Study On The Effect Of Ionic Liquid On The Lipid Production By Trichosporon Fermentans

Microbial lipid,which is synthesized and accumulated by oleaginous microorganisms under certain conditions,is a supplier of functional oils and feedstock for biodiesel production.Using lignocellulose as the substrate for microbial lipid production may reduce the production cost of microbial lipid and facilitate the commercialization of microbial lipid.Due to its natural complicated structure,lignocellulose is highly recalcitrant to chemical and biological degradation.Therefore,pretreatment is essential prior to degrading of lignocellulose to fermentable sugars.Lignocellulose pretreatment with ionic liquid(IL)has been emerged as a promising technology.However,it was found that IL remained in lignocellulosic hydrolysate can influence the cell growth and product formation of industrial microorganism.Up to now,the impact of IL on the cell growth and lipid accumulation of oleaginous microorganisms remains largely unknown.Trichosporon fermentans is an oleaginous that can use lignocellulose dilute acid hydrolysate for efficient lipid production.In this study,the feasibility of lipid production on IL pretreated rice straw hydrolysate by T.fermentans was evaluated.Then,two types of IL that usually used for lignocellulose pretreatment,namely imidazolium IL and cholinium IL,were investigated on their effects on the cell growth and lipid accumulation of T.fermentans.Meanwhile,the impact of the ILs on the sugar metabolism,key enzymes activities in fatty acid synthesis,membrane permeability,cell morphology,cell surface topography,and metabolite profiling were also studied to understand the mechanism of the effect of those ILs on the cell growth and lipid accumulation of the yeast.Based on this,the inhibition of ILs on the lipid production by T.fermentans was reduced by changing the culture condition.Rice straw was pretreated by 1-butyl-3-methylimidazolium acetate([Bmim][OAc]).After 5 times of water wash,the Bmim+ and OAc-concentration in the hydrolysate were 3.6,and 0.7 g/L.T.fermentans can use the rice straw hydrolysate based medium for cell growth and lipid accumulation.However,[Bmim][OAc] remained in the hydrolysate could inhibit the cell growth and lipid accumulation of T.fermentans.Seven imidazolium ILs,namely 1-ethyl-3-methylimidazolium diethyl phosphate([Emim][DEP]),1-ethyl-3-methylimidazolium chlorine([Emim]Cl),1-allyl-3-methylimidazolium chlorine([Amim]Cl),1-butyl-3-methylimidazolium chlorine([Bmim]Cl),1-benzyl-3-methylimidazolium chlorine([Bzmim]Cl),1-ethyl-3-methylimidazolium acetate([Emim][OAc]),and [Bmim][OAc],as well as three cholinium ILs,namely cholinium lysine([Ch][Lys]),cholinium serine([Ch][Ser]),and cholinium acetate([Ch][OAc]),were chose to systematically study their effects on the cell growth and lipid accumulation of T.fermentans.In most cases,seven selected imidazolium ILs all exhibited inhibition effect on the cell growth and lipid accumulation.The inhibition of IL on lipid production increased as the elongation of the alkyl side chain on the cation of the IL.While the depression effect of different anions on lipid production showed the following order: [OAc]-? Cl-> [DEP]-.For cholinium IL,[Ch][Lys] and [Ch][Ser] could significantly enhance the cell growth but inhibit the lipid accumulation,while [Ch][OAc] could inhibit both the cell growth and lipid accumulation of T.fermentans.In addition,anions showed major influence on the cell growth and lipid accumulation of T.fermentans.In a word,the inhibition of cholinium IL on the lipid production by T.fermentans was much weaker than that of imidazolium IL.In most cases,all these ILs showed inhibitory effect on the sugar utilization of the yeast.The 7 imidazolium ILs all showed inhibitory effect on the malic enzyme and ATP : citrate lyase activities,except [Emim][OAc] and [Bmim][OAc].Imidazolium IL,especially [Bzmim]Cl,[Emim][OAc],and [Bmim][OAc],could also increase the cell membrane permeability.In most cases,the impact of imidazolium IL on the cell morphology of the yeast was minor,however,cells mostly changed to short ellipsoidal or sub-globose shape in the presence of [Bmim]Cl,[Bzmim]Cl,and [Bmim][OAc].Study on the impact of IL on the cell surface topography of T.fermentans found that IL could induce fibrous structure on the cell