Kluyveromyces marxianus, an Attractive Yeast for Ethanolic Fermentation in the Presence of Imidazolium Ionic Liquids

Imidazolium ionic liquids (ILs) are promising solvents for lignocellulosic biomass (LCB) pretreatment and allow the achievement of higher ethanolic yields after enzymatic hydrolysis and ethanolic fermentation. However, residual ILs entrapped in pretreated biomass are often toxic for fermentative microorganisms, but interaction mechanisms between ILs and cells are still unknown. Here we studied the effects of 1-ethyl-3-methylimidazolium acetate [Emim][OAc] and 1-ethyl-3-methylimidazolium methylphosphonate [Emim][MeO(H)PO2] on Kluyveromyces marxianus, a thermotolerant ethanologenic yeast. Morphological impacts induced by ILs on K. marxianus were characterized by Scanning Electron Microscopy analysis and showed wrinkled, softened, and holed shapes. In Yeast-Malt-Dextrose (YMD) medium, K. marxianus tolerated IL additions up to 2% for [Emim][OAc] and 6% for [Emim][MeO(H)PO2]. Below these thresholds, some IL concentrations enhanced ethanolic yields up to +34% by switching the metabolic status from respiratory to fermentative. Finally, K. marxianus fermentation was applied on several substrates pretreated with [Emim][OAc] or [Emim][MeO(H)PO2] and enzymatically hydrolyzed: a model long fiber cellulose and two industrial LCBs, softwood (spruce) and hardwood (oak) sawdusts. The maximum ethanolic yields obtained were 1.8 to 3.9 times higher when substrates were pretreated with imidazolium ILs. Therefore K. marxianus is an interesting fermentative yeast in a second-generation bioethanol process implying IL pretreatment.


Introduction
Lignocellulosic biomass (LCB) is a promising sustainable raw material for second-generation bioethanol production. Whatever the feedstocks (agricultural or forest residues, dedicated crops), a first step to disorganize the LCB architecture, named pretreatment, is a prerequisite to make cellulose more accessible to cellulolytic enzymes and generate fermentiscible sugars [1][2][3]. Ionic liquids (ILs) are among the most efficient pretreatments to increase cellulose enzymatic digestibility while allowing a better recovery of lignin and hemicellulose for subsequent valorization in a biorefinery strategy [4][5][6]. However, detrimental effects could be observed due to residual IL entrapped in the pretreated LCB +22%). However, a small supplementary increase in IL concentration to 2% cancelled growth and ethanol production (Figure 2A,E). When 1% [Emim][MeO(H)PO2] was added, K. marxianus produced similar maximum ethanol concentration (5.5 g/L at 24 h) than standard condition without IL (5.7 g/L) ( Figure 2F). However, when 2%, 3% or 4% [Emim][MeO(H)PO2] were added, the maximum ethanol formation became significantly higher (from +13% to +34%) than without IL: 6.6, 7.7 or 6.5 g/L, respectively. Moreover, ethanol did not decrease to zero after glucose exhaustion which could be a supplementary advantage in the context of bioethanol production. However, a small another increase in [Emim][MeO(H)PO2] concentration reduced ethanol formation to 2.5 g/L with 5% IL and 1.0 g/L with 6% IL ( Figure 2F).

Oxygen Transfer Rate (OTR) and Carbon Dioxide Transfer Rate (CTR) Profiles
Then, the K. marxianus respirofermentative activity was studied in presence of [Emim][OAc] ( Figure S1) and [Emim][MeO(H)PO2] (Figure 3). For the control condition without IL, the oxygen transfer rate (OTR) ( Figure 3A) rapidly increased to a value of 7.5 mmol/L/h at 3 h, followed by a small wave at 7.0 mmol/L/h between 3 and 8 h. At the same time, the carbon dioxide transfer rate (CTR) increased to 57 mmol/L/h indicating a high release of CO2 coupled to biomass production and respiration metabolism. This important CO2 liberation limited in time is responsible for the downwards wave observed in OTR values. Then the OTR remained constant at 8 mmol/L/h up to 47 h, indicating a maximum oxygen transfer capacity and a clear O2 limitation [18,19]. In parallel, the                     (Figure 3). For the control condition without IL, the oxygen transfer rate (OTR) ( Figure 3A) rapidly increased to a value of 7.5 mmol/L/h at 3 h, followed by a small wave at 7.0 mmol/L/h between 3 and 8 h. At the same time, the carbon dioxide transfer rate (CTR) increased to 57 mmol/L/h indicating a high release of CO 2 coupled to biomass production and respiration metabolism. This important CO 2 liberation limited in time is responsible for the downwards wave observed in OTR values. Then the OTR remained constant at 8 mmol/L/h up to 47 h, indicating a maximum oxygen transfer capacity and a clear O 2 limitation [18,19]. In parallel, the CTR was constant at about 5.5 mmol/L/h, which reflected the consumption of ethanol by yeasts after depletion of glucose in accordance with the previous observations in Figure 2F. Then, at 45 h, the OTR dropped sharply to a value of 0.5 mmol/L/h, as well as the CTR to zero at the same time, which proved that all ethanol was consumed by K. marxianus, no more carbon substrate was available anymore.
When  Figure 2F). In addition, the CTR dropped directly to zero after glucose depletion, without the plateau to about 5 mmol/L/h. These results confirmed that ethanol was more weakly consumed by yeasts (or not at all depending on the CTR was constant at about 5.5 mmol/L/h, which reflected the consumption of ethanol by yeasts after depletion of glucose in accordance with the previous observations in Figure 2F. Then, at 45 h, the OTR dropped sharply to a value of 0.5 mmol/L/h, as well as the CTR to zero at the same time, which proved that all ethanol was consumed by K. marxianus, no more carbon substrate was available anymore.
When adding [Emim][MeO(H)PO2], the maximum OTR decreased progressively and O2 was no longer limiting from 2% IL. Concomitantly, the maximum CTR slowed down when [Emim][MeO(H)PO2] concentration rose. Altogether, these results indicated that the yeast metabolism switched to fermentative with lower O2 consumption and decreased CO2 liberation [17,18], which was consistent with ethanol formations higher with 2- Figure 2F). In addition, the CTR dropped directly to zero after glucose depletion, without the plateau to about 5 mmol/L/h. These results confirmed that ethanol was more weakly consumed by yeasts (

