Effect of water hardness on antibacterial activity of green tea extract was studied using tap water and mineral water. The agar medium containing green tea made with six samples of tap water or two samples of mineral water, were tested for Escherichia coli culture, and the colony forming number was compared with that on the agar medium containing each water. Increasing water hardness resulted in increasing antibacterial activity of green tea. Using the high hardness (1468 mg/L) mineral water, the ratio of colony forming number on the green tea medium to one on the water medium was 0.016, which is 43 times lower than that on the low hardness (18 mg/L) tap water. The principal component analysis based on the concentrations of mineral elements in water samples, suggested that the high concentrations of Ca and Mg in water, and low concentration of Si increased antibacterial activity of green tea. The water hardness increased the amount of (-)-epicatechin gallate, not the total amount of catechins in green tea extract. The results are discussed considering the formation of complexs with catechin with Ca and Mg and the change in antibacterial activity of green tea.

We developed a novel bioremediation system, symbiotic engineering, based on the symbiosis between leguminous plant, Astragalus sinicus and the recombinant rhizobium, Mesorhizobium huakuii subsp. rengei B3, by over expressing a synthetic tetrameric metallothionein gene (MTL4) and cDNA encoding the phytochelatin synthase (AtPCS) and the iron regulated transporter (AtIRT1) from Arabidopsis thaliana. In rice paddy soil, addition of recombinant strain B3 carrying a plasmid with the MTL4, AtPCS and/or AtIRT1 genes significantly increased the accumulation of cadmium in roots and nodules of A. sinicus. Thus, this system uses the advantages of both plants and rhizobium. In particular, the system can easily transform engineered genes to the host plant through infection with a recombinant rhizobium.

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.

The comprehensive changes in testicular gene expression before and after haploid germ cell differentiation were examined using microarray analysis. Approximately 14,000 expressed sequence tag (EST) clones of Mouse FANTOM Array ver.1 were hybridized with probes generated from mRNA of adult and juvenile (17 days postpartum) testes before the onset of spermiogenesis. Of 1315 genes that exhibited reproducible changes in expression (p < 0.05), 46% exhibited an increase of twofold or more in adults compared to juveniles, and 22% a decrease of twofold or more. The analysis not only confirmed the reported haploid-specific expression of several known genes, but also provided new information on the differential expression of various other genes, including upregulated genes such as Allc and Skd3 and downregulated genes such as hbb b1, before or after the onset of spermiogenesis. Based on the fundamental difference in expression profiles, and molecular functions of the encoded products, the genes were classified into several groups: postmeiotically upregulated genes encoding various enzymes, structural and regulatory proteins, and chaperones, and downregulated genes encoding haemoglobins and oxidation/reduction-related proteins or the machinery associated with protein synthesis, such as ribosomal proteins.

Cadmium contamination in rice grains is one of the important issues in Asian countries. We have developed a novel bio-remediation system based on the symbiosis between leguminous plant and genetically engineered rhizobia. We designed two types of recombinant rhizobia, carrying two genes, synthetic tetrameric metallothionein (MTL4) and cDNA encoding phytochelatin synthase from Arabidopsis thaliana (AtPCS). The MTL4 and AtPCS genes were transferred to Mesorhizobium huakuii subsp. rengei B3, which can infect and form nodules on Chinese milk vetch, Astragalus sinicus. The two genes were fused to the nolB or nifH promoter, which generated nodule specific expression of these genes in strain B3. The two recombinant strains, B3(pMPnolBMTL4nifHPCS) and B3::nifHMTL4(pMPnifHPCS), showed 25 and 12-fold increase in Cd concentration, in the free-living cells, respectively. When these recombinant strains established the symbiotic relationship with A. sinicus, the symbionts increased Cd accumulation in nodules by two-fold in hydroponic culture. The expression of the both MTL4 and AtPCS genes showed additive effect on cadmium accumulation in nodules. We also applied these recombinant bacteria to rice paddy soil polluted with Cd (1mgkg(-1) dry weight soil). The accumulation of Cd increased not only in nodules but also in the roots of A. sinicus infected by the recombinant rhizobia. The accumulation of Cd in the plant roots infected by B3(pMPnolBMTL4nifHPCS) achieved three-fold than that by the wild-type B3. After two months of cultivation of the symbiont, a maximum of 9% of Cd in paddy soil was removed. Thus, the symbiosis will be useful in phytoremediation for heavy metals.

