Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
KENJI SAKAI Last modified date:2019.06.14

Professor / Division of Systems Bioengineering / Department of Bioscience and Biotechnology / Faculty of Agriculture

1. Abdul-Mutalib NA, Osman M, Amin Nordin S, Ishida N, Tashiro K, Sakai K , Tashiro Y , Maeda T, Shirai Y, Prevalence of spoilage microorganism, Pseudomonas spp. on restaurants cutting boards collected in Seri Kembangan, Malaysia, J Food Sci Nutr, 2, 2, 7-12, 2019.05, Due to the tropical climate that promotes the growth of most microorganisms, the food service
industry in Malaysia faces a lot of challenges in order to ensure quality foods are served to the
customers. One of the bacteria that are associated with food spoilage is Pseudomonas spp., and
the presence of these bacteria on cutting boards could cross-contaminate food items during food
handling. This study performed pyrosequencing analysis to investigate the existence of microbial
communities on cutting boards collected from restaurants in Seri Kembangan, Malaysia. Most
of the samples were dominated by Flavobacteriales, Enterobacteriales, Lactobacillales, and
Pseudomonadales. This study discovered that, all samples of cutting boards were contaminated
with Pseudomonas spp. With the mean bacterial count of 1.4 × 106 cfu/cm2, 5.1 × 105 cfu/cm2
and 5.6 × 106 cfu/cm2 on samples collected from clean, moderately clean and less clean food
premises, respectively. The study shows that the contamination level of Pseudomonas spp. was
not significantly different in different grades of food premises. Regardless of the status and
grades of the food premises, they have the same likelihood to introduce spoilage bacteria like
Pseudomonas spp. from cutting board to food if they neglect correct food handling measures.
Therefore, all food handlers need to prioritize safe food handling to avoid food contamination
and spoilage..
2. Tao Zhao, Kento Yasuda, Yukihiro Tashiro, Rizki Fitria Darmayanti, Kenji Sakai, Kenji Sonomoto, Semi-hydrolysate of paper pulp without pretreatment enables a consolidated fermentation system with in situ product recovery for the production of butanol, Bioresource Technology, 10.1016/j.biortech.2019.01.043, 57-65, 2019.04, Utilization of lignocellulosic biomasses for biobutanol fermentation usually requires costly processes of pretreatment and enzymatic hydrolysis. In this study, paper pulp (93.2% glucan) was used as a starting biomass material to produce biobutanol. We conducted enzymatic semi-hydrolysis of paper pulp without pretreatment and with low enzyme loading, which produced high concentrations of cellobiose (13.9 g L
) and glucose (21.3 g L
). In addition, efficient fermentation of the semi-hydrolysate was achieved similar to that with the use of commercial sugars without inhibitors. Finally, we designed a novel non-isothermal simultaneous saccharification and fermentation with in situ butanol recovery, which was composed of a repeated semi-hydrolysis process and successive butanol-extractive fermentation process under the respective optimal conditions. The consolidated system improved butanol production, butanol yields, and butanol productivities and enabled repeated use of medium when compared with other integrated hydrolysis and fermentation processes..
3. Diana Mohd-Nor, Norhayati Ramli, Siti Suhailah Sharuddin, Mohd Ali Hassan, Nurul Asyifah Mustapha, Afzufira Amran, Kenji Sakai, Yoshihito Shirai, Toshinari Maeda, Alcaligenaceae and Chromatiaceae as reliable bioindicators present in palm oil mill effluent final discharge treated by different biotreatment processes, Ecological Indicators, 10.1016/j.ecolind.2018.08.007, 95, 468-473, 2018.12, This study was undertaken to confirm the reliability of the proposed potential bioindicators, Alcaligenaceae and Chromatiaceae to specifically indicate contamination in river water by palm oil mill effluent (POME) final discharge. The use of bioindicators could assist in determining the specific cause of contamination in water bodies. Alcaligenaceae and Chromatiaceae were shown by a previous study to be present in river water contaminated with the final discharge from the treatment of POME. In the present study, the compositions of the bacterial communities in the POME final discharge obtained from four different palm oil mills which used different biotreatments of POME were elucidated using high-throughput MiSeq. The four POME final discharges studied showed different species richness and evenness among them. However, the bacterial community compositions in the different final discharges exhibited almost similar patterns in that the phylum Proteobacteria was dominant in all the samples. The proposed bioindicators, the Alcaligenaceae and Chromatiaceae families, were found to be present in all the four final discharges despite the different characteristics of the mills and the different biotreatment processes used by them. These bioindicators were also strongly and positively correlated with the biochemical oxygen demand (BOD
) concentration. This makes them reliable bacterial indicators to detect the presence of POME final discharge in river water..
4. Rizki Fitria Darmayanti, Yukihiro Tashiro, Takuya Noguchi, Ming Gao, Kenji Sakai, Kenji Sonomoto, Novel biobutanol fermentation at a large extractant volume ratio using immobilized Clostridium saccharoperbutylacetonicum N1-4, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2018.06.006, 126, 6, 750-757, 2018.12, Product inhibition by butanol and acetone is a known drawback in acetone-butanol-ethanol (ABE) fermentation. Extractive fermentation improves butanol production by several ABE-producing Clostridium spp., but only low volume ratios (<4) of extractant to broth (Ve/Vb) have been studied. Here, a novel extractive fermentation process was developed using Clostridium saccharoperbutylacetonicum N1-4 and a large Ve/Vb ratio. A mixture of oleyl alcohol-tributyrin (1:1 (v/v)) yielded high distribution coefficients for both butanol (3.14) and acetone (0.660). Although a fed-batch culture using free cells and the oleyl alcohol-tributyrin mixture at a Ve/Vb ratio of 5 had a lag phase of >24 h, it produced a higher concentration of total butanol (i.e., butanol produced in all the phases per broth volume used) of 24.2 g/L-broth after 96 h compared with 14.4 g/L-broth at a Ve/Vb ratio of 1, resulting in a low butanol concentration in the aqueous phase. The use of cells immobilized with calcium alginate beads shortened the lag phase to <12 h. Butanol production was achieved not only in a 3-phase mode (extractant, beads, and tryptone-yeast extract-acetate (TYA) medium) but also in a 2-phase mode (extractant and beads containing TYA medium, without an aqueous phase) at a Ve/Vb ratio of 5, resulting butanol concentrations of 30.9 g/L-broth and 27.7 g/L-broth, respectively. The 3-phases fed-batch extractive fermentation at a Ve/Vb ratio of 10 showed a better performance compared with published reports: a total butanol concentration of 64.6 g/L-broth and a butanol yield to consumed sugar of 0.378 C-mol/C-mol..
5. Chuanfu Wu, Miao Yu, Qiqi Huang, Hongzhi Ma, Ming Gao, Qunhui Wang, Kenji Sakai, Stimulation of methane yield rate from food waste by aerobic pre-treatment, Bioresource Technology, 10.1016/j.biortech.2018.04.006, 261, 279-287, 2018.08, Aerobic pre-treatment (AP) was applied to enhance methane yield from food waste through anaerobic digestion. Different AP durations (i.e. 2, 5 and 8 days) prior to anaerobic digestion were tested. The results indicated that AP of food waste led to no significant differences (p > 0.05) in methane yield potential (ca. 418 mL/g-VS). However, a suitable AP duration (5 days) increased methane yield rates (ca. 18 mL/d/g-VS; 22.0% higher than the control) by anticipating methane generation and shortening the methanogenic phase via volatile fatty acid reduction and pH increase. Although AP induced chemical oxygen demand loss to some extent (i.e. by 2.6%–9.9%) in the AP stage via aerobic degradation, the methane yield potential could be recovered by enhancing organic matter hydrolysis. Therefore, maximisation of hydrolysis should be used as a basis for determining a suitable AP duration for various types of organic matter..
6. Tao Zhao, Yukihiro Tashiro, Jin Zheng, Kenji Sakai, Kenji Sonomoto, Semi-hydrolysis with low enzyme loading leads to highly effective butanol fermentation, Bioresource Technology, 10.1016/j.biortech.2018.05.056, 264, 335-342, 2018.09, To improve butanol fermentation efficiencies, semi-hydrolysate with low enzyme loading using H<sub>2</sub>SO<sub>4</sub> pretreated rice straw was designed, which preferably produced cellobiose with xylose (instead of glucose). Fermentation of semi-hydrolysates avoided carbon catabolite repression (CCR) and produced higher butanol yield to enzyme loading (0.0290 g U<sup>−1</sup>), a newly proposed parameter, than the conventional glucose-oriented hydrolysate (0.00197 g U<sup>−1</sup>). Further, overall butanol productivity was improved from 0.0628 g L<sup>−1</sup> h<sup>−1</sup> to 0.265 g L<sup>−1</sup> h<sup>−1</sup> during fermentation of undetoxified semi-hydrolysate by using high cell density. A novel simultaneously repeated hydrolysis and fermentation (SRHF) was constructed by recycling of enzymes and cells, which further improved butanol yield to enzyme loading by 183% and overall butanol productivity by 6.04%. Thus, semi-hydrolysate with SRHF is a smartly designed biomass for efficient butanol fermentation of lignocellulosic materials..
7. Winai Jamjan, Chanwit Suriyachadkun, Somboon Tanasupawat, Kenji Sakai, Yukihiro Tashiro, Yuki Okugawa, Saowanit Tongpim, Amycolatopsis silviterrae sp. Nov., isolated from forest soil, International Journal of Systematic and Evolutionary Microbiology, 10.1099/ijsem.0.002687, 68, 5, 1455-1460, 2018.05, A novel actinobacterial strain, designated C12CA1T, was isolated from forest soil in the conservation area of Chulabhorn dam, Thailand, and its taxonomic position was determined by using a polyphasic approach. Strain C12CA1T contained meso-2,6 diaminopimelic acid in the cell-wall peptidoglycan, and arabinose and galactose as diagnostic sugars of the whole-cell hydrolysate. On the basis of morphological and chemotaxonomic characteristics, strain C12CA1T was classified in the genus Amycolatopsis. It contained MK-9(H4) as the predominant menaquinone, C16:0, iso-C15:0 and iso-C16:0 as the major cellular fatty acids, and several phospholipids consisting of diphosphotidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, hydroxyphosphatidylethanolamine, phosphatidylinositol mannosides and an unidentified glucosamine-containing phospholipid. Based on 16S rRNA gene sequence and phylogenetic analyses, strain C12CA1T was closely related to Amycolatopsis vancoresmycina DSM 44592T (98.96%) and Amycolatopsis pretoriensis JCM 12673T (98.82%). The strain exhibited low DNA-DNA relatedness values with A. vancoresmycina DSM 44592T (6.9±0.2-11.6±1.9%) and A. pretoriensis JCM 12673T (8.8±0.3-9.2±1.8%). The DNA G+C content of strain C12CA1T was 69.8 mol%. Based on the results of polyphasic characterization, strain C12CA1T represents a novel species of the genus Amycolatopsis, for which the name Amycolatopsis silviterrae sp. nov. is proposed. The type strain is C12CA1T (=TBRC 1456T=NBRC 111116T)..
8. Yukihiro Tashiro, Kosuke Kanda, Yuya Asakura, Toshihiko Kii, Huijun Cheng, Pramod Poudel, Yuki Okugawa, Kosuke Tashiro, Kenji Sakai, A unique autothermal thermophilic aerobic digestion process showing a dynamic transition of physicochemical and bacterial characteristics from the mesophilic to the thermophilic phase, Applied and Environmental Microbiology, 10.1128/AEM.02537-17, 84, 6, 2018.03, A unique autothermal thermophilic aerobic digestion (ATAD) process has been used to convert human excreta to liquid fertilizer in Japan. This study investigated the changes in physicochemical and bacterial community characteristics during the full-scale ATAD process operated for approximately 3 weeks in 2 different years. After initiating simultaneous aeration and mixing using an air-inducing circulator (aerator), the temperature autothermally increased rapidly in the first 1 to 2 days with exhaustive oxygen consumption, leading to a drastic decrease and gradual increase in oxidation-reduction potential in the first 2 days, reached > 50°C in the middle 4 to 6 days, and remained steady in the final phase. Volatile fatty acids were rapidly consumed and diminished in the first 2 days, whereas the ammonia nitrogen concentration was relatively stable during the process, despite a gradual pH increase to 9.3. Principal-coordinate analysis of 16S rRNA gene amplicons using next-generation sequencing divided the bacterial community structures into distinct clusters corresponding to three phases, and they were similar in the final phase in both years despite different transitions in the middle phase. The predominant phyla (closest species, dominancy) in the initial, middle, and final phases were Proteobacteria (Arcobacter trophiarum, 19 to 43%; Acinetobacter towneri, 6.3 to 30%), Bacteroidetes (Moheibacter sediminis, 43 to 54%), and Firmicutes (Thermaerobacter composti, 11 to 28%; Heliorestis baculata, 2.1 to 16%), respectively. Two predominant operational taxonomic units (OTUs) in the final phase showed very low similarities to the closest species, indicating that the process is unique compared with previously published ones. This unique process with three distinctive phases would be caused by the aerator with complete aeration..
9. Chuanfu Wu, Qiqi Huang, Miao Yu, Yuanyuan Ren, Qunhui Wang, Kenji Sakai, Effects of digestate recirculation on a two-stage anaerobic digestion system, particularly focusing on metabolite correlation analysis, Bioresource Technology, 10.1016/j.biortech.2017.12.020, 251, 40-48, 2018.03, Single-stage (S-N treatment) and two-stage anaerobic digestion with (T-R treatment) and without digestate recirculation (T-N treatment) for methane production using food waste (FW) were comparatively evaluated to examine the effects of digestate recirculation on anaerobic digestion (AD). Digestate recirculation positively affected the methane yield and organic loading rate (OLR). Metabolite correlation analysis revealed that a systematic hydrolysis degree of greater than 75% is crucial to achieve the complete recoverable yield of methane from FW. Digestate recirculation also markedly increased the system alkalinity, maintaining an optimum pH for methanogens. However, the ammonium accumulated by T-R treatment would destroy the metabolic balance between the hydrolytic bacteria and methanogens, especially at a critical OLR. Therefore, the appropriate control of two-stage AD systems with digestate recirculation is limited not only to OLR regulation but also to the prevention of ammonium accumulation..
10. Ratchanu Meidong, Kulwadee Khotchanalekha, Sompong Doolgindachbaporn, Takahiro Nagasawa, Miki Nakao, Kenji Sakai, Saowanit Tongpim, Evaluation of probiotic Bacillus aerius B81e isolated from healthy hybrid catfish on growth, disease resistance and innate immunity of Pla-mong Pangasius bocourti, Fish and Shellfish Immunology, 10.1016/j.fsi.2017.11.032, 73, 1-10, 2018.02, Infectious diseases have been found to be a major cause of mortality in fish hatcheries. Probiotics have been introduced to replace antibiotics commonly used for treatment of bacterial infection in aquaculture. This study was conducted to isolate, screen, and evaluate the probiotic Bacillus spp. for potential use as a feed supplement to enhance fish growth, disease resistance and innate immunity of Pla-mong Pangasius bocourti. Bacillus aerius strain B81e was selectively isolated from the intestine of healthy catfish and chosen based on its probiotic properties both in vitro and in vivo. This bacterium produced a bacteriocin-like substance and exhibited a broad-spectrum antibacterial activity inhibiting both Gram-positive and Gram-negative bacteria especially the fish pathogens Aeromonas hydrophila and Streptococcus agalactiae. The susceptibility to all 8 antibiotics tested implies that it is unlikely to be an antibiotic-resistant bacterium. B. aerius strain B81e possessed interesting adhesion properties as shown by its high percentages of hydrophobicity, auto-aggregation, co-aggregation with fish pathogens A. hydrophila FW52 and S. agalactiae F3S and mucin binding. The strain B81e survived simulated gastrointestinal conditions, producing protease and lipase but not β-haemolysin. The study also evaluated the effects of dietary supplementation with strain B81e on growth performance, innate immunity, and the disease resistance of P. bocourti against A. hydrophila infection. Fish with a mean body weight of 69 g were fed strain B81e at 0 (control) and 107 CFU g−1 feed (test) for 60 days. Various growth and immune parameters were examined at 30 and 60 days post-feeding. Fish were challenged with A. hydrophila 60 days post-feeding and mortalities were recorded over 14 days post-infection. Results showed that the administration of strain B81e for 60 days had significant effects (p < 0.05) on weight gain, specific growth rate and feed utilization efficiency of P. bocourti. Dietary administration of strain B81e increased the serum lysozyme and bactericidal activities of P. bocourti significantly throughout the experimental period whereas the alternative complement, phagocytic and respiratory burst activities were significantly (p < 0.05) higher in the test fish compared to the control fish after 60 days of feeding. In addition, the fish fed a strain B81e supplemented diet had a significantly higher (p < 0.05) post-challenge survival rate than the control fish. The results in this study indicate that B. aerius B81e has beneficial effects on growth performance, innate immunity and disease resistance of P. bocourti. This is the first report on the probiotic roles of B. aerius in aquaculture..
11. Hoe Seng Tin, Kishneth Palaniveloo, Junia Anilik, Mathavan Vickneswaran, Yukihiro Tashiro, Charles S. Vairappan, Kenji Sakai, Impact of Land-use Change on Vertical Soil Bacterial Communities in Sabah, Microbial Ecology, 10.1007/s00248-017-1043-6, 75, 2, 459-467, 2018.02, Decline in forest productivity due to forest conversion is defining the Bornean landscape. Responses of bacterial communities due to land-use changes are vital and could define our understanding of ecosystem functions. This study reports the changes in bacterial community structure in organic soil (0–5 cm; O-Horizon) and organic-mineral soil (5–15 cm; A-Horizon) across Maliau Basin Conservation Area old growth forest (MBOG), Fragment E logged forest (FELF) located in Kalabakan Forest Reserve to Benta Wawasan oil palm plantation (BWOP) using two-step PCR amplicon analysis of bacteria DNA on Illumina Miseq next generation sequencing. A total of 30 soil samples yielded 893,752-OTU reads at ≥97% similarity from 5,446,512 good quality sequences. Soil from BWOP plantation showed highest unshared OTUs for organic (49.2%) and organic-mineral (50.9%) soil. MBOG soil showed a drop in unshared OTUs between organic (48.6%) and organic-mineral (33.9%). At phylum level, Proteobacteria dominated MBOG but shifted to Actinobacteria in logged and plantation soil. Present findings also indicated that only FELF exhibited change in bacterial communities along the soil depth, moving from the organic to the organic-mineral layer. Both layers of BWOP plantation soils deviated from other forests’ soil in β-diversity analysis. To our knowledge, this is the first report on transitions of bacterial community structures with different soil horizons in the tropical rainforest including Borneo, Sabah. Borneo tropical soils form a large reservoir for soil bacteria and future exploration is needed for fully understanding the diversity structure and their bacterial functional properties..
12. Siti Suhailah Sharuddin, Norhayati Ramli, Diana Mohd-Nor, Mohd Ali Hassan, Toshinari Maeda, Yoshihito Shirai, Kenji Sakai, Yukihiro Tashiro, Shift of low to high nucleic acid bacteria as a potential bioindicator for the screening of anthropogenic effects in a receiving river due to palm oil mill effluent final discharge, Ecological Indicators, 10.1016/j.ecolind.2017.10.020, 85, 79-84, 2018.02, The microbiological effects of palm oil mill effluent (POME) final discharge upon a receiving river were assessed in this study by using the nucleic acid double staining assay based on flow cytometry. The functional status of the bacterial community at the single-cell level was determined with regards to their abundance, viability and nucleic acid content to monitor the effects of POME final discharge on the affected river. The effluent resulted in the increment of the total cell concentration (TCC) and viable cells which were correlated with the increment of biological oxygen demand (BOD5) and total organic carbon (TOC) concentrations in the receiving river. The shift of low nucleic acid (LNA) to high nucleic acid (HNA) bacterial cells in the affected river suggested the transformation of dormant to active cells due to the POME final discharge. This is the first study to report on the shift of LNA/HNA ratios which may serves as a potential bioindicator in the screening of the anthropogenic effects due to POME final discharge in river water with originally high LNA proportions. Monitoring the effluent discharge at low trophic level using flow cytometry is a rapid and sensitive approach when compared to the current physicochemical assessment method. This approach allows for the screening of river water contamination caused by POME final discharge prior to a full assessment using the recently proposed specific bacterial indicators..
13. Kazunori Nakamura, Kenji Sakai, Gut microbiota of Pheretima heteropoda and allolobophorajaponica fed under the same laboratory conditions, Journal of the Faculty of Agriculture, Kyushu University, 63, 1, 71-74, 2018.02, Our previous study reported that the gut microbiota of Megascolecidae was distinguishable from that of Lumbricidae. In this study, two earthworms, Pheretima heteropoda (Megascolecidae) and Allolobophora japonica (Lumbricidae), were fed under the same in vitro conditions to investigate the effect of feed on gut microbiota. Their gut microbiota was analyzed by denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments. Most of the dominant bands were shared between both earthworm species, but some were specific to one species. These banding patterns corresponded to in situ earthworms, i.e. they were directly analyzed after collection from the field. The predominant bacterium found in P. heteropoda gut samples was Bacillus longiquaesitum (AM747042)-related, whereas the predominant bacteria found in A. japonica gut samples were bacterium TG141 (AB308367)-related and Rhizobium sp. BBCT66 (DQ337551)-related. These results indicated that the major bacterial species were not influenced by the surrounding environment, including the food source, even though a large portion of the bacterial population was opportunistic..
