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The down-flow hanging sponge (DHS) reactor is advantageous for sewage treatment since it produces an effluent quality that complies with the standards for reuse and there is little excess sludge. A full-scale DHS module was efficiently employed for the treatment of domestic sewage (200 m

Monoethanolamine (MEA), a toxic organic chemical, is widely used in industries and is found in their wastewater. Anaerobic MEA degradation is an appropriate strategy to reduce energy and cost for treatment. Industry wastewaters also contain sulfate, but information on the effects of sulfate on MEA degradation is limited. Here, an up-flow anaerobic sludge blanket (UASB) for MEA-containing wastewater treatment was operated under psychrophilic conditions (18-20 ºC) to investigate the effects of sulfate on the microbial characteristics of the retained sludge. To acclimatize the sludge, the proportion of MEA in the influent (containing sucrose, acetate, and propionate) was increased from 15% to 100% of total COD (1500 mg L

Unpasteurized draft sake has a potentially high market value, due to its fresh flavor and fruity taste, compared with conventional thermal-pasteurized sake. However, the shelf life of draft...

A sparkling-type draft cloudy sake (Japanese rice wine), AWANAMA, was recently developed using high hydrostatic pressure (HHP) treatment as a non-thermal pasteurization method. This prototype sake has a high potential market value, since it retains the fresh taste and flavor similar to draft sake while avoiding over-fermentation. From an economic point of view, a lower pressure level for HHP pasteurization is still required. In this study, we carried out a genome analysis of a pressure-sensitive (piezosensitive) mutant strain, a924E1, which was generated by UV mutagenesis from a laboratory haploid Saccharomyces cerevisiae strain, KA31a. This mutant strain had a deletion of the COX1 gene region in the mitochondrial DNA and had deficient aerobic respiration and mitochondrial functions. A metabolomic analysis revealed restricted flux in the TCA cycle of the strain. The results enabled us to use aerobic respiration deficiency as an indicator for screening a piezosensitive mutant. Thus, we generated piezosensitive mutants from a Niigata-sake yeast strain, S9arg, which produces high levels of ethyl caproate but does not produce urea and is consequently suitable for brewing a high-quality sake. The resultant piezosensitive mutants showed brewing characteristics similar to the S9arg strain. This study provides a screening method for generating a piezosensitive yeast mutant as well as insight on a new way of applying HHP pasteurization.

In this study, a lab-scale upflow anaerobic sludge blanket (UASB) reactor was applied to the treatment of artificial electronics industry wastewater containing tetramethylammonium-hydroxide (TMAH), monoethanolamine (MEA), and isopropyl-alcohol (IPA) in order to evaluate process performance and degradation properties. During 800 days of operation, 96% efficiency of chemical oxygen demand (COD) removal was stably achieved at an organic loading rate of 8.5 kgCOD/m

Draft (non-pasteurized) sake typically has a fresh flavor, but usually has to be consumed as soon as possible because of its short shelf life due to the potential for over-fermentation by residual yeast. In contrast, thermally pasteurized sake has a long shelf life, but the fresh flavor is lost during heat sterilization. High hydrostatic pressure (HHP) treatment can be used as a non-thermal pasteurization process while maintaining the characteristics of a draft sake. We evaluated the HHP inactivation behavior of sake yeast for the application of HHP pasteurization as an alternative to thermal pasteurization. The results showed complete pasteurization of sake yeast in the draft sake by HHP treatment at 400 MPa for 10 min. The viable cell count in HHP-pasteurized sake was below the detection limit during storage at 10°C for 3 months. Thus, we have established a HHP pasteurization technology to prevent over-fermentation and succeeded in producing a prototype of HHP-sparkling-cloudy sake, which we hav...

The use of untreated sewage in agriculture is gaining attention as a result of rapid urbanization and increased global water scarcity. However, the incidence of waterborne infections among farmers in such areas is increasing due to the exposure of contaminated sewage. The aim of this study was to estimate the risk and health impact related to waterborne infections in farmers using agricultural drainage wastewater (ADW) for irrigation in the West Nile Delta. A total of nine viruses were detected by quantitative PCR and quantitative real-time PCR in ADW samples collected from the Dishody drain, which was found to be the most polluted branch drain in the region through a water pollution survey. The results of quantitative microbial risk assessment and disability-adjusted life year evaluation indicated that ADW of the Dishody drain is not acceptable for irrigation reuse. A water recirculation system including appropriate sewage treatment technology must be quickly installed to reduce microbial risks for farmers in the West Nile Delta.

