| Microbial oils, which are similar to vegetable oils in fatty acid composition andsynthesized and accumulated by oleaginous microorganisms under certain conditions, havebeen considered as a good feedstock for biodiesel due to shorter production time and nolimitation by season or location. However, the high cost of synthetic media on which themicrobes are cultivated has restricted its large-scale production and application.Lignocellulosic biomass is the most abundant and available green regenerable resources innature, thus using lignocellulosic hydrolysate for microbial oil production is promising forreducing its cost. Glucose and xylose are the major component of lignocellulosic hydrolysate,however, xylose cannot be utilized by most of microorganisms. Moreover, many by-products,such as organic acids, aldehydes, and alcohol compounds, are generated during dilute acidpretreatment and recognized as inhibitors since they would inhibit cell growth and productformation of microorganisms. Therefore, the key point of efficient production of microbialoils with lignocellulosic hydrolysates is: Firstly, obtaining the microorganism strains whichcan efficiently use xylose for lipid production, in addition, understanding the inhibitory lawand mechanism of inhibitors on cell growth and lipid accumulation of oleaginousmicroorganisms and then adopting specific strategies to eliminate or alleviate the inhibition. Itis worth noting that there is little work focused on the microbial oil production onlignocellulosic hydrolysates and the inhibitory law and mechanism of inhibitors present inlignocellulosic hydrolysates on the cell growth and lipid accumulation of oleaginousmicroorganisms as well.Trichosporon fermentans, an oleaginous yeast strain screened by our lab, is able to useboth glucose and xylose for lipid production. Unfortunately, it could not grow well on thedilute acid treated rice straw hydrolysate without dexofication and only gave a lipid yield of1.7g/L, which was much lower than those on the medium containing glucose or xylose (13.6g/L and9.9g/L). As a result, in this dissertation, T. fermentans was firstly domesticated on thedilute acid treated rice straw hydrolysate to increase its tolerance to the inhibitors present inlignocellulosic hydrolysates. Then, the effect of different hydrolysis and detoxificationmethods on the composition of lignocellulosic hydrolysates was compared. And then, thepossibility of lipid production on the resulting lignocellulosic hydrolysates by thedomesticated T. fermentans was explored. Meanwhile, the effect of various factors on thelipid production was investigated and a lipid fermentation system based on lignocellulosichydrolysates was thus established. Subsequently, the influential rule of three kinds of representative inhibitors (organic acids, aldehydes, and alcohol compounds) in lignocellulosichydrolysates on the cell growth and lipid accumulation of T. fermentans was studied.Furthermore, in order to illustrate the inhibitory mechanism of inhibitors on the cell growthand lipid accumulation of T. fermentans, the effect of above-mentioned inhibitors on sugarmetabolism, malic enzyme activity, cell morphology, and cell membrane integrity of T.fermentans was investigated.Rice straw and bagasse, two kinds of typical lignocellulosic biomass in the southernChina, were used as feedstock for lipid fermentation. It was found that the inhibitors'concentration in the hydrolysates could be significantly reduced by choosing properdetoxification methods. T. fermentans could grow well on the dilute acid treated rice strawhydrolysate detoxified by a series of treatments (overliming, concentration, and absorption byresin Amberlite XAD-4), and give a lipid yield of11.5g/L after8days' fermentation. Therewas marginal alteration in the detoxification efficacy when low-cost activated charcoalsubstituted resin Amberlite XAD-4as adsorbent, leading to a great decrease in thedetoxification cost. It was revealed that fermentation time, inoculum size, and initial pH arethe key factors affecting the lipid production on the bagasse hydrolysate detoxified byactivated charcoal. Under the optimal conditions (inoculum size10%, peptone concentration1.8g/L, initial pH7.5,25oC, and fermentation time9days), the biomass, lipid content, andlipid yield of T. fermentans are33.7g/L,44.9%, and15.1g/L, respectively.In order to realize comprehensive