14. Huijun Cheng, Yuya Asakura, Kosuke Kanda, Ryo Fukui, Yoshihisa Kawano, Yuki Okugawa, Yukihiro Tashiro, Kenji Sakai, Dynamic bacterial community changes in the autothermal thermophilic aerobic digestion process with cell lysis activities, shaking and temperature increase, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2018.02.012, 2018.01, Autothermal thermophilic aerobic digestion (ATAD) is conducted for stabilization of sludge waste and is driven by the action of various microorganisms under aerobic conditions. However, the mechanism controlling bacterial community changes during ATAD via three (initial, middle and final) phases is currently unclear. To investigate this mechanism, activity analysis and a microcosm assay with shaking were performed on a bacterial community during the initial, middle, and final phases of incubation. Cell lysis activities toward gram-negative bacteria, but not gram-positive bacteria, were detected in the ATAD samples in the middle and final phases. During shaking incubation in initial-phase samples at 30 °C, major operational taxonomic units (OTUs) related to Acinetobacter indicus and Arcobacter cibarius dramatically increased along with decreases in several major OTUs. In middle-phase samples at 45 °C, we observed a major alteration of OTUs related to Caldicellulosiruptor bescii and Aciditerrimonas ferrireducens, together with distinct decreases in several other OTUs. Final-phase samples maintained a stable bacterial community with major OTUs showing limited similarities to Heliorestis baculata, Caldicellulosiruptor bescii, and Ornatilinea apprima. In conclusion, the changes in the bacterial community observed during ATAD could be partially attributed to the cell lysis activity toward gram-negative bacteria in the middle and final phases. The microcosm assay suggested that certain physical factors, such as a high oxygen supply and shearing forces, also might contribute to bacterial community changes in the initial and middle phases, and to the stable bacterial community in the final phase of ATAD..
15. Hirokuni Miyamoto, Hisashi Miyamoto, Yukihiro Tashiro, Kenji Sakai, Hiroaki Kodama, Studies on highly functional fermented-products made from unutilized biomass resources by thermophilic bacteria, Seibutsu-kogaku Kaishi, 96, 2, 56-63, 2018.01.
16. Siti Suhailah Sharuddin, Norhayati Ramli, Mohd Ali Hassan, Nurul Asyifah Mustapha, Afzufira Amran, Diana Mohd-Nor, Kenji Sakai, Yukihiro Tashiro, Yoshihito Shirai, Toshinari Maeda, Bacterial community shift revealed Chromatiaceae and Alcaligenaceae as potential bioindicators in the receiving river due to palm oil mill effluent final discharge, Ecological Indicators, 10.1016/j.ecolind.2017.07.038, 82, 526-529, 2017.11, A thorough outlook on the effect of palm oil mill effluent (POME) final discharge towards bacterial community dynamics in the receiving river is provided in this study by using a high-throughput MiSeq. The shift of bacterial composition could be used to determine the potential bacterial indicators to indicate contamination caused by POME. This study showed that the POME final discharge did not only alter the natural physicochemical properties of the river water but also caused the reduction of bacterial diversity in the receiving river. The Chromatiaceae and Alcaligenaceae which were not detected in the upstream but were detected in the downstream part of the river are proposed as the indicator bacteria to indicate the river water contamination caused by POME final discharge. The emergence of either one or both bacteria in the downstream part of the river were shown to be carried over by the effluent. Therefore, an accurate pollution monitoring approach using bacterial indicator is expected to complement the conventional POME pollution assessment method which is currently dependent on the physicochemical properties of the final discharge. This is the first study that reported on the potential indicator bacteria for the assessment of river water contamination caused by POME final discharge..
17. Clament Fui Seung Chin, Yoshihide Furuya, Mohd Huzairi Mohd Zainudin, Norhayati Ramli, Mohd Ali Hassan, Yukihiro Tashiro, Kenji Sakai, Novel multifunctional plant growth–promoting bacteria in co-compost of palm oil industry waste, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2017.05.016, 124, 5, 506-513, 2017.11, Previously, a unique co-compost produced by composting empty fruit bunch with anaerobic sludge from palm oil mill effluent, which contributed to establishing a zero-emission industry in Malaysia. Little was known about the bacterial functions during the composting process and fertilization capacity of this co-compost. We isolated 100 strains from the co-compost on 7 types of enumeration media and screened 25 strains using in vitro tests for 12 traits, grouping them according to three functions: plant growth promoting (fixation of nitrogen; solubilization of phosphorus, potassium, and silicate; production of 3-indoleacetic acid, ammonia, and siderophore), biocontrolling (production of chitinase and anti-Ganoderma activity), and composting (degradation of lignin, xylan, and cellulose). Using 16S rRNA gene sequence analysis, 25 strains with strong or multi-functional traits were found belong to the genera Bacillus, Paenibacillus, Citrobacter, Enterobacter, and Kosakonia. Furthermore, several strains of Citrobacter sedlakii exhibited a plant growth-stimulation in vivo komatsuna plant cultivation test. In addition, we isolated several multifunctional strains; Bacillus tequilensis CE4 (biocontrolling and composting), Enterobacter cloacae subsp. dissolvens B3 (plant growth promoting and biocontrolling), and C. sedlakii CESi7 (plant growth promoting and composting). Some bacteria in the co-compost play significant roles during the composting process and plant cultivation after fertilization, and some multifunctional strains have potential for use in accelerating the biodegradation of lignocellulosic biomass, protecting against Ganoderma boninense infection, and increasing the yield of palm oil..
18. Ratchanu Meidong, Sompong Doolgindachbaporn, Winai Jamjan, Kenji Sakai, Yukihiro Tashiro, Yuki Okugawa, Saowanit Tongpim, A novel probiotic Bacillus siamensis B44v isolated from Thai pickled vegetables (Phak-dong) for potential use as a feed supplement in aquaculture, Journal of General and Applied Microbiology, 10.2323/jgam.2016.12.002, 63, 4, 246-253, 2017.10, The use of probiotic bacteria to control bacterial infection in farmed fish is of clear practical interest. The aims of this study were to isolate and select a probiotic Bacillus sp. and to evaluate the effects of its supplementation on the growth and disease resistance of hybrid catfish. Bacillus siamensis strain B44v, selectively isolated from Thai pickled vegetables (Phak-dong), displayed a high potential as a probiotic in catfish culture. This bacterium produced a bacteriocin-like substance and exhibited a broad-spectrum antibacterial activity inhibiting both Gram-positive and Gram-negative bacteria, especially the fish pathogens Aeromonas hydrophila and Streptococcus agalactiae. The susceptibility to all 14 antibiotics tested implies its less possibility to be the antibiotic-resistant bacterium. Bacillus siamensis strain B44v possessed interesting adhesion properties, as shown by its high percentages of hydrophobicity (64.8%), auto-agglutination (73.8%), co-aggregation (67.2% with A. hydrophila FW52 and 63.5% with S. agalactiae F3S), and mucin binding (88.7%). The strain B44v survived simulated gastrointestinal conditions and produced protease and cellulase enzymes. Hybrid catfish (C. macrocephalus × C. gariepinus) were employed in the feed-trial experiments. Fish fed diet containing strain B44v (107 CFU/g feed) displayed not only no mortality but also growth improvement. At the end of the feed trial, fish were challenged by an intraperitoneal injection of Aeromonas hydrophila FW52. The Bacillus siamensis strain B44v fed fish survived (75.0%; p < 0.05) better than the controls (36.7%; p < 0.05) after a two week challenge. These collective results present for the first time the potential of Bacillus siamensis strain B44v for use as a bacterial probiotic in aquaculture..
19. Srisakul Trakarnpaiboon, Nantana Srisuk, Kuakoon Piyachomkwan, Kenji Sakai, Vichien Kitpreechavanich, Enhanced production of raw starch degrading enzyme using agro-industrial waste mixtures by thermotolerant Rhizopus microsporus for raw cassava chip saccharification in ethanol production, Preparative Biochemistry and Biotechnology, 10.1080/10826068.2017.1342264, 47, 8, 813-823, 2017.09, In the present study, solid-state fermentation for the production of raw starch degrading enzyme was investigated by thermotolerant Rhizopus microsporus TISTR 3531 using a combination of agro-industrial wastes as substrates. The obtained crude enzyme was applied for hydrolysis of raw cassava starch and chips at low temperature and subjected to nonsterile ethanol production using raw cassava chips. The agro-industrial waste ratio was optimized using a simplex axial mixture design. The results showed that the substrate mixture consisting of rice bran:corncob:cassava bagasse at 8 g:10 g:2 g yielded the highest enzyme production of 201.6 U/g dry solid. The optimized condition for solid-state fermentation was found as 65% initial moisture content, 35°C, initial pH of 6.0, and 5 × 106 spores/mL inoculum, which gave the highest enzyme activity of 389.5 U/g dry solid. The enzyme showed high efficiency on saccharification of raw cassava starch and chips with synergistic activities of commercial α-amylase at 50°C, which promotes low-temperature bioethanol production. A high ethanol concentration of 102.2 g/L with 78% fermentation efficiency was achieved from modified simultaneous saccharification and fermentation using cofermentation of the enzymatic hydrolysate of 300 g raw cassava chips/L with cane molasses..
20. Mohd Huzairi Mohd Zainudin, Norhayati Ramli, Mohd Ali Hassan, Yoshihito Shirai, Kosuke Tashiro, Kenji Sakai, Yukihiro Tashiro, Bacterial community shift for monitoring the co-composting of oil palm empty fruit bunch and palm oil mill effluent anaerobic sludge, Journal of Industrial Microbiology and Biotechnology, 10.1007/s10295-017-1916-1, 44, 6, 869-877, 2017.06, A recently developed rapid co-composting of oil palm empty fruit bunch (OPEFB) and palm oil mill effluent (POME) anaerobic sludge is beginning to attract attention from the palm oil industry in managing the disposal of these wastes. However, a deeper understanding of microbial diversity is required for the sustainable practice of the co-compositing process. In this study, an in-depth assessment of bacterial community succession at different stages of the pilot scale co-composting of OPEFB-POME anaerobic sludge was performed using 454-pyrosequencing, which was then correlated with the changes of physicochemical properties including temperature, oxygen level and moisture content. Approximately 58,122 of 16S rRNA gene amplicons with more than 500 operational taxonomy units (OTUs) were obtained. Alpha diversity and principal component analysis (PCoA) indicated that bacterial diversity and distributions were most influenced by the physicochemical properties of the co-composting stages, which showed remarkable shifts of dominant species throughout the process. Species related to Devosia yakushimensis and Desemzia incerta are shown to emerge as dominant bacteria in the thermophilic stage, while Planococcus rifietoensis correlated best with the later stage of co-composting. This study proved the bacterial community shifts in the co-composting stages corresponded with the changes of the physicochemical properties, and may, therefore, be useful in monitoring the progress of co-composting and compost maturity..
21. Eiji Nishi, Kota Watanabe, Yukihiro Tashiro, Kenji Sakai, Terminal restriction fragment length polymorphism profiling of bacterial flora derived from single human hair shafts can discriminate individuals, Legal Medicine, 10.1016/j.legalmed.2017.01.002, 25, 75-82, 2017.03, Human hairs are the trace evidence most commonly encountered at many crime scenes. However, they have not been effectively utilized for actual criminal investigations because of the low accuracy of their morphological inspection, low detection rate of short tandem repeat (STR) typing, and the problem of heteroplasmy in mitochondrial DNA analysis. Here, we examined the possibility of individual discrimination by comparing profiles of bacterial flora on hair. We carried out the profiling of terminal restriction fragment length polymorphisms (T-RFLP) of the amplified bacterial 16S ribosomal RNA (rRNA) gene from hair samples. Compared with existing STR typing methods that use hair roots, this method using hair shafts allowed the detection of stable bacterial DNA. We successfully obtained the T-RFLP profile from single hair shafts of all volunteers tested. The profiles were specific to each individual, and multiple profiles obtained from the individual him/herself showed higher similarity than those from different individuals. These individual-specific profiles were stably obtained from samples from most volunteers, when collected again after 6 months. Storage of the collected hair samples at −30 °C was effective for obtaining reproducible T-RF profiles. When unidentified hair samples collected in the laboratory were compared with a pre-constructed database, 17 of 22 hairs were assigned to a small group of people, including the corresponding individuals. These results show that T-RFLP analysis of bacterial flora on a hair shaft found at a crime scene could provide useful information for narrowing down a suspect..
22. Kathrina Mae Bienes, Minoru Ito, Kota Shiotsuka, Sachi Yamaguchi, Taiki Fujioka, Yukihiro Tashiro, Kenji Sakai, Ecological distribution of extremely thermophilic bacteria belonging to the genus Calditerricola using the novel enrichment MPN-PCR method, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2017.06.003, 2017.02, A unique compost called Satsuma soil is produced from sewage sludge by a hyperthermal composting process in Kagoshima City, Japan. The composting process is carried out at a controlled temperature of at least 80°C and the resulting compost might be useful for recycling sustainable agricultural products. The extremely thermophilic bacterial genus Calditerricola was initially isolated from the high-temperature compost. Likewise, the bacteria were previously isolated from material sludge. It is believed that bacteria in this genus might be involved in the hyperthermal composting process. Calditerricola bacteria are distributed not only in compost, but also in all of its material sludge, and are more abundant in material sludge than in compost. Moreover, based on investigations of samples near geothermal areas in high temperature conditions, such as volcanoes, Calditerricola was presumed to originate in the volcanic ash of Mt. Sakurajima in Kagoshima City, Japan. However, its precise origin and ecology are unclear. Thus, in this study, a new molecular biological method called enrichment most probable number (MPN)-PCR (eMPN-PCR) was established and used to quantitatively investigate the population and distribution of the extreme thermophile Calditerricola in environmental samples using genus-specific PCR primers. The eMPN-PCR method was an effective quantitative detection method with high sensitivity, yielding MPN estimates that were highly correlated with colony forming unit (CFU) estimates but a low detection threshold value..
23. Pramod Poudel, Yukihiro Tashiro, Hirokuni Miyamoto, Hisashi Miyamoto, Yuki Okugawa, Kenji Sakai, Development of a systematic feedback isolation approach for targeted strains from mixed culture systems, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2016.07.019, 123, 1, 63-70, 2017.01, Elucidation of functions of bacteria in a mixed culture system (MCS) such as composting, activated sludge system is difficult, since the system is complicating with many unisolated bacteria. Here, we developed a systematic feedback isolation strategy for the isolation and rapid screening of multiple targeted strains from MCS. Six major strains (Corynebacterium sphenisci, Bacillus thermocloacae, Bacillus thermoamylovorans, Bacillus smithii, Bacillus humi, and Bacillus coagulans), which are detected by denaturing gradient gel electrophoresis (DGGE) analysis in our previous study on MCS for L-lactic acid production, were targeted for isolation. Based on information of suitable cultivation conditions (e.g., media, pH, temperature) from the literature, feedback isolation was performed to form 136 colonies. The following direct colony matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was optimised as the second screening to narrow down 20 candidate colonies from similar spectra patterns with six closest type strains. This step could distinguish bacteria at the species level with distance similarity scores ≥0.55 corresponding to 16S rRNA gene sequence similarity ≥98.2%, suggesting that this is an effective technique to minimize isolates close to targeted type strains. Analysis of 16S rRNA gene sequences indicated that two targeted strains and one strain related to the target had successfully been isolated, showing high similarities (99.5–100%) with the sequences from the DGGE bands, and that the other candidates were affiliated with three strains that were closely related to the target species. This study proposes a new method for systematic feedback isolation that may be useful for isolating targeted strains from MCS for further investigation..
24. Jin Zheng, Yukihiro Tashiro, Tao Zhao, Qunhui Wang, Kenji Sakai, Kenji Sonomoto, Enhancement of acetone-butanol-ethanol fermentation from eucalyptus hydrolysate with optimized nutrient supplementation through statistical experimental designs, Renewable Energy, 10.1016/j.renene.2017.05.097, 113, 580-586, 2017.01, Eucalyptus has been previously suggested as a potential substrate for acetone-butanol-ethanol (ABE) production without nutrient supplementation; however, incomplete sugar consumption has prevented improvement of ABE production. Cellulase loading with 35 FPU g−1 was first optimized in terms of high hydrolysis efficiency (95%). However, only 0.43 g L−1 ABE production and 3.44% glucose consumption rate were achieved. To improve ABE production from eucalyptus hydrolysate, supplementation of 6 nutrients in common tryptone-yeast extract (TY) medium were investigated by statistical approaches. Three nutrients including yeast extract, tryptone, and FeSO4·7H2O were screened as significant nutrients for ABE production. Subsequently, use of a modified TY medium (MTY medium: yeast extract 3.04 g L−1, tryptone 7.64 g L−1, FeSO4·7H2O 15.3 mg L−1), which was subsequently predicted by Plackett-Burman and Box-Behnken designs to stimulate ABE production, resulted in ca. 40-fold increase in ABE concentration (16.9 g L−1) and a glucose consumption rate of 100%. We first examined previously uninvestigated nutrition combinations using Plackett-Burman and Box-Behnken designs for high ABE production from eucalyptus hydrolysate. This study shows that statistical method would be a powerful tool for the optimization and enhancement of ABE production from eucalyptus hydrolysate..
25. Ratchanu Meidong, Sompong Doolgindachbaporn, Kenji Sakai, Saowanit Tongpim, Isolation and selection of lactic acid bacteria from Thai indigenous fermented foods for use as probiotics in tilapia fish Oreochromis niloticus, AACL Bioflux, 10, 2, 455-463, 2017.01, In this study, 119 bacterial strains were isolated from various samples such as healthy tilapia fish (Oreochromis niloticus), water and sediment around the culture fish-cages, and several kinds of traditional fermented foods. These bacterial isolates were screened for antibacterial activities against bacterial fish pathogens i.e. Aeromonas hydrophila, A. caviae and Streptococcus agalactiae using an agar well diffusion assay. The isolate CR1T5 derived from fermented rice showed the highest antibacterial activity against all three fish pathogens tested. It was identified as Lactobacillus plantarum by using both conventional and molecular methods. The other probiotic properties were evaluated in vitro which revealed that strain CR1T5 tolerated the simulated gastrointestinal conditions well, showed high capacity to adhere intestinal mucosa and did not lyse red blood cells. The efficiency of L. plantarum CR1T5 was also examined in vivo. O. niloticus were employed in the feed-trial experiments. Fish fed a diet containing strain CR1T5 (108 CFU g-1 feed) displayed not only no mortality but also growth improvement. At the end of feed-trial, fish were challenged by intramuscularly injection of A. hydrophila (3.1x105 CFU) The L. plantarum CR1T5-fed fish survived (87.5%) better than the fish fed a control diet (12.5%) after a two week-challenge. This study clearly shows that L. plantarum strain CR1T5 is a promising probiotic candidate for farmed fish..
26. J. Tan, Mohamed Ali Sayed Mohamed Abdelrahman, M. Numaguchi, Yukihiro Tashiro, Takeshi Zendo, Kenji Sakai, Kenji Sonomoto, Thermophilic Enterococcus faecium QU 50 enabled open repeated batch fermentation for l-lactic acid production from mixed sugars without carbon catabolite repression, RSC Advances, 10.1039/c7ra03176a, 7, 39, 24233-24241, 2017.01, Enterococcus faecium QU 50, a novel thermophilic l-lactic acid (LA) producing strain, was used in this study to ferment sugar mixtures into LA. Under the optimal fermentation conditions (50 °C, pH 6.5), strain QU 50 could ferment both mixed glucose/xylose sugars with relaxed CCR and mixed cellobiose/xylose sugars simultaneously without CCR to produce homo l-LA. The activity of enzymes related to xylose metabolism was also investigated. In the cells grown in a medium containing cellobiose/xylose, the activity of xylose isomerase and xylulose kinase, was 3.22 and 1.91 times higher, respectively, as compared to that of cells grown in a glucose/xylose medium. Strain QU 50 produced 70.8 g L-1 of l-LA with a yield of 1.04 g g-1 and a productivity of 2.95 g L-1 h-1 from simulated energy cane hydrolysate in batch fermentation. Immobilisation of strain QU 50 improved the operational stability of open repeated fermentation (three cycles), resulting in 61.1-64.3 g L-1 of l-LA with a yield of 1.01-1.02 g g-1 and a productivity of 3.22-3.82 g L-1 h-1. Thus, an efficient and cost-effective fermentation system was successfully established for l-LA production from sugar mixtures..
