Evaluating the effects of biocompatible cholinium ionic liquids on microbial lipid production by Trichosporon fermentans

Background Microbial lipid is a potential raw material for large-scale biodiesel production and lignocellulosic hydrolysate has been considered as promising low-cost substrate for lipid fermentation. Lignocellulosic biomass needs to be pretreated before enzymatic hydrolysis, and biocompatible cholinium ionic liquids (ILs) have been demonstrated to be highly efficient for pretreatment. However, the impact of these ILs residues in hydrolysates on downstream biotransformation remains unknown. Therefore, the influence of three typical cholinium ILs on the lipid production by Trichosporon fermentans was first investigated. Results The cell growth of T. fermentans was stimulated in the presence of cholinium lysine ([Ch][Lys]) and cholinium serine ([Ch][Ser]), while the lipid accumulation was inhibited by [Ch][Lys]) and [Ch][Ser]. Both cell growth and lipid accumulation of T. fermentans were inhibited in the presence of cholinium acetate ([Ch][OAc]). Despite the reduction in lipid content, the lipid production by T. fermentans was improved in the presence of low concentrations of [Ch][Lys] (≤30 mM) and [Ch][Ser] (≤20 mM) due to the remarkable increase of biomass. It was found that cholinium cation had minor influence on lipid production. However, the anions of [Ch][Lys] and [Ch][Ser] could be assimilated as nitrogen source by T. fermentans and the reduced C/N ratio accounts for the inhibition of lipid accumulation, which could be alleviated by improving C/N ratio of medium. In addition, the anion of [Ch][OAc] could be metabolized by T. fermentans, leading to a rapid alkaline-pH shift and strong inhibition of lipid production. And this inhibitory effect on lipid production could be significantly reduced by controlling culture pH. Conclusions The anions of [Ch][Lys], [Ch][Ser] and [Ch][OAc] play an important role in affecting the cell growth and lipid accumulation of T. fermentans, and the inhibition of these three ILs on lipid production can be alleviated by careful fermentation condition control. Hence, T. fermentans is a promising strain for microbial lipid production from cholinium ILs-pretreated lignocellulosic hydrolysates.


Background
The rising cost of fossil fuels, coupled with concerns over the environmental impact of associated CO 2 emission, calls for renewable and low-cost energy alternatives. Biodiesel has been proved to be an attractive alternative to the conventional fossil diesel [1]. However, most of the biodiesel was converted from edible oils, which has brought about the food vs. biofuel debate. Recently, because of the similar fatty acid composition to vegetable oils, microbial lipid has gained increasing interest in its use as feedstock for biodiesel production. Yet, high production cost of microbial lipid hinders its further application. To solve this problem, various low-cost substrates were adopted to lower its fermentation cost [2,3]. Lignocellulosic biomass, the most abundant and renewable biomass resources in nature, is considered to be a promising low-cost raw material for biofuel production [4].

