Toru Matsui

Removal of siloxane from digestion gas of sewage sludge.

This paper studies the way to remove siloxanes from digestion gas of sewage sludge by adsorbents. Many adsorbents were tested by using the model gas of siloxane in nitrogen. The adsorption ratio was 0.056-0.192 for the activated carbons, 0.004-0.077 for the molecular sieve and 0.104 for the silica gel. An observed tendency was that the activated carbons showed high adsorption ratio, and that the adsorption ratio was increased when BET surface area was higher, and the pore volume was higher and the pH value was higher. Finally, we found a few kinds of activated carbons with the superior ability of adsorption, and we tested the ability of them by using the real digestion gas. All of the siloxanes were found to be removed in the 1000 h test.

Thermophilic anaerobic co-digestion of garbage, screened swine and dairy cattle manure.

Methane fermentation characteristics of garbage, swine manure (SM), dairy cattle manure (DCM) and mixtures of these wastes were studied. SM and DCM showed much lower volatile total solid (VTS) digestion efficiencies and methane yield than those of garbage. VTS digestion efficiency of SM was significantly increased when it was co-digested with garbage (Garbage: SM=1:1). Co-digestion of garbage, SM and DCM with respect to the relative quantity of each waste discharged in the Kikuchi (1: 16: 27) and Aso (1: 19: 12) areas indicated that co-digestion with garbage would improve the digestion characteristic of SM and DCM as far as the ratio of DCM in the wastes was maintained below a certain level. When the mixed waste (Garbage: SM: DCM=1:19:12) was treated using a thermophilic UAF reactor, methanogens responsible for the methane production were Methanoculleus and Methanosarcina species. Bacterial species in the phylum Firmicutes were dominant bacteria responsible for the digestion of these wastes. As the percentage of garbage in the mixed wastes used in this study was low (2-3%) and the digestion efficiency of DCM was obviously improved, the co-digestion of SM and DCM with limited garbage was a prospective method to treat the livestock waste effectively and was an attractive alternative technology for the construction of a sustainable environment and society in stock raising area.

Methane fermentation of a mixture of seaweed and milk at a pilot-scale plant.

In this study, a pilot-scale plant was built to examine the practicality of producing biogas from seaweeds, widely available in Japan. Laminaria sp. and Ulva sp. seaweeds were mixed with other organic waste (milk) and used as fermentation materials. Though quantities and ratios of the materials were varied, the ratio of generated methane to input chemical oxygen demand (COD) was largely stable (0.2-0.3m(3) methane/kg COD) and the organic acid concentration in the methane fermentation solution was low (<1200 ppm) during prolonged operation. These findings indicate that stable methane fermentation was achieved and that mixing with other organic material was effective in suppressing fluctuations in material amounts caused by the variable supply of seaweeds. Our results demonstrate the practical feasibility of biogas generation using seaweeds.

Effect of temperature on microbial community of a glucose-degrading methanogenic consortium under hyperthermophilic chemostat cultivation

We continuously fed an anaerobic chemostat with synthetic wastewater containing glucose as the sole source of carbon and energy to study the effects of temperature on the microbial community under hyperthermophilic (65-80 degrees C) conditions. Methane was produced normally up to 77.5 degrees C at a dilution rate of 0.025 d(-1). However, the concentration of microorganisms and the rate of gas production decreased with increasing operation temperature. The microbial community in the chemostat at various temperatures was analyzed based on the 16S rRNA gene using molecular biological techniques including clone library analysis and denaturing gradient gel electrophoresis (DGGE). Aceticlastic methanogens related to Methanosarcina thermophila were detected at 65 degrees C and hydrogenotrophilic methanogens related to Methanothermobacter thermautotrophicus were the dominant methanogens between 70 degrees C to 77.5 degrees C. Bacteria related to Clostridium stercorarium and Thermoanaerobacter subterraneus comprised the dominant glucose-fermenting bacteria at temperatures of 65 degrees C and above, respectively. Bacteria related to Thermacetogenium phaeum and to Tepidiphilus margaritifer and Petrobacter succinatimandens were the dominant acetate-oxidizing bacteria at 70 degrees C and at 75-77.5 degrees C, respectively. The results suggested that, at temperatures of 70 degrees C and above, methane production via the aceticlastic pathway was negligible and indirect methanogenesis from acetate was dominant. Since acetate oxidation is a rate limiting step and a higher temperature favors the hydrolysis and acid formation, a two stage fermentation process, acidogenic and methanogenic fermentation stages operated under different temperatures, should be more suitable for the thermophilic anaerobic treatment at temperatures above 65 degrees C.

A mutated hygromycin resistance gene is functional in the n-alkane-assimilating yeast Candida tropicalis.