Carbon and nitrogen stable isotope ratios (δ13C and δ15N) were determined in activated sludge, which was exposed to endogenous conditions for 36 days and contained a wide diversity of organisms across several trophic levels. The aim of this study was to elucidate the fluctuation of δ13C and δ15N through trophic transfer in the microbial consortia. The sludge was evaluated in view of sludge mass, bacterial community, higher trophic organisms, sludge δ13C and δ15N, and δ15N and δ18O of nitrate. The results show that the activated sludge became more enriched with 15N as degradation proceeded. Eventually, the mixed liquor volatile suspended solid concentrations in the activated sludge decreased from 1610 to 710 mg/L and the δ15N of the sludge increased from 8.3‰ to 10.8‰. In contrast, the δ13C values of the sludge were stable. Microscope observations confirmed that consumers such as Rotifera, Tardigrada and Annelida (Aelosoma sp.) were present in the activated sludge for the entire operational period. The abundance of those organisms drastically changed during the operational periods, and the diversity in bacterial community also changed, resulting in community succession. Changes in biotic community, reduction in sludge mass, and increase in δ15N of the sludge occurred during the sludge degradation processes. This implies that the sludge degradation was partly caused by the trophic conversion of the sludge-derived nitrogen in the food web. The δ15N of the sludge can be used as an indicator of the sludge degradation through trophic transfer in wastewater treatment reactors. These findings provide new insights into understanding trophic transfer during microbial community succession and the effects of the feeding process on sludge degradation.

Application of high hydrostatic pressure (HHP) treatment to food processing is expected as a non-thermal fermentation regulation technology that supresses over fermentation. However, the yeast Sacc...

The characteristics of sludge retained in a down-flow hanging sponge reactor were investigated to provide a better understanding of the sewage treatment process in the reactor. The organic removal and sulfur oxidation conditions were found to differ between the first layer and the following three layers. It was found that 63% and 59% of the organic matter was removed in the first layer, even though the hydraulic retention time was only 0.2 h. It is thought that the organic removal resulted from aerobic and anaerobic biodegradation on the sponge medium. The sulfate concentration increased 1.5–1.9-fold in the first layer, with almost no subsequent change in the second to fourth layers. It was shown that oxidation of sulfide in the influent was completed in the first layer. The result of the oxygen uptake rate test with an ammonium nitrogen substrate suggested that the ammonium oxidation rate was affected by the condition of dissolved oxygen (DO) or oxidation–reduction potential (ORP).

A down-flow hanging sponge (DHS) reactor has been developed as a cost-effective wastewater treatment system that is adaptable to local conditions in low-income countries. A pilot-scale DHS reactor previously demonstrated stable reduction efficiencies for chemical oxygen demand (COD) and ammonium nitrogen over a year at ambient temperature, but the pathogen reduction efficiency of the DHS reactor has yet to be investigated. In the present study, the reduction efficiency of a pilot-scale DHS reactor fed with municipal wastewater was investigated for 10 types of human pathogenic viruses (norovirus GI, GII and GIV, aichivirus, astrovirus, enterovirus, hepatitis A and E viruses, rotavirus, and sapovirus). DHS influent and effluent were collected weekly or biweekly for 337 days, and concentrations of viral genomes were determined by microfluidic quantitative PCR. Aichivirus, norovirus GI and GII, enterovirus, and sapovirus were frequently detected in DHS influent, and the log10 reduction (LR) of these viruses ranged from 1.5 to 3.7. The LR values for aichivirus and norovirus GII were also calculated using a Bayesian estimation model, and the average LR (±standard deviation) values for aichivirus and norovirus GII were estimated to be 1.4 (±1.5) and 1.8 (±2.5), respectively. Quantitative microbial risk assessment was conducted to calculate a threshold reduction level for norovirus GII that would be required for the use of DHS effluent for agricultural irrigation, and it was found that LRs of 2.6 and 3.7 for norovirus GII in the DHS effluent were required in order to not exceed the tolerable burden of disease at 10-4 and 10-6 disability-adjusted life years loss per person per year, respectively, for 95% of the exposed population during wastewater reuse for irrigation.

