Raw materials and pretreatment
Corn stover (CS) was grown in Jilin, China, and harvested in fall 2007. Rice straw and cotton stalk were grown in Hubei, China, and harvested in 2008. Wheat straw and rape straw were grown in Henan, China, and harvested in 2008. After collection, the materials were milled coarsely on a beater pulverizer (SF-300; Ketai Milling Equipment, Shanghai, China) and screened through mesh with a circle diameter of 10 mm. The milled raw materials were washed to remove the field dirt, stones and metals; dried; and then stored in sealed plastic bags for use.
The steam explosion pretreatment was performed on CS only. The milled CS materials were steam heated to 210°C, 2.2 MPa, for 4 minutes, then the pressure was released quickly . Only saturated steam was used, and no chemicals were added. The pretreated CS contained approximately 50% dry solid matter (DM) and was stored at 4°C for use.
The dilute sulfuric acid pretreatment was performed on all the materials, including CS, rice straw, wheat straw, rape straw and cotton stalk. The pretreatment reactor was a self-made stainless cylinder with the working volume of 10 L, and 800 g of each feedstock material were filled in each operation. The feedstock was presoaked with diluted sulfuric acid solution with the solid (dry material) to liquid (5.0% (wt/wt) sulfuric acid solution) ratio of 2:1 (wt/wt). The presoaked wet materials were fed into the reactor, and the hot steam was jetted directly into the reactor to 190°C, 1.2 MPa, for 3 minutes. All the sulfuric acid solution and the steam-condensed water were absorbed into the solid material to give a DM content of 50% (wt/wt). No free water was generated during the pretreatment. The pretreated materials were released from the reactor and stored at 4°C. The most frequently used pretreatment method in this work is the dilute sulfuric acid method unless mentioned otherwise.
Enzymes and ethanol fermentation strain
The cellulase enzyme used was Accellerase 1000 from Genencor International (Rochester, NY, USA). The filter paper activity and the cellobiase activity were determined to be 65.8 FPU/ml and 152.0 IU/ml, respectively. One unit of filter paper cellulase (FPU) was defined as the amount of enzyme which produces 2.0 mg of reducing sugar from 50.0 mg of filter paper within 1 h. The detailed procedures for determination of cellulase and cellobiase activities were described in Zhang et al. .
A thermo-and inhibitor-tolerant mutant strain Saccharomyces cerevisiae DQ1 was used for ethanol fermentation. The culture solution was aliquoted into 1-mL vials containing 30% (wt/wt) glycerol and stored in a -80°C. A vial of S. cerevisiae DQ1 was taken from the -80°C freezer and directly inoculated in the seeding culture for the purpose of keeping all the seeding strains the same. The adaptation procedure of the strain S. cerevisiae DQ1 was described in detail by Zhang et al. .
Isolation of detoxification strains
The steam explosion-pretreated CS samples were exposed to ambient air for 2 weeks and stored at 4°C in a refrigerator for months after being transported from the cold northeast China region to the warm southern Shanghai area. The original detoxification strains were isolated using a three-step screening procedure as described in the next three subsections.
Ten grams of the pretreated CS samples were diluted with 90 ml of sterilized water and incubated for 2 h at 30°C and 180 rpm to obtain the 1 × 10-1 suspension. The suspension was further diluted into 10-2, 10-3 and 10-4 suspensions. The 10-4 dilution was streaked onto the potato-dextrose-agar (PDA) plates containing 200 g/L potato extract juice, 20 g/L glucose and 20 g/L agar for enrichment culture. The plates were incubated for 5 days at 25°C, and the colonies were restreaked onto the PDA plates on the basis of the morphology and color of the colonies. Then the single colony was isolated, and each colony was restreaked for five generations to obtain the purified single colony.
The screening medium was prepared by adding the toxic compounds of 6.0 g/L acetic acid, 1.0 g/L furfural, and 1.5 g/L HMF into the PDA medium. The isolated colonies were streaked onto the screening medium and incubated for 5 days at 25°C. Then the toxin tolerant strains were selected from the PDA medium containing toxins.