Promoters, including neither TATA box nor initiator, have been frequently found in testicular germ cellspecific 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.

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.

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.

This chapter focuses on a system for converting algal biomass into H2, biologically. Algal biomass could be supplied from the oxidation pond in waste-water treatment plants, or from eutrophicated lakes. It can be converted to various energy media, such as methane and oil. Among various microorganisms which produce H2, photosynthetic bacteria have the advantage that they can carry out H2 production from organic substrates using solar energy. Therefore, a cost effective energy production process could be achieved using photosynthetic bacteria. For the photoproduction of H2 from a CO2-fixing algal biomass, a two-step process consisting of lactic acid fermentation as pretreatment preceding the H2 production, and single-step process in which a halotolerant bacterial community directly converted starch in the algal biomass to H2, are proposed. The bacterial community has designated BC1 contains three halophilic or halotolerant bacterial species, Vibrio fluvialis, Rhodobium marinum, and Proteus vulgaris. Among them, V. fluvialis and R. marinum play roles in the degradation of starch into acetic acid and ethanol, and the production of H2 from the degraded products, respectively. A study using the starch-rich microalgae, Chlamydomonas reinhardtii and Dunaliella tertiolecta demonstrated the H2 production efficiencies of the above two processes.

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.

A combined system for recovering air pollutants and converting them to H-2 was studied. CO2 and nitric oxide (NO) were removed simultaneously from a model flue gas using Dunnliella tertiolecta and Chlamydomonas reinhardtii cultures in a tubular photobioreactor. The algal biomass was converted to a substrate suitable for bacterial H-2 production by the starch-hydrolyzing lactic acid bacterium, Lactobacillus amylovorus. The photosynthetic bacterium Rhodobium marinum produced H-2 from lactic acid fermentation products, and a conversion yield of 6 mol H-2 per mole of starch-glucose in the algal biomass was observed in a system starting from biomass of D. tertiolecta. Such a combined system would give additional value and thereby improve practical efficiency of solar energy conversion to H-2.

To produce H-2 from CO2-fixing algal biomass, two-step systems including heat-HCl treatment or lactic acid fermentation followed by H-2 production, have been attempted. An alternate system involving direct conversion of algal starch to H-2 is proposed. Such a singlestep conversion has been achieved using a halotolerant bacterial community selected from night soil treatment sludge. H-2 yield from starch-glucose was lowest in the process via heat-HCl treatment, while the process with lactic acid fermentation followed by H-2 photoproduction of Rhodobacter sphaeroides RV gave the highest yield of 4.6 mol/mol starch-glucose from Chlamydamonas reinhardtii biomass. Although the single-step system gave a lower H-2 yield at present, it produced H-2 directly from starch in C. reinhardtii biomass.

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.

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 H-2 production by the photosynthetic bacterium Rhodobacter sphaeroides RV. Starch accumulated in the algal biomass was converted to H-2 with a high, conversion yield of 5 mol H-2/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.

(1) 固定化海水馴養硝化汚泥を用いて, NH4-N負荷量を2.0mg-N/g-pellet/d以下にとれば, 余水に含まれる窒素を90%の効率で硝化できた.又, 余水に無機炭素源を補填することで, 固定化海水馴養硝化汚泥の硝化能力が向上することを認めた.(2) 固定化海水馴養脱窒汚泥は余水中でCH3OHを脱窒の水素供与体とした場合, 8.0mg-NO3-N/g-pellet/d以下の負荷量で90%のNO3-N除去率が得られた.(3) 固定化海水馴養硝化活性汚泥と固定化海水馴養脱窒汚泥を用いる循環型の硝化-脱窒システムを構築し, 液循環比を3.0, 硝化ペレットと脱窒ペレットの湿重量比を1にとった条件で, CH3OHを余水のT-N濃度の2倍の濃度で添加することにより, 6.2時間の液滞留時間で余水の窒素を80%の効率で除去できた.

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 reactor's 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.

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.