In this study, a continuous feeding experiment was conducted with synthetic iso-plophyl alcohol (2-propanol)-containing wastewater using a lab-scale psychrophilic UASB reactor to evaluate process performance and retained sludge properties. For smooth acclimation, methanogenic granular sludge was seeded and a proportion of 2-propanol in the synthetic wastewater containing sucrose and volatile fatty acids was increased stepwise from 0% to 30%, 60% and then 90% of COD (chemical oxygen demand). As a result, after a 4-week period for acclimation to 2-propanol degradation, a COD removal rate of 95% was achieved at an organic loading rate (OLR) of 8.4 kg COD/m

Carbon and nitrogen stable isotope ratios (δ

High hydrostatic pressure (HP) inhibits growth and inactivates microorganisms by destabilizing non-covalent molecular interactions. Arginine contributes to stress resistance because it has a guanidine side chain, which assists in the refolding of aggregated proteins. We attempted to analyze the contribution of arginine to high HP stress using a pressure-sensitive mutant strain of Saccharomyces cerevisiae and a metabolomics approach. Our results showed that the content of 136 out of 250 detected metabolites differed in the mutant and parent strains. Decreased metabolites were involved in the tricarboxylic acid cycle and arginine biosynthesis. The expression of genes contributing to arginine biosynthesis was significantly lower in the mutant strain than in the parent strain. When arginine was supplemented to the medium, the mutant strain showed more tolerance to pressure. These results suggest that yeast cells survived due to the contribution of arginine to high pressure resistance. This indicates that depl...

High hydrostatic pressure causes physical stress to microorganisms; therefore, this technology may be applied to food pasteurization without introducing the unfavorable effects of thermal denaturation. However, its application is limited to high-value foods because the treatment requires a robust steel vessel and expensive pressurization equipment. To reduce these costs, we studied the pasteurization of Saccharomyces cerevisiae using relatively moderate high-pressure levels. A mutant strain isolated by ultraviolet mutagenesis showed significant loss of viability under high-pressure conditions. Gene expression analysis of the mutant strain revealed that it incurred a deletion of the COX1 gene. Our results suggest that the pressure-sensitivity can readily be introduced into industrial/food microorganisms by complementing a COX1 deleted mitochondria. Practical Application High pressure causes physical damages to microorganisms; therefore, this technology may be applied to food pasteurization without introducing the unfavorable effects of thermal denaturation. Our study suggests that pressure-sensitive variants of industrial strains can readily be constructed by complementing mitochondria. This would facilitate the use of pressure-pasteurization technology in controlling microbial fermentations using pressure-sensitive industrial strains.

The characteristics of the microbial community in a practical-scale down-flow hanging sponge (DHS) reactor, high in organic matter and sulfate ion concentration, and the seasonal variation of the microbial community composition were investigated. Microorganisms related to sulfur oxidation and reduction (2–27%), as well as Leucobacter (7.50%), were abundant in the reactor. Anaerobic bacteria (27–38% in the first layer) were also in abundance and were found to contribute to the removal of organic matter from the sewage in the reactor. By comparing the Simpson index, the abundance-based coverage estimator (ACE) index, and the species composition of the microbial community across seasons (summer/dry, summer/rainy, autumn/dry, and winter/dry), the microbial community was found to change in composition only during the winter season. In addition to the estimation of seasonal variation, the difference in the microbial community composition along the axes of the DHS reactor was investigated for the first time. Although the abundance of each bacterial species differed along both axes of the reactor, the change of the community composition in the reactor was found to be greater along the vertical axis than the horizontal axis of the DHS reactor.

The microbial community composition of a down-flow hanging sponge (DHS) reactor in an up-flow anaerobic sludge blanket (UASB)-DHS system used for the treatment of municipal sewage was investigated. The clone libraries showed marked differences in microbial community composition at different reactor heights and in different seasons. The dominant phylotypes residing in the upper part of the reactor were likely responsible for removing organic matters because a significant reduction in organic matter in the upper part was observed. Quantification of the amoA genes revealed that the proportions of ammonia oxidizing bacteria (AOB) varied along the vertical length of the reactor, with more AOB colonizing the middle and lower parts of the reactor than the top of the reactor. The findings indicated that sewage treatment was achieved by a separation of microbial habitats responsible for organic matter removal and nitrification in the DHS reactor.