Open Access
*Correspondence: btmhzong@scut.edu.cn; bbhwu@scut.edu.cn 1 State Key Laboratory of Pulp and Paper Engineering, College of Light Industry and Food Sciences, South China University of Technology, 381 Wushan Rd., Tianhe District, Guangzhou 510640, China Full list of author information is available at the end of the article Before being effectively exploited, lignocellulosic biomass needs to be pretreated in order to enhance its accessibility to enzymatic hydrolysis [5]. Up to now, the most commonly used pretreatment method is dilute acid hydrolysis. Although this method can give considerable monosaccharide yield, special reactors are needed to resist the corrosion effect of acid on equipment. Moreover, the acid used will degrade sugars to inhibitors that are harmful for downstream microbial growth and product formation [6,7]. Ionic liquids (ILs), a type of molten salts with melting points of <100°C, are composed of organic cations and organic or inorganic anions; and they are considered 'ecofriendly' because of negligible vapor pressure, non-flammability, high thermal, and chemical stability [8]. Since Rogers and coworkers [9] demonstrated the dissolving capacity of ILs to cellulose, ILs have emerged as promising solvents for lignocellulosic biomass pretreatment [10]. So far, imidazolium ILs have been proved to be the most effective ILs used in biomass pretreatment. Although this type of ILs can greatly increase the enzymatic hydrolysis rate of pretreated biomass, they are recently demonstrated to be harmful to microbes. For example, it was found that 1-butyl-3-methylimidazolium chloride ([Bmim] Cl) is about 300 times more toxic to Vibrio fischeri than acetone [11]. Additionally, when used for pretreatment of corn stover for bioethanol production, 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]) at 52.4 mM could significantly inhibit Saccharomyces cerevisiae's cell growth and ethanol production and there was a synergistic inhibitory effect between the anion and the cation [12]. Huang  [OAc] could inhibit lipid production of oleaginous yeast Rhodosporidium toruloides due to the assimilation of acetate by the yeast which led to a rapid alkaline-pH shift [13]. Very lately, it was found that the presence of [Emim][OAc] could induce morphological changes of S. cerevisiae, which exhibited wrinkled, softened, and holed shapes [14]. Irrespective of their cytotoxicity, the nonbiodegradable characteristics of imidazolium ILs would be another hamper to their wide application [15]. Therefore, it is of urgent need to find new solvents with more biocompatible and biodegradable properties for biomass pretreatment.
Recently, Hou et al. [16,17] and Ninomiya et al. [18] reported a type of novel renewable cholinium ILs as highly effective solvents for lignocellulosic biomass pretreatment. For example, when [Ch][Lys] was used for pretreatment of rice straw at 90°C for 5 h, sugar yields of 84% for glucose and 42.1% for xylose were achieved. As biomass pretreatment solvents, it is inevitable that ILs will be left over at various concentrations in the lignocellulosic hydrolysates. To understand the effect of ILs on the downstream biotransformation will be helpful in assessing the possibility of using ILs-pretreated lignocellulosic hydrolysates for biofuel production. However, to date, there is no report about the impacts of cholinium ILs on microbial production of bio-based products. Trichosporon fermentans is an oleaginous yeast which can efficiently produce lipid in detoxified lignocellulosic hydrolysates [19,20] [OAc], as shown in Scheme 1) on the cell growth and lipid accumulation of T. fermentans were firstly investigated. To give a deep insight into the influential mechanism, the sugar metabolism of cells and the effects of cation and anions of cholinium ILs on lipid production were further analyzed. This study will provide some valuable information for efficient application of cholinium ILs-pretreated lignocellulosic hydrolysates in biorefinery processes, particularly in microbial lipid production.

Effects of cholinium ILs on cell growth and lipid accumulation of T. fermentans
A series of cholinium ILs have been synthesized and tested for lignocellulosic biomass pretreatment [17]. [OAc] for lignocellulose pretreatment found that the concentrations of residual [Emim][OAc] remained in the subsequent enzymatic hydrolysates were up to 52 mM, depending on biomass regeneration process and washing conditions [12]. Accordingly, in this study, [ (Fig. 1b). When [Ch][Lys] was at 60 mM, the lipid content of T. fermentans was only 28.5%, reduced by 52.4% compared with that obtained in the absence of the IL (28.5 vs. 59.9%). As can be seen in Fig. 1c, despite of the reduction in lipid content, the lipid production was still improved in the presence of low concentrations of [Ch] [Lys] (≤30 mM), which was attributed to the remarkable increase of biomass. For [Ch][Ser], the impact of which on the cell growth and lipid accumulation of T. fermentans was quite similar to that of [Ch][Lys], with features of stimulating cell growth but inhibiting lipid accumulation, and the increase in biomass was significant except at 5 and 60 mM (p < 0.05), while the reduction in lipid content was significant except at 5 mM (p < 0.05). The lipid production was also improved in the presence of low concentrations of [ [21]. It was also demonstrated that lysine could enhance the cell growth and ethanol production by regulating the nitrogen metabolism of Saccharomyces pastorianus [22]. Therefore, the  [OAc] could be assimilated by T. fermentans, cells were cultured in the media containing 30 mM various ILs and 1 mL sample was taken daily to measure the anion concentration of the ILs. As can be seen in Fig. 1d, the concentrations of the three anions decreased with the increase of fermentation time and all the anions could be used up. The consumption rate of the anions followed the order: It is known that both the cation and the anion of ILs can contribute to their toxicity [11,12]. Hence, to understand the influence of cholinium cation on lipid production by T. fermentans, various concentrations of choline chloride ([Ch]Cl) were supplemented into the fermentation medium. As shown in Fig. 2, when [Ch]Cl was added at 10 mM, the biomass and lipid content were comparable to those of the control (14.9 vs. 14.2 g/L, 58.7 vs. 59.9%). Even in the presence of 60 mM [Ch]Cl, the biomass and lipid content still reached 12.7 g/L and 58.6%, respectively, indicating that cholinium cation has minor effect on cell growth and lipid accumulation of T. fermentans.
The effects of the selected cholinium ILs on the fatty acid composition of lipid produced by T. fermentans were also investigated, and the results were shown in Table 1. The major fatty acids of the lipid produced by T. fermentans in the absence of ILs were found to be oleic acid (C18:1), palmitic acid (C16:0), stearic acid (C18:0), and linoleic acid (C18:2), accounting for 60.6, 21.7, 11.  [13].

