Satoshi Wakai

Involvement of sulfide:quinone oxidoreductase in sulfur oxidation of an acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans NASF-1.

The effects of cyanide, azide, and 2-n-Heptyl-4-hydroxy-quinoline-N-oxide (HQNO) on the oxidation of ferrous ion or elemental sulfur with Acidithiobacillus ferrooxidans NASF-1 cells grown in iron- or sulfur-medium were examined. The iron oxidation of both iron- and sulfur-grown cells was strongly inhibited by cyanide and azide, but not by HQNO. Sulfur oxidation was relatively resistant to cyanide and azide, and inhibited by HQNO. Higher sulfide oxidation, ubiquinol dehydrogenase activity, and sulfide:quinone oxidoreductase (SQR) activity were observed in sulfur-grown cells more than in iron-grown cells. Sulfide oxidation in the presence of ubiquinone with the membrane fraction was inhibited by HQNO, but not by cyanide, azide, antimycin A, and myxothiazol. The transcription of three genes, encoding an aa3-type cytochrome c oxidase (coxB), a bd-type ubiquinol oxidase (cydA), and an sqr, were measured by real-time reverse transcription polymerase chain reaction. The transcriptional levels of coxB and cydA genes were similar in sulfur- and iron-grown cells, but that of sqr was 3-fold higher in sulfur-grown cells than in iron-grown cells. A model is proposed for the oxidation of reduced inorganic sulfur compounds in A. ferrooxidans NASF-1 cells.

Purification and Characterization of Sulfide : Quinone Oxidoreductase from an Acidophilic Iron-Oxidizing Bacterium, Acidithiobacillus ferrooxidans

Sulfide:quinone oxidoreductase (SQR) was purified from membrane of acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans NASF-1 cells grown on sulfur medium. It was composed of a single polypeptide with an apparent molecular mass of 47 kDa. The apparent K m values for sulfide and ubiquinone were 42 and 14 μM respectively. The apparent optimum pH for the SQR activity was about 7.0. A gene encoding a putative SQR of A. ferrooxidans NASF-1 was cloned and sequenced. The gene was expressed in Escherichia coli as a thioredoxin-fusion protein in inclusion bodies in an inactive form. A polyclonal antibody prepared against the recombinant protein reacted immunologically with the purified SQR. Western blotting analysis using the antibody revealed an increased level of SQR synthesis in sulfur-grown A. ferrooxidans NASF-1 cells, implying the involvement of SQR in elemental sulfur oxidation in sulfur-grown A. ferrooxidans NASF-1 cells.

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.

Development of bio-based fine chemical production through synthetic bioengineering

AbstractFine chemicals that are physiologically active, such as pharmaceuticals, cosmetics, nutritional supplements, flavoring agents as well as additives for foods, feed, and fertilizer are produced by enzymatically or through microbial fermentation. The identification of enzymes that catalyze the target reaction makes possible the enzymatic synthesis of the desired fine chemical. The genes encoding these enzymes are then introduced into suitable microbial hosts that are cultured with inexpensive, naturally abundant carbon sources, and other nutrients. Metabolic engineering create efficient microbial cell factories for producing chemicals at higher yields. Molecular genetic techniques are then used to optimize metabolic pathways of genetically and metabolically well-characterized hosts. Synthetic bioengineering represents a novel approach to employ a combination of computer simulation and metabolic analysis to design artificial metabolic pathways suitable for mass production of target chemicals in host strains. In the present review, we summarize recent studies on bio-based fine chemical production and assess the potential of synthetic bioengineering for further improving their productivity.

From mannan to bioethanol: cell surface co-display of β-mannanase and β-mannosidase on yeast Saccharomyces cerevisiae.

BackgroundMannans represent the largest hemicellulosic fraction in softwoods and also serve as carbohydrate stores in various plants. However, the utilization of mannans as sustainable resources has been less advanced in sustainable biofuel development. Based on a yeast cell surface-display technology that enables the immobilization of multiple enzymes on the yeast cell walls, we constructed a recombinant Saccharomyces cerevisiae strain that co-displays β-mannanase and β-mannosidase; this strain is expected to facilitate ethanol fermentation using mannan as a biomass source.ResultsParental yeast S. cerevisiae assimilated mannose and glucose as monomeric sugars, producing ethanol from mannose. We constructed yeast strains that express tethered β-mannanase and β-mannosidase; co-display of the two enzymes on the cell surface was confirmed by immunofluorescence staining and enzyme activity assays. The constructed yeast cells successfully hydrolyzed 1,4-β-d-mannan and produced ethanol by assimilating the resulting mannose without external addition of enzymes. Furthermore, the constructed strain produced ethanol from 1,4-β-d-mannan continually during the third batch of repeated fermentation. Additionally, the constructed strain produced ethanol from ivory nut mannan; ethanol yield was improved by NaOH pretreatment of the substrate.ConclusionsWe successfully displayed β-mannanase and β-mannosidase on the yeast cell surface. Our results clearly demonstrate the utility of the strain co-displaying β-mannanase and β-mannosidase for ethanol fermentation from mannan biomass. Thus, co-tethering β-mannanase and β-mannosidase on the yeast cell surface provides a powerful platform technology for yeast fermentation toward the production of bioethanol and other biochemicals from lignocellulosic materials containing mannan components.