27. Nurul Asyifah Mustapha, Kenji Sakai, Yoshihito Shirai, Toshinari Maeda, Impact of different antibiotics on methane production using waste-activated sludge
mechanisms and microbial community dynamics, Applied Microbiology and Biotechnology, 10.1007/s00253-016-7767-2, 100, 21, 9355-9364, 2016.11, Anaerobic digestion is an effective method for reducing the by-product of waste-activated sludge (WAS) from wastewater treatment plants and for producing bioenergy from WAS. However, only a limited number of studies have attempted to improve anaerobic digestion by targeting the microbial interactions in WAS. In this study, we examined whether different antibiotics positively, negatively, or neutrally influence methane fermentation by evaluating changes in the microbial community and functions in WAS. Addition of azithromycin promoted the microbial communities related to the acidogenic and acetogenic stages, and a high concentration of soluble proteins and a high activity of methanogens were detected. Chloramphenicol inhibited methane production but did not affect the bacteria that contribute to the hydrolysis, acidogenesis, and acetogenesis digestion stages. The addition of kanamycin, which exhibits the same methane productivity as a control (antibiotic-free WAS), did not affect all of the microbial communities during anaerobic digestion. This study demonstrates the simultaneous functions and interactions of diverse bacteria and methanogenic Archaea in different stages of the anaerobic digestion of WAS. The ratio of Caldilinea, Methanosarcina, and Clostridium may correspond closely to the trend of methane production in each antibiotic. The changes in microbial activities and function by antibiotics facilitate a better understanding of bioenergy production..
28. Yukihiro Tashiro, Hanae Tabata, Asuka Itahara, Natsuki Shimizu, Kosuke Tashiro, Kenji Sakai, Unique hyper-thermal composting process in Kagoshima City forms distinct bacterial community structures, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2016.04.006, 122, 5, 606-612, 2016.11, A unique compost, Satsuma soil, is produced from three types of wastewater sludge using hyper-thermal processes at temperatures much higher than that of general thermophilic processes in Kagoshima City, Japan. We analyzed the bacterial community structures of this hyper-thermal compost sample and other sludges and composts by a high-throughput barcoded pyrosequencing method targeting the 16S rRNA gene. In total, 621,076 reads were derived from 17 samples and filtered. Artificial sequences were deleted and the reads were clustered based on the operational taxonomic units (OTUs) at 97% similarity. Phylum-level analysis of the hyper-thermal compost revealed drastic changes of the sludge structures (each relative abundance) from Firmicutes (average 47.8%), Proteobacteria (average 22.3%), and Bacteroidetes (average 10.1%) to two main phyla including Firmicutes (73.6%) and Actinobacteria (25.0%) with less Proteobacteria (∼0.3%) and Bacteroidetes (∼0.1%). Furthermore, we determined the predominant species (each relative abundance) of the hyper-thermal compost including Firmicutes related to Staphylococcus cohnii (13.8%), Jeotgalicoccus coquinae (8.01%), and Staphylococcus lentus (5.96%), and Actinobacteria related to Corynebacterium stationis (6.41%), and found that these species were not predominant in wastewater sludge. In contrast, we did not observe any common structures among eight other composts produced, using the hyper-thermal composts as the inoculums, under thermophilic conditions from different materials. Principle coordinate analysis of the hyper-thermal compost indicated a large difference in bacterial community structures from material sludge and other composts. These results suggested that a distinct bacterial community structure was formed by hyper-thermal composting..
29. Yukihiro Tashiro, Shota Inokuchi, Pramod Poudel, Yuki Okugawa, Hirokuni Miyamoto, Hisashi Miayamoto, Kenji Sakai, Novel pH control strategy for efficient production of optically active l-lactic acid from kitchen refuse using a mixed culture system, Bioresource Technology, 10.1016/j.biortech.2016.05.031, 216, 52-59, 2016.09, Uninvestigated control factors of meta-fermentation, the fermentative production of pure chemicals and fuels in a mixed culture system, were examined for production of optically pure l-lactic acid (LA) from food waste. In meta-fermentations by pH swing control, l-LA production with 100% optical purity (OPl-LA) was achieved even using unsterilized model kitchen refuse medium with preferential proliferation of l-LA-producing Bacillus coagulans, a minor member in the seed, whereas agitation decreased OPl-LA drastically. pH constant control shortened the fermentation time but decreased OPl-LA and LA selectivity (SLA) by stimulating growth of heterofermentative Bacillus thermoamylovorans. Deliberately switching from pH swing control to constant control exhibited the best performance for l-LA production: maximum accumulation, 39.2 g L-1; OPl-LA, 100%; SLA, 96.6%; productivity, 1.09 g L-1 h-1. These results present a novel pH control strategy for efficient l-LA production in meta-fermentation based on a concept different from that of pure culture systems..
30. Ming Gao, Yukihiro Tashiro, Qunhui Wang, Kenji Sakai, Kenji Sonomoto, High acetone-butanol-ethanol production in pH-stat co-feeding of acetate and glucose, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2016.01.013, 122, 2, 176-182, 2016.08, We previously reported the metabolic analysis of butanol and acetone production from exogenous acetate by 13C tracer experiments (Gao et al., RSC Adv., 5, 8486-8495, 2015). To clarify the influence of acetate on acetone-butanol-ethanol (ABE) production, we first performed an enzyme assay in Clostridium saccharoperbutylacetonicum N1-4. Acetate addition was found to drastically increase the activities of key enzymes involved in the acetate uptake (phosphate acetyltransferase and CoA transferase), acetone formation (acetoacetate decarboxylase), and butanol formation (butanol dehydrogenase) pathways. Subsequently, supplementation of acetate during acidogenesis and early solventogenesis resulted in a significant increase in ABE production. To establish an efficient ABE production system using acetate as a co-substrate, several shot strategies were investigated in batch culture. Batch cultures with two substrate shots without pH control produced 14.20 g/L butanol and 23.27 g/L ABE with a maximum specific butanol production rate of 0.26 g/(g h). Furthermore, pH-controlled (at pH 5.5) batch cultures with two substrate shots resulted in not only improved acetate consumption but also a further increase in ABE production. Finally, we obtained 15.13 g/L butanol and 24.37 g/L ABE at the high specific butanol production rate of 0.34 g/(g h) using pH-stat co-feeding method. Thus, in this study, we established a high ABE production system using glucose and acetate as co-substrates in a pH-stat co-feeding system with C. saccharoperbutylacetonicum N1-4..
31. Vichien Kitpreechavanich, Arisa Hayami, Anfal Talek, Clament Fui Seung Chin, Yukihiro Tashiro, Kenji Sakai, Simultaneous production of l-lactic acid with high optical activity and a soil amendment with food waste that demonstrates plant growth promoting activity, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2015.12.017, 122, 1, 105-110, 2016.07, A unique method to produce highly optically-active l-lactic acid and soil amendments that promote plant growth from food waste was proposed. Three Bacillus strains Bacillus subtilis KBKU21, B. subtilis N3-9 and Bacillus coagulans T27, were used. Strain KBKU21 accumulated 36.9 g/L l-lactic acid with 95.7% optical activity and 98.2% l-lactic acid selectivity when fermented at 43°C for 84 h in a model kitchen refuse (MKR) medium. Residual precipitate fraction (anaerobically-fermented MKR (AFM) compost) analysis revealed 4.60%, 0.70% and 0.75% of nitrogen (as N), phosphorous (as P2O5), and potassium (as K2O), respectively. Additionally, the carbon to nitrogen ratio decreased from 13.3 to 10.6. AFM compost with KBKU21 promoted plant growth parameters, including leaf length, plant height and fresh weight of Brassica rapa (Komatsuna), than that by chemical fertilizers or commercial compost. The concept provides an incentive for the complete recycling of food waste, contributing towards a sustainable production system..
32. Pramod Poudel, Yukihiro Tashiro, Yuki Okugawa, Hisashi Miyamoto, KENJI SAKAI, Development of a systematic feedback isolation approach for targeted strains
from mixed culture systems
, Journal of Bioscience and Bioengineering, 122, 1-8, VOL. 122 No. 5, 606e612, 2016, 2016.05.
33. Nurul Asyifah Mustapha, Yoshihito Shirai, KENJI SAKAI, Yukihiro Tashiro, KOSUKE TASHIRO, Toshinari Maeda, The prevalence of foodborne pathogenic bacteria on cutting boards and their ecological correlation with background biota, AIMS Microbiology, 2, 2, 138-151, 2016.04.
34. Nao Murakami, Mana Oba, Mariko Iwamoto, Yukihiro Tashiro, Takuya Noguchi, Kaori Bonkohara, Mohamed Ali Sayed Mohamed Abdelrahman, Takeshi Zendo, Mitsuya Shimoda, Kenji Sakai, Kenji Sonomoto, L-Lactic acid production from glycerol coupled with acetic acid metabolism by Enterococcus faecalis without carbon loss, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2015.05.009, 121, 1, 89-95, 2016.01, Glycerol is a by-product in the biodiesel production process and considered as one of the prospective carbon sources for microbial fermentation including lactic acid fermentation, which has received considerable interest due to its potential application. Enterococcus faecalis isolated in our laboratory produced optically pure l-lactic acid from glycerol in the presence of acetic acid. Gas chromatography-mass spectrometry analysis using [1, 2-13C2] acetic acid proved that the E. faecalis strain QU 11 was capable of converting acetic acid to ethanol during lactic acid fermentation of glycerol. This indicated that strain QU 11 restored the redox balance by oxidizing excess NADH though acetic acid metabolism, during ethanol production, which resulted in lactic acid production from glycerol. The effects of pH control and substrate concentration on lactic acid fermentation were also investigated. Glycerol and acetic acid concentrations of 30 g/L and 10 g/L, respectively, were expected to be appropriate for lactic acid fermentation of glycerol by strain QU 11 at a pH of 6.5. Furthermore, fed-batch fermentation with 30 g/L glycerol and 10 g/L acetic acid wholly exhibited the best performance including lactic acid production (55.3 g/L), lactic acid yield (0.991 mol-lactic acid/mol-glycerol), total yield [1.08 mol-(lactic acid and ethanol)]/mol-(glycerol and acetic acid)], and total carbon yield [1.06 C-mol-(lactic acid and ethanol)/C-mol-(glycerol and acetic acid)] of lactic acid and ethanol. In summary, the strain QU 11 successfully produced lactic acid from glycerol with acetic acid metabolism, and an efficient fermentation system was established without carbon loss..
35. Pramod Poudel, Yukihiro Tashiro, Kenji Sakai, New application of Bacillus strains for optically pure L-lactic acid production
General overview and future prospects, Bioscience, Biotechnology and Biochemistry, 10.1080/09168451.2015.1095069, 80, 4, 642-654, 2016.01, Members of the genus Bacillus are considered to be both, among the best studied and most commonly used bacteria as well as the most still unexplored and the most wide-applicable potent bacteria because novel Bacillus strains are continuously being isolated and used in various areas. Production of optically pure L-lactic acid (L-LA), a feedstock for bioplastic synthesis, from renewable resources has recently attracted attention as a valuable application of Bacillus strains. L-LA fermentation by other producers, including lactic acid bacteria and Rhizopus strains (fungi) has already been addressed in several reviews. However, despite the advantages of L-LA fermentation by Bacillus strains, including its high growth rate, utilization of various carbon sources, tolerance to high temperature, and growth in simple nutritional conditions, it has not been reviewed. This review article discusses new findings on LA-producing Bacillus strains and compares them to other producers. The future prospects for LA-producing Bacillus strains are also discussed..
36. Mohamed Ali Sayed Mohamed Abdelrahman, Yukihiro Tashiro, Takeshi Zendo, Kenji Sakai, Kenji Sonomoto, Highly efficient L-lactic acid production from xylose in cell recycle continuous fermentation using Enterococcus mundtii QU 25, RSC Advances, 10.1039/c5ra27579b, 6, 21, 17659-17668, 2016.01, A few strains of lactic acid bacteria metabolise xylose into optically pure L-lactic acid (LA). This study achieved an effective homofermentative cell recycle continuous fermentation of xylose to L-LA with high concentration, productivity, and yield using Enterococcus mundtii QU 25. In conventional continuous fermentation, the optimal xylose concentration in the feeding solution is 50 g L-1, and the optimal dilution rate is 0.15 h-1. Continuous fermentation with cell recycling using a microfiltration membrane module produced an L-LA concentration of 32.3 g L-1 with a yield of 0.789 g g-1 and a productivity of 5.33 g L-1 h-1. Controlling pH and optimising the feeding medium were important for achieving a high L-LA yield with strain QU 25. Using corn steep liquor-containing medium at pH 6.2, a maximum L-LA concentration, yield and productivity were achieved at 41.0 g L-1, 1.01 g g-1, and 6.15 g L-1 h-1, respectively. This study is the first to report on continuous fermentation with cell recycling for lactic acid production from xylose..
37. Eiji Nishi, Yukihiro Tashiro, Kenji Sakai, Discrimination among individuals using terminal restriction fragment length polymorphism profiling of bacteria derived from forensic evidence, Zeitschrift fur Rechtsmedizin. Journal of legal medicine, 10.1007/s00414-014-1092-z, 129, 3, 425-433, 2015.05, DNA typing from forensic evidence is commonly used to identify individuals. However, when the quantity of the forensic evidence is insufficient, successful identification using DNA typing is impossible. Such evidence may also contain DNA from bacteria that occur naturally on the skin. In this study, we aimed to establish a profiling method using terminal restriction fragment length polymorphisms (T-RFLPs) of the amplified bacterial 16S ribosomal RNA (rRNA) gene. First, the extraction and digestion processes were investigated, and the T-RFLP profiling method using the 16S rRNA gene amplicon was optimized. We then used this method to compare the profiles of bacterial flora from the hands of 12 different individuals. We found that the T-RFLP profiles from one person on different days displayed higher similarity than those between individuals. In a principal component analysis (PCA), T-RFLPs from each individual were closely clustered in 11 out of 12 cases. The clusters could be distinguished from each other, even when the samples were collected from different conditions. No major change of the profile was observed after six months except in two cases. When handprints on glass plates were compared, 11 of 12 individuals were assigned to a few clusters including the cluster corresponding to the correct individual. In conclusion, a method for reproducible T-RFLP profiling of bacteria from trace amounts of handprints was established. The profiles were obtained for particular individuals clustered in PCA and were experimentally separable from other individuals in most cases. This technique could provide useful information for narrowing down a suspect in a criminal investigation..
38. Noor Azira Abdul-Mutalib, Syafinaz Amin Nordin, Malina Osman, Natsumi Ishida, Kosuke Tashiro, Kenji Sakai, Yukihiro Tashiro, Toshinari Maeda, Yoshihito Shirai, Pyrosequencing analysis of microbial community and food-borne bacteria on restaurant cutting boards collected in Seri Kembangan, Malaysia, and their correlation with grades of food premises, International Journal of Food Microbiology, 10.1016/j.ijfoodmicro.2015.01.022, 200, 57-65, 2015.05, This study adopts the pyrosequencing technique to identify bacteria present on 26 kitchen cutting boards collected from different grades of food premises around Seri Kembangan, a city in Malaysia. Pyrosequencing generated 452,401 of total reads of OTUs with an average of 1.4×107 bacterial cells/cm2. Proteobacteria, Firmicutes and Bacteroides were identified as the most abundant phyla in the samples. Taxonomic richness was generally high with >1000 operational taxonomic units (OTUs) observed across all samples. The highest appearance frequencies (100%) were OTUs closely related to Enterobacter sp., Enterobacter aerogenes, Pseudomonas sp. and Pseudomonas putida. Several OTUs were identified most closely related to known food-borne pathogens, including Bacillus cereus, Cronobacter sakazaki, Cronobacter turisensis, Escherichia coli, E. coli O157:H7, Hafnia alvei, Kurthia gibsonii, Salmonella bongori, Salmonella enterica, Salmonella paratyphi, Salmonella tyhpi, Salmonella typhimurium and Yersinia enterocolitica ranging from 0.005% to 0.68% relative abundance. The condition and grade of the food premises on a three point cleanliness scale did not correlate with the bacterial abundance and type. Regardless of the status and grades, all food premises have the same likelihood to introduce food-borne bacteria from cutting boards to their foods and must always prioritize the correct food handling procedure in order to avoid unwanted outbreak of food-borne illnesses..
39. Eiji Nishi, Yukihiro Tashiro, KENJI SAKAI, Discrimination among individuals using terminal restriction fragment length polymorphism profiling of bacteria derived from forensic evidence, International Journal of Legal Medicine, 2015.04.
40. Noor-Azira Abdul-Mutalib, Natsumi Ishida, KOSUKE TASHIRO, Yukihiro Tashiro, KENJI SAKAI, Yoshinari MAeda, Yoshihito Shirai, Pyrosequencing analysis of microbial community and foodborne bacteria on restaurant cutting boards collected in Seri Kembangan, Malaysia and their correlation with grades of food premises, International Journal of Food Microbiology,, 200, 57-65, 2015.03.
41. Jin Zheng, Yukihiro Tashiro, Qunhui Wang, Kenji Sakai, Kenji Sonomoto, Feasibility of acetone-butanol-ethanol fermentation from eucalyptus hydrolysate without nutrients supplementation, Applied Energy, 10.1016/j.apenergy.2014.11.037, 140, 113-119, 2015.02, The economic feasibility of acetone-butanol-ethanol (ABE) fermentation is greatly affected by the type of raw material used. The easy availability of eucalyptus from marginal environments is an alternative feedstock for use as raw material to reduce the production cost. In this study, hydrolyzed eucalyptus was used for ABE production without any nutrients supplementation. Increasing the solid concentration in the eucalyptus slurry from 6.7% (w-dry matter/. v) to 25% led to an increase in the initial glucose concentration from 33.7. g/L to 86.7. g/L after enzymatic hydrolysis. Dosed cellulases not only hydrolyzed cellulose but also supplied nitrogen source for ABE producing strain. However, ABE production from the obtained hydrolysate decreased when the solid concentration was increased to more than 10%. The maximum ABE of 12.3. g/L was obtained at 10% solid concentration, with an initial glucose concentration of approximately 40. g/L. In addition, the fermentation capability of eucalyptus hydrolysate was found to be improved by diluting the hydrolysate, which prevented inhibition by substrate and fermentation inhibitors. Finally, ABE concentration was improved to 13.1. g/L by diluting the hydrolysate from the initial solid concentration of 25% to an initial glucose concentration of 45. g/L, which resulted in ABE productivity of 0.109. g/L/h and ABE yield of 0.413. g/g. Thus, the high ABE production from eucalyptus makes it a potential feedstock for biofuel production..
42. Pramod Poudel, Yukihiro Tashiro, Hirokuni Miyamoto, Hisashi Miyamoto, Yuki Okugawa, Kenji Sakai, Direct starch fermentation to l-lactic acid by a newly isolated thermophilic strain, Bacillus sp. MC-07, Journal of Industrial Microbiology and Biotechnology, 10.1007/s10295-014-1534-0, 42, 1, 143-149, 2015.01, A newly isolated Bacillus sp. MC-07 showed 99.2 % 16S rRNA gene sequence similarity with the Bacillus thermoamylovorans LMG 18084T. It demonstrated optimum and maximum growth temperatures of 50 and 62 °C, respectively. The ability of MC-07 to produce optically pure l-lactic acid via direct fermentation of starch without enzymatic hydrolysis was investigated at different pH values (6.0–8.0) by intermittent adjustments every 12 h. During batch fermentation in mineral salt medium containing 0.001 % yeast extract at pH 7.0, 20 g/L of soluble starch was utilized to produce 16.6 g/L l-lactic acid at 50 °C within 24 h of fermentation, with 100 % optical purity, 92.1 % lactic acid selectivity, and an l-lactic acid yield of 0.977 g/g. Direct starch fermentation at pHs 6.0, 6.5, 7.5, and 8.0 resulted in considerably lower concentrations of lactic acid than did at pH 7.0. Compared with B. thermoamylovorans LMG 18084T, the ability of strain MC-07 to produce l-lactic acid was superior..
43. Mohamed Ali Sayed Mohamed Abdelrahman, Yaotian Xiao, Yukihiro Tashiro, Ying Wang, Takeshi Zendo, Kenji Sakai, Kenji Sonomoto, Fed-batch fermentation for enhanced lactic acid production from glucose/xylose mixture without carbon catabolite repression, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2014.07.007, 119, 2, 153-158, 2015.01, There has been tremendous growth in the production of optically pure l-lactic acid from lignocellulose-derived sugars. In this study, Enterococcus mundtii QU 25 was used to ferment a glucose/xylose mixture to l-lactic acid. Maintenance of the xylose concentration at greater than 10g/L achieved homo-lactic acid fermentation and reduced the formation of byproducts. Furthermore, carbon catabolite repression (CCR) was avoided by maintaining the glucose concentration below 25g/L; therefore, initial concentrations of 25g/L glucose and 50g/L xylose were selected. Supplementation with 5g/L yeast extract enhanced the maximum xylose consumption rate and consequently increased lactic acid production and productivity. Finally, a 129g/L lactic acid without byproducts was obtained with a maximum lactic acid productivity of 5.60g/(L·h) in fed-batch fermentation with feeding a glucose/xylose mixture using ammonium hydroxide as the neutralizing agent. These results indicate a potential for lactic acid production from glucose and xylose as the main components of lignocellulosic biomasses..
