| With the decline in reserves of oil and coal, and the rapid growth of oil prices, the development and production of fuel ethanol to instead of oil have a huge potential market. The bottleneck of cellulosic ethanol production is how to converted cellulose into glucose effectively, of which abtained highly efficient cellulase is important. The anaerobic cellulolytic degradation bacterium consitium SVY42 was enriched and identified. The fermentation characteristics of SVY42 were studied, and the metagenomics library of SVY42 was constructed. It laid a foundation for cloning and expressing efficient and new cellulase in the future.1. Identification of anaerobic cellulolytic bacterium consortium SVY42. A stable anaerobic group SVY42 degrading filter paper was obtained, which was isolated and enriched from the Great Basin of Nevada Hot Springs, with anaerobic medium of the filter paper as the sole carbon source. The 16S rDNA clone library of SVY42 was constructed,21 positive clones picked randomly were sequenced, then alignmented in the NCBI BLAST, and the microbial structure of SVY42 was determined finally. SVY42 mainly consisted of four genera, Clostridium sp., Ruminococcus sp., Cellulosilyticum sp. and Sporomusa sp. Clostridium sp. accounted for 38% of the entire consortium; Ruminococcus sp. accounted for 24% of the total consortium; Cellulosilyticum sp. accounted for 24%, Sporomusa sp.accounted for 14% of the entire consortium.2. Fermentation characteristics of anaerobic cellulolytic bacterium consortium SVY42. The fermentation condition of the conversion from cellulose to sugar was researched, the results showed that the optimal cultivation time was 7 d, optimal fermentation temperature was 42.5℃, optimal initial pH was 8.0, optimal carbon source was CMC, optimal nitrogen source was yeast extract. Under the optimum conditions, the reducing sugar yield reached 23%. The ability of the consortium SVY42 using different natural cellulosic feedstock was researched, and the results showed that SVY42 could produce cellulase using a variety of cellulosic materials under the optimum growth conditions. The highest filter paper enzyme activity was 9.41 U/mL using bagasse as substrate and the highest CMCase activity was 6.35 U/mL using waste mushroom barrel as substrate. In addition, SVY42 has the capacity of transforming a variety of cellulose to ethanol. The ethanol yield using CMC as substrate was the highest, which was up to 1.13 g/L; the capability of conversion filter paper and miscanthus to ethanol was slightly higher which up to 1.10 g/L; the ethanol yield was a minimum of 0.98 g/L using waste mushroom.3. Construction of Metagenomic Library of anaerobic cellulolytic bacterium consortium SVY42. The bacterium consortium SVY42 was screened and could anaerobic degrade cellulose into sugar (ethanol) efficiently. SVY42 was enriched and extracted the total genomic DNA, applied to Fosmid vector, and then its Fosmid metagenomic library was constructed. After tested and assessed the quality of library, it found that the inserted DNA fragment sizes were 30～50 Kb. The random restriction maps of recombinant plasmid were significantly different, which indicated that the gene of the Fosmid metagenomic library were diversity. The restriction map of the first generation and the 100 generation of plasmid DNA clones were no significant difference, which indicated that the library’s stability was good, and there were no insert fragment loss or rearrangement. Ten positive clones had been screened by the method of CMC substrate inducing gene expression combining with Congo red staining, which could generate CMCase transparent ring. It would benefit for cloning and expressing the cellulase gene, andobtain efficient cellulase. |
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