Recently, the industry and productions have been rapidly developed by the advancement of science and technology. On the other hand, the dwindling natural resources and the global warming increasingly pose a severe problem to our life in near future. Indeed, solar photovoltaic (PV) has been attractive as one of the alternative energy resource to oil. However, some problems such as the reduction of electromotive force and the degradation of back-sheet influence the properties of the long-term life of the PV panel system. In this research, we used the high- and low-molecular-weight PET film in order to evaluate the effect of molecular weight for the hydrolysis of PET under the acceleration degradation test. As results, the mechanical properties of PET film were decreased with increasing the acceleration degradation time. In addition, it was found that the dielectric breakdown strength of PET film indicated the similar tendency with the mechanical properties. Accordingly, the non-destructive analytical technique, i.e. the partial discharge measurement makes it easy to evaluate the degradation of PET film without any damage or cut out for the film.

We analyzed high hydrostatic pressure (HP)-induced inactivation of Escherichia coli in the presence of sucrose, NaCl, KCl, or LiCl under 250–400 MPa. Salt stress response (osmotic or cationic) mechanism was demonstrated to be evaluated by HP-induced inactivation kinetics. We selected nine mutant strains from the Keio collection; five strains each lacked one osmoregulatory gene (ΔmzrA, ΔenvZ, ΔompR, ΔompC or ΔompF), and four strains each lacked one cationic stress-response gene (ΔmscS, ΔmscL, ΔchaA or ΔnhaA). The nine mutants exhibited three different patterns of HP-induced inactivation. Of the nine genes, four (mzrA, mscS, nhaA and mscL) were involved in piezotolerance at NaCl concentration of 0.0–0.35 M.

A high pressure (HP) tolerant (barotolerant) mutant a2568D8 and a variably barotolerant mutant a1210H12 were generated from Saccharomyces cerevisiae using ultra-violet mutagenesis. The two mutants, a barosensitive mutant a924E1 and the wild-type strain, were pressurized (225 MPa), and pressure inactivation behavior was analyzed. In the wild-type strain, a proportion of the growth-delayed cells were detected after exposure to HP. In a924E1, the proportion of growth-delayed cells significantly decreased compared with the wild-type. In a2568D8, the proportion of growth-delayed cells increased and the proportion of inactivated cells decreased compared with the wild-type. In a1210H12, the growth-delayed cells could not be detected within 120 s of exposure to HP. The proportion of growth-delayed cells, which incurred the damage, would affect the survival ratio by HP. These results suggested that cellular changes in barotolerance caused by mutations are remarkably affected by the ability to recover from cellular...

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We analyzed the effect of high hydrostatic pressure (HHP) treatment on reducing sugar production in the tuberous root of sweet potato (Ipomoea batatas), based on pressure-gelatinization of starch and subsequent saccharification by internal amylases. HHP treatment at up to 600 MPa at ambient temperature for 10 min did not apparently affect the reducing sugar concentration in tuberous root. However, HHP treatment at 100 to 500 MPa and 60 °C or 70 °C for 10 min increased reducing sugar concentration as both the pressure and temperature increased. The reducing sugar concentration after HHP treatment at 500 MPa and 70 °C for 10 min was roughly comparable to that of the thermal treatment control (80 °C for 10 min under atmospheric pressure). HHP treatment enabled the gelatinization and enzymatic saccharification of starch in the tuberous root of sweet potato, at a lower temperature than required by thermal treatment at atmospheric pressure.

Profile analysis of the down-flow hanging sponge (DHS) reactor was conducted under various temperature and organic load conditions to understand the organic removal and nitrification process for sewage treatment. Under high organic load conditions (3.21–7.89 kg-COD m−3 day−1), dissolved oxygen (DO) on the upper layer of the reactor was affected by organic matter concentration and water temperature, and sometimes reaches around zero. Almost half of the CODCr was removed by the first layer, which could be attributed to the adsorption of organic matter on sponge media. After the first layer, organic removal proceeded along the first-order reaction equation from the second to the fourth layers. The ammoniacal nitrogen removal ratio decreased under high organic matter concentration (above 100 mg L−1) and low DO (less than 1 mg L−1) condition. Ammoniacal nitrogen removal proceeded via a zero-order reaction equation along the reactor height. In addition, the profile results of DO, CODCr, and NH3-N were different in the horizontal direction. Thus, it is thought the concentration of these items and microbial activities were not in a uniform state even in the same sponge layer of the DHS reactor.

