Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6521-5

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Simultaneous saccharification and fermentation of cellulose in ionic liquid for efficient production of α-ketoglutaric acid by Yarrowia lipolytica Seunghyun Ryu & Nicole Labbé & Cong T. Trinh

Received: 13 January 2015 / Revised: 1 March 2015 / Accepted: 2 March 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Ionic liquids (ILs) are benign solvents that are highly effective for biomass pretreatment. However, their applications for scale-up biorefinery are limited due to multiple expensive IL recovery and separation steps that are required. To overcome this limitation, it is very critical to develop a compatible enzymatic and microbial biocatalyst system to carry the simultaneous saccharification and fermentation in IL environments (SSF-IL). While enzymatic biocatalysts have been demonstrated to be compatible with various IL environments, it is challenging to develop microbial biocatalysts that can thrive and perform efficient biotransformation under the same conditions (pH and temperature). In this study, we harnessed the robust metabolism of Yarrowia lipolytica as a microbial platform highly compatible with the IL environments such as 1-ethyl3-methylimidazolium acetate ([EMIM][OAc]). We optimized the enzymatic and microbial biocatalyst system using commercial cellulases and demonstrated the capability of Y. lipolytica to convert cellulose into high-value organics such as α-ketoglutaric acid (KGA) in the SSF-IL process at relatively low temperature 28 °C and high pH 6.3. We showed that SSF-IL not only enhanced the enzymatic saccharification but also produced KGA up to 92 % of the maximum theoretical yield.

Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6521-5) contains supplementary material, which is available to authorized users. S. Ryu : C. T. Trinh (*) Department of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, USA e-mail: [email protected] N. Labbé Center of Renewable Carbon, University of Tennessee, Knoxville, USA

Keywords Yarrowia lipolytica . α-Ketoglutaric acid . Simultaneous saccharification and fermentation in ionic liquid . SSF-IL . 1-Ethyl-3-methylimidazolium acetate . Ionic liquid

Introduction Most chemicals and transportation fuels are currently produced from petroleum-based feedstocks, which are neither renewable nor sustainable. Their use has adversely affected market prices, environment, and national energy security. In recent years, extensive research has explored microbial bioconversion routes to produce these chemicals and fuels from renewable and sustainable lignocellulosic biomass (Buschke et al. 2013). The key challenges to achieve this goal are to overcome biomass recalcitrance, engineer novel microbial biocatalysts that can yield high production of target products, and, at the same time, replace those synthesized from petroleum-based feedstocks in a competitive manner (Himmel et al. 2007; Peralta-Yahya et al. 2012; Stephanopoulos 2007). Lignocellulosic biomass is mainly composed of cellulose, hemicellulose, and lignin. Cellulose and hemicellulose are carbohydrate polymers consisting of glucose and mixture of hexose and pentose sugars, respectively, which can be degraded into mono-sugars by glycoside hydrolases (Chundawat et al. 2011; Himmel et al. 2007; Lynd et al. 2002). These polysaccharides together with lignin are tightly cross-linked, which causes biomass recalcitrance and hence decreases saccharification efficiency. However, the recalcitrant structure of biomass can be deconstructed to enhance enzyme accessibility for efficient saccharification by various pretreatment processes such as dilute acid, ammonium fiber explosion, steam explosion, organosolv, and wet oxidation (Haghighi Mood et al. 2013; Mosier et al. 2005; Yang and Wyman 2008).

