Akiko Ike

Photoproduction of hydrogen from raw starch using a halophilic bacterial community

The bacterial community designated BC1, which originates from night soil treatment sludge, exhibited a strong ability to produce H2 from raw starch in the light in the presence of 3% NaC1. Three halophilic or halotolerant bacterial species, Vibrio fluvialis, Rhodobium marinum, and Proteus vulgaris, were isolated from BC1 and identified. The level of H2 production from starch by coculture of V. fluvialis and R. marinum was nearly equal to that by BC1, indicating that these two strains play roles in starch degradation and H2 production from the degraded products in BC1, respectively. Acetic acid and ethanol, which were detected as the major products of degradation of starch by V. fluvialis in pure culture, seemed to be mainly utilized for H2 production by R. marinum in BC1 and the coculture. However, R. marinum in pure culture could not produce H2 from a synthetic medium containing acetic acid and ethanol, suggesting that V. fluvialis supplied not only substrates but also some unknown factors capable of inducing H2 production from these substrates by R. marinum. A study using the starch-rich microalgae, Chlamydomonas reinhardtii and Dunaliella tertiolecta, demonstrated that the above coculture could be applied to the production of H2 at high yield from raw starch in an algal biomass.

Hydrogen photoproduction from CO2-fixing microalgal biomass: Application of halotolerant photosynthetic bacteria

Lactic acid fermentates of Chlamydomonas reinhardtii and Dunaliella tertiolecta biomass resulting from fermentation by a starch-hydrolyzing lactic acid bacterium, Lactobacillus amylovorus, were converted to H2 by various photosynthetic bacteria. Using L. amylovorus and a halotolerant bacterial strain Rhodobium marinum, H2 was produced at 8 mol/mol starch-glucose from C. reinhardtii biomass, while from D. tertiolecta biomass, the H2 production was 6 mol/mol starch-glucose even under the condition of 3% NaCl.

Hydrogen photoproduction from CO2-fixing microalgal biomass: Application of lactic acid fermentation by Lactobacillus amylovorus

Intact and/or freeze-thawed microalgal biomass of Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Dunaliella tertiolecta were liquefied using a starch-hydrolyzing lactic acid bacterium, Lactobacillus amylovorus, in order to obtain an ideal substrate for H2 production by the photosynthetic bacterium Rhodobacter sphaeroides RV. Starch accumulated in the algal biomass was converted to H2 with a high conversion yield of 5 mol H2/mol of starch glucose. In this system, the rigid algal cell wall structure could be degraded without the need for any physicochemical or enzymic pretreatment; L. amylovorus appeared to play a role in this degradation.

Transient expression analysis of the mouse ornithine decarboxylase antizyme haploid-specific promoter using in vivo electroporation

The testicular isoform of the ornithine decarboxylase antizyme (OAZt) gene is expressed exclusively in the haploid spermatids of mice. The 357‐bp region, which includes a TATA‐less promoter and an untranslated region, is sufficient for OAZt gene expression in the spermatids of transgenic mice. In this study, in vivo transient transfection to living mouse testes was used to define the transcriptional regulatory elements of the OAZt gene promoter. We found that the 10‐bp element that contains an initiator (Inr) plays a central role as the core promoter, in combination with a downstream element, while two cyclic adenosine monophosphate‐responsive element (CRE)‐like sites in the upstream region also contribute to promoter activity. The electrophoretic mobility shift assay showed binding of the testis‐specific factors to these elements. Our results show that the in vivo DNA transfer technique enables detailed analysis of haploid germ cell‐specific gene regulation in mice.

Nitrification of NH4-N polluted sea water by immobilized acclimated marine nitrifying sludge (AMNS)

Abstract Acclimated marine nitrifying sludge (AMNS) prepared from activated sludge obtained from a night soil treatment plant equipped with a sea water dilution system for controlling the reactors temperature was successfully immobilized using a polyvinyl alcohol (PVA) freezing method. About 2 weeks of recovery culture was required before continuous treatment could be carried out. Transmittance electron microscopic observations of sliced immobilized AMNS pellets indicated that bacteria with an intracytoplasmic membrane dominated the AMNS colony. Continuous nitrification experiments of NH4-N containing synthetic sea water were carried out in a 1.2 l bioreactor containing 57.2 g (wet weight) of immobilized AMNS pellets. The NH4-N removal rate reached a saturation level above an NH4-N loading rate of 1.5 mg-NH4-N/g-pellet/d. The maximum allowable NH4-N loading rate necessary to obtain 90% NH4-N removal was found to be 1.0 mg-NH4-N/g-pellet/d. It was possible to store the immobilized AMNS pellets in a refrigerator for at least 1 week without loss of nitrifying capability. Inorganic carbon source was shown to be a limiting factor in the continuous nitrification experiments. The maximum allowable NH4-N loading rate needed to obtain 90% NH4-N removal increased to 2.0 mg-NH4-N/g-pellet/d through supplementation of an inorganic carbon (IC) source to the influent synthetic sea water.

cAMP-responsive element in TATA-less core promoter is essential for haploid-specific gene expression in mouse testis.