Cross-linked polyethylene (PEX) pipes used in hot water supply are required for high mechanical strength and high creep resistance at high temperature. Especially PEX-a pipes which are made by peroxide cross-linking have better performance, such as creep resistance and thermal shock resistance than the pipes made by the other cross-linking method. Because the PEX-a pipes indicate the higher cross-link degree as compared with the other PEX pipes. In this study, the PEX-a pipes which were mixed with several stabilizers were tested to evaluate the effects on cross-link degree and the oxygen induction time. And also they are evaluated with the chlorine aqueous solution by the performance of the long-term hydrostatic pressure test and the long-term hydro dynamic pressure test. As a result, it was found that the combination of antioxidants for PEX-a pipes plays an important role to prolong the oxygen induction time without inhibiting the cross-linking. From the results of the 1H pulsed NMR measurement over the melting point of polyethylene, it was found that each peroxide PEX pipe with different antioxidant combinations indicated the different proportion and crosslink density of cross-linked region, in addition, that these pipes had the effective structure of cross-linking for the hydrostatic and hydrodynamic pressure test with the chlorine aqueous solution. Therefore, it was considered to be useful results for studies of the stricture of cross-linking of polyethylene.

We previously obtained a pressure-tolerant (piezotolerant) and a pressure sensitive (piezosensitive) mutant strain, under ambient temperature, from Saccharomyces cerevisiae strain KA31a. The inactivation kinetics of these mutants were analyzed at 150 to 250 MPa with 4 to 40 °C. By a multiple regression analysis, the pressure and temperature dependency of the inactivation rate constants k values of both mutants, as well as the parent strain KA31a, were well approximated with high correlation coefficients (0.92 to 0.95). For both mutants, as well as strain KA31a, the lowest k value was shown at a low pressure levels with around ambient temperature. The k value approximately increased with increase in pressure level, and with increase and decrease in temperature. The piezosensitive mutant strain a924E1 showed piezosensitivity at all pressure and temperature levels, compared with the parent strain KA31a. In contrast, the piezotolerant mutant strain a2568D8 showed piezotolerance at 4 to 20 °C, but did not show significant piezotolerance at 40 °C. These results of the variable influence of temperature on pressure inactivation of these strains would be important for better understanding of piezosensitive and piezotolerant mechanisms, as well as the pressure inactivation mechanism of S. cerevisiae .

In upflow anaerobic sludge blanket (UASB) reactors treating high-strength organic wastewaters, uncultured phylotypes belonging to the phylum Nitrospirae were assumed to play a key role in the treatment processes because of their high abundance as determined by 16S rRNA gene-based clone library analyses (Narihiro et al. (27)). A comparative nucleotide sequence analysis of the phylotypes, commonly found in anaerobic mesophilic sludges, revealed that these phylotypes formed a coherent clade at the family to genus levels with no cultured representatives. To determine the abundance and distribution in methanogenic waste/wastewater treatment sludges, 16S rRNA gene-based quantitative PCR measurements and 16S rRNA-targeted fluorescence in situ hybridization (FISH) were carried out. A specific gene primer/probe for the uncultured clade was developed and used for quantitative PCR and in situ detection of the targeted cells within the sludge granules. In all the six UASB sludges treating food-processing organic wastewaters, the phylotypes were detected at a range of 0.1 to 10.9% of total 16S rRNA genes. FISH revealed that the gene probe reacted with coccoid or irregular coccoid-like cells in the middle and inner layers of sludge granules of a UASB reactor, in which the Nitrospirae phylotypes were found in abundance by real-time PCR analyses.

A comprehensive survey of bacterial and archaeal community structures within granular sludges taken from twelve different types of full-scale, food-processing wastewater-treating, upflow anaerobic sludge blanket (UASB) reactors was performed with a 16S rRNA gene-based clone library method. In total, 1,282 bacterial 16S rRNA gene clones and 722 archaeal clones were analyzed, and their identities were determined by phylogenetic analyses. Overall, clones belonging to the bacterial phyla Proteobacteria (the class Deltaproteobacteria in particular), Firmicutes, Spirochaetes, and Bacteroidetes were observed in abundance within the bacterial clone libraries examined, indicating common bacterial denominators in such treatment systems. Within the domain Archaea, clones affiliated with the classes Methanomicrobia and Methanobacteria were found to be abundant in the archaeal libraries. In relation to features of reactor performance (such as chemical oxygen demand removal, fatty acid accumulation, and sludge bulking), possible representative phylotypes likely to be associated with process failures, such as sludge bulking and the accumulation of propionate, were found in comparative analyses of the distribution of phylotypes in the sludge libraries.

