| Due to consumption of fossil fuels,seeking new renewable energy is one of the effective measures to alleviate the current energy crisis.As an important renewable energy source,biodiesel is recognized as a high-quality substitute for fossil fuels due to its low aromatic content and high energy density.However,biodiesel refining raw materials mainly include waste food oil,animal and vegetable fats,etc.,whose sources are very limited,and it is difficult to meet the vast market demand of biodiesel.Microbial lipid has similar components to vegetable lipid.The production process is not limited by season and arable land.It can be produced by abundant natural lignocellulosic biomass,is an ideal raw material for biodiesel production.However,there are still some challenges in the production of lignocellulosic microbial lipids,including low lipid titer,high content of inhibitors produced in the pretreatment process,low sugar utilization efficiency,and high enzyme dosage in the hydrolysis process.In view of the above issues,Trichosporon dermatis 32903 with high lipid producing and xylose utilization capacity was screened from oleaginous yeast strains for further study.Through the development and application of densifying dignocellulosic biomass with chemicals（DLC）and densifying dignocellulosic biomass with chemicals followed by autoclave（DLCA）pretreatment technology,the generation of inhibitors in the pretreatment process was reduced,and the lipid production efficiency was improved.Based on the characteristics of sugar consumption and inhibitor tolerance of T.dermatis 32903,a novel sugar-targeted-utilization cycle fermentation process was established to improve lipid production.Rapid bioconversion with integrated recycle technology of DLCA-RS was further studied to reduce the enzyme dosage and the cost of microbial lipid refining process.The main research contents and results are as follows:（1）Study on lipid fermentation based on traditional acid/alkali pretreatmentStrains and carbon to nitrogen ratio（C/N）of lipid fermentation were important factors affecting lipid synthesis.T.dermatis 32903 with high lipid synthesis and xylose utilization capacity（lipid concentration 11.48 g/L,xylose consumption rate 85.92%）was screened from oleaginous yeast strains and used for further study.The lipid fermentation performance of T.dermatis 32903 was further improved by optimizing the C/N ratio（C/N）of the medium（the optimal C/N was 110,the lipid concentration was improved to 16.33 g/L）.The lipid fermentation potential from lignocellulosic biomass of T.dermatis 32903 and the effects of inhibitors on the lipid fermentation were investigated by using hydrolysates of traditional acid/alkali pretreated biomass with or without washing detoxification,resptectively.The results showed that the lipid concentration in hydrolyzates of detoxificated traditional acid/alkali pretreated biomass was 11.43 and 20.36 g/L with the lipid yield of 0.119 and 0.212 g/g of consumed sugar while these number decreased to 6.81 and 7.46 g/L,0.104 and 0.101 g/g consumed sugar in hydrolyzates of non-detoxificated biomass.Pretreatment inhibitors significantly inhibited the lipid fermentation process.The effects of different inhibitors on the fermentation performance of T.dermatis 32903 were further analyzed.The results showed that T.dermatis 32903 exhibited high tolerance to furans and weak acids and low tolerance to phenols.（2）Study on rice straw pretreatment of DLC（A）alkali and lipid fermentationReducing the generation of inhibitors in the pretreatment is an important strategy to improve the efficiency of lignocellulosic lipid refining.DLC and DLCA alkali pretreatment were further developed and studied on rice straw（RS）.Firstly,the effects of DLC alkali pretreatment on RS were investigated.The results showed that the digestibility of RS was greatly improved.The glucan and xylan conversion of RS were imprvoed to 86.45,62.25%（DLC calcium hydroxide pretreatment）and 91.90,66.59%（DLC sodium hydroxide pretreatment）from 15.31、4.08%（without pretreatment）.The DLCA alkali pretreatment were further studied to relieve the limitation of storage time.The pretreatment conditions were opitimized by response surface methodology.The effect of solid loading on enzymatic hydrolysis of DLC（A）alkali pretreated RS was further analyzed.The results showed that the sugar concentrations in hydrolysate increased while the conversions decreased slightly with solid loading.The inhibition content analysis and lipid fermentation results showed that inhibitors concentrations in DLC（A）sodium hydroxide pretreated RS were higher than DLC（A）calcium hydroxide.Lipid titer reached the maximum of 29.5 g/L in hydrolysate of DLCA calcium hydroxide pretreated RS.Through the development and application of DLC（A）alkali pretreatment technology,the fermentation lipid titer of T.dermatis 32903 from lignocellulosic biomass was significantly improved.