utilization of lignocellulosic biomass, the enzymatichydrolysis of the remaining cellulose composition in rice straw resulted from dilute acidpretreatment was carried out and yielded the complete hydrolysate. Compared with dilute acidtreated hydrolysate, the rice straw complete hydrolysate has higher sugar concentration butlower inhibitor concentration, and its detoxification needs only overliming, thus simplifyingthe detoxification process. The resulting complete hydrolysate could be used for lipidfermentation without adding other nutrients except for a small amount of nitrogen source andtrace CuSO4·5H2O. Upon optimization (inoculum size5%, initial pH7.0,25oC, andfermentation7dyas), the biomass, lipid content and lipid yield reached26.4g/L,52.2%and13.8g/L, respectively. The shorter fermentation time on the rice straw complete hydrolysate isdue to the presence of higher ratio of glucose in the medum which are favorable formicroorganism metabolism thus increasing the fermentation efficacy.It was shown that the lipid coefficient of T. fermentans in either dilute acid treatedbagasse hydrolysate or rice straw complete hydrolysate is both lower than that on the mediumwithout inhibitors (13.9%,17.0%vs.20.6%), indicating that the lipid production is inhibited by the inhibitors in lignocellulosic hydrolysates. On the other hand, the effect of fermentationconditions, including inoculum size and initial pH, etc., on the lipid production by T.fermentans varies with medium, suggesting that the fermentation conditions are also keyfactors affecting the inhibitory effect of inhibitors.To understand the influential rule of different kinds of inhibitors on the cell growth andlipid accumulation of T. fermentans,10organic acids (acetic acid, formic acid, levulinic acid,4-hydroxybenzoic acid, furoic acid, caproic acid, gallic acid, ferulic acid, syringic acid, andvanillic acid),5aldehydes (furfural,5-hydroxymethylfurfural (HMF), vanillin,syringaldehyde and4-hydroxybenzaldehyde), and4alcohol compounds (catechol,hydroquinone, furfuryl alcohol, and vanillyl alcohol), totally19inhibitors were chosen toinvestigate the effect of them on the lipid production by T. fermentans. It was found that thereis no correlation between the inhibitory effect of inhibitors and their hydrophobicity, which isdifferent from the results obtained in other work, in which the toxicity of inhibitors on theethanologenic microorganism or other oleaginous microorganism is positively related to theirhydrophobicity, indicating that the hydrophobicity is not the unique factor determining thetoxicity of inhibitors. Among the three kinds of inhibitors, organic acids are the least toxic toT. fermentans, followed by alcohols and aldehydes. However, the fatty acid composition oflipid produced by T. fermentans is marginally affected by inhibitors. Most of inhibitors showlittle influence on the growth and lipid accumulation of T. fermentans at their lowconcentration. Interestingly, some organic acids, such as acetic acid, formic acid, levulinicacid,4-hydroxybenzoic acid, and gallic acid, could even stimulate cell growth of T.fermentans at their low concentration, and furoic acid and vanillyl alcohol are able to boostlipid synthesis at a certain concentration. A comparative study showed that among the threefuran analogues of furfuryl alcohol, furoic acid, and furfural, furoic acid is the least toxic, andfurfural is less toxic than furfuryl alcohol at low concentration, but T. fermentans could sufferhigher concentration of furfuryl alcohol. For aromatic analogues, vanillyl alcohol, vanillicacid, and vanillin, vanillic acid was the least toxic while vanillin shows the strongestinhibitory effect.There exists more than one inhibitor in lignocellulosic hydrolysates; as a result, thesynergistic action of various inhibitors on the growth and lipid accumulation of T. fermentanswas further studied. Except for the binary combination of aromatic aldehydes, catechol andhydroquinone or vanillyl alcohol, acetic acid and gallic acid, other binary combinationshowed no synergistic effect. By using response surface methodology, we found thatindividual acetic acid, furfural or catechol would decrease the biomass, lipid yield and sugar consumption of T. fermentans. Interestingly, the binary combination of acetic acid andcatechol would increase the values of these variables instead. But other binary or ternarycombination