Influence of 1-butyl-3-methylimidazolium Chloride on the Ethanol Fermentation Process of Pichia pastoris GS115

To evaluate the influence of 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) on the ethanol fermentation process of Pichia pastoris GS115, this paper investigated the yeast growth, ethanol formation and the fermentable sugars consumption during the ethanol fermentation process of Pichia pastoris GS115 at different [Bmim]Cl concentrations in the medium. The results indicated that the [Bmim]Cl had no influence on the ethanol fermentation process at its concentration less than 0.0001 g.L -1 . The [Bmim]Cl inhibited the yeast growth and had a negative effect on ethanol formation at its concentration higher than 0.0001 g.L -1 . The final biomass and ethanol concentration, and the overall ethanol yield from the fermentable sugars all decreased with its concentration increasing. The yeast growth was very slow and nearly no ethanol formed when its concentration reached 5 g.L -1 . Compared to Saccharomyces cerevisiae, the growth of Pichia pastoris GS115 was more sensitive to the [Bmim]Cl, and its ethanol fermentation had lower final ethanol concentration and overall ethanol yield from fermentable sugars at the same [Bmim]Cl concentration. This work provides useful information on selecting suitable strains for ethanol fermentation containing the [Bmim]Cl in the medium.


INTRODUCTION
Lignocellulosic materials are the most economical and highly renewable natural resources in the world. Lignocellulosic ethanol production has drawn much attention in recent years [1]. Ethanol is not only used as a clean and renewable energy but also as a versatile chemical. Its consumption is to keep increasing steadily. It is now one of the most widely used transport bio-fuels. In general, production of lignocellulosic ethanol needs to firstly convert the carbohydrates in lignocellulosic materials to the fermentable sugars, and then ferment the obtained fermentable sugars to ethanol. Due to the complex structure of lignin and hemicellulose with cellulose in lignocellulosic materials, the conversion of carbohydrates in lignocellulosic materials to the fermentable sugars becomes the control procedure in the lignocellulosic ethanol production. Although lots of studies have been carried out, there are still facing great challenges in converting the carbohydrates in lignocellulosic materials to the fermentable sugars in an industrial scale based on economical and environmental consideration [1,2]. Use of ionic liquids has provided a new technical tool to convert the carbohydrates in lignocellulosic materials to the fermentable sugars for ethanol production [3]. Some studies have indicated that the *Address correspondence to this author at the Key Laboratory for Green Chemical Process of Ministry of Education, Hubei Key Laboratory of Novel Chemical Reactor and Green Chemical Technology, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430073, P.R. China; Tel: +86-27-87195671; Fax: +86-27-87195671; E-mail: zhusd2003@21cn.com carbohydrates in lignocellulosic materials can be efficiently converted to the fermentable sugars using ionic liquid technology [4]. The conversion of carbohydrates in lignocellulosic materials to the fermentable sugars using ionic liquid technology has three technical routes: ionic liquid pretreatment of lignocellulosic materials [5], enzymatic hydrolysis of lignocellulosic materials in ionic liquid medium, and chemical hydrolysis of of lignocellulosic materials in ionic liquid medium [6][7][8]. Whatever technical route was employed, some ionic liquids remained in the obtained fermentable sugars were inevitable. Therefore, it is extremely important to know how the residual of ionic liquids in fermentable sugars will affect the subsequent ethanol fermentation process, because the 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) is one of the most widely-used and cheapest ionic liquid in conversion the carbohydrates in lignocellulosic materials to the fermentable sugars for ethanol production, it was often chosen as a model ionic liquid to study the influence of its residual in fermentable sugars on the subsequent ethanol fermentation process. Some studies have been carried out on the effects of [Bmim]Cl on the growth and ethanol fermentation of Saccharomyces cerevisiae in our previous work [9,10]. Apart from Saccharomyces cerevisiae, Pichia pastoris is also often used for ethanol fermentation [11,12]. In order to select the suitable strains for ethanol fermentation containing the [Bmim]Cl in the medium, the influence of [Bmim]Cl on the growth and ethanol fermentation of Pichia pastoris GS115 will be investigated in this work and compared with our previous studies.

