Reagents and strain
Ionic liquid 1-ethyl-3-methylimidazolium acetate (EmimOAc) was supplied by Lanzhou Greenchem ILs, LIPC, CAS (Lanzhou, China) and used without further purification. Cellulose (sigmacell cellulose Type101, moisture content 10% (wt/wt)), cellulase and cellobiase were purchased from Sigma. The activities of cellulase and cellobiase were determined as 161.0 FPU/mL and 674.7 CBU/mL, respectively [31, 32]. Xylanase was obtained from Imperial Jade Bio-Technology Co., Ltd. (Yinchuan, China), and the activity was 5000 kU/g. One unit of xylanase (U) was defined as the amount of enzyme which produces 1.0 μg of xylose from 1% xylan solution at pH 5.0, 50°C within 1 min. N-Methylpyrrolidone (NMP) and other reagents were analytical grade and purchased from local company.
The yeast C. curvatus ATCC 20509 was obtained from the American Type Culture Collection, and maintained at 4°C every two weeks on yeast peptone dextrose (YPD) agar slant (yeast extract 10 g/L, peptone 10 g/L, glucose 20 g/L, agar 15 g/L, pH 6.0).
Pretreatment of corn stover with the EmimOAc–NMP solution
Corn stover harvested from countryside of Changchun, China, was milled to a particle size of 1–2 mm. The milled materials were washed to remove the field dirt, stones and metals, dried at 105°C until the weight was constant, and stored in desiccate before use. Analysis of the corn stover sample according to the procedures of the National Renewable Energy Laboratory revealed a composition (dry weight basis) of 37.9% glucan, 20.1% xylan, 2.3% arabinan, and 20.8% lignin.
Corn stover was pretreated by the EmimOAc–NMP solution . Briefly, to a solution of EmimOAc (60 g) and NMP (140 g) preheated in an oil bath at 140°C was added corn stover sample (20 g). The suspension was held at the same temperature with stirring for 1 h, until a viscous black yellow solution formed. The solution was cooled down to 50°C, and methanol (500 mL) was added with vigorously stirring. The precipitates were filtrated, washed with methanol (500 mL) and water (2 × 500 mL), and freeze-dried to obtain regenerated corn stover samples.
Enzymatic hydrolysis of cellulose and regenerated corn stover
Cellulose hydrolysates were made by enzymatic hydrolysis of 3.6% (w/v) sigmacell cellulose in 0.3 M phosphate buffer (pH 5.2) at 37°C, 200 rpm. Cellulase and cellobiase per gram cellulose were loaded at 7.5 FPU and 15 CBU, respectively. Corn stover hydrolysates were made by enzymatic hydrolysis of 5.0% (w/v) regenerated corn stover in 50 mM phosphate buffer (pH 4.8) at 50°C, 200 rpm. Cellulase, cellobiase and xylanase per gram dry materials were loaded at 10 FPU, 20 CBU and 10 mg, respectively.
General procedure for the SSELP process
C. curvatus cells were cultivated in YPD liquid medium (yeast extract 10 g/L, peptone 10 g/L, glucose 20 g/L, pH 6.0) at 30°C, 200 rpm for 24 h. Cells were collected by centrifugation, washed with sterilized water, and then used as inocula for all two-stagedd culture processes. One unit optical absorbance at 600 nm (OD600 nm) for such inocula equaled to 0.36 g/L of cell dry weight (CDW). About 0.27 g of CDW equivalent inocula were transferred into a suspension of hydrolytic enzymes and 2.0 g of cellulose in 50 mL of 0.3 M phosphate buffer (pH 5.2), and other experimental details are summarized in Table 2.
Time course of lipid production with different culture processes
A two-stagedd lipid production process was carried out at 30°C, 200 rpm. About 0.27 g of CDW equivalent inocula were resuspended in 50 mL of 0.05 M phosphate buffer (pH 5.5) contained 2.08 g glucose · H2O and 1% (V/V) a trace element solution . The composition of the trace element solution contained (g/L): CaCl2
.2H2O 4.0, FeSO4
.7H2O 0.55, citric acid.H2O 0.52, ZnSO4
.7H2O 0.10, MnSO4
.H2O 0.076 and 100 uL of 18 M H2SO4.
For the SSELP process, 0.27 g of CDW equivalent inocula were transferred into a suspension contained 2.0 g of cellulose, 15 FPU cellulase and 30 CBU cellobiase in 50 mL of 0.3 M KH2PO4-Na2HPO4 buffer (pH 5.2), and the suspension was held at 37°C, 200 rpm for lipid production.
Lipid production from regenerated corn stover
Experiments were first done with the SHELP process. To 50 mL of the corn stover hydrolysates contained 31.9 g/L of glucose and xylose was inoculated with 0.27 g of CDW equivalent inocula, and the culture was held at 30°C, 200 rpm for lipid production.
For the SSELP process, 0.27 g of CDW equivalent inocula were transferred into a suspension contained 2.5 g of regenerated corn stover, 10 FPU cellulase, 20 CBU cellobiase and 12.5 mg xylanase in 50 mL of 0.3 M KH2PO4-Na2HPO4 buffer (pH 5.2), and the suspension was held at 37°C, 200 rpm for lipid production.
Glucose was determined using an SBA-50B glucose analyzer (Shandong Academy of Sciences, Jinan, China). Sugar mixtures were analyzed by ion chromatography (IC) on the Dionex ICS2500 system with a CarboPac PA10 analytical column (Dionex Co.) and an ED50 electrochemical detector (Dionex Co.). The column was washed with isocratic elution of NaOH at a speed of 1 mL/min at 30°C. The concentration of NaOH was 22 mM from 0 min to 20 min, retention time for glucose and xylose were 10.9 and 12.1 min, respectively. Cellulose concentration was determined as described . Residual cellulose was collected by repeated precipitation and washing with water to remove soluble carbohydrates. Precipitated sample was further washed using acetic acid-nitric acid reagent and water to remove non-cellulosic materials  and quantified by using the phenol-sulfuric acid method with glucose as the standard . Cellulose conversion was calculated based on the initial cellulose and residual cellulose.
Samples containing cellulose and yeast cells from 30 mL of culture broth were collected by centrifugation and washed twice with distilled water. Cell mass, expressed as CDW, was determined gravimetrically after drying the wet sample at 105°C overnight and deducting cellulose from the sample. Fat-free cell mass was calculated after subtraction of lipids from CDW.
Dried samples containing cellulose and yeast cells were digested with 4 M HCl at 78°C for 1 h before extraction with chloroform/methanol (1: 1, vol/vol). The extracts were washed with 0.1% NaCl, dried over anhydrous Na2SO4, evaporated in vacuo, and the residue was dried at 105°C for 24 h to give the total lipids . Lipid content was expressed as gram lipids per gram CDW. Lipid coefficient was expressed as gram lipids produced per gram cellulose.
The fatty acid compositional profiles of lipid samples were determined using a 7890F gas chromatography instrument after transmethylation according to a published procedure with minor modifications . Briefly, 70 mg of lipids were treated with 0.5 mL of 5% KOH solution in methanol at 65°C for 50 min, followed by the addition of 0.2 mL BF3 diethyletherate and 0.5 ml methanol. The mixture was refluxed for 10 min, cooled, and extracted with n-hexane. The organic layer was washed twice with distilled water, and used for fatty acid compositional analysis.
All data in this study were the averages of three independent experiments.