of these three inhibitors showed no significant effect on the biomass, lipid yieldand sugar utilization of T. fermentans.For the purpose of efficient reducing or eliminating the toxicity of inhibitors, theinfluence of fermentation conditions on the inhibitory effect of inhibitors was investigated. Itwas shown that larger inoculum size could reduce the inhibitory effect by most of inhibitors,including aliphatic acids, aldehydes, and catechol. Albeit there is no rule for the influence oftemperature and initial pH on the inhibition, alteration of temperature and initial pH couldalso alleviate or eliminate the inhibition and the efficiency varies with the inhibitor. Therefore,in practical application, the temperature and initial pH should be optimized regarding thespecific lignocellulosic hydrolysate.The effect of above-mentioned inhibitors on the sugar metabolism, malic enzymeactivity, cell membrane integrity, and cell morphology of T. fermentans, was further studied toget a depth insight into inhibitory mechanism of inhibitors. It was found that some organicacids and alcohol compounds could enhance the sugar utilization of T. fermentans at their lowconcentration, which partly explains the phenomenon that these inhibitors could stimulate thegrowth or lipid synthesis of T. fermentans at their low concentration. At IC25concentration(the concentration required to inhibit25%lipid yield of T. fermentans), all the inhibitorstested would inhibit glucose and xylose utilization at the early phase of fermentation, leadingto a longer lag phase and a delay of lipid synthesis as well. At the later period of fermentation,some aromatic or furan inhibitors are able to stimulate xylose consumption, but the increaseof xylose utilization does not give a higher lipid yield. Malic enzyme is the key enzyme in thelipid synthesis pathway and it offers reduced force NADPH for lipid synthesis. Among the19inhibitors tested, all the inhibitors except furoic acid, vanillyl alcohol, vanillin, and HMFshowed inhibition on malic enzyme activity of T. fermentans. Particularly, furoic acid andvanillyl alcohol enhanced the activity of malic enzyme; which partly explains that they couldboost the lipid accumulation of T. fermentans at a certain concentration.Except for the influence on sugar utilization and key enzyme's activity, inhibitors wouldattack the hydrophobic sites on cell membrane, damage its integrity thus affecting thephysiological activity of microorganisms. Propidium iodide (PI) staining and flow cytometrydetection were used to analyze the effect of inhibitors on the cell membrane integrity of T.fermentans. Among the19inhibitors tested, all the inhibitors except ferulic acid, caproic acid,and vanillyl alcohol would damage the cell membrane integrity of T. fermentans. There is no correlation between the damage of cell membrane integrity and inhibitors' hydrophobicity,which partly explains the above phenomenon that the toxicity of inhibitors is not directlyrelated to their hydrophobicity. Besides, inhibitors would change cell morphology of T.fermentans, but there is also no correlation between their inhibition and the alteration of cellmorphology. During ethanol fermentation by yeast, furfural is reduced to furfuryl alcohol andfurfuryl alcohol is the final product of in vivo detoxification, however, there is a specificdetoxification mechanism for furfural by T. fermentans. Furfural is quickly reduced to furfurylalcohol at a short time, followed by a slow oxidation of furfuryl alcohol into furoic acid, andfuroic acid is the final product of in vivo detoxification. In conclusion, the inhibitorymechanism of inhibitors on the cell growth and lipid accumulation of T. fermentans mainlyincludes the inhibition of sugar metabolism and malic enzyme's activity, and the damage ofcell membrane integrity, etc.This work reveals the influential rule and mechanism of different kinds of inhibitors inlignocellulosic hydrolysates on the cell growth and lipid accumulation of T. fermentans,which will play the theoretical foundation for later strain modification, process optimizationof fermentation and detoxification. In addition, the establishment of lipid fermentation systembased on lignocellulosic hydrolysates by T. fermentans, is promising in decreasing the cost ofmicrobial oil, thus providing sustainable lipid feedstock for biodiesel production.
|
PDF Full Text Request