| At present, the second generation of bioethanol using hemicellulose as feedstocks has become a hot research topic. The high costs of cellulase and the lack of strains fermenting the pentose were two main obstacles for the ethanol production. Thermophilic anaerobic bacteria can metabolize almost all the sugars of lignocellulose to ethanol, but can’t degrade hemicellulose into ethanol at high yield. In the study, we isolated a novel strain to produce ethanol efficiently, and then determined its taxonomic status, and sequenced to get the whole genome draft. This paper also described its carbon-centred metabolic pathway and fermentation features. At the same time, the strain was used to ferment corn cob hemicellulose hydrolysates to provide theoretical basis for the industrialized lignocellulosic ethanol.1. A novel Gram-positive, thermophilic anaerobes strain Rxl was isolated, and the strain is one such hemicellulolytic organism with the ability to hydrolyze xylan. Based on its physiological and biochemical tests and DNA-DNA hybridization analyses, the isolate was considered to represent a novel species in the genus Thermoanaerobacterium, for which the name Thermoanaerobacterium calidifontis sp. nov. was proposed. The main advantages of T.calidifontis Rx1were able to rapidly grow and ferment xylan, with the highest xylase activity16.2U/ml during the process of fermentation,150-703times higher than the known strains of the genus, and T.calidifontis Rx1had the highest xylanase activity among the reported the genus Thermoanaerobacterium.2. Draft genome sequence of T. calidifontis Rx1has achieved by sequencing. The genome has a size of2.64Mb. a G+C content of35.03%, and it is predicted to contain2,715coding sequences, in which2,569genes have homologous sequences and2,069genes have definite function.43tRNA gene and2rRNA gene. All these homologous proteins are from121other species, of which T. xylanolyticum LX-11 has the highest similarity up to43.64%. The datas enriches genetic information of strains of the genus Thermoanaerobacterium, and provide important molecular informations for biology and genetic engineering of the thermophilic anaerobic bacteria.3. We investigated genes encoding the key enzymes related to glycolysis, pentose phosphate and fermentation pathway. By homology analysis of the gene sequences and amino acid sequences, we found only a few enzymes showed the highest identity with the known strains. Based on enzymatic assays and genome sequence analyses, pathways of T. calidifontis Rx1were identified that would lead to the generate of all major products. Lactate dehydrogenase(ldh) and phosphate acetyltransferase-acetate kinase (pta-ack) gene of T. calidiforntis Rxl were cloned, to constructe suicide plasmids pPSkn-ldh and pBSkpa, to reveal the influence to central carbon metabolism of T. calidiforntis Rxl by knockout metabolic bypass and lay a good foundation to genetic engineering of the strain.4. Fermentation characteristics of T.calidifontis Rxl were studied. T. calidifontis Rxl was able to ferment the majority of biomass-derived sugars to produce ethanol. The strain has been shown to produce up to1.37mol ethanol/xylose mol, which was the highest pentose conversion efficiency in known wild-type thermophilic anaerobic bacteria. Additionally. Ethanol production of pentose and polysaccharides were significantly higher than that of hexose, and the tolerance for disaccharide and polysaccharides (≥40g/L) of the strain were higher than that of monosaccharide (≤10g/L). In addition, highest ethanol concentration were obtained at10%inoculum concentration,3g/L yeast extract and4g/L tryptone, with0.75g/L cysteine and shaking (150rpm). All these would have a value to achieve industrial lignocellose-ethanol production.5. We studied the differences of metabolic regulation mechanism of T.calidifontis Rxl between fermenting mannitol and glucose. When fermentation was performed by T. calidiforntis Rx1using mannitol as substrate, ethanol yield was1.8mol/mol mannitol, with90.5%of the theoretical yield, which was168.9times as much yield as fermentation with glucose. There are two reasons for this phenomenon:first, when using mannitol, the intracellular content of NADH/NAD+was higher than that using glucose, which led the carbon resources to produce ethanol. Second, when using mannitol as substrate, there was a passway of converting acetic acid to ethanol in T. calidiforntis Rxl, so that the acetic acid can be completely changed into ethanol. But this pathway has not been found when fermenting glucose or other carbohydrate. Therefore, we supposed that the key enzymes related to convention acetic acid to ethanol can be induced by mannitol. However, no study has been done to demonstrate this passway.6. The ethanol production capacity with corn cob hemicellulose hydrolysates by T.calidifontis Rxl was studied. In this study, we optimized the conditions for dilute H2SO4pretreatment of corn cob by the central composite design of response surface method. The results showed that pretreatment conditions for the highest xylose production was0.70%sulfuric acid for45min at126.7℃, corresponding xylose yield82.1%. Fermenting of corn cob hydrolysate and synthetic medium by T.calidifontis Rx1, it was found that ethanol production was higher using corn cob hydrolysate. Apparently, the toxicant in hydrolysate of corn cob did not inhibit the ethanol production of strain Rxl. Additionally, trace amounts of acetic acid in hydrolysate showed an unexpected stimulatory effect on ethanol fermentation of corn cob hydrolysate by strain Rx1, which may have an active significance to realize the ethanol industrial production with lignocellulose as raw material. |
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