44. Ming Gao, Yukihiro Tashiro, Tsuyoshi Yoshida, Jin Zheng, Qunhui Wang, Kenji Sakai, Kenji Sonomoto, Metabolic analysis of butanol production from acetate in Clostridium saccharoperbutylacetonicum N1-4 using 13C tracer experiments, RSC Advances, 10.1039/c4ra09571e, 5, 11, 8486-8495, 2015.01, During acetone-butanol-ethanol (ABE) fermentation by clostridia, acetate is reutilised for butanol production. In this study, we investigated the characteristics of ABE production from acetate and analysed the metabolism of exogenously added acetate by Clostridium saccharoperbutylacetonicum N1-4. Supplementation of 4 g L-1 exogenous acetate, to media containing glucose, increased not only concentrations of butanol (48.3%) and acetone (90.5%), but also the ratio of acetone to butanol (27.1%), which suggested that acetate addition altered the metabolic flux. Acetate could not be metabolised in the absence of glucose, thus glycolysis appeared to be necessary for acetate utilisation. In order to clarify the metabolism of exogenous acetate, 13C tracer experiments were performed by supplementing [1,2-13C2] acetate in a culture broth. Based on the results of gas chromatography-mass spectroscopy analysis, we first confirmed both butanol and acetone formation from acetate. Further, the acetate-to-butanol efficiency will significantly decrease when more acetate than 2-4 g L-1 is added to the fermentation, while acetate-to-acetone efficiency may remain high (up to a ratio of 2 mol acetate per 1 mol glucose fed). Moreover, the culture supplemented with acetate exhibited an increase in conversion efficiency of glucose to butanol and acetone, from 0.196% to 19.5% and from 0 to 7.64%, respectively, even during acidogenesis. Thus, we first revealed quantitatively that acetate addition induced solvent production during the early growth phase, and increased metabolic flux to acetone and butanol production from both acetate and glucose..
45. N. A. Abdul-Mutalib, A. N. Syafinaz, Kenji Sakai, Y. Shirai, An overview of foodborne illness and food safety in Malaysia, International Food Research Journal, 22, 3, 896-901, 2015.01, Foodborne disease has been associated with microorganisms like bacteria, fungi, viruses and parasites. Most commonly, the outbreaks take place due to the ingestion of pathogenic bacteria like Salmonella Typhi, Escherichia coli, Staphylococcus aureus, Vibrio cholera, Campylobacter jejuni, and Listeria monocytogenes. The disease usually happens as a result of toxin secretion of the microorganisms in the intestinal tract of the infected person. Usually, the level of hygiene in the food premises reflect the quality of the food item, hence restaurant or stall with poor sanitary condition is said to be the contributor to food poisoning outbreak. In Malaysia, food poisoning cases are not rare because the hot and humid climate of this country is very suitable for the growth of the foodborne bacteria. The government is also implementing strict rules to ensure workers and owners of food premises prioritize the cleanliness of their working area. Training programme for food handlers can also help them to implement hygiene as a routine in a daily basis. A lot of studies have been done to reduce foodborne diseases. The results can give information about the types of microorganisms, and other components that affect their growth. The result is crucial to determine how the spread of foodborne bacteria can be controlled safely and the outbreak can be reduced..
46. Mohamed Ali Sayed Mohamed Abdelrahman, Yukihiro Tashiro, Takeshi Zendo, Kenji Sakai, Kenji Sonomoto, Enterococcus faecium QU 50
A novel thermophilic lactic acid bacterium for high-yield l-lactic acid production from xylose, FEMS Microbiology Letters, 10.1093/femsle/fnu030, 362, 2, 2015.01, Production of optically pure lactic acid from lignocellulosic material for commercial purposes is hampered by several difficulties, including heterofermentation of pentose sugars and high energy consumption by mesophilic lactic acid bacteria. Here, we report a novel lactic acid bacterium, strain QU 50, that has the potential to produce optically pure l-lactic acid (≥99.2%) in a homofermentative manner from xylose under thermophilic conditions. Strain QU 50 was isolated from Egyptian fertile soil and identified as Enterococcus faecium QU 50 by analyzing its sugar fermentation pattern and 16S rRNA gene sequence. Enterococcus faecium QU 50 fermented xylose efficiently to produce lactic acid over wide pH (6.0-10.0) and temperature ranges (30-52°C), with a pH of 6.5 and temperature of 50°C being optimal. To our knowledge, this is the first report of homofermentative lactic acid production from xylose by a thermophilic lactic acid bacterium..
47. Saowanit Tongpim, Kenji Sakai, Isolation and study of thermotolerant Bacillus strains including L-lactic acid production from kitchen refuse, Chiang Mai Journal of Science, 42, 1, 63, 2015.01, Seven thermotolerant, lactic acid-producing bacteria were isolated from tapioca factory waste and, liquid and solid organic composts in the northeastern Thailand. The)' were able to grow at temperatures ranging from 30 to 60°C, with maximum growth observed at approximately 42 °C, and within a pH ranging of 5.2 to 7.5, with maximum growth observed at approximately pH 6.5. These bacteria were identified as the genus Bacillus based on their phenotypic characteristics. The fermentation of saccharified kitchen refuse by these strains was also evaluated. Using glucoamylase-pretreated model kitchen refuse as a substrate medium, all seven strains produced I.-lactic acid (15.12-24.07 g/1). Among the seven strains tested, strain N15 produced the largest amount of L-lactic acid (24.07 g/1), achieving a 149.3% yield from glucose, 144.9% yield from total sugar, 97% optical activity, 95.5% lactic acid selectivity and 0.31 g/l/h L-lactic acid productivity. By using 16S rDNA sequence analysis, strain N15 displayed 99.45% homology' to Bacillus coagulans..
48. Mohd Huzairi Mohd Zainudin, Mohd Ali Hassan, Umi Kalsom Md Shah, Norhani Abdullah, Mitsunori Tokura, Hisashi Yasueda, Yoshihito Shirai, Kenji Sakai, Azhari Samsu Baharuddin, Bacterial community structure and biochemical changes associated with composting of lignocellulosic oil palm empty fruit bunch, BioResources, 9, 1, 316-335, 2014.10, Bacterial community structure and biochemical changes during the composting of lignocellulosic oil palm empty bunch (EFB) and palm oil mill effluent (POME) anaerobic sludge were studied by examining the succession of the bacterial community and its association with changes in lignocellulosic components by denaturing gradient gel electrophoresis (DGGE) and the 16S rRNA gene clone library. During composting, a major reduction in cellulose after 10 days from 50% to 19% and the carbon content from 44% to 27% towards the end of the 40-day composting period were observed. The C/N ratio also decreased. A drastic change in the bacterial community structure and diversity throughout the composting process was clearly observed using PCR-DGGE banding patterns. The bacterial community drastically shifted between the thermophilic and maturing stages. 16s rRNA clones belonging to the genera Bacillus, Exiguobacterium, Desemzia, and Planococcus were the dominant groups throughout composting. The species closely related to Solibacillus silvestris were found to be major contributors to changes in the lignocellulosic component. Clones identified as Thermobacillus xylanilyticus, Brachybacterium faecium, Cellulosimicrobium cellulans, Cellulomonas sp., and Thermobifida fusca, which are known to be lignocellulosic-degrading bacteria, were also detected and are believed to support the lignocellulose degradation..
49. Pramod Poudel, Hirokuni Miyamoto, Hisashi Miyamoto, Yuki Okugawa, Yukihiro Tashiro, Kenji Sakai, Thermotolerant Bacillus kokeshiiformis sp. nov. isolated from marine animal resources compost, International Journal of Systematic and Evolutionary Microbiology, 10.1099/ijs.0.059329-0, 64, PART 8, 2668-2674, 2014.08, A novel Gram-staining-positive, endospore-forming, rod-shaped, facultatively anaerobic, thermotolerant bacterium, designated strain MO-04T, was isolated from a marine animal resources (MAR) compost. The 16S rRNA gene sequence of strain MO-04T showed 99.4 % similarity with Bacillus thermolactis R-6488T, 94.1 % similarity with Bacillus thermoamylovorans CNCM I-1378T, 93.3 % similarity with Bacillus humi LMG 22167T, 93.2 % similarity with Bacillus niacini IFO 15566T and the similarities with other species were less than 93 %. DNA-DNA relatedness between strain MO-04Tand B. thermolactis DSM 23332T was 45 %. The DNA G+C content of strain MO-04T was 33.4 mol%, comparatively lower than that of B. thermolactis R-6488T (35.0 mol%). Strain MO-04T grew at 35-61 °C (optimum 50 °C), pH 4.5-9.0 (optimum pH 7.2) and tolerated up to 8.0 % (w/v) NaCl (optimum 2 %). The MO-04T cell wall peptidoglycan type was meso-2, 6-diaminopimelic acid, and the major fatty acids were C16: 1, C14: 1, C17: 0 and C17: 1. The major polar lipids were represented by diphosphatidylglycerol and phosphatidylglycerol and two unidentified phospholipids. The analysed polyphasic data presented here clearly indicate that the isolate MO-04T is considered to represent a novel species within the genus Bacillus for which the name Bacillus kokeshiiformissp. nov. is proposed. The type strain of B. kokeshiiformis is MO-04T (= JCM 19325T = KCTC 33163T)..
50. Saowanit Tongpim, Ratchanu Meidong, Pramod Poudel, Satoshi Yoshino, Yuki Okugawa, Yukihiro Tashiro, Masayuki Taniguchi, Kenji Sakai, Isolation of thermophilic l-lactic acid producing bacteria showing homo-fermentative manner under high aeration condition, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2013.08.017, 117, 3, 318-324, 2014.03, By applying non-sterile open fermentation of food waste, various thermotolerant l-lactic acid-producing bacteria were isolated and identified. The predominant bacterial isolates showing higher accumulation of l-lactic acid belong to 3 groups of Bacillus coagulans, according to their 16S rRNA gene sequence similarities. B.coagulans strains M21 and M36 produced high amounts of l-lactic acid of high optical purity and lactic acid selectivity in model kitchen refuse medium and glucose-yeast extract-peptone medium. Other thermotolerant isolates resembling to Bacillus humi, B. ruris, B.subtilis, B. niacini and B. soli were also identified. These bacteria produced low amounts of l-lactic acid of more than 99% optical purity. All isolated strains showed the highest growth rate at temperatures around 55-60°C. They showed unique responses to various oxygen supply conditions. The majority of isolates produced l-lactic acid at a low overall oxygen transfer coefficient (KLa); however, acetic acid was produced instead of l-lactic acid at a high KLa. B.coagulans M21 was the only strain that produced high, consistent, and reproducible amounts of optically pure l-lactic acid (>99% optical purity) under high and low KLa conditions in a homo-fermentative manner..
51. Tashiro Saiwai Hiroshi, Kenji Sakai, Kyushu Branch
Efforts aggressive biomass recycling in Fukuoka Prefecture, Seibutsu-kogaku Kaishi, 92, 10, 565, 2014.03.
52. Ying Wang, Mohamed Ali Sayed Mohamed Abdelrahman, Yukihiro Tashiro, Yaotian Xiao, Takeshi Zendo, Kenji Sakai, Kenji Sonomoto, L-(+)-Lactic acid production by co-fermentation of cellobiose and xylose without carbon catabolite repression using Enterococcus mundtii QU 25, RSC Advances, 10.1039/c4ra02764g, 4, 42, 22013-22021, 2014.01, The use of lignocellulosic biomass for the production of optically pure lactic acid remains challenging because it requires efficient utilisation of mixed sugars without carbon catabolite repression (CCR). Enterococcus mundtii QU 25, a novel l-lactic acid-producing strain, was used in this study to ferment mixed sugars. This strain exhibited apparent CCR in a glucose-xylose mixture; however, replacement of glucose by cellobiose (cellobiose-xylose mixture) led to simultaneous consumption of both sugars without CCR. The production of lactic acid and activity of enzymes related to xylose metabolism were also investigated. Xylose isomerase and xylulokinase specific activity in cellobiose-xylose grown cells was three times higher than that in glucose-xylose grown cells. The addition of yeast extract and ammonium hydroxide effectively improved sugar utilisation and cell growth. Under the optimal conditions with simulated lignocellulosic hydrolysates, a high l-lactic acid concentration (up to 163 g L-1) was produced with a yield of 0.870 g g-1 and maximum productivity of 7.21 g L-1 h-1 without CCR in the fed-batch fermentation. Thus, we could establish rapid and simultaneous consumption of hexose and pentose sugars by using a lactic acid bacterium strain, which significantly increased production of high-purity l-lactic acid. This journal is.
53. Takuya Noguchi, Yukihiro Tashiro, Tsuyoshi Yoshida, Jin Zheng, Kenji Sakai, Kenji Sonomoto, Efficient butanol production without carbon catabolite repression from mixed sugars with Clostridium saccharoperbutylacetonicum N1-4, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2013.05.030, 116, 6, 716-721, 2013.12, Acetone-butanol-ethanol (ABE) fermentation using Clostridium saccharoperbutylacetonicum N1-4 and mixed sugars containing cellobiose and xylose was studied to establish efficient butanol production process without carbon catabolite repression (CCR). Although batch culture with glucose and xylose exhibited apparent CCR, we achieved simultaneous consumption of cellobiose and xylose. Moreover, preculture of the N1-4 strain with xylose yielded maximum butanol and solvent concentrations (16 and 23g/L, respectively). Thus, we succeeded in ABE fermentation with mixed sugars of hexose and pentose, without CCR, by using wild-type ABE-producing clostridia. We also investigated the effect of various ratios of cellobiose and xylose on the fermentation process and yield. Increasing initial xylose concentration improved butanol and solvent concentrations and maximum xylose consumption rate. Fed-batch culture with cellobiose and xylose showed rapid and simultaneous sugar consumption and improved maximum consumption rate of both sugars..
54. Yukihiro Tashiro, Hiroko Matsumoto, Hirokuni Miyamoto, Yuki Okugawa, Poudel Pramod, Hisashi Miyamoto, Kenji Sakai, A novel production process for optically pure l-lactic acid from kitchen refuse using a bacterial consortium at high temperatures, Bioresource Technology, 10.1016/j.biortech.2013.07.102, 146, 672-681, 2013.01, We investigated l-lactic acid production in static batch fermentation of kitchen refuse using a bacterial consortium from marine-animal-resource (MAR) composts at temperatures ranging from 30 to 65°C. At relatively low temperatures butyric acid accumulated, whereas at higher temperatures l-lactic acid was produced. In particular, fermentation at 50°C produced 34.5gL-1 l-lactic acid with 90% lactic acid selectivity and 100% optical purity. Denaturing gradient gel electrophoresis indicated that dominant bacteria present in the original MAR composts diminished rapidly and Bacillus coagulans strains became the dominant contributors to l-lactic acid production at 45, 50 and 55°C. This is the first report of the achievement of 100% optical purity of l-lactic acid using a bacterial consortium..
55. Kazunori Nakamura, Kenji Sakai, Denaturing gradient gel electrophoresis analysis of gut bacterial community for the Japanese earthworms, Soil Science and Plant Nutrition, 10.1080/00380768.2011.594965, 57, 4, 519-528, 2011.12, The gut bacterial community structure for Pheretima hilgendorfi and P. heteropoda (Family Megascolecidae), and Allolobophora japonica (Family Lumbricidae) collected from agricultural grasslands in Japan was analyzed by denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16S rRNA gene fragments (PCR-DGGE) and compared with those in the surrounding soils. Denaturing gradient gel electrophoresis (DGGE) profiles indicated that each earthworm species had their own specific bacterial communities, and multidimentional scaling analysis grouped the DGGE profiles into three groups: gut samples from P. hilgendorfi and P. heteropoda, gut samples from A. japonica and samples from the surrounding soils. Nine dominant bands were identified by their direct sequencing and cloning. Major three bands from P. hilgendorfi and P. heteropoda were closely related to Bacillus species belonging to the phylum Firmicutes. Major four and two bands from A. japonica were closely related to the phyla Proteobacteria and Bacteroidetes, respectively..
56. Utilization and Recycle of Waste Biomass Resources.
57. Norjan Yusof, Ali Hassan Mohd Ali Hassan, Lai Yee Phang Lai Yee, Meisam Tabatabaei, Ridzuan Othman Mohd Ridzuan Othman, Masatsugu Mori, Minato Wakisaka, Kenji Sakai, Yoshihito Shirai, Nitrification of high-strength ammonium landfill leachate with microbial community analysis using fluorescence in situ hybridization (FISH), Waste Management and Research, 10.1177/0734242X10397581, 29, 6, 602-611, 2011.06, Nitrification of mature sanitary landfill leachate with high-strength of N-NH4 + (1080-2350 mg L-1) was performed in a 10 L continuous nitrification activated sludge reactor. The nitrification system was acclimatized with synthetic leachate during feed batch operation to avoid substrate inhibition before being fed with actual mature leachate. Successful nitrification was achieved with an approximately complete ammonium removal (99%) and 96% of N-NH4 + conversion to N-NO- 3. The maximum volumetric and specific nitrification rates obtained were 2.56 kg N-NH4 + m-3 day-1 and 0.23 g N-NH4 + g-1 volatile suspended solid (VSS) day-1, respectively, at hydraulic retention time (HRT) of 12.7 h and solid retention time of 50 days. Incomplete nitrification was encountered when operating at a higher nitrogen loading rate of 3.14 kg N-NH4 + m-3 day-1. The substrate overloading and nitrifiers competition with heterotrophs were believed to trigger the incomplete nitrification. Fluorescence in situ hybridization (FISH) results supported the syntrophic association between the ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria. FISH results also revealed the heterotrophs as the dominant and disintegration of some AOB cell aggregates into single cells which further supported the incomplete nitrification phenomenon..
58. Masayuki Taniguchi, Daisuke Takahashi, Daisuke Watanabe, Kenji Sakai, Kazuhiro Hoshino, Tomoaki Kouya, Takaaki Tanaka, Effect of steam explosion pretreatment on treatment with Pleurotus ostreatus for the enzymatic hydrolysis of rice straw, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2010.04.014, 110, 4, 449-452, 2010.10, The effects of steam explosion (1.5. MPa, 1. min) on the treatment of rice straw with Pleurotus ostreatus were evaluated in terms of the change in composition of the components and the susceptibility to enzymatic hydrolysis. When rice straw was pretreated with a steam explosion prior to biological treatment, the treatment time required for obtaining a 33% net glucose yield was reduced to 36. days from 60. days. The reduction is probably due to loosening of networks of Klason lignin with sugar moieties and partial collapse of the structure during the biological treatment..
59. Jantima Arnthong, Boonpa Wanitchaploy, Kenji Sakai, Jean Jacques Sanglie, Vichien Kitpreechavanich, Statistical screening of factors affecting glucoamylase production by a thermotolerant rhizopus microsporus tistr 3518 using plackett-burman design, African Journal of Biotechnology, 9, 43, 7312-7316, 2010.10, Glucoamylase is a key enzyme used in food processing as well as in commercial production of glucose from starch. The use of thermotolerant strain of Rhizopus microsporus TISTR 3518 offers the advantages of cooling-costs reduction during fermentation and high thermostable enzyme production. The effect of various carbon and nitrogen sources on glucoamylase production was evaluated. It was found that α-amylase treated liquefied cassava starch and CH3COONH4 gave the highest enzyme activity. The influence of various medium components and culture parameters were investigated using Plackett-Burman. It was shown that CH3COONH4, FeSO4.7H2O, ZnSO4.7H2O, CaCl2, temperature and pH are significant factors affecting the glucoamylase production. The medium with the initial pH of 6.5 which consisted of α-amylase treated liquefied cassava starch, 10 gl-1; CH3COONH4, 5 gl-1; K2HPO4, 0.5 gl-1; KCl, 1.5 gl-1; MgSO4.7H2O, 0.5 gl-1; FeSO4.7H2O, 0.06 gl-1; ZnSO4.7H2O, 0.035 gl-1; CaCl2, 0.05 gl-1 and C6H8O7.H2O, 5.6 gl-1 yielded the highest enzyme production (948 U ml-1) after cultivation at 40°C for 48 h..
60. Shiotsuka K, Tanaka A, and Sakai K , Occurrence of Extreme Thermophiles, Thermaerobacter spp., in Sewage Sludge , J Jap Soc Extremophiles , 9, 67-71, 2010.09.