MATERIALS AND METHODS
All experiments were carried out three times, and the given numbers are the mean values, whose relative errors are within .

Micro-Organism, Medium and Culture Conditions
The Pichia pastoris GS115 was used throughout this study. The medium and culture conditions were the same as our previous work for ethanol fermentation of Saccharomyces cerevisiae [9,10]. During the ethanol fermentation process of Pichia pastoris GS115, some samples were taken at regular intervals for later analysis.

Analytical Methods
The samples taken from the ethanol fermentation process of Pichia pastoris GS115 were used to determine the concentration of biomass, ethanol and the fermentable sugars. Biomass concentration was determined by the dry weight method [13]. Ethanol content was determined by gas chromatography [14] and the fermentable sugars concentration was estimated using the 3,5-dinitrosalicylic acid method [15].

Effect of the [Bmim]Cl on the Growth of Pichia pastoris GS115
In order to evaluate the influence of the [Bmim]Cl on the ethanol fermentation process of Pichia pastoris GS115, the growth of Pichia pastoris GS115 was first investigated at different [Bmim]Cl concentrations in the fermentation medium. Fig. (1) shows the growth curves of Pichia pastoris GS115 at different [Bmim]Cl concentrations form 0.0001 to 5 g.L -1 during the ethanol fermentation process. As indicated in Fig. (1), the [Bmim]Cl had no influence on the growth of Pichia pastoris GS115 at its concentration less than 0.0001 g.L -1 . However, the [Bmim]Cl inhibited the growth of Pichia pastoris GS115 at its concentration higher than 0.0001 g.L -1 . Moreover, this inhibition became stronger with its concentration increasing. When the [Bmim]Cl in the medium reached 5 g.L -1 , the yeast had almost no growth and its biomass concentration increased very slowly. Compared to our previous studies [9,10], the influence of [Bmim]Cl on the growth of Pichia pastoris GS115 and Saccharomyces cerevisiae had the same characteristic, but Pichia pastoris GS115 was was more sensitive to the [Bmim]Cl than Saccharomyces cerevisiae. Under the same [BMIM]Cl concentration, the [BMIM]Cl had more serious inhibition on the growth of Pichia pastoris GS115. The inhibition mechanism of [BMIM]Cl on the growth of Pichia pastoris GS115 might be similar with its inhibition on Saccharomyces cerevisiae, which comes from the interaction between [BMIM]Cl and its cytomembrane.

Effect of [Bmim]Cl on the Ethanol Fermentation Process of Pichia pastoris GS115
Apart from yeast growth, the [Bmim]Cl also affects the ethanol formation and fermentable sugars consumption during the ethanol fermentation process of Pichia pastoris GS115. The time courses of ethanol formation and fermentable sugars consumption were measured at different [Bmim]Cl concentrations in the fermentation medium and the results are shown in Figs. (2 and 3) respectively. As shown in Figs. (2 and 3), the [Bmim]Cl had no influence on the ethanol formation and fermentable sugars consumption during the ethanol fermentation process of Pichia pastoris GS115 at its concentration is less than 0.0001 g.L -1 . However, the [Bimim]Cl negatively affected the ethanol formation and fermentable sugars consumption at its concentration is higher than 0.0001 g.L -1 . With the [Bmim]Cl concentration  increasing, the ethanol formation rate and the consumption rate of fermentable sugars both decreased. When the [Bmim]Cl in the medium reached 5 g.L -1 , there was almost no ethanol formation and fermentable sugars consumption.  Ci represents the [Bmim]Cl concentration (g.L -1 ), Cb represents the final biomass concentration (g.L -1 ), Cp represents the final ethanol concentration (g.L -1 ), Cs represents the final fermentable sugars concentration (g.L -1 ), Y represents the ethanol overall yield from the fermentable sugars.