Appl Microbiol Biotechnol

The ionic liquid (IL) pretreatment of lignocellulosic biomass has recently emerged as a promising technology (Da Costa Lopes et al. 2013; Mora‐Pale et al. 2011). An IL is a salt that can exist in the liquid form at room temperature and can significantly reduce biomass recalcitrance, effective for efficient biomass fractionation and saccharification (KleinMarcuschamer et al. 2011; Liu et al. 2012; Park and Kazlauskas 2003; Van Rantwijk and Sheldon 2007). Recent studies demonstrated that IL pretreatment of lignocellulosic biomass achieved a better enzymatic hydrolysis with higher sugar yields, higher hydrolysis rates, and lower enzyme loadings than other pretreatment methods (e.g., dilute acid and ammonium fiber explosion) where IL removed significant amounts of lignin and hemicellulose from cellulose and reduced cellulose crystallinity (Lee et al. 2009; Li et al. 2010, 2011, 2013). In addition, lignin from the IL pretreatment can be converted to high-value products (e.g., lignin-carbon fibers, lignin-starch films, and vanillin), which is very advantageous for economics of biorefinery (Klein-Marcuschamer et al. 2011; Varanasi et al. 2013). Despite these advantages, many challenges have hindered the IL pretreatment process from being deployed for scale-up biorefineries due to the following reasons. First, ILs are expensive and require many separation and recovery steps where a large amount of water is used to wash ILs from biomass, followed by their separation from water. Second, ILs can significantly inhibit the catalytic activity of enzymatic and microbial biocatalysts, limiting the deployment of the cost-effective simultaneous saccharification and fermentation (SSF) or consolidated bioprocessing process (CBP) in the presence of ILs (Konda et al. 2014). Most microbes have been reported to suffer from growth inhibition in media of 10 % (w/v) IL (Docherty and Kulpa 2005; Khudyakov et al. 2012; Santos et al. 2014; Thi et al. 2010). For instance, an industrial workhorse Saccharomyces cerevisiae was significantly inhibited in a defined medium containing 1 % 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]) (Ouellet et al. 2011) and exhibited moderate tolerance in a complex medium containing up to 5 % 1-ethyl-3-methylimidazolium diethylphosphate ([EMIM][DEP]) or 5 % 1-ethyl-3-methylimidazolium chloride ([EMIM][Cl]) (Nakashima et al. 2011). Even for the IL-tolerant lignin-degrading bacterium Enterobacter lignolyticus SCF1 recently isolated, it suffered a significant reduction in growth rate from 0.15 h−1 in 0 % [EMIM][Cl] to 0.06 h−1 in 5 % [EMIM][Cl] (Khudyakov et al. 2012). To develop a cost-effective IL pretreatment process, it is very critical to engineer a compatible enzymatic and microbial biocatalyst system that can function in ILs and hence reduce multiple downstream separation and recovery steps of ILs, biocatalysts, and desirable products. Recently, an enzymatic saccharification of lignocellulosic biomass in ILs was successfully demonstrated (Liu et al. 2014; Wang et al. 2011b). Specifically, the IL-pretreated switchgrass containing 10–

20 % [EMIM][OAc] could be directly hydrolyzed by a thermophilic, IL-tolerant cellulase cocktail (Park et al. 2012; Shi et al. 2013). However, the key challenge to develop compatible microbial and enzymatic biocatalysts for SSF-IL still remains (Ruegg et al. 2014). In this study, we tackled the challenge of IL pretreatment process by developing a novel enzymatic and microbial biocatalyst system that is highly compatible for SSF-IL or CBPIL. Our biocatalyst system employed Yarrowia lipolytica as a novel microbial biocatalyst platform coupled with commercial cellulases. Y. lipolytica is a nonconventional oleaginous yeast capable of metabolizing various substrates to produce valuable molecules (Beopoulos et al. 2009; Coelho et al. 2010). It also exhibits unique characteristics of extremophiles that can grow in a wide range of pH 2–11 and even high salt concentrations up to 12 % (v/v) NaCl (Andreishcheva et al. 1999; Epova et al. 2012). Since ILs are liquid salts at room temperature, we hypothesized that Y. lipolytica can tolerate high concentration of ILs and can be deployed as a novel microbial platform to perform bioconversion in high concentration of ILs. Here, we investigated the effect of [EMIM][OAc], one of the commonly used ILs for effective biomass pretreatment, on growth of Y. lipolytica, and exploited the capability of Y. lipolytica as a novel microbial platform for converting ILpretreated cellulose into α-ketoglutaric acid (KGA) at high yields by SSF-IL (Fig. 1). KGA is a high-value organic that can extend the organismal lifespan (Chin et al. 2014) and has a broad application such as a building block for synthesis of dietary supplements, pharmaceuticals, cosmetics, and heterocycles (Aurich et al. 2012; Otto et al. 2011; Stottmeister et al. 2005; Zhou et al. 2010).

