Isolation of thermophilic anaerobes from paddy field soils and ethanol production by isolates
| Project/Area Number |
15580295
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Boundary agriculture
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| Research Institution | Yamagata University |
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Principal Investigator |
UEKI Katsuji Yamagata Univ., Faculty of Agriculture, Professor, 農学部, 教授 (10111337)
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| Co-Investigator(Kenkyū-buntansha) |
UEKI Atsuko Yamagata University, Faculty of Agriculture, Professor, 農学部, 教授 (60143088)
KAKU Nobuo Yamagata University, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (80359570)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Thermophilic anaerobes / Starch-degrading thermophilic anaerobes / Cellulose-degrading thermophilic anaerobes / MPN counting and isolation / Diversity of thermophiles / Thermostable amylolytic enzymes / Ethanol production / Mesophilic anoxic environments / MPN係数と分離 / 計数と分離 / 水田土壌 |
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| Research Abstract |
1.Enumeration of thermophilic anaerobes in anaerobic environments at the moderate temperature.
Thermophilic anaerobes in paddy field soil and freshwater sediment were enumerated by the MPN counting method with three tubes. Paddy field soils were sampled from a paddy field, which had been continuously fertilized with rice straw and N-, P-, K-and Si-fertilizers over 20 years. MPN cultures using PY medium as the basal medium were incubated at temperatures in the range from 30 to 80℃. Mesophilic anaerobes were counted with MPN cultures incubated at 30℃, and MPN values at the level of 10^6-10^71MPN/cm^3 fresh soil were estimated. MPN values counted by the culture incubated at 50, 65 or 75℃ were at the level of 10^5-10^6, 10^5 or 10^4, respectively.
On the other hand, mesophilic anaerobes in the freshwater sediment, which was sampled from a pond with the catchment area designated as the area for the protection of nature, were counted at 10^6 MPN/cm^3 fresh sediment. Anaerobes were also counted
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with the culture incubated at the temperature over 50℃. However, MPN values counted with the culture at 50 or 65℃ were at the level of 10^3 or 10^2 MPN/cm^3.
Seventy strains of fermentative anaerobes were isolated from MPN-counting cultures of paddy field soils incubated at 65℃. All the isolates were thermophilic and did not grow at 30℃. According to the growth at 65℃ and fermentation products from glucose, isolates fell into more than 4 groups. Besides, DGGE analyses of PCR-amplified fragments of 16 S rDNA sequences suggested that the isolates were divided into at least 8 phylogenetic groups.
2.Utilization of thermophilic anaerobic isolates for biomass conversion
(1)Thermophilic anaerobes fermenting starch to ethanol
Six strains of thermophilic anaerobes, which were isolated from soil samples from paddy fields distantly located in three main islands of Japan, were most closely related to Thermoanaerobacter wiegelil with the similarity value of 16S rDNA sequence of more than 98% and affiliated with the genus Thermoanaerobacter Six strains produced and secreted amylolytic enzymes, when cultured on starch. The strains isolated from different soil samples produced amylolytic enzymes with different properties, i.e., the pH-profile, temperature-profile and thermal stability.
(2)Cellulose-degrading thermophilic anaerobes
Six strains of cellulose-degrading anaerobes were isolated from a paddy field soil. The isolates were obligate anaerobes, had the temperature optima at 70-75℃ and did not grow at the temperature below 45℃ or above 85℃. The isolates degraded filter paper in 24-72 hours of culture at 65℃. According to the 16S rDNA phylogeny, six isolates were most closely related to Calcicellulosyruptor saccharolyticus and Thermoanaerobacter cellulolyticus with the similarity value of 90% or 96-97%. Less
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Report
(3 results)
Research Products
(2 results)