（3）Study on rice straw pretreatment of DLC（A）acid and lipid fermentationDLC（A）acid and alkali pretreatment may exhibited different pretreatment effects on RS due to the different mechanisms of acid and alkali pretreatment.Therefore, DLC（A）acid pretreatment of RS was further studied.The results of chemical dosage and storage time of DLC sulfuric acid pretreatment showed that the digestibility of DLC sulfuric acid pretreated RS increased with the increasing of chemical dosage and prolonging of storage time.Compared to DLC alkali pretreatment,DLC sulfuric acid pretreated RS exhibited higher xylan conversion（84.94%）and lower glucan conversion（61.94%）.To further improve the digestibility of RS and relieve the limitation of storage time,DLCA sulfuric acid pretreatment conditions were optimized by response surface methodology:0.05 g sulfuric acid/g biomass,treatment time of 26.59 min,dry biomass loading of 40%.After DLCA sulfuric acid pretreatment,the glucan and xylan conversions of RS were improved to 70.53,94.21%,respectively.The glucose and xylose concentrations in hydrolysate of DLCA sulfuric acid pretreated RS reached122.00 and 63.28 g/L,respectively at 35wt.%solid loading,Both DLC and DLCA sulfuric acid pretreated RS showed high fermentability.Lipid titer reached maximum of 31 g/L in hydrolysate of DLCA sulfuric acid pretreated RS.（4）Establishment and application of sugar-targeted-utilization cycle fermentationThe sugar utilization efficiency and the tolerance of microorganism to inhibitors are essential for lipid synthesis.The sugar consumption and inhibitors tolerance characteristics of T.dermatis 32903 during lipid fermentation were investigated.Based on the sugar consumption and inhibitors tolerance characteristics,sugar-targeted-utilization cycle fermentation process was established.The results showed that the increase of xylose ratio in the medium decreased the lipid yield of T.dermatis 32903and showed no significant effect on cell growth during fermentation.The conversion efficiency of mixed sugar could be improved by the sugar-targeted-utilization（xylose targeted for cell growth and glucose targeted for lipid fermentation）.Lipid fermentation in inhibitor meium results showed that the maximum OD600 and lipid concentration in fermentation increased from 43.1,0.53 g/L to 110.1,19.06 g/L as initial inoculum size increased from 0.25 to 64,respectively.T.dermatis 32903 tolerance to inhibitors was improved with the increasing of initial inoculation size.Based on the above characteristics,sugar-targeted-utilization cycle fermentation strategy was established.In the process,the tolerance of inoculum size of T.dermatis 32903 to inhibitor was utilized,glucose was targeted for lipid fermentation and xylose was targeted for cell growth,so as to the lipid production efficiency during fermentation was improved.Lipid fermentation in mixed suagr medium showed that lipid titer increased from 29.3g/L（common fermentation）to 40.3 g/L（sugar-targeted-utilization cycle fermentation） at 190 g/L total sugar concentration.The lipid titer in hydrolysates of DLCA calcium hydroxide and sulfuric acid pretreated RS were further improved to 33.9 g/L and 38.4g/L by sugar-targeted-utilization cycle fermentation,respectively.（5）Study on rapid bioconversion with integrated recycle technology of DLCA-RSThe reduction of enzyme dosage is an important aspect to reduce the cost of lignocellulosic microbial lipids refining.Rapid bioconversion with integrated recycle technology of DLCA-RS were studied.The feasibility of improving enzymatic hydrolysis efficiency of DLCA-RS through rapid process were verified by comparing rapid enzymatic hydrolysis with common enzymatic hydrolysis.The results showed that the glucan and xylan conversion of DLCA calcium hydroxide pretreated RS increased from 77.47 and 87.21%（common enzymatic hydrolysis）to 85.70 and 90.47%（rapid enzymatic hydrolysis）,respectively;the glucan and xylan conversion of DLCA sulfuric acid pretreated RS increased from 83.94 and 96.92%（common enzymatic hydrolysis）to 88.86 and 97.10%（rapid enzymatic hydrolysis）,respectively,under enzyme dosage of 7 mg pretein/g glucan.The rapid enzymatic hydrolysis process of DLCA-RS at high solid loading（20-30wt.%）was further studied.The effect of solid residue recovery and enzyme dosage on enzymatic hydrolysis efficiency at different solid loading was investigated.The results showed that the improvement of solid residue recovery significantly improved the enzymatic hydrolysis efficiency of DLCA-RS at high solid loading within the recovery range of 50-100%.At solid residue recovery of 100%,enzymatic hydrolysis efficiency was not significantly affected as enzyme dosage was reduced to 12 and 8 mg protein/g glucan（for DLCA calcium hydroxide and sulfuric acid pretreated RS,respectively）.The stability of the rapid process were further verified by increasing the enzymatic hydrolysis cycles.The mass balance analysis results showed that enzyme dosage of DLCA calcium hydroxide and sulfuric acid pretreated RS were reduced to 3.2 and 2.8 g protein/kg DLCA-RS through rapid bioconversion with integrated recycle technology with lipid yield of 58.9 and 69.1g/kg DLCA-RS,respectively. |
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