surface of the yeast.GC-MS based metabonomics analysis showed that [Emim][OAc] and [Bmim][OAc] could inhibit glucose and xylose assimilation of the yeast and thus inhibit the central carbon metabolism,while the impact of [Bmim]Cl on the central carbon metabolism was minor.Thus,imidazolium IL inhibited the cell growth and lipid accumulation of T.fermentans mainly by the following ways: 1.Inhibiting sugar assimilation as well as sugar metabolism,2.Inhibiting the activity of key enzymes in lipid accumulation,3.Increasing cell membrane permeability,and 4.Influencing the way of budding of T.fermentans.For cholinium IL,[Ch][Lys]and [Ch][Ser] could significantly enhance the glucose and xylose consumption of the yeast,while [Ch][OAc] at most cases could inhibit the glucose and xylose consumption of the yeast.It is worth noting that the 3 cholinium ILs all promoted the malic enzyme activity.Except for [Ch][OAc],[Ch][Lys]and [Ch][Ser] both could suppress the ATP : citrate lyase activity of T.fermentans,while [Ch][OAc] could enhance the ATP : citrate lyase activity.[Ch][Lys]and [Ch][Ser] could also increase the cell membrane permeability of T.fermentans,while [Ch][OAc] had little impact on the cell membrane permeability of T.fermentans.[Ch][Lys] and [Ch][Ser] could induce the yeast cells into longer expanding hyphae,while [Ch][OAc] could induce the cells into sub-globose shape.Study on the effect of cholinium IL on the cell surface topography of T.fermentans found that [Ch][Lys] and [Ch][OAc] could also induce fibrous structure on the cell surface of the yeast.Further study on the impact of [Ch][Lys] and [Ch][OAc] on the metabolic process of T.fermentans found that [Ch][Lys] could enhance citric acid metabolism and hinder its accumulation and flow into fatty acid synthesis,while [Ch][OAc] could inhibit glucose and xylose assimilation and thus inhibit the central carbon metabolism.Hence,[Ch][Lys] and [Ch][Ser] enhanced the cell growth but inhibited the lipid accumulation of T.fermentans mainly by the following ways: 1.Promoting the glucose and xylose consumption as well as citric acid metabolism,but inhibiting the accumulation of citric acid,2.Inhibiting the ATP : citrate lyase activity,3.Influencing the way of cell budding,and 4.Increasing cell membrane permeability.While the inhibitory mechanism of [Ch][OAc] on the cell growth and lipid accumulation of T.fermentans mainly due to its inhibition on sugar metabolism.Based on the knowledge obtained above,we try to remove or alleviate the inhibition effect of ILs on the lipid production by T.fermentans by simply changing the fermentation conditions.The results showed that the inhibition of imidazolium IL and [Ch][OAc] on the cell growth and lipid accumulation of the yeast could be reduced by improving the inoculum concentration.Elevating the culture temperature could alleviate the inhibition on the lipid production by most of the imidazolium ILs.Decreasing the medium p H reduced the depression effect of most imidazolium ILs and [Ch][OAc] on the cell growth and lipid accumulation of T.fermentans,while elevating the medium p H could enhance the beneficial effect of [Ch][Lys] and [Ch][Ser] on the cell growth by the yeast and meanwhile alleviating their inhibition effect on the lipid accumulation.Moreover,we found that the anion of IL with an acetate anion could be assimilated by T.fermentans and induced the medium p H elevated into alkaline conditions,which increased the inhibitory effect of the IL on the cell growth and lipid accumulation by the yeast.Thus,by controlling the medium p H during fermentation,the inhibition on the lipid production by T.fermentans of IL with an acetate anion could be alleviated or even removed.Similarly,we also found that the anions of [Ch][Lys] and [Ch][Ser] could be utilized by T.fermentans as nitrogen source,which reduced the C/N ratio of the fermentation medium.Hence,the inhibition of [Ch][Lys] and [Ch][Ser] on the lipid accumulation could be alleviated by controlling the C/N ratio of the fermentation medium.This study reveals the influential rule and mechanism of ILs that commonly used for lignocellulosic biomass pretreatment on the cell growth and lipid accumulation of T.fermentans,which will play the theoretical foundation for later process optimization of lignocellulose pretreatment and lipid fermentation.