Sugar consumption profile of T. fermentans in the presence of cholinium ILs
To better understand the effect of the tested cholinium ILs on the cell growth and lipid accumulation of T. fermentans, the concentrations of residual sugars in the fermentation media after 4 days' cultivation were measured. As shown in Fig. 3a , the extra sugar consumed by T. fermentans were not transformed into lipid indicated by the lower lipid coefficient in most cases compared with the control (Fig. 3c). In contrast, except that at its low concentration (≤10 mM), [Ch][OAc] showed inhibitory effect on both glucose and xylose metabolism of T. fermentans, and the inhibition increased with the increase of which concentration. Albeit more glucose and xylose were consumed when the concentration of [Ch][OAc] was below 10 mM, there was no improvement in the biomass and lipid content of T. fermentans, suggesting that the extra sugar consumed was not used for cell growth and lipid synthesis. Similar phenomenon was also observed in studying the influence of organic acids on T. fermentans' lipid production [23]. It was reported that acetic acid (acetate) could interfere with yeast metabolism, which increased in the ATP requirement for cell maintenance [24,25]. Hence, it is possible that the extra consumed sugars were used for synthesis of ATP. However, the actual mechanism still needs further investigation.

Effect of C/N ratio on lipid production by T. fermentans in the presence of [Ch][Lys] and [Ch][Ser]
Previous reports showed that amino acids could be used as nitrogen source by yeasts [21,22]   Residual glucose (g/L)  Generally, an excess of carbon substrate and a limiting amount of nitrogen in the medium are necessary for achieving high lipid accumulation in a microorganism [26]. It was found that 163 was the most suitable C/N ratio for lipid production by T. fermentans [27] 27.8 and 47.4, respectively. Therefore, the inhibition of these two ILs on lipid accumulation of T. fermentans might be mainly due to the drastic reduction of C/N ratio. To testify this hypothesis, extra sugar was supplemented into the medium for elevating the C/N ratio. As indicated in Fig. 4b, a  [Ser] on the cell growth of T. fermentans was attributed to the assimilation of amino acid anions of ILs as nitrogen source. Whereas, the inhibitory effect of these two ILs on the lipid accumulation of T. fermentans was due to the reduction of C/N ratio. And this inhibition could be efficiently relieved by regulating the C/N ratio of medium.