The selected colonies were further quantitatively screened by their toxin degradation abilities on the steam explosion-pretreated CS samples. Ten grams of the pretreated CS were inoculated with 1 ml of diluted suspension (about 1 × 106 spores/mL) and incubated in the 250-mL flasks at 25°C for 4 days. A quantity of 44 ml of the citrate acid buffer (100 mM, pH 4.8) and 1.25 ml of Accellerase 1000 (15.0 FPU/g DM) were added to the flasks to reach solid loading of 10% (wt/wt). The enzymatic hydrolysis lasted for 12 h at 50°C and 150 rpm in a water bath shaking incubator. The steam explosion-pretreated CS without strain inoculation was used as the control. The initially selected strains with better inhibitor-degrading performance were selected for further experimentation.
Molecular identification of the detoxification strain
The selected strains were cultured in the PDA medium, and 8 mg of the dry mycelia were collected. The genomic DNA was extracted using the Qiagen DNeasy Tissue Kit (Qiagen, Valencia, CA, USA) and purified following the "Yeast Genomic DNA Purification Protocol" discussed in the attached handbook. The 18 S rDNA internal transcribed spacer (ITS) sequence was amplified by the universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTAGATATGC-3'). The polymerase chain reaction (PCR) products were purified using the PCR Purification Mini Kit (Omega Biotek, Norcross, GA) and sequenced by Shanghai Biotech Service (Shanghai, China). The ITS sequences were blasted in the National Center for Biotechnology Information database http://www.ncbi.nlm.nih.gov/, and the phylogenetic trees were constructed using Bioedit 7.0 http://www.mbio.ncsu.edu/bioedit/bioedit.html and Mega 4 software http://www.megasoftware.net/ using the neighbor-joining method.
Growth assay of isolates on pretreated CS material
The growth performance under different pH and temperature conditions was assayed by counting the colony numbers. One milliliter of the selected strain (1 × 106 spores) suspension was inoculated onto 10 g of the pretreated CS and incubated for 3 days at 25°C. The CS was washed with 90 ml of sterilized water, shaken for 2 hours at 25°C and 180 rpm and then diluted to 10-3 or 10-4 fold. A quantity of 0.1 ml of the suspension was taken, spread onto the PDA plates and incubated for 4 days at 25°C. Then the colony numbers on PDA plates were counted. The growth assay under different oxygen levels was operated using two flasks, one filled with nitrogen and sealed with a rubber stopper and the other covered only with a cotton stopper.
Growth assays of isolates using toxins as the sole carbon sources
The detoxification performance of the isolates using the inhibitor substances as the sole carbon sources was assayed by observation of the mycelia growth on the pretreated CS solids. The dilute sulfuric acid-pretreated CS was thoroughly washed with deionized water until no inhibitor substances from the washout solution were found on performing high performance liquid chromatography (HPLC). Three grams of such thoroughly washed, dried CS were mixed with various toxins (each toxin was dissolved in 10 ml of sterilized water) and used for the solid-state culture. Two milliliters of the spore suspension (1 × 106 ml) were inoculated onto the 3 g of the mixed CS in 250-ml flasks and cultured for 9 days at 25°C. The mycelia formation in the flask was observed periodically.
Detoxification assays of toxins by the isolates
The biodetoxification performance of the isolates was assayed by mixing an extra amount of toxic inhibitor substances onto the pretreated CS and then inoculated with the spore suspension of the isolates. Five grams of the pretreated CS were mixed with each of the inhibitor substances, and then 8 ml of sterilized deionized water was added. Next, 1 ml of the isolate suspension (1 × 106) was inoculated onto the CS. The toxic inhibitors supplemented included acetic acid, formic acid, levulinic acid, furfural and HMF. First, the substances were added separately, and then the toxin mixtures were added. The culture was carried out for 5 days at 25°C, then the cellulase enzyme was added for simultaneous saccharification and ethanol fermentation (SSF) assay at the conditions of 15.0 FPU/g DM, 10% (wt/wt) of the detoxified CS solid loading, pH 4.8 (100 mM citrate buffer), 50°C and 150 rpm in the water bath shaking incubator for 12 h. Then S. cerevisiae DQ1 was added at a 10% (vol/vol) inoculation ratio for another 12 h at 37°C. The samples were taken at 12-h intervals for HPLC analysis of toxins, glucose and ethanol.