Fig. 1 Simplified central metabolism of Y. lipolytica for conversion of fermentable sugars into α-ketoglutaric acid (KGA). Under thiaminelimited growth conditions, the thiamine-dependent α-ketoglutarate dehydrogenase (KGDH) is inhibited, causing the extracellular secretion of KGA

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Materials and methods Plasmid, strain, and medium Wild-type Y. lipolytica (ATCC MYA-2613), a thiamine, leucine, and uracil auxotroph, was purchased from American Type Culture Collection. The plasmid pSL16-CEN1-1(227) containing leucine gene (LEU2) as a selection marker was provided by Dr. Matsuoka (Sojo University, Japan) (Yamane et al. 2008) as a kind gift. This plasmid was transferred into MYA-2613 by electroporation (Wang et al. 2011a) to create Y. lipolytica (leu+). Y. lipolytica strains were cultured in either SC-LEU or MpA defined medium. The SC-LEU defined medium is a yeast nitrogen base containing synthetic dropout amino acid mixture without leucine (cat# Y0626, Sigma Inc., St. Louis, MO, USA) and 10 g/L glucose. The MpA defined medium contains the following: 3 g/L (NH4)2SO4, 2 g/L KH2PO4, 1 g/L MgSO4·7H2O, 10 mg/L FeSO4·7H2O, 44 mg/L ZnSO4·7 H2O, 79 mg/L CaCl2·2 H2O, 0.8 mg/L biotin, trace elements (0.4 mg/L ZnSO4·7 H2O, 6 mg/L FeCl3·6H2O, 0.04 mg/L CuSO4·5 H2O, 0.4 mg/L MnCl2·4 H2O, 0.2 mg/L Na2MoO4·2 H2O, 0.1 mg/L KI, 0.5 mg/L H3BO3), and 20 mg/L uracil. Unless specified, 10 g/L glucose was used as a carbon source together with 0.5 μg/L thiamine-HCl and 1 g/L peptone as supplements. Ionic liquid toxicity test Y. lipolytica (leu+) was used to test the toxicity of [EMIM][OAc] (>95 % purity, IoLiTec Ionic Liquids Technologies Inc., Tuscaloosa, AL, USA) on cell growth in the SC-LEU medium. When reaching the exponential phase, cells were pelleted at 4500×g and 22 °C for 7 min; washed twice with 10 mM phosphate-buffered saline (PBS); and suspended in fresh SC-LEU medium containing 0, 5, 10, and 20 % (v/v) [EMIM][OAc] with an initial loading of 1×107 viable cells/mL. Viable cell concentration was determined by a hematocytometer after cells were stained with 0.1 % methylene blue using a Fisher Scientific Micromaster Brightfield Microscope (Fisher Scientific, Pittsburgh, PA, USA). Enzymatic saccharification in 1-ethyl-3-methylimidazolium acetate A solution containing 30 mg Avicel PH-101 and 300 μL [EMIM][OAc] were well-mixed and incubated at 50 °C for 24 h (Wang et al. 2011b). Avicel PH-101, a microcrystalline cellulose, was purchased from Sigma-Aldrich (St. Louis, NO, USA). The final concentration of [EMIM][OAc] was then adjusted to 10 % (v/v) by addition of 2.7 mL of MpA medium. The pH of the IL-cellulose solution was about 6.3. Saccharification experiments were conducted by adding

