**3. Results and discussion**

of pH 4.9 sodium citrate buffer, and 180 ml of Millipore filtered water. The reactor was stirred continuously at a slow speed as reported by Martin et al. (2010) and maintained at 50 °C for

Aliquots from pretreatment hydrolyzates, wash waters and enzyme hydrolyzates were analyzed by high-pressure liquid chromatography (HPLC) for carbohydrates and inhibitory byproducts. Two instruments were used to conduct these analyses. Carbohydrates were analyzed with Waters 2695 Separations module (Milford, MA) equipped with Shodex (Waters, Milford, MA) precolumn (SP-G, 8 µm, 6 x 50 mm) and Shodex column (SP0810, 8 µm x 300 mm). Millipore filtered water (0.2 mL/min) was the mobile phase and the column was heated to 85 ˚C with an external heater. Carbohydrates were detected with a Waters 2414 Refractive Index Detector (Milford, MA) as described by Djioleu et al. (2012). Inhibitory byproducts were analyzed on a Waters 2695 Separations module equipped with a Bio-Rad (Hercules, CA) Aminex HPX-87H Ion Exclusion 7.8 mm X 30 mm column, heated to 55˚C. The mobile phase was 0.005 M H2SO4, flowing at 0.6 ml/min. Compounds were detected with a UV index using the Waters 2996 Photodiode Array detector. Furfural and hydroxymethylfurfural (HMF) were

24 hours. The entire sample was collected at the end of the run and stored at 4 °C.

108 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

detected at 280 nm; whereas, formic acid and acetic acid were detected at 210 nm.

of each hydrolysate to reach a yeast cell concentration of 8×107

µl of 0.1 mg/ml n-butanol as an internal standard.

Fermentation was carried out in 50 ml shake flasks with two strains of yeast, self-flocculating SPSC01 and ATCC4126. The SPSC01 strain was provided by Dalian University of Technology, China (Bai et al. 2004). Preculture of both yeast strains was carried out in medium consisting of 30 g/L glucose, 5 g/L yeast extract and 5 g/L peptone. The overnight grown yeasts were harvested by centrifugation at 4,100 *g* for 30 min. The pellets of yeast cells were washed twice with de-ionized water, and then re-suspended in 50 mM sodium citrate buffer (pH 4.8) to reach a cell concentration of 2 to 4×109 /ml. The re-suspended yeast cells were inoculated into 10 ml

performed at 30°C on a rotary shaker at 150 rpm for 8 hours. Glucose content of the samples was assayed using a glucose colorimetric assay kit (Cayman Chemical, MI). Produced ethanol was quantified by gas chromatography (GC) on the Shimadzu GC-2010 equipped with a flame ionization detector (FID) and a Stabilwax®-DA column (cross-bond polyethylene glycol, 0.25 mm ×0.25 µm ×30 m), as described early by Ge et al. (2011). Before injection into the GC, 50 µl of fermentation broth was diluted 10 times with de-ionized water and supplemented with 50

Experiments were conducted in duplicate (pretreatment and enzymatic saccharification) or triplicate (fermentation). Calculations of carbohydrate and degradation compounds, including HMF, furfural, formic acid, and acetic acid, were calculated using Microsoft Office Excel 2007. Analysis of the variance (ANOVA) was determined using JMP 9.0, LSMeans Differences

/ml. Ethanol fermentations were

**2.4. HPLC analysis**

**2.5. Fermentation**

**2.6. Statistical analysis**

Student's t, with α= 0.10.