SSF at high solids loading
Different lignocellulose materials, including CS, rice straw, wheat straw, rape straw and cotton stalk were pretreated using steam explosion or dilute acid methods and then detoxified at 25°C for 3 days with the selected detoxification strain. The SSF at the solid loading of 30% (wt/wt, dry base) was operated in a 5-L helical stirring bioreactor as described by Zhang et al. . In the prehydrolysis stage, Accellerase 1000 was fed into the bioreactor at the dosage of 15.0 FPU/g DM, followed by feeding of the detoxified feedstocks into the bioreactor within 12 h at 50°C and 150 rpm. Then the temperature was reduced to 37°C, and the S. cerevisiae DQ1 seeds were inoculated into the hydrolysate at the ratio of 10% (vol/vol). pH was maintained at 5.0 using a 5 M NaOH solution and a 1 M H2SO4 solution. The SSF operation continued for 60 h, and the samples were withdrawn at regular intervals.
The procedure for the water-washing detoxification was operated as follows. First, different amounts of tap water were added to the pretreated CS and stirred for 1 h at 25°C. Then squeezed liquid out of the slurry until the solid content rose to 50% (wt/wt, dry base) by a hydraulic press machine at 15 MPa (P-204; Dazhang Filter Equipment, Shanghai, China) for SSF use.
Cellulose/hemicellulose measurement and yield calculation
The contents of cellulose and hemicellulose were determined according to a two-step H2SO4 hydrolysis method put forth by the National Renewable Energy Laboratory (NREL) , with minor modifications. A quantity of 100 mg of thoroughly washed and dried CS were added with 1 ml of 72% (wt/wt) H2SO4, and the mixture was stirred using a glass rod until the sample was completely mixed with the acid solution. After incubation at 30°C for 1 h, the mixture was diluted by adding 28 ml of deionized water, and the diluted mixture was hydrolyzed at 121°C for 1 h. The mixture was neutralized with CaCO3 powder and then centrifuged at 10,000 rpm for 5 min, and the supernatant was used for HPLC analysis.
The glucose yields were calculated according to NREL LAP-009  as follows:
Glucose yield = ([glucose] + 1.053 × [cellobiose])/(1.111 × [fraction] × [biomass]) × 100%,
where [glucose] is the glucose concentration in the broth after enzymatic hydrolysis, [cellobiose] is the cellobiose concentration in the broth after enzymatic hydrolysis, [biomass] is the dry biomass weight concentration at the beginning of the enzymatic hydrolysis, [fraction] is the cellulose fraction of the dry biomass (g/g), 1.053 is the conversion factor for cellobiose to equivalent glucose and 1.111 is the conversion factor for cellulose to equivalent glucose. The ethanol yields were calculated using the method described by Zhang et al. .
Analysis of sugars, ethanol and inhibitors using HPLC
Glucose, ethanol and toxins such as furfural, HMF, acetic acid, formic acid and levulinic acid were analyzed using HPLC (LC-20AD, refractive index detector RID-10A; Shimadzu, Kyoto, Japan) with a Bio-Rad Aminex HPX-87 H column (Bio-Rad, Hercules, CA, USA) at the column temperature of 65°C. The mobile phase was 5 mM H2SO4 at the rate of 0.6 mL/min. All samples were centrifuged at 10,000 rpm for 5 min and then filtered through a 0.22-μm filter before analysis.