Abstract. Development of a transformation system in the n-alkane-assimilating diploid yeast Candida tropicalis requires an antibiotic resistance gene in order to establish a selectable marker. The resistance gene for hygromycin B has often been used as a selectable marker in yeast transformation. However, C. tropicalis harboring the hygromycin resistance gene (HYG) was as sensitive to hygromycin B as the wild-type strain. Nine CTG codons were found in the ORF of the HYG gene. This codon has been reported to be translated as serine rather than leucine in Candida species. Analysis of the tRNA gene in C. tropicalis with the anticodon CAG [tRNA(CAG) gene], which is complementary to the codon CTG, showed that the sequence was highly similar to that of the C. maltosa tRNA(CAG) gene. In C. maltosa, the codon CTG is read as serine and not leucine. These results suggested that the HYG gene was not functional due to the nonuniversal usage of the CTG codon. Each of the nine CTG codons in the ORF of the HYG gene was changed to a CTC codon, which is read as leucine, by site-directed mutagenesis. When a plasmid containing the mutated HYG gene (HYG#) was constructed and introduced into C. tropicalis, hygromycin-resistant transformants were successfully obtained. This mutated hygromycin resistance gene may be useful for direct selection of C. tropicalis transformants.

Novel and convenient methods for Candida tropicalis gene disruption using a mutated hygromycin B resistance gene

Abstract. We established a novel and convenient method to construct a ura3 strain (ura3/ura3) of the asporogenous and diploid yeast, Candida tropicalis, that produces dicarboxylic acid. One copy of the URA3 gene was disrupted using a mutated hygromycin B resistance gene (HYG#). The obtained hygromycin-resistant strain was further transformed with a URA3 disruption cassette and selected on a plate containing 5-fluoroorotic acid. The obtained strains were analyzed and the disruption of the gene was confirmed by PCR and Southern blot analysis. The results showed that the strains were obtained in which allelic URA3 genes were simultaneously disrupted. Furthermore, we established a cotransformation method for this gene disruption, using HYG# in C. tropicalis. In order to disrupt the allelic POX4 genes (encoding acyl-CoA oxidase) of dicarboxylic acid-producing strains, the ARS plasmid (which contained HYG#) and a POX4 disruption cassette (which carried the LAC4 gene encoding β-galactosidase of Kluyveromyces lactis) were simultaneously introduced by transformation. As a result, the allelic POX4 gene was successfully disrupted.

Construction of an autonomously replicating plasmid in n-alkane- assimilating yeast, Candida tropicalis

A transformation system using the autonomously replicating plasmid in the n-alkane-assimilating and asporogenic diploid yeast, Candida tropicalis, was developed. For the cloning of a DNA fragment containing a potential autonomously replicating sequence (ARS) from the genomic DNA of C. tropicalis, the ura3 mutant obtained using ethylmethane sulfonate as the host and the URA3 gene amplified by PCR using the C. tropicalis genomic DNA as a selectable marker were prepared. Comparison of ARSs among yeasts revealed that the consensus sequence found in S. cerevisiae was also present in C. tropicalis. The autonomously replicating plasmid containing the putative ARS as the shuttle vector, capable of replicating in both E. coli and C. tropicalis, was first constructed. The transformation system using this plasmid, in addition to the integrative transformation system, will be applicable to genetic studies of C. tropicalis.

Specific DNA Binding of a Potential Transcriptional Regulator, Inosine 5′-Monophosphate Dehydrogenase-Related Protein VII, to the Promoter Region of a Methyl Coenzyme M Reductase I-Encoding Operon Retrieved from Methanothermobacter thermautotrophicus Strain ΔH

ABSTRACT Two methyl coenzyme M reductases (MCRs) encoded by the mcr and mrt operons of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus ΔH are expressed in response to H2 availability. In the present study, cis elements and trans-acting factors responsible for the gene expression of MCRs were investigated by using electrophoretic mobility shift assay (EMSA) and affinity particle purification. A survey of their operator regions by EMSA with protein extracts from mrt-expressing cultures restricted them to 46- and 41-bp-long mcr and mrt upstream regions, respectively. Affinity particle purification of DNA-binding proteins conjugated with putative operator regions resulted in the retrieval of a protein attributed to IMP dehydrogenase-related protein VII (IMPDH VII). IMPDH VII is predicted to have a winged helix-turn-helix DNA-binding motif and two cystathionine β-synthase domains, and it has been suspected to be an energy-sensing module. EMSA with oligonucleotide probes with unusual sequences showed that the binding site of IMPDH VII mostly overlaps the factor B-responsible element-TATA box of the mcr operon. The results presented here suggest that IMPDH VII encoded by MTH126 is a plausible candidate for the transcriptional regulator of the mcr operon in this methanogen.