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.

Increased ethanol production from sweet sorghum juice concentrated by a membrane separation process

The aim of this investigation was to attain high ethanol concentration by concentrating sweet sorghum juice using a two-step membrane separation process. Ultrafiltration permeation of the juice was used to remove residues, followed by nanofiltration concentration to increase the sugar concentration. The concentrated juice containing 180.0 g L(-1) sucrose, 59.3 g L(-1) glucose and 49.3 g L(-1) fructose supplemented with nitrogen sources (10 and 20 g L(-1) of yeast extract and polypeptone, respectively) was fermented by Saccharomyces cerevisiae BY4741 to produce 133.5 g L(-1) of ethanol (87.6% of theoretical yield) after 48 h fermentation. Importantly, the addition of lower concentrations of exogenous nitrogen sources (3 and 6 g L(-1) of yeast extract and polypeptone, respectively) or no exogenous nitrogen sources resulted in the production of 131.4 and 132.8 g L(-1) of ethanol (84.8% and 86.0% of theoretical yield), respectively, after 48 h fermentation.

Comparative Analysis of Highly Homologous Shewanella Cytochromes c5 for Stability and Function

Homologous cytochromes c 5 from a mesophile, Shewanella amazonensis (SA cytc 5), and a psychrophile, Shewanella violacea (SV cytc 5), were compared to elucidate the molecular mechanisms underlying protein stability and function. Cyclic voltammetry revealed that the two proteins had the same redox potential value. Differential scanning calorimetry showed that SV cytc 5 was more stable than SA cytc 5 in an enthalpic manner. These results and the structure model of Shewanella oneidensis cytochrome c 5 indicated that hydrophobic heme environments in the two proteins are the same to maintain the same redox potential value, and that the intra-molecular interactions in SV cytc 5, perhaps involved in Lys-50 and Tyr-73, account for its higher stability. Electron transfer from SV cytc 5 to membrane proteins of S. violacea and S. amazonensis was faster than that from SA cytc 5, suggesting that solvent-exposed Lys-4 in SV cytc 5 is responsible for the faster association and dissociation between SV cytc 5 and its redox partner.

l-lactic acid production from starch by simultaneous saccharification and fermentation in a genetically engineered Aspergillus oryzae pure culture

Lactic acid is a commodity chemical that can be produced biologically. Lactic acid-producing Aspergillus oryzae strains were constructed by genetic engineering. The A. oryzae LDH strain with the bovine L-lactate dehydrogenase gene produced 38 g/L of lactate from 100g/L of glucose. Disruption of the wild-type lactate dehydrogenase gene in A. oryzae LDH improved lactate production. The resulting strain A. oryzae LDHΔ871 produced 49 g/L of lactate from 100g/L of glucose. Because A. oryzae strains innately secrete amylases, A. oryzae LDHΔ871 produced approximately 30 g/L of lactate from various starches, dextrin, or maltose (all at 100 g/L). To our knowledge, this is the first report describing the simultaneous saccharification and fermentation of lactate from starch using a pure culture of transgenic A. oryzae. Our results indicate that A. oryzae could be a promising host for the bioproduction of useful compounds such as lactic acid.

Some properties of a novel obligately autotrophic iron-oxidizing bacterium isolated from seawater

Abstract An iron-oxidizing bacterium obligately requiring NaCl for growth was isolated from seawater in Seto Inland Sea, Japan, and designated as strain KU2-11. Strain KU2-11 was a Gram negative, non-spore-forming and rod-shaped bacterium, and can grow autotrophically by using ferrous ion and elemental sulfur as sole energy sources. The optimum growth pH and temperature were 2 and 30°C, respectively. The G+C content of the DNA was 59 mol%. The bacterium could not grow in the medium without NaCl and obligately required NaCl for growth. The optimum NaCl concentration for the growth was 2%. Strain KU2-11 seemed to be identified as a member of Thiobacillus ferrooxidans on the basis of morphological and physiological properties. However, the phylogenetic analysis based on 16S rDNA gene sequences indicated that strain KU2-11 is distinct from T. ferrooxidans . Iron- and sulfur-oxidizing activity of the bacterium were not detected in a reaction mixture without NaCl, and depended on the presence of NaCl. The optimum NaCl concentrations for the iron- and sulfur-oxidizing activities were 2–4% and 1%, respectively. On the basis of the phenotypic characteristics of strain KU2-11 and its phylogenetic position, we suggested that this bacterium should be placed in a new species of the genus Thiobacillus .