61. Meisam Tabatabaei, Raha Abdul Rahim, Norhani Abdullah, André Denis G. Wright, Yoshihito Shirai, Kenji Sakai, Alawi Sulaiman, Mohd Ali Hassan, Importance of the methanogenic archaea populations in anaerobic wastewater treatments, Process Biochemistry, 10.1016/j.procbio.2010.05.017, 45, 8, 1214-1225, 2010.08, Methane derived from anaerobic treatment of organic wastes has a great potential to be an alternative fuel. Abundant biomass from various industries could be a source for biomethane production where combination of waste treatment and energy production would be an advantage. This article summarizes the importance of the microbial population, with a focus on the methanogenic archaea, on the anaerobic fermentative biomethane production from biomass. Types of major wastewaters that could be the source for biomethane generation such as brewery wastewater, palm oil mill effluent, dairy wastes, cheese whey and dairy wastewater, pulp and paper wastewaters and olive oil mill wastewaters in relevance to their dominant methanogenic population are fully discussed in this article..
62. Azhari S. Baharuddin, K. Nakamura, S. A-Aziz, N. A. Rahman, M. Tabatabaei, M. A. Hassan, M. Wakisaka, , K. Sakai,Y. Shirai, , Characteristics and Microbial Succession in Co-composting of Oil Palm Empty Fruit Bunch and Partially Treated Palm Oil Mill Effluent , , The Open Biotechnology Journal, 3, 26, 87-95 , 2010.07.
63. Azhari Samsu Baharuddin, Lim Siong Hock, Mohd Zulkhairi Md Yusof, Nor' Aini Abdul Rahman, Umi Kalsom Md Shah, Mohd Ali Hassan, Minato Wakisaka, Kenji Sakai, Yoshihito Shirai, Effects of palm oil mill effluent (POME) anaerobic sludge from 500 m3 of closed anaerobic methane digested tank on pressed-shredded empty fruit bunch (EFB) composting process, African Journal of Biotechnology, 9, 16, 2427-2436, 2010.04, In this study, co-composting of pressed-shredded empty fruit bunches (EFB) and palm oil mill effluent (POME) anaerobic sludge from 500 m3 closed anaerobic methane digested tank was carried out. High nitrogen and nutrients content were observed in the POME anaerobic sludge. The sludge was subjected to the pressed-shredded EFB to accelerate the co-composting treatment. In the present study, changes in the physicochemical characteristics of co-composting process were recorded and evaluated. The co-composting treatment was completed in a short time within 40 days with a final C/N ratio of 12.4. The co-composting process exhibited a higher temperature (60 - 67°C) in the thermophilic phase followed by curing phase after four weeks of treatment. Meanwhile, pH of the composting pile (8.1 - 8.6) was almost constant during the process and moisture content was reduced from 64.5% (initial treatment) to 52.0% (final matured compost). The use of pressed-shredded EFB as a main carbon source and bulking agent contributed to the optimum oxygen level in the composting piles (10 - 15%). The biodegradation of composting materials is shown by the reduction of cellulose (34.0%) and hemicellulose (27.0%) content towards the end of treatment. In addition, considerable amount of nutrients and low level of heavy metals were detected in the final matured compost. It can be concluded that the addition of POME anaerobic sludge into the pressed-shredded EFB composting process could produce acceptable and consistent quality of compost product in a short time..
64. Masayuki Taniguchi, Daisuke Takahashi, Daisuke Watanabe, Kenji Sakai, Kazuhiro Hoshino, Tomoaki Kouya, Takaaki Tanaka, Evaluation of fungal pretreatments for enzymatic saccharification of rice straw, Journal of Chemical Engineering of Japan, 10.1252/jcej.09We193, 43, 4, 401-405, 2010.04, To enzymatically hydrolyze cellulose of rice straw to glucose, the effect of pretreatment of rice straw with 15 strains of basidiomycetes is evaluated in terms of the quantitative changes in the components of pretreated rice straw and their susceptibility to enzymatic hydrolysis. Pleurotus ostreatus was found to be one of the most suitable white-rot fungi for biological pretreatment of rice straw. Of 11 strains of P. ostreatus tested, ATCC 66376 was found to have the properties superior to the other strains. Thus, in the pretreatment for 48d, P. ostreatus ATCC 66376 degraded 39% Klason lignin and retained 79% cellulose in the pretreated rice straw.When the rice straw pretreated with this fungus was hydrolyzed with a commercial cellulase, the net yield of glucose determined on the basis of the weight of cellulose fraction of the untreated rice straw was the highest..
65. Masatsugu Mori, Yuko Iwami, Kenji Sakai, Enhancement of biodegradability of paperboard in soil by supporting Trichoderma cells and nutritional constituents, Journal of the Faculty of Agriculture, Kyushu University, 55, 1, 97-99, 2010.02, When paperboard made from used paper was supported by Trichoderma reesei NBRC31137, a cellulolytic basidiomyces, the weight decrease of the paperboard in soil was enhanced. Number of viable microorganisms around the paperboard in the soil increased and reached 10 7 after 2 weeks. Nutritional medium by itself, constituted of yeast extract and peptone, also stimulated the degradation in soil. Cells of T. viride NBRC31326 and Trichoderma reesei NBRC 31327 were less effective. These results suggested that we could control biodegradation rare of cellulosic material in an agricultural field..
66. Kedong Ma, Minato Wakisaka, Kenji Sakai, Yoshihito Shirai, Flocculation phenomenon of a mutant flocculent Saccharomyces cerevisiae strain
Effects of metal ions, sugars, temperature, pH, protein-denaturants and enzyme treatments, African Journal of Biotechnology, 9, 7, 1037-1045, 2010.02, The flocculation mechanism of a stable mutant flocculent yeast strain Saccharomyces cerevisiae KRM-1 was quantitatively investigated for potential industrial interest. It was found that the mutant flocculent strain was NewFlo phenotype by means of sugar inhibition test. The flocculation was completely inhibited by treatment with proteinase K, protein-denaturants and carbohydrate modifier. The absence of calcium ions significantly inhibited the flocculation, indicating that Ca2+ was specifically required for flocculation. The flocculation was stable when temperature below 70° C and pH was in the range of 3.0 - 6.0. The flocculation onset of the mutant flocculent strain was in the early stationary growth phase, which coincided with glucose depletion in the batch fermentation for the production of ethanol from kitchen refuse medium. The results are expected to help develop better strategies for the control of mutant flocculent yeast for future large-scale industrial ethanol fermentation..
67. Meisam Tabatabaei, Mohd Rafein Zakaria, Raha Abdul Rahim, Norhani Abdullah, André Denis G Wright, Yoshihito Shirai, Mehdi Shamsara, Kenji Sakai, Mohd Ali Hassan, Comparative study of methods for extraction and purification of environmental DNA from high-strength wastewater sludge, African Journal of Biotechnology, 9, 31, 4926-4937, 2010.02, DNA extraction from wastewater sludge (COD 50000 and BOD 25000 mg/l) was conducted using nine different methods normally used for environmental samples including a procedure used in this study and the results obtained were compared. The quality of the differently extracted DNAs was subsequently assessed by measuring humic acid concentration, cell lysis efficiency, polymerase chain reaction (PCR) amplification of methanogenic and eubacterial 16S rDNA. The protocol developed in this study was further evaluated by extracting DNA from various high-strength wastewater sludge samples, denaturing gradient gel electrophoresis (DGGE) and fluorescent in situ hybridization (FISH) analyses. The results revealed that great differences existed among the nine procedures and only a few produced satisfactory results when applied to high-strength wastewater sludge. Thermal shock alone was shown inefficient to disrupt the methanogenic cell wall to release the DNA. The method presented in this study (Procedure 9) is generally recommended because of the low concentration of contaminants and its high efficiency despite its simplicity..
68. Azhari Samsu Baharuddin, Mohamad Nafis Abd Razak, Lim Siong Hock, Mohd Najib Ahmad, Suraini Abd-Aziz, Nor' Aini Abdul Rahman, Umi Kalsom Md Shah, Mohd Ali Hassan, Kenji Sakai, Yoshihito Shirai, Isolation and characterization of thermophilic cellulase-producing bacteria from empty fruit bunches-palm oil mill effluent compost, American Journal of Applied Sciences, 7, 1, 56-62, 2010.01, Problems statement: Lack of information on locally isolated cellulase-producing bacterium in thermophilic compost using a mixture of Empty Fruit Bunch (EFB) and Palm Oil Mill Effluent (POME) as composting materials. Approach: The isolation of microbes from compost heap was conducted at day 7 of composting process where the mixture of composting materials consisted of 45.8% cellulose, 17.1% hemicellulose and 28.3% lignin content. The temperature, pH and moisture content of the composting pile at day 7 treatment were 58.3, 8.1 and 65.5°C, respectively. The morphological analysis of the isolated microbes was conducted using Scanning Electron Microscope (SEM) and Gram stain method. The congo red test was conducted in order to detect 1% CMC agar degradation activities. Total genomic DNAs were extracted from approximately 1.0 g of mixed compost and amplified by using PCR primers. The PCR product was sequent to identify the nearest relatives of 16S rRNA genes. The localization of bacteria chromosomes was determined by Fluorescence In Situ Hybridization (FISH) analysis. Results: Single isolated bacteria species was successfully isolated from Empty Fruit Bunch (EFB)-Palm Oil Mill Effluent (POME) compost at thermophilic stage. Restriction fragment length polymorphism profiles of the DNAs coding for the 16S rRNAs with the phylogenetic analysis showed that the isolated bacteria from EFB-POME thermophilic compost gave the highest homology (99%) with similarity to Geobacillus pallidus. The strain was spore forming bacteria and able to grow at 60°C with pH 7. Conclusion: Thermophilic bacteria strain, Geobacillus pallidus was successfully isolated from Empty Fruit Bunch (EFB) and Palm Oil Mil Effluent (POME) compost and characterized..
69. Meisam Tabatabaei, Mohd Rafein Zakaria, Raha Abdul Rahim, André Denis G. Wright, Yoshihito Shirai, Norhani Abdullah, Kenji Sakai, Shinya Ikeno, Masatsugu Mori, Nakamura Kazunori, Alawi Sulaiman, Mohd Ali Hassan, PCR-based DGGE and FISH analysis of methanogens in an anaerobic closed digester tank for treating palm oil mill effluent, Electronic Journal of Biotechnology, 10.2225/vol12-issue3-fulltext-4, 12, 3, 2009.07, 16S ribosomal RNA (rRNA)-targeted fluorescent in situ hybridization combined with polymerase chain reaction (PCR)-cloning, light microscopy using Gram stains, scanning electron microscopy and denatured gradient gel electrophoresis were used to reveal the distribution of methanogens within an anaerobic closed digester tank fed with palm oil mill effluent. For specific detection of methanogens, 16S rRNA-cloning analysis was conducted followed by restriction fragment length polymorphism (RFLP) for presumptive identification of methanogens. To cover the drawbacks of the PCRcloning study, the organization of the microorganisms was visualized in the activated sludge sample by using fluorescent oligonucleotide probes specific to several different methanogens, and a probe for bacteria. In situ hybridization with methanogens and bacterial probes and denatured gradient gel electrophoresis within activated sludge clearly confirmed the presence of Methanosaeta sp. and Methanosarcina sp. cells. Methanosaeta concilii was found to be the dominant species in the bioreactor. These results revealed the presence of possibly new strain of Methanosaeta in the bioreactor for treating palm oil mill effluent called Methanosaeta concilii SamaliEB (Gene bank accession number: EU580025). In addition, fluorescent hybridization pictured the close association between the methanogens and bacteria and that the number of methanogens was greater than the number of bacteria..
70. Kedong Ma, Minato Wakisaka, Kenji Sakai, Yoshihito Shirai, Flocculation characteristics of an isolated mutant flocculent Saccharomyces cerevisiae strain and its application for fuel ethanol production from kitchen refuse, Bioresource Technology, 10.1016/j.biortech.2008.11.010, 100, 7, 2289-2292, 2009.04, A stable mutant flocculent yeast strain of Saccharomyces cerevisiae KRM-1 was isolated during repeated-batch ethanol fermentation using kitchen refuse as the medium. The mechanism of flocculation and interaction with the medium was investigated. According to sugar inhibition assay, it was found that the mutant flocculent strain was a NewFlo phenotype. Flocculation was completely inhibited by protease, proteinase K and partially reduced by treatments with carbohydrate-hydrolyzing enzymes. Flocculation ability showed no difference for pH 3.0-6.0. Furthermore, the mutant flocculent yeast provided repeated-batch cultivations employing cell recycles by flocculation over 10 rounds of cultivation for the production of ethanol from kitchen refuse medium, resulting in relatively high productivity averaging 8.25 g/L/h over 10 batches and with a maximal of 10.08 g/L/h in the final batch. Cell recycle by flocculation was fast and convenient, and could therefore be applicable for industrial-scale ethanol production..
71. Vichien Kitpreechavanich, Thanapoom Maneeboon, Youichi Kayano, Kenji Sakai, Comparative Characterization of l-Lactic Acid-Producing Thermotolerant Rhizopus Fungi, Journal of Bioscience and Bioengineering, 10.1263/jbb.106.541, 106, 6, 541-546, 2008.12, Acid-producing Rhizopus fungi from loog-pang, a traditional Thai fermented food, was screened to investigate its potential for use in industrial lactic acid production from starch. A thermotolerant strain, TISTR 3518, was isolated and characterized by its morphological, physiological, genetic and fermentation properties, and compared with its mesophilic isolates, TISTR 3514 and TISTR 3523. TISTR 3518 was characterized by shorter sporangiophores and smaller sporangia than the other isolates; however, apparent differences between the mesophilic isolates and the strain could not be clarified. Moreover, TISTR 3518 grew at 45°C, whereas the others did not. The three isolates showed different profiles of oligosaccharide assimilation and organic acid production. Their rDNA ITS sequences indicated that TISTR 3518 is a strain of Rhizopus microsporus, and TISTR 3514 and TISTR 3523 are strains of Rhizopus oryzae. TISTR 3523 and TISTR 3518 mainly formed l-lactic acid from glucose, while TISTR 3514 primarily formed fumaric acid. Under thermotolerant conditions, R. microsporus TISTR 3518 showed higher glucoamylase activity than the others, suggesting this enzyme from TISTR 3518 is more thermostable than that from TISTR 3523. The strain formed higher amounts of l-lactic acid from starch at 40°C compared to R. oryzae TISTR 3523. This is the first report on the production of optically active l-lactic acid from starch by a thermotolerant fungus and could potentially provide a good tool for transforming biomass resources to chemical materials..
72. Kota Shiotsuka, Shinjiro Kanazawa, Kenji Sakai, Direct analysis of thermophilic bacteria in sewage sludge compost, Journal of the Faculty of Agriculture, Kyushu University, 53, 2, 471-477, 2008.10, To analyze metabolically active thermophilic bacteria, a high-temperature direct viable count (HT-DVC) method was developed and applied to sewage sludge compost made by a hyperthermal composting method. When the HT-DVC method was conducted at 60°C and 80°C, maximum numbers of 23.3×108 and 2.62×108 cells/(g of dry sample) of elongated cells (length > 4μm), respectively, were detected. These results indicate that the HT-DVC method can be used to enumerate even metabolically active extreme thermopniles. Strain TH, a Gram-negative, spore-forming, and extremely thermophilic bacterium, which showed growth at 55-78°C, was isolated from the sewage sludge compost. Strain TH is closely related to Caldaterra satsumae YM081. The HT-DVC method could detect strain TH inoculated into sewage sludge compost with autoclaving, but could not selectively detect the strain inoculated into the compost without autoclaving..
73. Jiro Nakayama, Hiroyuki Hoshiko, Mizuki Fukuda, Hidetoshi Tanaka, Naoshige Sakamoto, Shigemitsu Tanaka, Kazutoshi Ohue, Kenji Sakai, Kenji Sonomoto, Molecular Monitoring of Bacterial Community Structure in Long-Aged Nukadoko
Pickling Bed of Fermented Rice Bran Dominated by Slow-Growing Lactobacilli, Journal of Bioscience and Bioengineering, 10.1263/jbb.104.481, 104, 6, 481-489, 2007.12, Nukadoko is the fermented rice bran bed traditionally used for pickling vegetables in Japan. Here, we investigate the bacterial community structure of nukadoko using several culture-independent methods. Denaturing gradient gel electrophoresis (DGGE) and sequence analysis of V2-V3 16S rRNA gene (16S rDNA) fragments amplified from a long-aged nukadoko bacterial community indicated seven predominant operational taxonomic units (OTUs) closely related to known Lactobacillus species. Phylogenetic analysis of these OTUs indicated a major cluster consisting of six OTUs including a dominant OTU closely related to Lactobacillus acidifarinae and one distinct OTU corresponding to Lactobacillus acetotolerans. L. acetotolerans was commonly detected as a dominant species in samples from different seasons. The succession of microbial community structure in the fermentation and ripening processes was investigated using a laboratory model nukadoko. The L. acidifarinae-like bacteria grew rapidly with a pH decrease in the first few days after inoculation, whereas L. acetotolerans grew slowly and became dominant after one week. Real-time quantitative polymerase chain reaction (Q-PCR) showed that the doubling time of L. acetotolerans was 12 h, while that of total bacteria was 4 h. Real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) targeting 16S rRNA showed a low metabolic activity of L. acetotolerans throughout the fermentation and ripening processes. Fluorescence in situ hybridization (FISH) showed that L. acetotolerans was a dominant bacterium in the ripening period and had a low metabolic activity. These results indicate that the slow-growing L. acetotolerans stably dominated nukadoko microbiota after the L. acidifarinae-like bacteria mainly contributed to the lactic acid fermentation of the rice bran..
74. Kenji Sakai, Norihisa Fujii, Ekachai Chukeatirote, Racemization of l-lactic acid in pH-swing open fermentation of kitchen refuse by selective proliferation of Lactobacillus plantarum, Journal of Bioscience and Bioengineering, 10.1263/jbb.102.227, 102, 3, 227-232, 2006.09, We have shown that stable lactic acid fermentation of model kitchen refuse occurs with intermittent pH adjustment under nonsterilized conditions. Nonetheless, the optical activity of the accumulated lactic acid was low, which is disadvantageous for the production of high-quality poly-l-lactic acid. Here, we attempt to increase optical purity by introducing l-lactic acid-producing strains under nonsterilized conditions and demonstrate that the inoculation of Lactobacillus rhamnosus or Lactococcus lactis, both of which are l-lactic acid producers, is partially effective in the early fermentation stage, but does not improve the final optical purity of the accumulated lactic acid. We confirmed by fluorescence in situ hybridization using group-specific and species-specific 16S rDNA probes that this is due to the selective proliferation of naturally existing L. plantarum. L. plantarum KY-1, which is isolated from model kichen refuse, showing lactic acid racemase activity, as well as d-lactate dehydrogenase activity, in its membrane fraction. We conclude that racemase activity associated with L. plantarum is the main cause of decreased optical purity in the accumulated lactic acid..
75. Kenji Sakai, Tetsuya Yamanami, Thermotolerant Bacillus licheniformis TY7 produces optically active l-lactic acid from kitchen refuse under open condition, Journal of Bioscience and Bioengineering, 10.1263/jbb.102.132, 102, 2, 132-134, 2006.08, A thermotolerant l-lactic-acid-producing bacterium was isolated and identified as Bacillus licheniformis TY7. TY7 shows optimum growth at pH 6.5 at 30°C and normal growth up to 65°C. Using nonsterile kitchen refuse at 50°C, the strain produced 40 g/l l-lactic acid with 97% optical activity and 2.5 g/l·h productivity..
76. Kenji Sakai, Yutaka Ezaki, Open L-lactic acid fermentation of food refuse using thermophilic Bacillus coagulans and fluorescence in situ hybridization analysis of microflora, Journal of Bioscience and Bioengineering, 10.1263/jbb.101.457, 101, 6, 457-463, 2006.06, In the production of commercially useful poly-L-lactic acid plastic from biomass wastes, a feasible fermentation process to produce optically active L-lactic acid would be required. Here, model kitchen refuse (MKR) was inoculated with Bacillus coagulans NBRC12583 under nonsterilized openculture conditions. At temperatures below 45°C, a racemic mixture of D- and L-lactic acids was accumulated, whereas only L-lactic acid was selectively accumulated by incubation at 50-65°C. At 45°C, the results of fermentation could not be consistently reproduced. To analyze microflora in this type of mixed culture system, whole-cell fluorescence in situ hybridization (FISH) using 16S rRNA-targeted oligonucleotide probes for B. coagulans, Bcoa191, and LAC722(L), a group-specific probe for a wide range of mesophilic lactic acid bacteria was applied. The dominancy of mesophilic lactic acid bacteria at lower temperatures, and that of B. coagulans at higher temperatures were confirmed. By using a saccharified liquid of collected kitchen refuse, 86 g/l of L-lactic acid was accumulated under nonsterile conditions by a 5-d incubation at 55°C, pH 6.5, with 53% carbon yield and 97% optical purity. To conclude, high temperature open lactic acid fermentation is a simple and promising method for producing high-grade L-lactic acid from biomass waste, and FISH analysis of such mixed-culture systems is helpful for monitoring the microflora in these cultures..
