetrafluoroethylene (PTFE) membrane sheet superimposed on a cellulosic sheet at a density of 3.5-5.0gdry weight per m2, and the sheet was adhered to an inverted V-shaped acrylic plate of 10cm in height. Several acrylic plates were placed side by side on a tray containing liquid medium at a depth of 0.6cm, and illuminated from above with a light intensity of 300-340μmolm-2s-1. Water and nutrients were supplied to cells by capillary action through the cellulosic sheet. Footprint biomass productivities of cells grown in atmospheric CO2 on this photobioreactor were 8-10gm-2day-1. This cultivation system is strongly energy- and labor-saving as it does not require mixing of culture fluid, irrigation of medium, and delivery of CO2-enriched air.

Construction of Marker-Free Transgenic Strains of Chlamydomonas reinhardtii Using a Cre/loxP-Mediated Recombinase System

The Escherichia coli bacteriophage P1 encodes a site-specific recombinase called Cre and two 34-bp target sites of Cre recombinase called loxP. The Cre/loxP system has been used to achieve targeted insertion and precise deletion in many animal and plant genomes. The Cre/loxP system has particularly been used for the removal of selectable marker genes to create marker-free transgenic organisms. For the first time, we applied the Cre/loxP-mediated site-specific recombination system to Chlamydomonas reinhardtii to construct marker-free transgenic strains. Specifically, C. reinhardtii strains cc4350 and cc124 carrying an aphVIII expression cassette flanked by two direct repeats of loxP were constructed. Separately, a synthetic Cre recombinase gene (CrCRE), the codons of which were optimized for expression in C. reinhardtii, was synthesized, and a CrCRE expression cassette was introduced into strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette. Among 46 transformants carrying the CrCRE expression cassette stably, the excision of aphVIII by CrCre recombinase was observed only in one transformant. We then constructed an expression cassette of an in-frame fusion of ble to CrCRE via a short linker peptide. The product of ble (Ble) is a bleomycin-binding protein that confers resistance to bleomycin-related antibiotics such as Zeocin and localizes in the nucleus. Therefore, the ble-(linker)-CrCRE fusion protein is expected to localize in the nucleus. When the ble-(linker)-CrCRE expression cassette was integrated into the genome of strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette, CrCre recombinase-mediated excision of the aphVIII expression cassette was observed at a frequency higher than that in stable transformants of the CrCRE expression cassette. Similarly, from strain cc124 carrying a single loxP-flanked aphVIII expression cassette, the aphVIII expression cassette was successfully excised after introduction of the ble-(linker)-CrCRE expression cassette. The ble-(linker)-CrCRE expression cassette remained in the genome after excision of the aphVIII expression cassette, and it was subsequently removed by crossing with the wild-type strain. This precise Cre-mediated deletion method applicable to transgenic C. reinhardtii could further increase the potential of this organism for use in basic and applied research.