Studies On Cellulose Degradation And Hydrogen Production Characteristics Of Thermophilic Anaerobic Clostridium

Cellulose is the most abundant renewable resource that can be turned into fuel. Effective utilization of cellulose effectively is of great significance.However. cellulose is very difficult to hydrolyze because of lack of cellulases with high activities,which has limited its uses.At present,cellulases are mainly from fungi. Some anaerobic bacteria such as Clostridium thermocellum produce cellulases which can hydrolyze cellulose effectively.C.thermocellum can produce ethanol and H₂. However,the use of C.thermocellum for ethanol fermentation often has organic acids as by-products.The highest concentration of ethanol is only about 3%. Recombinant DNA technology was used to re-design the glycolysis pathway to block the synthetic pathway of by-products and to improve the cell tolerance to ethanol.Hydrogen is a clean and sustainable energy carrier for the future because of its high conversion efficiency and nonpolluting nature when used in fuel cells.Anaerobic fermentation has been demonstrated as a technically feasible way to produce hydrogen.Some anaerobic bacteria can use for H₂ production.However.the cost of hydrogen production and low hydrogen yield could limit its widespread application as an energy source in the future.Cellulose is the promising economical source for hydrogen production.Hydrogen production by fermentation can use only 15% energy of the organic matter.Therefore,to improve the H₂ production is becoming the focus of energy research.Combination of hydrogen production by fermentation with photosynthetic hydrogen production or methane production can improve the efficiency of the use of biomass.Thermophilic anaerobic bacteria can effectively utilize cellulose and have great potential for H₂ production.The study of cellulose degradation characteristics of thermophilic anaerobic bacteria can help us have a better understanding of their cellulases and regulation mechanisms.Furthermore,we can make use of cellulose fermentation to product hydrogen.The main results of the thesis are as follows: 1.Screening and identification of thermophilic anaerobic bacteriaUsing cellulose as the sole carbon source,the thermophilic anaerobic bacteria that can utilize cellulose were enriched from rotten wheat straw.Single colonies were isolated by using agar roll tubes containing cellobiose.A cellulose-degrading bacterium JN4 was obtained and identified as C.thermocellum by 16S rDNA analysis and morphological observation.Another bacterium that was the most abundant in the sample was isolated and identified as Thermoanaerobacterium thermosaccharolyticum.2.Growth and H₂ production properties of C.thermocellum and T. thermosaccharolyticumThe optimal growth temperature of C.thermocellum JN4 and T. thermosaccharolyticum GD 17 was 60℃by using cellobiose as carbon source.The growth curves of the two strains were determined.GC and HPLC analysis showed that the fermentation products of C.thermocellum JN4 were H₂,acetate,lactate and ethanol using cellulose as carbon source.C.thermocellum JN4 could utilize microcrystalline cellulose,filter paper and several kinds of natural substrates such as corn cob powder and corn stalk powder.The extracellular proteins of C. thermocellum JN4 showed CMCase,PASCase and cellobiohydrolase activity when grown on microcrystalline cellulose.The fermentation products of T. thermosaccharolyticum GD17 were H₂,acetate,butyrate and ethanol using reducing sugar as carbon source.The production of hydrogen by both strains was investigated. The hydrogen yield of C.thermocellum was 0.7mol H₂(mol cellobiose)^-1.The hydrogen yield of T.thermosaccharolyticum GD17 was 4mol H₂(mol cellobiose)_-1, 2.2 tool H₂(mol glucose)^-1 and 1.7mol H₂(mol xylose)^-1.3.The growth kinetics and hydrogen production of C.thermocellum JN4 and co-cultures of C.thermocellum JN4 and T.thermosaccharolyticum GD17 using microcrystalline cellulose as carbon source Using microcrystalline cellulose as carbon source and Hungate tubes,batch fermentation was performed to investigate the growth characteristics and H₂ production of C.thermocellum JN4 cultures and co-cultures of C.thermocellum JN4 and T.thermosaccharolyticum GD17.The results showed that C.thermocellum JN4 can degrade microcrystalline cellulose to produce hydrogen,ethanol,acetate and lactate,but cannot completely utilize cellobiose and glucose produced by the cellulose degradation.Its hydrogen yield was about 0.8 mol H₂(mol glucose)^-1,with lactate as the main product.When C.thermocellum JN4 was co-cultured with T. thermosaccharolyticum GD17,hydrogen production increased about 2-fold and H₂ yield reached a high level of 1.8 mol H₂(mol glucose)^-1.Butyrate was the most abundant byproduct and lactate was not detected at the end of the co-culture process.4.The growth and H₂ production of C.thermocellum JN4 and co-cultures of C. thermocellum JN4 and T.thermosaccharolyticum GD17 in 2L anaerobic reactorUsing filter paper as carbon source in 2L anaerobic reactor.C.thermocellum JN4 can produce 18mmol H₂/1 culture.Other fermentation products are ethanol,acetic acetate and lactate.The higher ethanol content of 24 mmol/1 culture indicated that the strain had the potential of cellulosic ethanol production.Differences from cultures in Hungate tubes,cellobiose and glucose were not detected.That means that the scale-up of cultivation may benefit the growth and metabolism of C thermocellum JN4.When C thermocellum JN4 was co-cultured with T. thermosaccharolyticum GD17 without pH control,the H₂ production increased to 35mmol/1 culture.Other fermentation products are ethanol,acetate,lactate and butyrate.Lactate increased until 50h and then decreased.At the same time, butyrate increased,which was similar to the cultures in Hungate tubes.When C. thermocellum JN4 was co-cultured with T.thermosaccharolyticum GD17 with pH control at pH 6.0～6.5,the H₂ production was 25mmol/l culture.Lactate was the predominant fermentation product.Because no H₂ was produced in lactate fermentation,the H₂ production decreased. 5.Cloning and expression of Clostridium thermocellum JN4 celS gene in Escherichia coliThe gene was cloned by using the genome of Clostridium thermocellum JN4 as template.The primers were designed according to C.thermocellum ATCC 27405 celS gene.The nucleotide sequence data were submitted to GenBank.There were only 3 bases' difference between the submitted sequence and C.thermocellum ATCC 27405 celS gene.So the fragment we cloned should be celS gene.The PCR-amplified gene fragment,following restriction-enzyme digestions,was ligated with the similarly digested plasmid pET-22b.The ligated plasmid was introduced into E.coli BL21(DE3).The recombinant protein rCelS had PASCase activity. But the activity was low.6.Site-directed mutagenesis of Clostridium thermocellum JN4 celS gene and expression in Escherichia coliAccording to protein structural information,two sites were site-directed mutagenesis.The one was D255N and the other was Y351F.The sites were chosed because they were key sites of catalytic function.The mutations were expressed in Escherichia coli but neither of them had PASCase activity.So we speculated that Asp255 may be the base group of the catalytic center.Tyr351 is a conservative site and the change to Phe may impact the structure of CelS catalytic center.