The putative autolysin Atl(WM) of Staphylococcus warneri M is a modular protein exhibiting two enzyme activities, an N-terminal side amidase (ami(atlwm)-R1-R2) and a C-terminal side glucosaminidase (R3-glu(atlwm)). Zymographic analysis of the protein overproduced in Escherichia coli showed that both enzymes were active toward 17 Gram-positive bacteria, including staphylococci, lactobacilli, lactococci, enterococci, and micrococci. The purified enzyme core ami(atlwm) (or glu(atlwm)) had the pH and temperature optima of about 7.0 (5.5) and 41 (50) degrees C, respectively. ami(atlwm) was inactivated by EDTA, and was stimulated by such salts as CoCl(2), MnCl(2), CaCl(2), or ZnCl(2). Six mutations within ami(atlwm), (H362A, E421A, H467A, H479, D481A, and Y491D) drastically reduced cell-lytic activity. Comparative analysis with other related amidases suggested that the three residues H362, H467, and D481 likely act as ligands (and/or active sites). The lytic activity of glu(atlwm) markedly declined in four mutants (E1238A, E1238Q, T1239A, and Y1332A). For determination of the putative cell-recognition regions, four domains (R1-R2, R1, R2, and R3) were purified; all the proteins substantially bound to S. warneri M cells from exponential to stationary growth phases, and R1-R2 aggregated the cells. Protein sequencing and immunoblot analysis suggested that the extacellular Atl(WM) might be primarily processed at two specific sites (one between pro and ami(atlwm), and the other between R2 and R3) to yield the mature amidase and glucosaminidase.

At present, 95 % of the total population of the Egypt country takes up residency in areas around of Nile, irrigation agriculture caused by using raw sewage exacerbate the issue of environmental health hazard risk in arid regions of Egypt. So, Egypt country goes ahead with effective utilization of treated sewage for irrigation, our international research consortium has proposed a novel concept of sewage treatment technology for risk reduction of health hazard and we focus on down-flow hanging sponge reactor (DHS) as sewage treatment technology. DHS behaved extremely well as sewage treatment technology in India and it is widely expected to dominate in a developing country. The method is a combination of the DHS for posttreatment and primary sedimentation tank for pretreatment. This method went live in Egypt in 2014. So, KNCT tried demonstrating experiment in Japan with the use of share of the same design as method. Preliminary experiment has confirmed the feasibility to satisfy a criterion for irrigation in EGYPT as an objective. Irrigation standard (TSS < 60 mg/L, BOD < 60 mg/L, CODtotal < 80 mg/L, \( {\text{NO}}_{3}^{ - } \) < 50 mg/L) set by Egypt government has achieved effective utilization of treated sewage for irrigation as objective. When hydraulic retention time (HRT) set 1 h as DHS’s volume load of target, condition of operation of experiment is controlled by varying treatment quantity of sewage. We started up the experiment provided that treatment quantity of sewage: 5 m3/day, HRT: 4 h. The experiment is performed in five stages. It has continued since March 2013 (Phase 1: HRT 4 h, Phase 2: HRT 2 h, Phase 3: HRT 1 h, Phase 4: HRT 2 h, and Phase 5: HRT 4 h). The preliminary experiment indicates the possibility of effective utilization of treated sewage for irrigation. The removal rate of biochemical oxygen demand (BOD) and suspended solids (SS) retains sufficient strength to meet irrigation standards. (Phase 1: 82 %(BOD) 97 %(SS), Phase 2: 92 %(BOD) 81 %(SS), Phase 3: 76 %(BOD) 65 %(SS) Phase 4: 81 %(BOD) 68 %(SS), Phase 5: 89 %(BOD) 88 %(SS)). The result of the profile analysis indicated that organics destruction occurred from the top of the reactor (1st stage) to the 3rd stage and the nitrification process proceed from the 4th stage to the 5th stage. On the other hand, the nitrification process of \( {\text{NH}}_{3}^{ - } \)–N was poor at Phase 3. We believe that this related to a blocked gap of media sponge. At Phase 4 and Phase 5, the removal rate of BOD and nitrification of the water was provided so as to lengthen the processing time. Improvement in the ability to remove \( {\text{NH}}_{3}^{ - } \)–N is vital to use the primary settled-DHS process for irrigation, because \( {\text{NH}}_{3}^{ - } \)–N is a toxic substance for agricultural irrigation.