Application to Lignocellulosic Biomasses (LCBs)
These first results obtained with ILs in a medium containing glucose as the sole carbon source were transposed to various LCBs: the model long fiber cellulose and two industrial wood residues, spruce sawdust (softwood) and oak sawdust (hardwood). These LCBs were all pretreated with [Emim][OAc] or [Emim][MeO(H)PO 2 ] following a protocol already described [13,20,21], except that the pretreatment temperature was 45 • C. The IL-pretreated LCBs were recovered and intensively washed with ultra-pure water, but residual ILs estimated to about 10% (w/w) remained entrapped in the pretreated matrix [13,20,21]. The  Then the IL-pretreated LCBs were enzymatically hydrolyzed with cellulases from Trichoderma reesei during 80 h. The enzymatic hydrolysis liberated various glucose yields depending on the pretreatment ( Table 2).  (Table 1). However, in both cases it remained superior to the non-pretreated oak sawdust indicating that IL-pretreatment allowed a better cellulase accessibility to the cellulose fibers.
After LCB enzymatic hydrolysis, the liberated glucose units were fermented by K. marxianus in presence of the residual ILs still remaining in the reaction medium since the pretreatment step. A concentrated Yeast-Malt (YM) medium was added to the hydrolysis buffer to obtain good final YM component concentrations. The only ethanologenic substrate was the LCB enzymatic hydrolysate [17]. Results of yeast growth, glucose consumption and ethanol production are presented in Figure 4 for model cellulose, in Figure 5 for spruce sawdust and in Figure 6 for oak sawdust, respectively.   (Table 1). However, in both cases it remained superior to the non-pretreated oak sawdust indicating that IL-pretreatment allowed a better cellulase accessibility to the cellulose fibers. After LCB enzymatic hydrolysis, the liberated glucose units were fermented by K. marxianus in presence of the residual ILs still remaining in the reaction medium since the pretreatment step. A concentrated Yeast-Malt (YM) medium was added to the hydrolysis buffer to obtain good final YM component concentrations. The only ethanologenic substrate was the LCB enzymatic hydrolysate [17]. Results of yeast growth, glucose consumption and ethanol production are presented in Figure 4 for model cellulose, in Figure 5 for spruce sawdust and in Figure 6

Discussion
Although ILs are now recognized as very promising solvents for efficient LCB pretreatments, their negative impacts on hydrolytic enzymes and fermentative cells remain a major drawback in their extensive use [4][5][6]. Nowadays, recent developments deal with genetically engineered yeast strains able to ferment both pentoses and hexoses, or consolidated bioprocessing with cellulase-displaying strains able to hydrolyze and ferment simultaneously the cellulosic part of LCBs [23][24][25]. More, performance optimizations of microorganisms tolerant to ILs naturally or genetically induced is gaining increasing interest [26][27][28][29][30]. However, very few works are exploring impact of ILs on fermenting microorganism physiology in a fundamental way and it remains very difficult to understand the mechanisms of IL toxicity on cells.
Here, we studied K. marxianus fermentation in diluted [Emim][OAc] and [Emim][MeO(H)PO2] ILs in YMD medium (glucose as the sole carbon source) and in enzymatically hydrolyzed LCBs (cellulose, spruce sawdust, and oak sawdust). We showed for the first time SEM micrographs of K. marxianus cells in presence of low IL concentrations, which exhibited wrinkled, softened, and holed cell surfaces. Thus, both imidazolium ILs interacted with the yeast cell walls, composed essentially of polysaccharides [31], and disorganized the cell wall network probably in the same manner as ILs deconstruct the LCB architecture by disrupting the hydrogen bondings [4,31] [13]. These wrinkled and holed shapes were already