77. Kenji Sakai, Kazutoshi Oue, Miki Umeki, Masatsugu Mori, Mari Kuribayashi, Satoshi Mochizuki, Species-specific FISH analysis of cecal microflora in rats administered with lactic acid bacteria, World Journal of Microbiology and Biotechnology, 10.1007/s11274-005-9062-8, 22, 5, 493-499, 2006.05, We investigated the effects of lactic acid bacterial (LAB) cells in rats using fluorescence in situ hybridization (FISH) targeting 16S rRNA to identify the cecal microbial community. We designed a novel species-specific 16S rDNA probe to detect Lactobacillus rhamnosus (Lrham454). Subtractive technique using the LAC722 probe (Sakai et al. 2004 Journal of Bioscience and Bioengineering 98, 48) under different hybridization stringency (LAC722(L-H)) was applied to identify Lactococcus lactis. We also applied Lplan447 and LAC722(L) to detect Lactobacillus plantarum and a wide range of LAB (total LAB), respectively. We optimized the hybridization and washing conditions and then quantified L. rhamnosus, L. plantarum, and L. lactis cells in rat cecal contents. We monitored increases in individual bacterial populations and in total LAB caused by the administration of the corresponding LAB cells. Growth, food efficiency and internal disorders did not significantly differ among the rats administered with LABs. Rats administered with polydextrose (POL) developed diarrhea, which decreased the total numbers of cecal bacteria, whereas the simultaneous administration of POL and L. rhamnosus KY-3 eased this symptom, and recovered the numbers of total LAB and of L. rhamnosus..
78. Takaaki Tanaka, Masahiro Hoshina, Suguru Tanabe, Kenji Sakai, Sadami Ohtsubo, Masayuki Taniguchi, Production of D-lactic acid from defatted rice bran by simultaneous saccharification and fermentation, Bioresource Technology, 10.1016/j.biortech.2005.02.025, 97, 2, 211-217, 2006.01, Production of d-lactic acid from rice bran, one of the most abundant agricultural by-products in Japan, is studied. Lactobacillus delbrueckii subsp. delbrueckii IFO 3202 and defatted rice bran powder after squeezing rice oil were used for the production. Since the rice bran contains polysaccharides as starch and cellulose, we coupled saccharification with amylase and cellulase to lactic acid fermentation. The indigenous bacteria in the rice bran produced racemic lactic acid in the saccharification at pH 6.0-6.8. Thus the pH was controlled at 5.0 to suppress the growth of the indigenous bacteria. L. delbrueckii IFO 3202 produced 28 kg m-3 lactic acid from 100 kg m-3 rice bran after 36 h at 37°C. The yield based on the amount of sugars soluble after 36-h hydrolysis of the bran by amylase and cellulase (36 kg m-3 from 100 kg m-3 of the bran) was 78%. The optical purity of produced d-lactic acid was 95% e.e..
79. Masayuki Taniguchi, Hiroyuki Suzuki, Daisuke Watanabe, Kenji Sakai, Kazuhiro Hoshino, Takaaki Tanaka, Evaluation of pretreatment with Pleurotus ostreatus for enzymatic hydrolysis of rice straw, Journal of Bioscience and Bioengineering, 10.1263/jbb.100.637, 100, 6, 637-643, 2005.12, The effects of biological pretreatment of rice straw using four white-rot fungi (Phanerochaete chrysosporium, Trametes versicolor, Ceriporiopsis subvermispora, and Pleurotus ostreatus) were evaluated on the basis of quantitative and structural changes in the components of the pretreated rice straw as well as susceptibility to enzymatic hydrolysis. Of these white-rot fungi, P. ostreatus selectively degraded the lignin fraction of rice straw rather than the holocellulose component. When rice straw (water content of 60%) was pretreated with P. ostreatus for 60 d, the total weight loss and the degree of Klason lignin degraded were 25% and 41%, respectively. After the pretreatment, the residual amounts of cellulose and hemicellulose were 83% and 52% of those in untreated rice straw, respectively. By enzymatic hydrolysis with a commercial cellulase preparation for 48 h, 52% holocellulose and 44% cellulose in the pretreated rice straw were solubilized. The net sugar yields based on the amounts of holocellulose and cellulose of untreated rice straw were 33% for total soluble sugar from holocellulose and 32% for glucose from cellulose. The SEM observations showed that the increase in susceptibility of rice straw to enzymatic hydrolysis by pretreatment with P. ostreatus is caused by partial degradation of the lignin seal. When the content of Klason lignin was less than 15% of the total weight of the pretreated straw, enhanced degrees of enzymatic solubilization of holocellulose and cellulose fractions were observed as the content of Klason lignin decreased..
80. Masayuki Taniguchi, Masahiro Hoshina, Suguru Tanabe, Yuki Higuchi, Kenji Sakai, Sadami Ohtsubo, Kazuhiro Hoshino, Takaaki Tanaka, Production of L-lactic acid by simultaneous saccharification and fermentation using unsterilized defatted rice bran as a carbon source and nutrient components, Food Science and Technology Research, 10.3136/fstr.11.400, 11, 4, 400-406, 2005.12, On the basis of growth rate at low pH, yield of lactic acid from glucose, and optical purity of lactic acid produced, we selected lactic acid bacteria favorable for production of optically pure L-lactic acid from defatted rice bran without sterilization. Of 21 strains tested, strains Nos. 13 and 16 produced 27-29 kg m-3 of lactic acid with high optical purity from 100 kg m-3 of unsterilized defatted rice bran in simultaneous saccharification and fermentation (SSF) with MRS medium at pH 4.5, a level at which the growth of indigenous lactic acid bacteria in defatted rice bran was suppressed. In a SSF process using strain No. 16 in which Mcllvaine buffer (pH 4.5) was used instead of MRS medium, no growth of indigenous lactic acid bacteria was observed in defatted rice bran, and 28 kg m -3 of lactic acid with 92% L-type content was produced from 100 kg m-3 of unsterilized defatted rice bran. In SSF using McIlvaine buffer (pH 4.5), the protein fraction of defatted rice bran was found to play a significant role as a nitrogen source for the growth of lactic acid bacteria. By increasing the initial cell concentration to OD660 = 1.0 for SSF using strain No. 16 and McIlvaine buffer (pH 4.5), the proportion of L-lactic acid produced was enhanced to 95%..
81. Miki Umeki, Kazutoshi Oue, Masatsugu Mori, Satoshi Mochizuki, Kenji Sakai, Fluorescent in situ hybridization analysis of cecal microflora in rats simultaneously administrated lactobacillus rhamnosus KY-3 and cellobiose, Food Science and Technology Research, 10.3136/fstr.11.168, 11, 2, 168-170, 2005.05, The cecal microflora of rats coadministered Lactobacillus rhamnosus KY-3 (L. rhamnosus KY-3) and cellobiose was analyzed by fluorescence in situ hybridization. When compared with L. rhamnosus KY-3 administration alone, simultaneous administration of L. rhamnosus KY-3 and cellobiose led to an increase in the number of lactic acid bacteria (LAB), particularly L. rhamnosus, and a significant decrease in the number of Gammaproteobacteria in the cecum. These results indicate that administration of L. rhamnosus KY-3 cells passed through the upper digestive tract to the cecum where it is likely that they proliferated through the assimilation of cellobiose..
82. Kazuaki Yoshimune, Masanori Kanda, Mamoru Wakayama, Shun Ichi Kanda, Akiko Sato, Kenji Sakai, Mitsuaki Moriguchi, Role of arginine residues of D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6, Protein and Peptide Letters, 10.2174/0929866053587192, 12, 3, 289-294, 2005.04, To investigate the role of arginine in the folding of D-aminoacylase, seven arginine residues, R26, R152, R296, R302, R354, R377, and R391, among twelve arginine residues highly conserved in D-aminoacylase, N-acyl-D-aspartatc amidohydrolase (D-AAase), and N-acyl-D-glutamate amidohydrolase (D-AGase) from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) were substituted with lysine by site-directed mutagenesis. The mutants, R26K, R152K, R296K, and R302K were identified as mutations that increase partitioning of the enzyme into inclusion bodies. No mutants with substitutions within the carboxyterminal segment were found to increase partitioning into inclusion bodies (R354K, R377K, and R392K). These results suggest that arginine residues that position between the N-terminus and central region can play an important role in facilitating folding or stabilizing the structure of D-aminoacylase. By anaerobic cultivation, the production level of R302K in the soluble fraction was improved. Coexpression of the DnaK-DnaJ-GrpE chaperone assisted the folding of R302K, and reduced the effect of the aeration conditions on the solubility of R302K. We hypothesized that R302K requires a larger amount of chaperones for efficient folding than the wild type enzyme..
83. Kenji Sakai, Yumiko Sadamitsu, Mami Sakurai, Kumiko Sakai, Release from Regulations of β-Glucosidase I Production in Bifidobacterium breve 203 by Acclimation to Cellobiose, Japan Journal of Food Engineering, 10.11301/jsfe2000.6.205, 6, 3, 205-208, 2005.01, Development of prebiotics and probiotics would be beneficial for human health, and several kinds of oligosaccharides have been commercially available recently, as food additives with a promoting effect of Bifidobacterial growth in gut. But few studies on regulation of oligosaccharide assimilation by the bacteria have been reported. In this report we have shown that utilization of cellobiose by Bifidobacterium breve 203 is under plural regulations: production of β-glucosidase I of the strain was repressed by glucose and induced by cellobiose, hardly assimilable oligosaccharide by this strain. While after long-term acclimation to cellobiose the strain came to be assimilable of cellobiose and a mutant showed higher β-glucosidase I activity than that of the parental strain constitutively, indicating both of the regulation have released in the acclimated mutant..
84. M. Taniguchi, T. Tokunaga, K. Horiuchi, K. Hoshino, Kenji Sakai, T. Tanaka, Production of L-lactic acid from a mixture of xylose and glucose by co-cultivation of lactic acid bacteria, Applied Microbiology and Biotechnology, 10.1007/s00253-004-1671-x, 66, 2, 160-165, 2004.12, The production of optically pure lactic acid in a high yield from xylose or a mixture of xylose and glucose, which is a model hydrolysate of lignocellulose, is described. In a single cultivation, Enterococcus casseliflavus produced 38 g/l of lactic acid with an optical purity of 96% enantiomeric excess (ee) and 6.4 g/l of acetic acid from 50 g/l of xylose when MRS medium was used. When a mixture of 50 g/l of xylose and 100 g/l of glucose was used as the carbon source in a cultivation of E. casseliflavus alone, glucose was converted to lactic acid in the early phase of the cultivation but xylose was hardly consumed. In a co-cultivation where E. casseliflavus and Lactobacillus casei specific for glucose were simultaneously inoculated, little or no lactic acid was produced after the glucose was almost consumed. A co-cultivation with two-stage inoculation (in which E. casseliflavus was added at a cultivation time of 40 h after L. casei cells were inoculated) resulted in complete consumption of 50 g/l of xylose and 100 g/l of glucose. In the co-cultivation, 95 g/l of lactic acid with a high optical purity of 96% ee was obtained at 192 h. Such a co-cultivation using two microorganisms specific for each sugar is considered to be one promising cultivation technique for the efficient production of lactic acid from a sugar mixture derived from lignocellulose..
85. Kenji Sakai, Masayuki Taniguchi, Shigenobu Miura, Hitomi Ohara, Toru Matsumoto, Yoshihito Shirai, Making plastics from garbage
A novel process for poly-L-lactate production from municipal food waste, Journal of Industrial Ecology, 10.1162/108819803323059406, 7, 3-4, 63-74, 2004.05, We propose a novel recycling system for municipal food waste that combines fermentation and chemical processes to produce high-quality poly-L-lactate (PLLA) biodegradable plastics. The process consists of removal of endogenous D,L-lactic acid from minced food waste by a propionibacterium, L-lactic acid fermentation under semisolid conditions, L-lactic acid purification via butyl esterification, and L-lactic acid polymerization via LL-lactide. The total design of the process enables a high yield of PLLA with high optical activity (i.e., a high proportion of optical isomers) and novel recycling of all materials produced at each step, with energy savings and minimal emissions. Approximately 50% of the total carbon was removed, mostly as L-lactic acid, and 100 kg of collected food waste yielded 7.0 kg PLLA (about 34% of the total carbon). The physical properties of the PLLA yielded in this manner were comparable to those of PLLA generated from commercially available L-lactic acid. Evaluation of the process is also discussed from the viewpoints of material and energy balances and environmental impact..
86. Mild Umeki, Kazutoshi Oue, Satoshi Mochizuki, Yoshihito Shirai, Kenji Sakai, Effect of Lactobacillus rhamnosus KY-3 and cellobiose as synbiotics on lipid metabolism in rats, Journal of Nutritional Science and Vitaminology, 10.3177/jnsv.50.330, 50, 5, 330-334, 2004.01, Lactobacillus rhamnosus KY-3 is a fermentative bacterium that is used for the industrial production of L-lactic acid, We have examined the effect of L. rhamnosus KY-3 and cellobiose as synbiotics on lipid metabolism in rats. Rats were fed on a 20% casein diet (C) supplemented with either 1.7% L rhamnosus KY-3 (KY-3). 10% cellobiose (CEB). or 1.7% L. rhamnosus KY-3 and 10% cellobiose (KY-3 + CEB) for 13d. The concentrations of serum total lipids. triacylglycerol, total cholesterol, and phospholipids were significantly reduced in rats fed a KY-3 + CEB diet in comparison to those on the C, KY-3 and CEB diets. There was an increase in the weight of cecal contents and a significant increase in the amount of cecal short-chain fatty acids (SCFA). The dry weight of excretion increased additively upon the simultaneous administration of L. rhamnosus KY-3 and cellobiose (KY-3 + CEB). The amount of excreted fecal bile acids did not differ among the groups in this study. These findings support the hypothesis that the promotion of cecal fermentation can lower the level of serum lipids. These results suggest that simultaneous administration of L. rhamnosus KY-3 and cellobiose as synbiotics has a beneficial effect on lipid metabolism..
87. Kenji Sakai, Masatsugu Mori, Akira Fujii, Yuko Iwami, Ekachai Chukeatirote, Yoshihito Shirai, Fluorescent in situ hybridization analysis of open lactic acid fermentation of kitchen refuse using rRNA-targeted oligonucleotide probes, Journal of Bioscience and Bioengineering, 10.1016/S1389-1723(04)70241-8, 98, 1, 48-56, 2004.01, Reproducible amounts of lactic acid accumulate in minced kitchen refuse under open conditions with intermittent pH neutralization [Sakai et al., Food Sci. Technol. Res., 6, 140 (2000)]. Here, we showed that such pH-controlled open fermentation of kitchen refuse reproducibly resulted a selective proliferation of a major lactic acid bacterial (LAB) species. In one experiment, the predominant microorganisms isolated during the early phase (6 h) were Gammaproteobacteria. In contrast, those that predominated during the late phase (48 h) were always Lactobacillus plantarum in three independent experiments. To further quantify the microbial community within open lactic acid fermentation, we performed fluorescent in situ hybridization (FISH) analysis targeting 16S (23S) rRNA. We designed two new group-specific DNA probes: LAC722(L) was active for most LAB including the genera Lactobacillus, Pediococcus, Leuconostoc and Weisella, whereas Lplan477 was specific for L. plantarum and its related species. We then optimized sample preparation using lysozyme and hybridization conditions including temperature, as well as the formamide concentration and the salt concentration in the washing buffer. We succeeded in quantification of microorganisms in semi-solid, complex biological materials sach as minced kitchen refuse by taking color microphotographs in modified RGB balance on pre-coated slides. FISH analysis of the fermentation of kitchen refuse indicated that control of the pH swing leads to domination by the LAB population in minced kitchen refuse under open conditions. We also confirmed that L. plantarum, which generates lactic acid in high quantities but with low optical activity, became the dominant microorganism in kitchen refuse during the late phase of open fermentation..
88. Kenji Sakai, Hiroyuki Kawano, Akihiko Iwami, Masakazu Nakamura, Mitsuaki Moriguchi, Isolation of a thermophilic poly-L-lactide degrading bacterium from compost and its enzymatic characterization, Journal of Bioscience and Bioengineering, 10.1016/S1389-1723(01)80266-8, 92, 3, 298-300, 2001.01, Thermophilic poly-L-lactide-degrading bacteria were isolated from a garbage fermentor. One of the isolates, strain PL21, was identified as Bacillus smithii based on its physiological properties, sugar assimilation pattern, and partial 16S rDNA sequence. The degradation activity of poly-L-lactide exibited by the culture fluid was parallel to the esterase activity, and the purified enzyme was active against various fatty acid esters and poly-L-lactide, at 60°C and pH 5..
89. Kenji Sakai, Hiroto Nisijima, Yoshihito Ikenaga, Mamoru Wakayama, Mitsuaki Moriguchi, Purification and characterization of nitrite-oxidizing enzyme from heterotrophic bacillus badius i-73, with special concern to catalase, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.64.2727, 64, 12, 2727-2730, 2000.01, Nitrite-oxidizing enzyme I (NiOx I) was purified from a heterotrophic bacterium, Bacillus badius I-73. The enzyme was a homotetramer of a heme-containing peptide, and was similar to catalases from various sources in its N-terminal amino acid sequence. The purified enzyme also catalyzed H2O2 degradation. The nitrite oxidation reaction required ascorbic acid and oxygen. Successive H2O2 feeding could be substituted for ascorbic acid. These indicated that NiOx I is a catalase and nitrite was oxidized by a peroxidase-like reaction..
90. Kenji Sakai, Yoshihiro Murata, Hiroshi Yamazumi, Yuko Tau, Masatsugu Mori, Mitsuaki Moriguchi, Yoshihito Shirai, Selective Proliferation of Lactic Acid Bacteria and Accumulation of Lactic Acid during Open Fermentation of Kitchen Refuse with Intermittent pH Adjustment, Food Science and Technology Research, 10.3136/fstr.6.140, 6, 2, 140-145, 2000.01, When minced and autodaved model kitchen refuse was inoculated with a small amount of non-autoclaved model kitchen refuse as seed culture, incubated at 37°C for 3-5 days and intermittently pH neutralized, 27-45 g/l of lactic acid was accumulated with a small amount of acetic acid and ethanol. The highest accumulation and highest productivity levels of lactic acid were observed at an initial and adjusted pH of 7.0 and a 6 h interval of pH adjustment. After several hours of lag, the lactic acid bacteria became the dominant cell type during the incubation, while the number of coliform bacteria and clostridia decreased. Such selective and stable accumulation of lactic acid was achieved hi dozens of different experiments with various refuse preparations. In contrast, with continuous pH adjustment, lactic acid once accumulated was labile and a small amount of butyric acid was produced, increasing the number of clostridia. The dominant bacteria isolated from the fermentation with intermittent pH adjustment were identified as Lactobacillus plantarum and L. brevis..
91. Kenji Sakai, Eiko Kudoh, Mamoru Wakayama, Mitsuaki Moriguchi, Analysis of the Microbial Community in an Activated Sludge Enriched with an Inorganic Nitrite Medium, Microbes and Environments, 10.1264/jsme2.2000.103, 15, 2, 103-112, 2000.01, To enrich nitrite-oxidizing bacteria, an activated sludge sample was transferred serially into an inorganic nitrite medium. Following the transfer, the culture maintained nitrite-oxidizing activity for over three months. Nitrite-oxidizing bacteria slightly decreased, but maintained their number at 104 MPN/ml, and 106-107 cfu/ml of heterotrophic bacteria were also detected. Random cloning and analysis of amplified 16S rDNA using a universal primer set for bacteria showed that a culturable Pseudomonas putida-related strain was dominant in the culture, though the bacterium did not oxidize nitrite. The most dominant bacterial group estimated from the proportion of clones that showed identical pattern of restriction fragment length polymorphism belonged to the γ-subdivision of Proteobacteria. This was partly consistent with the results from whole-cell hybridization using group-specific fluorescent probes. Further limiting dilutions of the enriched culture produced a nitrite-oxidizing system with low numbers of heterotrophs. Molecular analysis suggested that the members were different from those in the enriched culture, and several kinds of Proteobacteria belonging to the β-, γ-, and α-subdivision, as well as bacteria in the high G + C Gram-positive phylum, existed. But in 16S rDNA sequence, none showed close similarity to any known autotrophic nitrite oxidizers. These results indicated that the population in serially transferred culture and limiting dilution culture is rather diverse, with some heterotrophic bacteria, and suggested the occurrence of an unidentified species of nitrite-oxidizing bacteria..
92. Renu Nandakumar, Mamoru Wakayama, Yoshio Nagano, Tatsuro Kawamura, Kenji Sakai, Mitsuaki Moriguchi, Overexpression of salt-tolerant glutaminase from Micrococcus luteus K-3 in Escherichia coli and its purification, Protein Expression and Purification, 10.1006/prep.1998.1005, 15, 2, 155-161, 1999.01, A high-expression plasmid, pKSGHE3-1, containing the salt-tolerant glutaminase (EC from marine bacterium Micrococcus luteus K-3 was constructed, pKSGHE3-1 was made by inserting the DNA fragment (1.43 kb) containing the structural gene synthesized by polymerase chain reaction into the downstream region of the tac promoter of expression vector pKK223-3. The translational start codon was located 10 bases downstream of the Shine-Dalgarno sequence (AGGA) of pKK223-3. Escherichia coli JM109 transformed with pKSGHE3-1 exhibited more than 190-fold higher glutaminase activity than M. luteus K-3 under optimal culture conditions. The enzyme was purified to homogeneity through three column chromatography steps with a final yield of 17.1%. The recombinant enzyme showed the same enzymatic properties, including salt tolerance, as those of M. luteus K-3. This glutaminase expression system allows the production of sufficient quantities of glutaminase for basic structure-function studies including chemical modification and future X-ray crystallization analysis..
