||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..
||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..
||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..
||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..
||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..
||Shun ichi Baba, Yukihiro Tashiro, Hideaki Shinto, Kenji Sonomoto, Development of high-speed and highly efficient butanol production systems from butyric acid with high density of living cells of Clostridium saccharoperbutylacetonicum, Journal of Biotechnology, 10.1016/j.jbiotec.2011.06.004, 157, 4, 605-612, 2012.02, Living cells are alive and have the butanol-producing ability but not much proliferation under nitrogen source-limited condition. We investigated various butanol production systems with high density of living cells of Clostridium saccharoperbutylacetonicum N1-4 supplemented with methyl viologen (MV) as an electron carrier and nutrient dosing for activity regeneration. In continuous butanol production with high density of living cells, butanol yield was drastically increased from 0.365C-mol/C-mol with growing cells to 0.528C-mol/C-mol at a dilution rate of 0.85h -1, being increased with the butanol to total solvent ratio. This yield was increased to 0.591C-mol/C-mol by adding 0.01mM MV. MV addition increased not only butanol yield but also butanol concentration and productivity as compared to those without MV addition. However, living cells lost their activity with incubation time, which lowered the operational stability of the system. Therefore, to maintain constant stability, activity regeneration was carried out with high density of living cells and MV. This system produced butanol at high concentration (9.40gl -1) and productivity (7.99gl -1h -1) for approximately 100h with maintenance of considerably high yield of butanol (0.686C-mol/C-mol). Thus, we established a high-speed and highly efficient butanol production system..
||Yukihiro Tashiro, Wataru Kaneko, Yanqi Sun, Keisuke Shibata, Kentaro Inokuma, Takeshi Zendo, Kenji Sonomoto, Continuous D-lactic acid production by a novelthermotolerant Lactobacillus delbrueckii subsp. lactis QU 41, Applied Microbiology and Biotechnology, 10.1007/s00253-010-3011-7, 89, 6, 1741-1750, 2011.03, We isolated and characterized a d-lactic acid-producing lactic acid bacterium (d-LAB), identified as Lactobacillus delbrueckii subsp. lactis QU 41. When compared to Lactobacillus coryniformis subsp. torquens JCM 1166T and L. delbrueckii subsp. lactis JCM 1248T, which are also known as d-LAB, the QU 41 strain exhibited a high thermotolerance and produced d-lactic acid at temperatures of 50°C and higher. In order to optimize the culture conditions of the QU 41 strain, we examined the effects of pH control, temperature, neutralizing reagent, and initial glucose concentration on d-lactic acid production in batch cultures. It was found that the optimal production of 20.1 g/l d-lactic acid was acquired with high optical purity (>99.9% of d-lactic acid) in a pH 6.0-controlled batch culture, by adding ammonium hydroxide as a neutralizing reagent, at 43°C in MRS medium containing 20 g/l glucose. As a result of product inhibition and low cell density, continuous cultures were investigated using a microfiltration membrane module to recycle flow-through cells in order to improve d-lactic acid productivity. At a dilution rate of 0.87 h-1, the high cell density continuous culture exhibited the highest d-lactic acid productivity of 18.0 g/l/h with a high yield (ca. 1.0 g/g consumed glucose) and a low residual glucose (<0.1 g/l) in comparison with systems published to date..
||Yukihiro Tashiro, Hideaki Shinto, Miki Hayashi, Shun ichi Baba, Genta Kobayashi, Kenji Sonomoto, Novel high-efficient butanol production from butyrate by non-growing Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) with methyl viologen, Journal of Bioscience and Bioengineering, 10.1263/jbb.104.238, 104, 3, 238-240, 2007.09, Non-growing Clostridium saccharoperbutylacetonicum N1-4 hardly produced butanol from only butyrate. As adding glucose to the medium, butyrate utilization and butanol production were stimulated. Addition of 0.1 mM methyl viologen as electron carrier resulted in the highest yield of butanol of 0.671 mol/mol to butyrate and glucose..
||Yukihiro Tashiro, Katsuhisa Takeda, Genta Kobayashi, Kenji Sonomoto, Ayaaki Ishizaki, Sadazo Yoshino, High butanol production by Clostridium saccharoperbutylacetonicum N1-4 in fed-batch culture with pH-stat continuous butyric acid and glucose feeding method, Journal of Bioscience and Bioengineering, 10.1263/jbb.98.263, 98, 4, 263-268, 2004.10, A pH-stat fed-batch culture by feeding butyric acid and glucose has been studied in an acetone-butanol-ethanol (ABE) fermentation using Clostridium saccharoperbutylacetonicum N1-4. The specific butanol production rate increased from 0.10 g-butanol/g-cells/h with no feeding of butyric acid to 0.42 g-butanol/g-cells/h with 5.0 g/l butyric acid. The pH value in broth decreases with butyric acid production during acidogenesis, and them batyric acid reutilization and butanol prodnction result in a pH increase during solventogensis. The pH-stat fed-batch culture was performed to maintain a constant pH and butyric acid concentration in the culture broth, but feeding only butyric acid could not support butyric acid utilization and butanol production. Subsequently, when a mixture of butyric acid and glucose was fed, butyric acid was utilized and butanol was produced. To investigate the effect of the feeding ratio of butyric acid to glucose (B/G ratio), several B/G ratio solutions were fed. The maximum butanol production was 16 g/7 and the residual glucose concentration in broth was very low at a B/G ratio of 1.4. Moreover, yields of butanol in relation to cell mass and glucose utilization were 54% and 72% higher in pH-stat fed-batch culture with butyric acid than that of conventional batch culture, respectively..