| Microbial lipid derived from renewable biomass feedstock for the biodiesel production is an important way to solve the shortage of oil resources. Trichosporon cutaneum, one typical oleaginous microbe, possesses both of the capabilities of metabolism various monosaccharides derived from lignocellulose for lipid accumulation and excellent tolerance to lignocellulose derived inhibitors, that it is considered to be a microbial lipid production strain using lignocellulosic biomass in biorefinery way.Firstly, the highest potential of microbial lipid production of T. cutaneum using lignocellulose material in biorefinery way was analyzed, and biodiesel was synthesized. The tolerance and degradation of T. cutaneum to lignocellulose derived inhibitor were studied. Secondly, with genome sequencing, the molecular biological mechanism of high lipid accumulation capability and robust inhibitor tolerance was predict, central metabolism, lipid accumulation and inhibitors degradation pathways were speculated. Thirdly, explored to reduce the COD concentration of cellulosic ethanol fermentation wastewater by T. cutaneum in fermentation way, and accumulate lipid simmultaneously. Moreover, the characteristics of inulin hydrolysis enzyme, the possibility of inulin hydrolysis and consolidated microbial lipid production by T. cutaneum using inulin were studied.The inhibitors generated during lignocellulose process exhibit the subsequent enzyme hydrolysis, the activity and metabolism of oleaginous microbe, that the biotransformation could not be carry out normally. Therefore an ideal oleaginous fermenting strain should accumulate high lipid content, and also tolerate the toxic inhibitors when lignocellulose is used as feedstock. The maximum potential of lipid accumulation capacity of T. cutaneum ACCC 20271 was investigated using lignocellulosic biomass in dry biorefinery way firstly. Dry dilute acid pretreated corn stover was hydrolyzed by cellulase in different solid loading to obtain the corn stover hydrolysate wich was rich in glucose, xylose and other fermentable sugars. T. cutaneum ACCC 20271 exhibited excellent performance in growth and lipid production within corn stover hydrolysate. The dry cell weight and the lipid titer of T. cutaneum ACCC 20271 cultured in corn stover hydrolysate obtained in dry biorefinery way, were 31.31 g/L,8.57 g/L respectively.Microbial inhibitor tolerance is mainly reflected in its performance of degradation inhibitor. The tolerance, degradation potential and metabolism mechanism of T. cutaneum ACCC 20271 to lignocellulose derived inhibitors were firstly systematically analyzed. Weak acids, furan aldehydes and phenolic aldehydes were the major inhibitors generated during the pretreatment process of lignocellulose. Formic acid, acetic acid, levulinic acid, were representative of weak aicd; furfural,5-hydroxymethylfurfural, were representative of furan aldehyde; and 4-hydroxybenzaldehyde, vanillin, syringaldehyde, were representative of phenolic aldehyde. These inhibitors would inhibit various microorganisms, in the way of inhibiting their growth and subsequent fermentation. It was found that T. cutaneum ACCC 20271 exhibited the capability of tolerance to 6 g/L of formic acid,25 g/L of aceitic acid,10 g/L of levulinic acid,5 g/L of 5-hydroxymethylfurfural,2 g/L of vanillin,2.5 g/L of syringaldehyde, while it was sensitive to furfural which was obviously inhibited the cell growth in low concentration (0.5 g/L), followed by 4-hydroxybenzaldehyde (2.5 g/L). Formic acid, acetic acid could ultimately converted by T. cutaneum ACCC 20271, furfural, HMF, vanillin, syringaldehyde and 4-hydroxybenzaldehyde could transform into corresponding alcohols followed by less toxic acids which were reverse transformed into low concentration of aldehydes and corresponding acids. Vanillin and 4-hydroxybenzaldehy could be ultimately degraded by T. cutaneum ACCC 20271. While the degradation of furfural, HMF and syringaldehyde stop in the corresponding alcohols and acids instead of ultimate degrdation. The lipid titer of the adapted strain T. cutaneum 277 which obtained after long term adaption culture was 5.17 g/L in 15% freshly preteated corn stover hydrolysate, while the original strain was only 0.15 g/L.Based on the inhibitor metabolism expriments, the genome of T. cutaneum ACCC 20271 was sequenced on Illumina-Miseq platform to reveal the mechanism of inhibitor tolerance and degradation. The central metabolism and lipid biosynthesis pathways were studied based on genome annotation. The genome length of T. cutaneum ACCC 20271 was 30.45 Mb. A number of 5704 protein-coding genes and 925 non-coding RNA genes were predicted. The central metabolism and lipid accumulatioan pathways were speculated according to the genome annotation. Glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP), fatty acid synthesis, phospholipids and triglyceride synthesis, ?