High hydrostatic pressure causes physical stress to microorganisms; therefore, this technology may be applied to food pasteurization without introducing the unfavorable effects of thermal denaturation. However, its application is limited to high-value foods because the treatment requires a robust steel vessel and expensive pressurization equipment. To reduce these costs, we studied the pasteurization of Saccharomyces cerevisiae using relatively moderate high-pressure levels. A mutant strain isolated by ultraviolet mutagenesis showed significant loss of viability under high-pressure conditions. Gene expression analysis of the mutant strain revealed that it incurred a deletion of the COX1 gene. Our results suggest that the pressure-sensitivity can readily be introduced into industrial/food microorganisms by complementing a COX1 deleted mitochondria.

We established an enrichment culture of anammox bacteria by seeding a batch reactor with sediments from the Banzu mudflat in Tokyo Bay. The reactor was intermittently fed with an anammox substrate containing NH4Cl, NaNO2 and artificial seawater (3.03% salinity) for more than 800 days. An anammox enrichment culture capable of simultaneous reduction of approximately equivalent amounts of ammonium nitrogen and nitrite nitrogen was established. Batch assays of the enrichment culture revealed that anammox activity was similar for all batches at salinities ranging from 1.01 to 3.03%, with lower activity observed at a salinity of 0.02%. We identified three 16S rRNA genes in DNA fragments amplified from the enriched culture. One of these genes, mudflat sediment clone_K01 was highly homologous with Candidatus “Kuenenia stuttgartiensis” (identity 97%) being the most abundant (85 out of isolated 94 clones). In most of the studies conducted to date, anammox species belonging to the Candidatus “Scalindua” genus have been found to be the dominant species in enrichment cultures obtained from estuarine and marine environments. The results reported here are unusual, in that, a freshwater anammox bacterium, Candidatus "K. stuttgartiensis", was the dominant bacterium in a sediment sample from a marine environment. Key words: Anammox, mudflat sediment, salinity, Candidatus “Kuenenia stuttgartiensis”,Candidatus “Scalindua wagneri”

Using microplates as pressure and cultivation vessels, a high-throughput method was developed for analyzing the high-pressure inactivation kinetics of microorganisms. The loss of viability from a high-pressure treatment, measured based on the growth delay during microplate cultivation, showed reproducibility with the conventional agar plate method and was applicable for the kinetics analysis.

The major autolysin AcmA of Lactococcus lactis ssp. cremoris MG1363 is a modular protein consisting of an N-terminal signal sequence, a central enzymatic region (glu(acma) as a glucosaminidase), and a C-terminal cell-recognition domain (LysM123). glu(acma) (about 160 amino acids) belongs to the glycoside hydrolase (GH) 73 family, and the two acidic residues E128 and D153 have been thought to be catalytically important. In this study, amino-acid substitution analysis of AcmA was first carried out in the Escherichia coli system. Point mutations E94A, E94Q, E128A, D153A, and Y191A markedly reduced cell-lytic activity (3.8%, 1.1%, 4.2%, 4.8%, and 2.4%, respectively), whereas E128Q and D153N retained significant residual activities (32.1% and 44.0%, respectively). On the other hand, Y191F and Y191W mutations retained high activities (66.2% and 46.0%, respectively). These results showed that E94 (rather than E128 and D153) and the aromatic residue Y191 probably play important roles in catalysis of AcmA. Together with mutational analysis of another GH73 glucoaminidase Glu(atlwm) from the Staphylococcus warneri M autolysin Atl(WM), these results suggested that the GH73 members cleave a glycosidic bond via a substrate-assisted mechanism, as postulated in the GH20 members. AcmA and Glu(atlwm) were purified from E. coli recombinant cells, and their enzymatic properties were studied.

Heat polynomials have been applied to study the analytic solution of the initial value problem of the heat equation. Since the polynomials satisfy the heat equation the approximate solution of the initial value problem by using the polynomials become an interpolation problem. Therefore the approximate solution is determined from the given initial data since the solution method involves no integration and no discretization with respect to the time-space domain. In order to apply the approach to an initial-boundary value problem, we present an approximation method with Chebyshev heat polynomials which are proposed in this paper. A recurrence formula to generate Chebyshev heat polynomials is presented. Moreover we show that the present method give accurate approximate solutions by numerical experiments.