Celluclast 1.5 L with various loadings (0, 15, 30, 50, and 75 filter paper unit (FPU)/g glucan) and Novozyme 188 with 2:1 (cellobiase unit (CBU)/FPU) ratio of Novozyme 188 to Celluclast 1.5 L, where FPU represents filter paper unit. Celluclast 1.5 L and Novozyme 188 were kindly provided by Novozymes North America (Franklinton, NC, USA). The IL-pretreated cellulose and cellulase mixtures were incubated at 28 °C and 190 rpm for 96 h, and saccharification efficiency was determined by a 3,5-dinitrosalicylic acid (DNS) assay (Ghose 1987). Briefly, 100 μL of DNS solution was mixed with 100 μL of samples and boiled for 5 min. The absorbance of samples was recorded at 540 nm, and the amount of reducing sugars produced was quantified by using a glucose standard. No signal interference in DNS assay by [EMIM][OAc] was observed. Simultaneous saccharification and fermentation in 1-ethyl-3-methylimidazolium acetate Simultaneous saccharification and fermentation (SSF) was conducted in the defined MpA medium containing 10 % (v/v) [EMIM][OAc] at 28 °C and pH 6.3. Like the enzymatic saccharification, IL-pretreated Avicel PH-101 was generated and diluted to 10 % (v/v) in the MpA medium to yield a final cellulose concentration of 10 g/L. Next, 50 FPU/g glucan of Celluclast 1.5 L and 100 CBU/g glucan of Novozyme 188 were added to the diluted cellulose-IL-MpA solution, and fermentation was initiated by inoculating the exponentially grown Y. lipolytica (leu+) with an initial OD600 nm of 2.0 (~1×107 cells/mL). Experiments were conducted in 250-mL baffled flasks with 25-mL working volume at 28 °C and 190 rpm, and samples were collected for metabolite analysis. All experiments were carried out in at least biological triplicates. To determine residual cellulose at the end of SSF-IL, the fermentation broth was centrifuged at 4500×g for 10 min, and the pellet including cells plus cellulose was collected, washed with water to remove residual [EMIM][OAc], resuspended in 25 mM citrate buffer (pH 4.5), and boiled for 3 min to lyse the cell. A mixture of 350 FPU/g glucan Celluclast 1.5 L and 700 CBU/g glucan Novozyme 188 was then added into the reaction mixture, and the completed saccharification experiments were conducted at 50 °C and 150 rpm for 48 h. The amount of residual cellulose was determined by measuring the amount of glucose produced via high-performance liquid chromatography (HPLC). High-performance liquid chromatography The amounts of sugars and organic acids were quantified by using the Shimadzu HPLC system equipped with RID and UV detectors (Shimadzu Scientific Instruments Inc., Columbia, MD, USA) and Aminex 87H column

Appl Microbiol Biotechnol

(Bio-Rad Inc., Hercules, CA, USA). The method used 10 mN H2SO4 as a mobile phase operated at a flow rate of 0.6 mL/min and oven temperature set at 48 °C (Trinh et al. 2008).

Results Y. lipolytica can thrive in 1-ethyl-3-methylimidazolium acetate Development of a SSF-IL process can significantly reduce the cost of IL separation and recovery. However, not many microbes are known to thrive in IL environments while efficiently producing target products. Since Y. lipolytica has a robust metabolism that can not only thrive in high salt concentration up to 12 % NaCl (Andreishcheva et al. 1999; Epova et al. 2012) but also capable of producing valuable products such as organic acids, lipids, and enzymes (Coelho et al. 2010; Finogenova et al. 2005; Groenewald et al. 2014), Y. lipolytica can be a potential novel microbial platform for SSF-IL in the presence of ILs, such as [EMIM][OAc]. To test this, we first examined the IL effect on growth of Y. lipolytica in the MpA medium containing 0, 5, 10, and 20 % (v/v) [EMIM][OAc] and used glucose as a carbon and energy source. Here, we used [EMIM][OAc] because it is efficient for reducing biomass recalcitrance (Li et al. 2010) and highly compatible with commercial cellulases used for saccharification of IL-pretreated biomass (Wang et al. 2011b). The result shows that Y. lipolytica could grow in at least 10 % [EMIM][OAc] with a 60 % reduction in the specific growth rate (Fig. 2). In the 5 % IL medium, the relative specific growth rate of Y. lipolytica decreased about 30 % while slight growth was observed in the 20 % IL medium. Y. lipolytica can perform biotransformation in ionic liquids After discovering that Y. lipolytica could thrive in at least 10 % [EMIM][OAc], we tested its biotransformation capability to

Fig. 2 Growth study of wild-type Y. lipolytica in the [EMIM][OAc] media. a Effect of different concentrations of [EMIM][OAc] on cell growth. b Growth kinetics of Y. lipolytica in 10 % [EMIM][OAc]

produce organic acids such as α-ketoglutaric acid (KGA) (Fig. 1). We chose KGA as the target product because it is a high-value chemical with broad applications (Otto et al. 2011), and also, Y. lipolytica is a native KGA overproducer at low pH (3.5–4.5) and under thiamine limitation (