93. Mamoru Wakayama, Harutaka Yada, Shun Ichi Kanda, Shin Ichi Hayashi, Yukinori Yatsuda, Kenji Sakai, Mitsuaki Moriguchi, Role of conserved histidine residues in d-aminoacylase from alcaligenes xylosoxydans subsp. Xylosoxydans A-6, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.64.1, 63, 7, 1-8, 1999.01, D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was strongly inactivated by diethylpyrocarbonate (DEPC). An H67N mutant was barely active, with a kcat/Km 6.3×104 times lower than that of the recombinant wild-type enzyme, while the H67I mutant lost detectable activity. The H67N mutant had almost constant Km, but greatly decreased kcat. These results suggested that His67 is essential to the catalytic event. Both H69N and H69I mutants were overproduced in the insoluble fraction. The kcat/Km of H250N mutant was reduced by a factor of 2.5×104-fold as compared with the wild-type enzyme. No significant difference between H251N mutant and wild-type enzymes in the Km and kcat was found. The Zn content of H250N mutant was nearly half of that of wild-type enzyme. These results suggest that the His250 residue might be essential to catalysis via Zn binding..
94. Mamoru Wakayama, Eiichi Shiiba, Kenji Sakai, Mitsuaki Moriguchi, Purification and characterization of L-aminoacylase from Pseudomonas maltophila B1, Journal of Bioscience and Bioengineering, 10.1016/S0922-338X(97)85675-9, 85, 3, 278-282, 1998.01, The constitutive L-aminoacylase, which is used for optical resolution of DL-α-aminosuberic acid (DL-Asu), has been purified and characterized from Pseudomonas maltophila B1. The crude enzyme showed a specific activity of 0.062 units/mg for N-acetyl(Ac)-L-Asu. This value is very high compared with those from Aspergillus melleus, porcine kidney, and Bacillus stearothermophilus. Molecular masses of 108 kDa for the native enzyme and 50 kDa for the subunit were determined, indicating a dimer. The enzyme activity was optimal at pH 8.0 and at 55°C. The enzyme hydrolyzed N-acyl derivatives of various neutral L-amino acids and acidic L-amino acids, L-glutamate and L-Asu. The enzyme also had dipeptidase activity. The Km values for N-Ac-L-alanine and N-Ac-DL-Asu were determined at 2.32 and 12.7 mM, respectively. The apoenzyme was activated using Zn2+, Ca2+, and Co2+. Glyoxylate, DL-lactate, phenylboronic acid (PBA), butaneboronic acid (BBA), diethylpyrocarbonate (DEP), and phenylglyoxal (PGO) inhibited enzyme activity..
95. Kenji Sakai, Akira Yokota, Hajime Kurokawa, Mamoru Wakayama, Mitsuaki Moriguchi, Purification and characterization of three thermostable endochitinases of a noble Bacillus strain, MH-1, isolated from chitin-containing compost, Applied and Environmental Microbiology, 64, 9, 3397-3402, 1998.01, A thermophilic and actinic bacterium strain, MH-1, which produced three different endochitinases in its culture fluid was isolated from chitin- containing compost. The microorganism did not grow in any of the usual media for actinomyces but only in colloidal chitin supplemented with yeast extract and (2,6-O-dimethyl)-β-cyclodextrin. Compost extract enhanced its growth. In spite of the formation of branched mycelia, other properties of the strain, such as the formation of endospores, the presence of meso-diaminopimelic acid in the cell wall, the percent G+C of DNA (55%), and the partial 16S ribosomal DNA sequence, indicated that strain MH-1 should belong to the genus Bacillus. Three isoforms of endochitinase (L, M, and S) were purified to homogeneity and characterized from Bacillus sp. strain MH-1. They had different molecular masses (71, 62, and 53 kDa), pIs (5.3, 4.8, and 4.7), and N-terminal amino acid sequences. Chitinases L, M, and S showed relatively high temperature optima (75, 65, and 75°C) and stabilities and showed pH optima in an acidic range (pH 6.5, 5.5, and 5.5, respectively). When reacted with acetylchitohexaose [(GlcNAc)6], chitinases L and S produced (GlcNAc)2 at the highest rate while chitinase M produced (GlcNAc)3 at the highest rate. None of the three chitinases hydrolyzed (GlcNAc)2. Chitinase L produced (GlcNAc)2 and (GlcNAc)3 in most abundance from 66 and 11% partially acetylated chitosan. The p-nitrophenol (pNP)-releasing activity of chitinase L was highest toward pNP-(GlcNAc)2, and those of chitinases M and S were highest toward pNP-(GlcNAc)3. All three enzymes were inert to pNP-GlcNAc. AgCl, HgCl2, and (GlcNAc)2 inhibited the activities of all three enzymes, while MnCl2 and CaCl2 slightly activated all of the enzymes..
96. Mamoru Wakayama, Kazue Takashima, Yuko Tau, Sadatoshi Nakashima, Kenji Sakai, Mitsuaki Moriguchi, Spectrophotometric assay of D-aspartate and D-glutamate using D- aspartate oxidase with malate dehydrogenase and glutamate dehydrogenase, Analytical Biochemistry, 10.1006/abio.1997.2230, 250, 2, 252-253, 1997.08.
97. Kenji Sakai, Kazuhiko Nakamura, Mamoru Wakayama, Mitsuaki Moriguchi, Change in nitrite conversion direction from oxidation to reduction in heterotrophic bacteria depending on the aeration conditions, Journal of Bioscience and Bioengineering, 10.1016/S0922-338X(97)82785-7, 84, 1, 47-52, 1997.01, For investigation of the effects of aeration on nitrite- and nitrate-transforming activities of various heterotrophic bacteria, a series of coefficients of the oxygen absorption rate (Kd, 8-99 × 10-7 mol/ml·min·atm) in 500-ml shaking flasks were determined by varying plug types and culture volumes. Bacillus badius I-73, which neither shows denitrification activity nor utilize nitrate as a nitrogen source, consumed nitrite and accumulated nitrate at all Kd values at which experiments were conducted. In B. subtilis I-41, which does show denitrification activity, the manner of nitrite and nitrate conversion was influenced by the culture time and Kd, and the direction of conversion was changed from reduction to oxidation, as the Kd of the culture increased. Pseudomonas pavonaceae, another denitrification-positive strain, metabolized both nitrite and nitrate to more reduced compounds at low Kd, and the direction of conversion changed from reduction to oxidation at Kd = 20 × 1017 mol/ml·min·atm. Such switching behavior was also observed when P. pavonaceae was cultured continuously during variation of the aeration conditions with supply of pure oxygen. Many other denitrification-positive strains behaved similarly to P. pavonaceae, and showed their own critical Kd, the point at which the direction of nitrite metabolism changed. The results of intact-cell reaction experiments indicate that this switching might be caused by inhibition and repression of nitrite-reducing activity, and by stimulation of nitrite-oxidizing activity by oxygen..
98. Kenji Sakai, Yutaka Miyasako, Hiroshi Nagatomo, Hiroki Watanabe, Mamoru Wakayama, Mitsuaki Moriguchi, L-ornithine decarboxylase from Hafnia alvei has a novel L-ornithine oxidase activity, Journal of Biochemistry, 10.1093/oxfordjournals.jbchem.a021858, 122, 5, 961-968, 1997.01, A novel activity producing γ-aminobutyric acid (GABA) from L-ornithine in the presence of NAD(P)+ was found in the crude extract of L-ornithine-induced Hafnia alvei, in addition to L-ornithine decarboxylase (ODC) activity. The reaction system for the former activity consisted of two enzymes, L-ornithine oxidase (decarboxylating, OOD) and γ-aminobutyraldehyde (GABL) dehydrogenase (GDH). OOD catalyzed the conversion of L-ornithine into GABL, CO2, NH3, and H2O2 in the presence of O2, and GDH dehydrogenated GABL to GABA in the presence of NAD(P)+. OOD, purified to homogeneity, had a high ODC activity and the activity ratio of ODC to OOD was almost constant throughout the purification (ODC/OOD = 160:1). The molecular mass of the OOD was about 230 kDa, probably consisting of three identical subunits of a 77 kDa peptide, and OOD had an absorption maximum at 420 nm as well as at 278 nm, the specific absorption for an enzyme containing pyridoxal phosphate (PLP). The content of PLP was estimated at about 1 mol per subunit. OOD was specific to L-ornithine, and other L-amino acids and polyamines including putrescine were inert. The enzyme was activated by PLP, but not by pyridoxamine 5'-phosphate, FAD, FMN, or pyrroloquinoline quinone, and it was inactivated by hydrazine, semicarbazide, and hydroxylamine. The holoenzyme can be resolved to the apoenzyme by incubation with hydroxylamine, and reconstituted with PLP. These properties of OOD were almost the same as those of ODC separately purified to homogeneity from H. alvei. Zn2+ and Cu2+, butanedione, and sodium borohydride inhibited both OOD and ODC in a similar manner. The OOD reaction required O2 and only the ODC reaction proceeded under anaerobic conditions. The substitution of air for oxygen in the reaction vessel and the addition of catalase-H2O2 enhanced only the OOD reaction, resulting in an increase of the ratio of OOD/ODC to 1:30 and 1:4.1, respectively. These results suggested that OOD and ODC are identical and that the former is a side reaction of the latter in the presence of O2..
99. Mamoru Wakayama, Tetsuo Tsutsumi, Harutaka Yada, Kenji Sakai, Mitsuaki Moriguchi, Chemical Modification of Histidine Residue of N-Acyl-D-Glutamate Amidohydrolase from Pseudomonas sp. 5f-1;, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.60.650, 60, 4, 650-653, 1996.01, N-Acyl-D-glutamate amidohydrolase (D-AGase) from Pseudomonas sp. 5f-1 was inactivated by diethyl pyrocarbonate (DEP). The chemical modification by DEP showed a difference spectrum at 246 nm due to the N-carbethoxyhistidine residue. Removal of the carbethoxy group from inactivated enzyme with hydroxylamine restored enzyme activity. The inactivation by DEP proceeded with pseudo-first-order kinetics, and was protected in the presence of the substrate N-acetyl-D-glutamate (Glu), or the competitive inhibitor sodium α-ketoglutarate (α-KGA). These results suggest the presence of an essential histidine residue at or near of the active site of the enzyme..
100. Mamoru Wakayama, Yoshio Nagano, Nandakumar Renu, Tatsuro Kawamura, Kenji Sakai, Mitsuaki Moriguchi, Molecular cloning and determination of the nucleotide sequence of a gene encoding salt-tolerant glutaminase from Micrococcus luteus K-3, Journal of Bioscience and Bioengineering, 10.1016/S0922-338X(97)81259-7, 82, 6, 592-597, 1996.01, The gene encoding the salt-tolerant glutaminase I from marine Micrococcus luteus K-3 (M. luteus K-3) was cloned in Escherichia coli (E. coli) JM109. Clones were screened by hybridization with degenerate oligonucleotide probes designed using the known N-terminal amino acid sequence of glutaminase I from M. luteus K-3. A 2.4-kb HincII fragment from a 8-kb primary cloned DNA fragment was subcloned and sequenced. This fragment had an open reading frame of 1,368 nucleotides encoding 456 amino acids. The molecular weight of the deduced amino acid sequence of glutaminase I was determined to be 48,247. Comparison of the amino acid sequence of glutaminase I with those of kidney, brain, liver, and Caenorhabditis elegans glutaminases revealed high degrees of homology in several local regions..
101. Mamoru Wakayama, Shin ichi Hayashi, Yukinori Yatsuda, Yutaka Katsuno, Kenji Sakai, Mitsuaki Moriguchi, Overproduction of D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 in Escherichia coli and its purification, Protein Expression and Purification, 10.1006/prep.1996.0059, 7, 4, 395-399, 1996.01, We constructed the high-expression plasmid for D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. The appropriate Shine- Dalgarno sequence (AAGGAG) was introduced to the eight bases upstream of start codon (ATG) of D-aminoacylase structural gene by site- directed mutagenesis, and then the 1.75-kb DNA fragment including the open reading frame was inserted into the downstream of the tac promoter of plasmid vector pKK223-3. The resultant plasmid, which was named pKNSD2, showed a high D- aminoacylase activity in Escherichia coli JM109 cells transformed with it. The enzyme was purified to homogeneity in only two steps with a final yield of 24% (sp act, 2023 U/mg)..
102. Mamoru Wakayama, Yuka Takeuchi, Katsuyuki Tasaka, Kenji Sakai, Mitsuaki Moriguchi, Production of D-amino acid oxidase from Aspergillus sojae, Journal of Bioscience and Bioengineering, 10.1016/0922-338X(96)85045-8, 82, 2, 177-179, 1996.01, D-Amino acid oxidase activities for D-glutamate (D-Glu), D-aspartate (D- Asp) and D-alanine (D-Ala) were found in cell-free extract of Aspergillus sojae (A. sojae). The enzyme activities for these three substrates increased over 30-fold by the addition of 0.25% D-Ala to the culture medium. Glycerol was an effective carbon source for increasing the enzyme activities. D-Ala, D-serine (D-Ser), and D-tryptophan (D-Trp) were better inducers than other D- amino acids. D-Glu and D-Asp were oxidized at rates of 70 and 6%, respectively, relative to the rate of oxidation of D-Ala which was taken as 100%. A. sojae D-amino acid oxidase showed no inhibition by sodium benzoate or dicarboxylates and had a molecular weight of 129,000, which differed substantially from those of D-amino acid oxidases of porcine and rabbit kidney..
103. Kenji Sakai, Yoshitomo Ikehata, Yoshihiro Ikenaga, Mamoru Wakayama, Mitsuaki Moriguchi, Nitrite oxidation by heterotrophic bacteria under various nutritional and aerobic conditions, Journal of Bioscience and Bioengineering, 10.1016/S0922-338X(97)81265-2, 82, 6, 613-617, 1996.01, The nitrite transforming activities of heterotrophic bacteria from culture collections and isolates from activated sludge were studied under various nutritional and aerobic conditions. Among the 48 organisms tested, 17 strains, many of which are reported as denitrification negatives, consumed 1-5 mM of nitrite and accumulated corresponding amounts of nitrate. Twelve strains, many of which are denitrification-positive, consumed nitrite with the accumulation of less nitrate, while more than 1 mM nitrite was consumed but little nitrate was accumulated by 14 strains, many of which are Enterobacteriaceae or lactic acid bacteria. None of the organisms formed significant nitrate in the medium without nitrite, though a considerable amount of ammonia was also accumulated by most strains. Although the growth and nitrate accumulation of Bacillus badius I-73 was affected by the concentrations of sodium nitrite and peptone and by the culture volume, the amount of nitrate accumulated was always proportional to that of the nitrite consumed. Intact cells of B. badius I-73 produced almost the same amount of nitrate as the decrease in nitrite. On the other hand, in B. subtilis I-41, a denitrification-positive isolate, the ratio of the amount of nitrate accumulated to that of nitrite consumed varied from 0-100% depending on the culture conditions..
104. Mamoru Wakayama, Eiki Watanabe, Yasuhiro Takenaka, Yoshiro Miyamoto, Yuko Tau, Kenji Sakai, Mitsuaki Moriguchi, Cloning, expression, and nucleotide sequence of the N-acyl-d-aspartate amidohydrolase gene from Alcaligenes xylosoxydans subsp. xylosoxydans A-6, Journal of Bioscience and Bioengineering, 10.1016/0922-338X(95)94197-Y, 80, 4, 311-317, 1995.01, The gene (termed daa) encoding N-acyl-d-aspartate (d-Asp) amidohydrolase (d-AAase) from the Alcaligenes xylosoxydans subsp. xylosoxydans (Alcaligenes A-6) was cloned in Escherichia coli (E. coli) JM109. The daa gene consists of 1,494 nucleotides and encodes 498 amino acid residues. The molecular weight of d-AAase was calculated to be 53,581. The N-terminal amino acid sequence (NH2-TDRSTLDDAP-) predicted by the nucleotide sequence matched exactly those of the purified d-AAase from both Alcaligenes A-6 and cloned E. coli, with the exception of the removal of the N-terminal methionine processed after translation. A comparison of the amino acid sequence of d-AAase with that of d-aminoacylase from Alcaligenes A-6 showed high overall homology (56%). d-AAase from Alcaligenes A-6 showed 25∼29% homology with Bacillus stearothermophilus, porcine, and human l-aminoacylases. The daa was highly expressed in E. coli, and the recombinant enzyme was purified to homogeneity with 17.8% yield..
105. Mamoru Wakayama, Yasushi Miura, Koji Oshima, Kenji Sakai, Mitsuaki Moriguchi, Metal-characterization of N-Acyl-D-glutamate Amidohydrolase from Pseudomonas sp. Strain 5f-1, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.59.1489, 59, 8, 1489-1492, 1995.01, N-Acyl-D-glutamate amidohydrolase (D-AGase) from Pseudomonas sp. 5f-1 was a zinc-metalloenzyme which contained 2.06-2.61 g·atom of Zn per mole of enzyme. The zinc atom was required for the catalytic activity and stability of the enzyme. The N-terminal amino acid sequence of Pseudomonas sp. 5f-1 D-AGase showed 32% identity to that of Alcaligenes xylosoxydans subsp. xylosoxydans A-6..
106. Mamoru Wakayama, Toshiyuki Ashika, Yoshiro Miyamoto, Tomoya Yoshikawa, Yuji Sonoda, Kenji Sakai, Mitsuaki Moriguchi, Primary structure of N-acyl-D-glutamate amidohydrolase from Alcaligenes xylosoxydans subsp. Xylosoxydans A-6, Journal of Biochemistry, 10.1093/oxfordjournals.jbchem.a124879, 118, 1, 204-209, 1995.01, The gene coding the N-acyl-D-glutamate amidohydrolase of Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was cloned and its complete DNA sequence was determined. The N-acyl-D-glutamate amidohydrolase structural gene consists of 1, 464 nucleotides and encodes 488 amino acid residues. The molecular weight of the enzyme was calculated to be 51, 490. This value is close to the apparent molecular weight of 59, 000 determined for the purified enzyme from Alcaligenes A-6 by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). The N-terminal amino acid sequence of the recombinant protein exactly matches the amino acid sequence derived from the DNA sequence and that determined from the Alcaligenes A-6 enzyme (NH2-MQEKLDLVTEGGW-VTDGLGG). The deduced amino acid sequence of the cloned N-acyl-D-glutamate amidohydrolase showed high sequence homology with those of N-acyl-D-aspartate amidohydrolase (46%) and D-aminoacylase (47%) from Alcaligenes A-6. This fact strongly suggests that these three enzymes have evolved from a common ancestral gene..
107. Mamoru Wakayama, Yutaka Katsuno, Shin ichi Hayashi, Yoshiro Miyamoto, Kenji Sakai, Mitsuaki Moriguchi, Cloning and Sequencing of a Gene Encoding D-Aminoacylase from Alcaligenes xylosoxydans subsp. Xylosoxydans A-6 and Expression of the Gene in Escherichia Coli, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.59.2115, 59, 11, 2115-2119, 1995.01, The gene encoding the D-aminoacylase of Alcaligenes xylosoxydans subsp. xylosoxydans A-6(Alcaligenes A-6) was cloned and its complete nucleotide sequence was identified. The D-aminoacylase structural gene consists of 1452 nucleotides and encodes 484 amino acid residues. The molecular weight of D-aminoacylase was calculated to be 51, 918. This value agreed well with the apparent molecular weight of 52, 000 found for the purified enzyme from Alcaligenes A-6 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The N-terminal amino acid sequence (NH2-SQSDSQPFDLLRAG-) predicted by the nucleotide sequence exactly matched those of the purified D-aminoacylase both from Alcaligenes A-6 and from cloned Escherichia coli (E. coli), with the exception of the removal of the N-terminal methionine processed after translation. The purified recombinant enzyme showed almost the same enzymatic properties as the native enzyme from Alcaligenes A-6. Alcaligenes A-6 D-aminoacylase showed 25-29% homology with L-aminoacylases from Bacillus stearothermophilus, porcine, and humans..