Discussion
Although ILs are now recognized as very promising solvents for efficient LCB pretreatments, their negative impacts on hydrolytic enzymes and fermentative cells remain a major drawback in their extensive use [4][5][6]. Nowadays, recent developments deal with genetically engineered yeast strains able to ferment both pentoses and hexoses, or consolidated bioprocessing with cellulase-displaying strains able to hydrolyze and ferment simultaneously the cellulosic part of LCBs [23][24][25]. More, performance optimizations of microorganisms tolerant to ILs naturally or genetically induced is gaining increasing interest [26][27][28][29][30]. However, very few works are exploring impact of ILs on fermenting microorganism physiology in a fundamental way and it remains very difficult to understand the mechanisms of IL toxicity on cells.
Here, we studied K. marxianus fermentation in diluted [Emim][OAc] and [Emim][MeO(H)PO 2 ] ILs in YMD medium (glucose as the sole carbon source) and in enzymatically hydrolyzed LCBs (cellulose, spruce sawdust, and oak sawdust). We showed for the first time SEM micrographs of K. marxianus cells in presence of low IL concentrations, which exhibited wrinkled, softened, and holed cell surfaces. Thus, both imidazolium ILs interacted with the yeast cell walls, composed essentially of polysaccharides [31], and disorganized the cell wall network probably in the same manner as ILs deconstruct the LCB architecture by disrupting the hydrogen bondings [4,31] [17], but both species showed very similar behaviors despite metabolic divergences as S. cerevisiae is Crabtree-positive and K. marxianus is Crabtree-negative [14]. The increase in ethanol yields seemed to be linked to the oxygen limitation [14,15] [4,13]. Glucose yields obtained after IL pretreatment and enzymatic hydrolysis are closely linked to the experimental conditions: the initial LCB and the cellulosic percentage, the IL nature (commercially provided or lab-synthesized, presence of impurities), the substrate loading rate, the pretreatment temperature and duration, the enzyme origin (pure enzyme or enzymatic preparation containing several hydrolytic activities)... [4,6,12,13,20,21]. Some authors reported very high enzymatic performances after [Emim][OAc] pretreatment, such as 98.2% glucose yield on sugarcane bagasse [33] or 96% on switchgrass [34], but other works are weaker: 40% glucose yield on wheat straw [35], 34.8% on oak and 40.7% on spruce [21], pointing the importance of optimizing all the pretreatment parameters to the lignocellulosic substrate.
When K. marxianus was grown on LCB cellulase hydrolyzates, no major inhibition was observed by the other components of the woody biomasses: lignin, hemicellulose, or extractives, or by a co-product issued from the IL pretreatment or the hydrolysis step, which could sometimes prevent the ethanolic fermentation [10,32,36,37]. An ethanolic production occurred in all the tested conditions regardless of the LCB (model cellulose, spruce sawdust, or oak sawdust) or the IL used for pretreatment ( 2 ] generated a smaller raise by a factor 1.8. These results point the influence of substrate architecture on the IL pretreatment efficiency, in agreement with hypotheses from previous studies [4,13,20,21], showing again the difficulty to generalize the impacts of ILs on initial LCB, hydrolytic enzymes and fermentative cells.

IL Pretreatment of LCBs
Pretreatment of LCBs (long fiber cellulose, spruce sawdust, or oak sawdust) with imidazolium ILs was realized as already described [13,20,21], except temperature which was here 45 • C. After the pretreatment step, the LCB was precipitated by adding ultra-pure water.

Enzymatic Hydrolysis of LCBs
The hydrolysis of LCBs pretreated with ILs or not was realized with the cellulase from Trichoderma reesei (EC 3.2.1.4) during 80 h in acetate buffer (50 mM, pH 4.8): one unit liberates 1.0 µmol of glucose from cellulose in 1 h at pH 5.0 at 37 • C, from Sigma-Aldrich (Steinheim, Germany). The enzymatic hydrolysis protocol and the calculation of glucose yields were previously described [13,20,21]. After the enzymatic hydrolysis, the reaction medium was centrifuged and the liquid fraction rich in glucose was recovered for ethanolic fermentation.

Fermentation of Enzymatically Hydrolysed LCBs
The liquid fraction issued from enzymatic hydrolysis was sterilized by autoclave and transferred in an Erlenmeyer flask. A concentrate YM medium was added in the goal to obtain a final medium containing YM components at the good final value, i.e., yeast extract 3 g/L, malt extract 3 g/L, peptone 3 g/L, pH 4.8. A mid-log phase preculture of K. marxianus cells was then inoculated to start the culture.

Conclusions
K. marxianus is an emerging yeast offering huge biotechnological potential, from biofuels to heterologous protein production. Our results highlighted that K. marxianus is an excellent candidate for a prospective one-pot process from LCB to second-generation bioethanol grouping IL pretreatment, enzymatic hydrolysis and ethanolic fermentation. However, future lignocellulosic biorefinery still needs a deeper understanding of the mechanisms implied in IL toxicity for fermentative cells to further design rational genetically modified microorganisms with good IL-tolerance and high ethanolic yield ability.