Effect of pH on lipid production by T. fermentans in the presence of [Ch][OAc]
It was reported that the inhibition effect of [Emim][OAc] on lipid production by R. toruloides was mainly due to a rapid alkaline-pH shift resulted from the assimilation of [OAc] − [13]. In this study, the [OAc] − of [Ch] [OAc] was also metabolized by T. fermentans. Hence, the evolution of culture pH in the presence or absence of [Ch] [OAc] was detected during fermentation. As can be seen in Fig. 5a, the culture pH shifted from 6.5 to 7. . For shake-flask fermentation of T. fermentans without pH control, the maximal values of biomass, lipid content, and lipid yield were found to be obtained at initial pH of 6.5 [27], which can be explained from the variation of culture pH as indicated in Fig. 5a (at approximately 5.0 during most of the time). The biomass, lipid content, and lipid yield decreased with the increase of controlled pH from 5.0 to 7.5, and the reduction in biomass and lipid yield was significant (p < 0.05) while it was insignificant for lipid content (p > 0.05). Therefore, the alkaline-pH change through assimilation Huang et al. previous demonstrated that R. toruloides is a robust lipid producer tolerating residual imidazolium ILs at low concentrations [13]. The results achieved here suggest that T. fermentans is a potential strain for microbial lipid production from cholinium ILs-pretreated lignocellulosic hydrolysates. Meanwhile, this work provides additional information for efficient application of cholinium ILs-pretreated lignocellulosic hydrolysates in biorefinery processes. It was reported that some yeasts tolerant to [Emim][OAc] were screened by using the media containing this IL [28], indicating that it is feasible to obtain IL-tolerant microorganism strains in nature.
In addition, the tolerance of microorganisms to ILs could be also improved by domestication and/or genetic modification.

Conclusions
Three cholinium ILs were investigated for their influences on lipid production by T. fermentans. Cholinium cation had minor influence on lipid production but the anions of [ Cl was bought from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Yeast extract (containing 4.0% ammonium-N and 10.0% total nitrogen) and peptone (containing 2.0% ammonium-N and 14.5% total nitrogen) were purchased from Huankai Biotech (Guangzhou, China). Lysine and serine were obtained from Yuanju Biotech (Shanghai, China). All other chemicals used were of analytical grade or chromatographically pure.

Medium, precultivation, and cultivation
The precultivation medium contained glucose and xylose 20 g/L (ratio 2:1, wt/wt), yeast extract 10 g/L, and peptone 10 g/L. The composition of fermentation medium was as follows: glucose and xylose 60 g/L (ratio 2:1, wt/ wt), peptone 1. [Ser] could be used as sole nitrogen source by T. fermentans, 0.218 g lysine or 0.157 g serine instead of peptone and yeast extract was added into 50 mL fermentation medium, and the medium without any nitrogen source was used as the control. To test the effect of C/N ratio  [Lys] were used as nitrogen source by T. fermentans, cells in seed culture were collected by centrifugation and washed with sterile saline for three times before inoculation. Fermentation was carried out in a rotary shaker at 25°C and 160 rpm for 4 days. Experiments were done at least in duplicate and data were presented as mean ± standard error of mean of duplicate experiments.

Analytical methods
The medium pH was detected by pH meter (Sartorius, Germany). Cells were harvested by centrifugation, washed twice with distilled water and dried at 105°C for 24 h to get a constant dry cell weight. Cellular lipid from dry biomass was extracted as described by Huang et al. [19]. Lipid yield was defined as the amount of lipid extracted from the cells in per liter fermentation broth (g/L). Lipid content was calculated as g lipid per g dry cell weight. Lipid coefficient was defined as g lipid produced per g sugar consumed and then multiplied by 100%. The fatty acid profile of the lipid was determined as described by Morrison and Smith [29]. The fatty acid methyl esters produced by saponifying followed by methylation of the lipid were analyzed by gas chromatography (GC-2010, Shimadzu Corporation, Japan) with flameionization detector and a DB-Wax capillary column (30 m × 0.25 mm × 0.25 µm, Agilent Technologies Inc., USA). The column temperature was maintained at 180°C for 2 min and then upgraded to 210°C at a rate of 5°C/ min and kept for 11 min. Nitrogen was used as the carrier gas at 1.5 mL/min. Split ratio was 1:50 (v/v). The injector and the detector temperatures were set at 260 and 280°C, respectively.
Glucose, xylose, and acetate were measured by HPLC as described by Huang et al. [19].

Statistical analysis
All the experiments were performed at least in duplicate, and their average values with standard deviations were used for statistical analysis with SPSS 17.0 software for Windows (SPSS Statistics Inc., Chicago, IL, USA). Oneway analysis of variance (ANOVA) and Tukey's honestly significant differences (HSD) test were used to determine the significant differences of data at a 95% confidence interval.