108. Mitsuaki Moriguchi, Kenji Sakai, Ryoji Tateyama, Yoichi Furuta, Mamoru Wakayama, Isolation and characterization of salt-tolerant glutaminases from marine Micrococcus luteus K-3, Journal of Bioscience and Bioengineering, 10.1016/0922-338X(94)90143-0, 77, 6, 621-625, 1994.01, Marine Micrococcus luteus K-3 constitutively produced two salt-tolerant glutaminases, designated glutaminase I and II. Glutaminase I was homogeneously purified about approximately, 1620-fold with a 4% yield, and was a dimer with a molecular weight of about 86,000. Glutaminase II was partially purified about 190-fold with a 0.04% yield. The molecular weight of glutaminase II was also 86,000. Maximum activity of glutaminase I was observed at pH 8.0, 50°C and 8-16% NaCl. The optimal pH and temperature of glutaminase II were 8.5 and 50°C. The activity of glutaminase II was not affected by the presence of 8 to 16% NaCl. The presence of 10% NaCl enhanced thermal stability of glutaminase I. Both enzymes catalyzed the hydrolysis of l-glutamine, but not its hydroxylaminolysis. The Km values for l-glutamine were 4.4 (glutaminase I) and 6.5 mM (glutaminase II). Neither of the glutaminases were activated by the addition of 2 mM phosphate or 2 mM sulfate. p-Chloromercuribenzoate (0.01 mM) significantly inhibited glutaminase I, but not glutaminase II. The conserved sequences LA**V and V**GGT*A were observed in the N-terminal amino acid sequences of glutaminase I, similar to that for other glutaminases..
109. Mamoru Wakayama, Sadatoshi Nakashima, Kenji Sakai, Mitsuaki Moriguchi, Isolation, enzyme production and characterization of d-aspartate oxidase from Fusarium sacchari var. elongatum Y-105, Journal of Bioscience and Bioengineering, 10.1016/0922-338X(94)90284-4, 78, 5, 377-379, 1994.01, A microorganism that produces d-aspartate-oxidizing enzyme by induction was isolated from soil, and identified as Fusarium sacchari var. elongatum Y-105. The enzyme catalyzed the oxidative deamination of d-aspartate (d-Asp) and produced oxaloacetate, ammonia, and hydrogen peroxide, stoichiometrically. The enzyme is designated "d-Asp oxidase" (EC In addition to d-Asp, the enzyme oxidized d-glutamate (d-Glu) and N-methyl-d-aspartate (NMDA). N-Acetyl-d-Asp and other d- or l-amino acids, however, were inert as substrates. The optimum pH and temperature were 7.5 and 40°C, respectively. The enzyme was stable at pH 9.0 and temperature of 50°C, respectively. The enzyme activity was not inhibited by sodium benzoate which is a specific inhibitor of d-amino acid oxidase from mammals. The enzyme activity was also not affected by carboxylates such as meso- or d-tartarate, citrate, and fumarate which inhibit d-Asp oxidase from rabbits..
110. Kenji Sakai, M. Narihara, Y. Kasama, M. Wakayama, M. Moriguchi, Purification and characterization of thermostable β-N- acetylhexosaminidase of Bacillus stearothermophilus CH-4 isolated from chitin-containing compost, Applied and Environmental Microbiology, 60, 8, 2911-2915, 1994.01, Thermostable exochitinase was purified to homogeneity from the culture fluid of Bacillus stearothermophilus CH-4, which was isolated from agricultural compost containing shrimp and crabs. The enzyme was a single polypeptide with a molecular mass of 74 kDa, and the N-terminal amino acid sequence was WDKVGVTDLI ISLNIPEADAVVVGMTLQLQALHLY. The enzyme specifically hydrolyzed C-4 β-anomeric bonding of N-acetylchitooligosaccharides, as well as their p-nitrophenyl (pNP) derivatives. The enzyme also hydrolyzed pNP-β- N-acetyl-D-galactosaminide (26% of the activity of pNP-β-B-acetyl-D- glucosaminide). These results indicated that the enzyme is a β-N- acetylhexosaminidase (EC K(m)s for acetylchitooligosaccharides were 1 x 10-4 to 6 x 10-4 M, while those for the pNP derivatives were 4 x 10-3 to 8 x 10-3 M. The optimum temperature of the enzyme was 75°C, and it retained 100 and 28% reactivity after heating at 60 and 80°C, respectively. The enzyme exhibited 15 to 20% activity in a reaction mixture containing 80% organic solvents and maintained 91% of its original activity after exposure to 8 M urea. The optimum and stable pH was around 6.5, Fe2+, Zn2+, and Ca2+ activated the enzyme, but Hg2+ was inhibitory. N- Acetyl-D-glucosamine inhibited the enzyme competitively (K(i) = 4.3 x 10-4 M), whereas N-acetyl-D-galactosamine did not; in contrast, D-glucosamine and D-galactosamine activated it..
111. Mitsuaki Moriguchi, Kenji Sakai, Yoshiro Miyamoto, Mamoru Wakayama, Production, Purification, and Characterization of D-Aminoacylase from Alcaligenes xylosoxydans subsp. Xylosoxydans A-6, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.57.1149, 57, 7, 1149-1152, 1993.04, The best inducers for D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6(Alcaligenes A-6) were a poor substrate, N-acetyl-γ-methyl-D-leucine, and an inhibitor, N-acetyl-D-alloisoleucine. The enzyme has been homogeneously purified. The molecular weight of the native enzyme was estimated to be 58, 000 by gel filtration. A subunit molecular weight of 52, 000 was measured by SDS-PAGE, indicating that the native protein is a monomer. The isoelectric point was 5.2. The enzyme was specific to the D-isomer and hydrolyzed N-acetyl derivatives of D-leucine, D-phenylalanine, D-norleucine, D-methionine, and D-valine, and also N-formyl, N-butyryl, and N-propionyl derivatives of D-leucine. The Km for N-acetyl-D-leucine was 9.8 mM. The optimum pH and temperature were 7.0 and 50°C, respectively. The stabilities of pH and temperature were 8.1 and 40°C. D-Aminoacylases from three species of the genus Alcaligenes differ in inducer and substrate specificities, but are similar with respect to molecular weight and N-terminal amino acid sequence..
112. Mitsuaki Moriguchi, Kenji Sakai, Yutaka Katsuno, Tetsuyoshi Maki, Mamoru Wakayama, Purification and Characterization of Novel N-Acyl-D-aspartate Amidohydrolase from Alcaligenes xylosoxydans subsp. Xylosoxydans A-6, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.57.1145, 57, 7, 1145-1148, 1993.01, Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) produced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N-acetyl-D-aspartate as an inducer. The enzyme was purified to homogeneity. The enzyme had a molecular mass of 56 kDa and was shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to be a monomer. The isoelectric point was 4.8. The enzyme had maximal activity at pH 7.5 to 8.0 and 50°C, and was stable at pH 8.0 and up to 45°C. N-Formyl (Km=12.5mM), N-acetyl (Km=2.52mM), N-propionyl (Km=0.194mM), N-butyryl (Km=0.033mM), and N-glycyl (Km=1.11mM) derivatives of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glutamate were not substrates. The enzyme was inhibited by both divalent cations (Hg2+, Ni2+, Cu2+) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-terminal amino acid sequence and amino acid composition were analyzed..
113. Kenji Sakai, Kazuyuki Imamura, Yuji Sonoda, Hiroyuki Kido, Mitsuaki Moriguchi, Purification and characterization of N-acyl-D-glutamate deacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6., FEBS Letters, 10.1016/0014-5793(91)80904-H, 289, 1, 44-46, 1991.09, The purification and properties of N-acyl-D-glutamate deacylase from the cell extracts of Alcaligenes xylosoxydans subsp. xylosoxydans A-6 were studied. The two active fractions (peaks I and II) were obtained by a Mono Q column chromatography. The predominant enzyme (peak I) has been purified, 1960-fold to homogeneity and characterized. The enzyme was a monomer with a molecular weight of 59 000. The optimum pH and the isoelectric point were 8.0 and 5.5, respectively. The enzyme catalyzed the hydrolysis of N-acyl derivatives of D-glutamate. The Kms for N-acetyl, N-butyryl and N-propionyl derivatives of D-glutamate were 0.129, 0.066 and 0.01 mM, respectively..
114. Kenji Sakai, Taketeru Obata, Kohtaro Ideta, Mitsuaki Moriguchi, Purification and properties of d-aminoacylase from Alcaligenes denitrificans subsp. xylosoxydans MI-4, Journal of Bioscience and Bioengineering, 10.1016/0922-338X(91)90227-8, 71, 2, 79-82, 1991.01, d-Aminoacylase has been purified 144-fold to electrophoretic homogeneity by ammonium sulfate fractionation, DEAE-Toyopearl and affinity column chromatographies, and Sephadex G-100 gel filtration from the crude extracts of Alcaligenes denitrificans subsp. xylosoxydans MI-4. The enzyme was composed of a single polypeptide of about 51,000. The enzyme catalyzed hydrolysis of N-acyl-derivatives of neutral d-amino acids. Optimal pH and temperature were 7.8 and 50°C. The apparent Km and the Vmax for N-acetyl-d-phenylalanine were 14.1 mM and 1331 units/mg protein, respectively. The activity of the enzyme was inhibited by N-acetyl-d-valine (Ki=2.15 mM) and N-acetyl-d-alloisoleucine (Ki=1.47 mM), but not by its products (i.e., amino acids and acetate). The enzyme also had dipeptidase activity. Activation by metal ions was not observed..
115. Kenji Sakai, K. Oshima, M. Moriguchi, Production and characterization of N-acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1, Applied and Environmental Microbiology, 57, 9, 2540-2543, 1991.01, N-Acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1 was inducibly produced by D isomers of N-acetylglutamate, glutamate, aspartate, and asparagine. The enzyme has been purified to homogeneity by DEAE-cellulose, (NH4)2SO4 fractionation, and chromatofocusing followed by gel filtration on a Sephadex G-100 column. The enzyme was a monomer with molecular weight of 55,000. The enzyme activity was optimal at pH 6.5 to 7.5 and 45°C. The isoelectric point and the pH stability were 8.8 and 9.0, respectively. N-Formyl, N-acetyl, N-butyryl, N-propionyl, N-chloroacetyl derivatives of D-glutamate and glycyl-D-glutamate were substrates for the enzyme. At pH 6.5 in 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at 30°C, a K(m) of 6.67 mM and a V(max) of 662 μmol/min/mg of protein for N-acetyl-D-glutamate were obtained. None of the metal ions stimulated the enzyme activity. Na+, K+, Mg2+, and Ba2+ acted as stabilizers. Hg2+, Cu2+, Zn2+, Fe3+, and EDTA were strongly inhibitory..
116. Kenji Sakai, Kazuyuki Imamura, Makoto Goto, Isamu Hirashiki, Mitsuaki Moriguchi, Occurrence of Novel Enzymes, N-Acetyl-D-glutamate Deacetylase and N-Acetyl-D-aspartate Deacetylase, in Alcaligenes xylosoxydans Subsp. Xylosoxydans A-6, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb1961.54.841, 54, 3, 841-844, 1990.03, N-Acetyl-D-glutamate deacetylase and N-acetyl-D-aspartate deacetylase were found in cell extracts from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. N-Acetyl-D-glutamate deacetylase was produced inducibly by N-acetyl-D-glutamate and was highly specific to N-acetyl-D-glutamate. N-Acetyl-D-aspartate deacetylase was produced inducibly by N-acetyl-D-aspartate and was highly specific to N-acetyl-D-aspartate..
117. Kenji Sakai, Taketeru Obata, Susumu Takano, Mitsuaki Moriguchi, A Novel Inducer, y-Methyl-D-leucine, of D-Aminoacylase from Alcaligenes denitrificans subsp. xylosoxydans MI-4, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb1961.53.2285, 53, 8, 2285-2286, 1989.09.
118. Kenji Sakai, Takashi Tachiki, Hidehiko Kumagai, Tatsurokuro Tochikura, Synthesis of β-D-Fucosylglucose by β-D-Glucosidase I of Bifidobacterium breve clb and Assimilation by Bifidobacteria, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb1961.53.313, 53, 2, 313-318, 1989.01, The p-nitrophenol-releasing activity, from/Miitrophenyl (p-NP) β-d-fucoside, of β-d-glucosidase I from Bifidobacterium breve clb was enhanced by the addition of many kinds of sugars and alcohols, suggesting the occurrence of a β-d-fucosyl transferring reaction. The enhancement on glucose addition was dependent on the reaction pH, and the concentrations of p-NP β-d-fucoside and glucose, and the activity reached 430 % when 100 mM glucose was added to the mixture containing 20 mM p-NP β-d-fucoside at pH 4.5. A mixture of transfer products was separated from the other constituents by activated charcoal column chromatography. Further purification of the mixture by paper and thinlayer chromatographies gave four oligosaccharides, which were identified as β-d-fucosylglucoses with β 1→2, β 1→3, β 1→4 and β 1→6, linkages from the absorption spectra obtained with the phenolsulfuric acid method, and the results of acid and enzymatic hydrolyses, and methylation analyses. The mixture of oligosaccharides was well assimilated by many bifidobacteria but not by any other intestinal bacteria tested..
119. Kenji Sakai, Tatsurokuro Tochikura, Koji Takano, Takashi Tachiki, Purification and Properties of an Enzyme Oxidizing Nitrite to Nitrate form Candida rugosa, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb1961.52.2783, 52, 11, 2783-2789, 1988.04, A nitrite-oxidizing enzyme was isolated (1300-fold purification, 15% yield) from Candida rugosa IFO 0591 using a reaction mixture containing glucose oxidase and glucose. The enzyme (molecular weight 220, 000) consisted of four identical subunits with molecular weight of 58, 000. It showed absorption maxima at 277 and 405 nm with small peaks at 492, 535, and 625 nm. The spectrum was not altered by the addition of dithionite. These and other properties suggested that the enzyme was catalase and that the nitrite-oxidizing reaction was dependent on its peroxidase activity. Some aspects of the nitrite oxidation by the purified enzyme and various preparations of C. rugosa are described..
120. Takashi Tachiki, Kenji Sakai, Katsu Yamamoto, Masayuki Hatanaka, Tatsurokuro Tochikura, Conversion of Nitrite of Nitrate by Nitrite-Resistant Yeasts, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb1961.52.1999, 52, 8, 1999-2005, 1988.03, Candida species YK 11 and YK 92 and Geotrichum candidum YK 57, which were isolated as nitrite-resistants, converted nitrite in the culture medium to nitrate stoichiometrically during growth. The nitrite-oxidizing reaction was confirmed under aerobic conditions in the intact cell system with 15 mMnitrite, 150 mm glucose, and 100 mm Tris-HCl buffer (pH 7.0). Glucose or other carbohydrate which supported the microbial growth was indispensable for the reaction. The rate of oxidation (0.9~1.3 } 105 µg-N/g of YK 92 cells-day) and the maximum amounts of nitrate formed in the culture medium (200 mm, 2800 µg-N/ml) were much larger than those of other heterotrophic nitrifiers and almost the same as those of Nitrobacter. The nitrite-oxidizing activity was demonstrated in many types of yeast species..
121. Kenji Sakai, Takashi Tachiki, Hidehiko Kumagai, Tatsurokuro Tochikura, Hydrolysis of α-D-Galactosyl Oligosaccharides in Soymilk by α-D-Galactosidase of Bifidobacterium breve 203, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb1961.51.315, 51, 2, 315-322, 1987.03, Bifidobacterium breve 203 grew well on soymilk, reaching a cell number of 2x 109 cells/ml on 24 hr cultivation, at which time the medium had solidified and its pH had decreased to 4.0. Stachyose and raffinose in the medium were assimilated in preference to sucrose. A crude extract of B. breve 203 hydrolyzed the oligosaccharides in soymilk almost completely into their constituent monosaccharides. The a-D-galactosidase responsible for the first reaction in the degradation of the a-D-galactosyl oligosaccharides was isolated from a crude extract of the organism (500-fold purification, 2% yield) by means of ammonium sulfate-fractionation and column chromatographies on DEAE-cellulose, DEAE-cellulofine, Sepharose 6B, hydroxylapatite, Sephacryl S-300 and Phenyl-Sepharose 6B. The enzyme was a homo-octamer with a molecular weight of about 330,000, and its isoelectric point was 3.7. It reacted optimally at pH 5.5 and hydrolyzed stachyose (Km 8.6 mM), raffinose (Km 4.4 mM) and melibiose (Km 3.0 mM), with 50 ~ 60% of its reactivity toward p-nitrophenyl a-D-galactoside (Km 0.16mM). The reaction of the purified a-D-galactosidase with soymilk and guar gum was also investigated..
122. Kenji Sakai, Chizuru Mishima, Takashi Tachiki, Hidehiko Kumagai, Tatsurokuro Tochikura, Mortality of bifidobacteria in boiled yogurt, Journal of Bioscience and Bioengineering, 10.1016/0385-6380(87)90167-1, 65, 2, 215-220, 1987.01, Bifidobacterial strains showed various mortalities during storage at 30°C in boiled yogurt prepared with Streptococcus sp. and Lactobacillus sp. Bifidobacterium breve 203 was most stable, maintaining its initial cell number for more than 5 days. It was also stable at 4 or 10°C in fresh yogurt. B. longum 401 rapidly lost viability in the boiled yogurt and was unstable in the fresh yogurt at any temperature. Lactobacillus sp. remained fully viable for one week or more at 4-30°C while Streptococcus sp. lost viability at 20°C or above. The difference in mortality between B. breve 203 and B. longum 401 was mainly due to their sensitivity to the acidic environment, with temperature during storage having a secondary effect. Effects of lysozyme, pepsin and bile acids on the two strains were also investigated..
123. Kenji Sakai, Masaru Tanaka, Takashi Tachiki, Hidehiko Kumagai, Tatsurokuro Tochikura, Increase in D-Glucoside-assimilating Ability of Bifidobacterium breve 203 Due to Acclimation to jS-D-Glucoside, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb1961.51.699, 51, 3, 699-705, 1987.01, Bifidobacterium breve 203, an isolate which assimilates weakly, came to grow well on cellobiose (B. breve clb strain) or gentiobiose (B. breve gnt strain) after successive transfers in medium containing cellobiose or gentiobiose as a carbon source. The elevation of the assimilating ability of B. breve clb, which might reflect both higher cellobiose consuming- and I activities than those of the original B. breve 203, was specific to cellobiose. In the case of B. breve gnt, the elevated assimilating ability was specific to gentiobiose, with higher gentiobiose consuming-and II activities. Subsequent transfers (10 times) of B. breve clb in medium with glucose as a carbon source resulted only in a decrease in cellobiose consumption by the intact cells, while similar treatment of B. breve gnt resulted in decreases in both gentiobiose consumption and II activity. I partially purified from B. breve clb grown on glucose as a carbon source showed the same properties as the enzyme from B. breve 203 in molecular weight, optimum pH for the reaction, effects of divalent cations and sulfhydryl reagents, substrate specificity, activity and so on..
124. Kenji Sakai, Takashi Tachiki, Hidehiko Kumagai, Tatsurokuro Tochikura, Isolation and Characterization of Two β-D-Glucosidases from Bifidobacterium breve 203, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb1961.50.2287, 50, 9, 2287-2293, 1986.01, Two β-D-glucosidases were purified to homogeneity from Bifidobacterium breve 203: one β-D-glucosidase I; molecular weight, 96,000) showed reactivity toward p-nitrophenyl O-NP) P-D-fucoside, 74% of that to p-NP β-D-glucoside, and the other (β-D-glucosidase II; molecular weight, 450,000) did not. They also differed in their thermal and pH stabilities. Laminaribiose, cellobiose and gentiobiose were hydrolyzed by β-D-glucosidase I, with 53%, 34% and 3% of the reactivity in the case of p-NP β-D-glucoside, and by β-D-glucosidase II, with 53%, 6% and 107% of the reactivity. The reaction of β-D-glucosidase I with p-NP β-D-fucoside was enhanced by the addition of glucose and other monosaccharides to the reaction mixture, whereas that with p-NP β-D-glucoside was not affected. The activity of β-D-glucosidase II with p-NP β-D-glucoside was inhibited by glucose..
125. Tatsurokuro Tochikura, Kenji Sakai, Takako Fujiyoshi, Takashi Tachdci, Hidehiko Kumagai, ρ-Nitrophenyl Glycoside-hydrolyzing Activities in Bifidobacteria and Characterization of β-D-Galactosidase of Bifidobacterium longum 401, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb1961.50.2279, 50, 9, 2279-2286, 1986.01, Bifidobacteria showed higher hydrolyzing activity toward various p-nitrophenyl glycosides (p-NP glycosides) than some other intestinal bacteria. The reactions commonly found in the organisms involved p-NP β-D-galactoside, ρ-NP a-D-glucoside and p-NP β-D-fucoside. Analysis of the enzyme species suggested that the β-D-fucoside-hydrolyzing reaction, which is undetectable in other bacteria, was catalyzed by β-D-galactosidase in many bifidobacteria and by- β-D-glucosidase in some strains. β-D-Galactosidase, which hydrolyzed ρ-NP β-D-fucoside (with 12% of its reactivity to p-NP β-D-galactoside), was purified to homogeneity from Bifidobacterium longum 401. The enzyme was distinct from other bacterial β-D-galactosidases in its higher activity toward lactulose than lactose and the insensitivity of its formation to the carbon source in the culture medium. Some properties of the β-D-galactosidase are described and compared with those of the lactase from the same organism..