-oxidation of fatty acid, glyoxylate cycle, were involved. The metabolic pathways of the lignocellulse derived inhibitor bioconversion and biodetoxification in T. cutaneum ACCC 20271, which was a representative of oleaginous yeasts, were firstly constructed based on experiment results, the genome annotation and previous relative reports. Putative genes involved in the biodegradation of furan aldehydes and phenolic aldehydes in T. cutaneum ACCC 20271 were collection and compared with other three oleaginous yeasts:R. toruloides NP11, Y. lipolytica CLIB122 and T. oleaginous ICB0246. Perhaps the rich source of alcohol dehydrogenase, aldehyde reductase, aldehyde dehydrogenase and oxidases genes totally in 101 in T. cutaneum ACCC 20271 was one of the reasons of high tolerance to inhibitors than these three oleaginous yeasts.Thirdly, the methond of reducing the chemical oxygen demand (COD) concentration of cellulosic ethanol fermentation wastewater by oleaginous yeast in the way of fermentation was analyzed in experient and its feasibility was assessed. The wastewater after solid-liquid separation of the bottom discharge of rectifying column for cellulosic ethanol rectification was containing large amount of monosaccharides, organic compounds, and high COD concentration of 112.41 g/L. T. cutaneum ACCC 20271, as the fermentation strain, inoculated into the cellulosic ethanol fermentation wastewater for lipid fermentation. The COD concentration was reduced to 53.31 g/L from 118.58 g/L after 120 h culture, and the dry cell weight and lipid titer reached to 16.20,2.16 g/L. The composition of microbial lipid produced by T. cutaneum ACCC 20271 using cellulosic ethanol fermentation wastewater was similar with vegetiable oil indicating that it could be used as raw material for biodiesel production. In this way the microbial lipid accumulated, simultaneously the COD concention of the wastewater was reduced 55.05%, partly achieced the target of COD reduction of cellulosic ethanol fermentation wastewater in the way of lipid fermentation.Furthermore, the microbial lipid production by T. cutaneum ACCC 20271 using inulin was studied to achieve coslidated bioprocessing inulin hydrolysis and lipid fermentation. It was found that T. cutaneum ACCC 20271 could utilize inulin directly for cell growth and microbial lipid accumulation, which proved the existence of inulin hydrolysis enzyme in cellular. The inulin hydrolysis enzyme of T. cutaneum ACCC 20271 was characterized in this part. The optimal temperature and pH were analyzed. The stability of the enzyme declined with temperature increase and in the range of 30-70?, while it did not obviously effected by pH in the range of 3.5-6.5. Combined with the genome annotation, it was proposed that the inulin hydrolysis enzyme of T. cutaneum ACCC 20271 was ?-fructofuranosidase.Conclusively, T. cutaneum ACCC 20271 possesses the capability of ultimately degradation or transformation the inhibitors into less toxic corresponding alcohols or acids, and lipid fermentation using monosaccharides derived from lignocellulose. The metabolism pathway of biotransform and biodetoxification of the inhibitors was firstly speculated in oleaginous yeast. The COD concentration of cellulosic ethanol fermentation wastewater could be obeviously reduced by T. cutaneum ACCC 20271, and obtained certain amount of lipid for biodiesel production. The existence of the inulin hydrolysis enzyme was important biological basis for the consolidated bioprocessing of microbial lipid production by T. cutaneum ACCC 20271. All of the theses results will provide a basic theoretical support for the further study of microbial lipid, genetic engineering and industrial production development by T. cutaneum. |
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