| Pretreatment is the core step of lignocellulose biorefinery process.Furan aldehydes and phenolic aldehydes are the two major inhibitor groups derived from lignocellulose pretreatment,which harshly inhibit not only the hydrolysis efficiency of cellulase in the enzymatic saccharification step but also the fermentation metabolism of microbes in the fermentation step.Therefore,the effective removal of furan aldehyde and phenolic aldehyde inhibitors is the prerequisite condition for highly efficient utilization of lignocellulose.Biological detoxification method is a kind of way to degrade or convert the toxic inhibitors by the metabolism of microorganisms.A biodetoxification method using specific microorganisms to remove aldehyde inhibitors prior to fermentation step was considered as an efficient strategy.However,the complete removal of aldehyde inhibitors by this method requires a long time and then leads to the loss of fermentable sugars such as the xylose from hemicellulose.To the completely remove of aldehyde inhibitors,effectively hold of fermentable sugars and then achieve the highly efficient lignocellulose biorefinery,an optimized biological detoxification strategy was proposed that strengthen the inhibitors degradation or conversion ability of biorefinery fermenting strains.In this thesis,the dry biodetoxification process was applied for the lignocellulose citric acid fermentation firstly.Then the molecular biodetoxification mechanism of furan and phenolic aldehydes were elucidated in two biorefinery fermenting strains Gluconobacter oxydans DSM 2003 and Corynebacteriurm glutamicum S9114 with high inhibitor tolerance.Finally,the synthesis pathway of fatty hydrocarbon was constructed in C.glutamicum S9114.The first part was study on the lignocellulose citric acid production in biodetoxified corn stover by a widely applied industrial strain Aspergillus niger SIIM M288.Amorphotheca resinae ZN1 is a specific robust biodetoxification fungus isolated from our previous study which playing important role in the dry lignocellulose detoxification process.This biodetoxification method has been successfully applied for the production of lignocellulose ethanol,lipid,lactic acid,gluconic acid and xylonic acid.No obvious cell growth and citric acid accumulation were observed when using the freshly pretreated corn stover hydrolysate as the substrate because the A.niger SIIM M288 was highly sensitive to the existence of furan and phenolic aldehyde inhibitors in the corn stover hydrolysate.After the removal of these inhibitors from the pretreated corn stover by A.resinae ZN1,A.niger SIIM M288 was able to achieve high concentration citric acid fermentation in the biodetoxified lignocellulose system.Various fermentation parameters such as the component of the medium,temperature,initial pH and inducer were tested and only minimum regulation was required during the fermentation.Citric acid at the titer of 100.0 g/L with the yield of 94.1%based on glucose were obtained in the 25%(w/w)solid content detoxification corn stover hydrolysate,reaching the separation and purification standard of industrial citric acid fermentation and also providing a very important feasibility basis for the industrial production of lignocellulosic citric acid.The second part elucidated the molecular biodetoxification mechanism of furan and phenolic aldehyde inhibitors of G.oxydans DSM 2003.The DNA microarray of G.oxydans DSM 2003 was carried out under the stress of furfural,5-hydroxymethylfurfural(HMF),4-hydroxybenzaldehyde(HBA),vanillin and syringaldehyde,respectively.The results showed that furan and phenolic aldehydes triggered not only the expression of corresponding intracellular and cell membrane bound oxidoreductase genes but also the expression of relevant genes involved in intracellular pentose phosphate pathway(PPP)and membrane bound respiratory chain,promoting the conversion of furan and phenolic aldehydes.In addition,a large number of transporter protein encoded genes were also significantly up-regulated expression,which might played an important role in the conversion process of aldehyde inhibitors on the intracellular space.The mining of membrane bound dehydrogenases will provide a new idea for construction of high inhibitor tolerance biorefinery fermenting strains.The third part analyzed the conversion mechanism of furan and phenolic aldehydes in C..glutamicum S9114.The real-time quantitative PCR(qRT-PCR)method was applied to investigate the transcription levels of 93 putative genes involved in the conversion of aldehydes under the stress of furfural,HMF,HBA,vanillin and syringaldehyde.C.glutamicum S9114 could quickly convert furan and phenolic aldehydes into corresponding alcohols and acids,the corresponding alcohols then continue to be converted into furan acids and phenolic acids.The qRT-PCR results showed that several alcohol dehydrogenase genes,aldehyde dehydrogenase genes and oxidoreductase genes were responsible for the conversion of furan and phenolic aldehydes.Some significant up-regulation genes were over-expressed in C.glutamicum S9114.The conversion rate of five aldehydes was significantly enhanced by the over-expression of alcohol dehydrogenase gene CGS9114_RS01115 and CGS9114_RS10340,aldehyde dehydrogenase CGS9114_RS04340 which up-regulated expression under both furan aldehyde and phenolic aldehdyde inhibitors.The above results have important reference value for improving the comprehensive tolerance ability of biorefinery fermenting microorganisms to various aldehydes inhibitors.The fourth part was the preliminary studies on the synthesis pathway construction and evaluation of fatty hydrocarbon in bacterial microorganisms.Lignocellulose biorefinery process has been successfully applied for the synthesis of the biofuels such as ethanol and lipid,while hydrocarbon is another biofuel with greater added value and potential for production,and the current research on the synthesis of fatty hydrocarbons by microbial using lignocellulose has attracted more attention.In this thesis,the long-chain acyl-(acyl-carrier-protein)reductase gene and aldehyde decarbonylation gene from Synechococcus elongatus PCC7942 were codon optimized and overexpressed in Escherichia coli BL21 and C.glutamicum S9114.The linear fatty hydrocarbons consisted mainly of pentadecane and heptadecene were formed by the recombinant E.coli.Various strategies to promote the production of fatty hydrocarbon were investigated and the results showed that increasing the solubility of long-chain acyl-(acyl-carrier-protein)reductase protein and blocking the oxidation pathway of fatty acid all could promote the synthesis of fatty hydrocarbon.Interestingly,the recombinant C.glutamicum S9114 could synthesize the very long chain hydrocarbon(C>23),and the synthesis pathway of very long chain hydrocarbon in C.glutamicum S9114 was speculated to be involved with the fatty acid and mycolic acid synthesis pathways.Overall,in view of the different performances of different biorefinery fermenting strains response to the furan and phenolic aldehydes,different biodetoxification strategies were adopted accordingly in order to achieve the efficient lignocellulose biorefinery and the extended application of the high inhibitor tolerance fermenting strains.In this thesis,the dry biodetoxification process was successfully applied for the production of lignocellulose citric acid and achieved the goal of high titer fermentation.The molecular detoxification mechanism of furan aldehyde and phenolic aldehyde inhibitors in two biorefinery fermenting strains G.oxydans DSM 2003 and C.glutamicum S9114 were elucidated.Key genes responsible for the conversion of aldehydes were selected and identified.The biorefinery fermenting strain C.glutamicum S9114 was firstly used as host bacterium for long chain fatty hydrocarbon synthesize and its synthesis pathway was also speculated.The thesis provides a theoretical foundation for the industrialization and application of lignocellulose biorefinery process. |
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