Promoters, including neither TATA box nor initiator, have been frequently found in testicular germ cell-specific genes in mice. These investigations imply that unique forms of the polymerase II transcription initiation machinery play a role in selective activation of germ cell-specific gene expression programs during spermatogenesis. However, there is little information about testis-specific core promoters, because useful germ cell culture system is not available. In this study, we characterize the regulatory region of the haploid-specific Oxct2b gene in detail by using in vivo transient transfection assay in combination with a transgenic approach, with electrophoretic mobility shift and chromatin immunoprecipitation assays. Expression studies using mutant constructs demonstrate that a 34 bp region, which extends from −49 to −16, acts as a core promoter in an orientation-dependent manner. This promoter region includes the cAMP-responsive element (CRE)-like sequence TGACGCAG, but contains no other motifs, such as a TATA box or initiator. The CRE-like element is indispensable for the core promoter activity, but not for activator in testicular germ cells, through the binding of a testis-specific CRE modulator transcription factor. These results indicate the presence of alternative transcriptional initiation machinery for cell-type-specific gene expression in testicular germ cells.

Structure and promoter activity of the gene encoding ornithine decarboxylase antizyme expressed exclusively in haploid germ cells in testis (OAZt/Oaz3)

Ornithine decarboxylase antizyme 1 and 2 (OAZ1 and OAZ2) are expressed ubiquitously, and control the intracellular concentration of polyamines. Their testicular isoform, OAZt/Oaz3, is specifically expressed in differentiated haploid germ cells. We have identified and characterized the gene encoding OAZt in mice. The mouse OAZt gene contains, as does the human ortholog and paralogs, five exons and four introns. Comparison of the mouse OAZt with the human ortholog gene revealed that exon sizes are identical and nucleotide sequences in exons are highly homologous (83% identity). The major transcriptional start site was determined by primer extension assay. Promoter activity was confirmed by transgenic mouse assays, using the upstream region of the mouse OAZt gene fused to a EGFP reporter gene. The OAZt essential promoter located between -133 and +242, has two CREs and an Inr, and lacks a TATA box. These elements are conserved in the human ortholog but not in the paralogs, indicating that such a short upstream region including two CREs and Inr is sufficient to drive endogenous OAZt mRNA expression in the haploid testicular germ cells.

Preparation of marine nitrifying sludge

Activated sludge taken from night soil treatment plants which employ a sea-water dilation system to decrease the temperature elevation caused by fermentative heat during operation was proved to be a good seed source for the preparation of acclimated marine nitrifying sludge (AMNS). The preparation of AMNS with high nitrifying activity was successful from seed activated sludge obtained from a night soil treatment plant which uses a sea-water dilution system throughout the year, within only a two-month acclimatizing period, by applying the fill and draw cultivation method. The nitrifying activity of our newly prepared AMNS was 13.1 mg-NH4-N/g-MLSS/h, which is comparable to that of fresh water nitrifying sludge. The AMNS was composed of 50 to 100 μm bacterial flocs and showed an excellent settling property. The AMNS exhibited high nitrifying activity, even at a low sodium concentration, but its activity decreased at sodium concentrations above 10g/l. The optimum pH for nitrification by the AMNS was 8.5 and its nitrifying activity did not greatly change within the pH range of 7.0 to 9.0. The optimum temperature for nitrification by the AMNS was 35°C. The nitrifying reaction of the AMNS was proved to be less sensitive to temperature change.

Promotion of metal accumulation in nodule of Astragalus sinicus by the expression of the iron-regulated transporter gene in Mesorhizobium huakuii subsp. rengei B3

Toxic metal contamination in agricultural fields is an important worldwide problem. In previous studies, we developed a bioremediation system based on the symbiosis between Astragalus sinicus and the recombinant rhizobium, Mesorhizobium huakuii subsp. rengei B3 developed by overexpressing a synthetic tetrameric metallothionein gene (MTL4) and cDNA encoding the phytochelatin synthase from Arabidopsis thaliana (AtPCS). To promote the transport of metals into the nodules of the rhizobium and the accumulation of metals, the iron-regulated transporter 1 gene from A. thaliana (AtIRT1) was introduced into recombinant strain B3 containing MTL4 or AtPCS in its chromosome. The fused AtIRT1-alkaline phosphatase was expressed in the free-living recombinant rhizobium and the nodule of A. sinicus. The recombinant strain B3 carrying AtIRT1 showed a higher Cd sensitivity and a higher amount of Cd accumulated in free-living culture than the wild-type strain B3. When the recombinant strain B3 established symbiosis with A. sinicus, the introduction of AtIRT1 in the recombinant strain B3 advantaged the accumulation of Cu and As in the nodules of A. sinicus, compared with that of Cd and Zn.

Hydrogen Photoproduction from Starch in CO2-Fixing Microalgal Biomass by A Halotolerant Bacterial Community

To produce H2 from CO2-fixing algal biomass, two-step systems including heat-HCl treatment or lactic acid fermentation followed by H2 production, have been attempted. An alternate system involving direct conversion of algal starch to H2 is proposed. Such a single-step conversion has been achieved using a halotolerant bacterial community selected from night soil treatment sludge. H2 yield from starch-glucose was lowest in the process via heat-HCl treatment, while the process with lactic acid fermentation followed by H2 photoproduction of Rhodobacter sphaeroides RV gave the highest yield of 4.6 mol/mol starch-glucose from Chlamydomonas reinhardtii biomass. Although the single-step system gave a lower H2 yield at present, it produced H2 directly from starch in C. reinhardtii biomass.