Chemicals and Microorganisms
Glucose and NaOH were purchased from VWR (West Chester, PA). Ca(OH)2, p-aminobenzoic acid and CH3COONH4 were purchased from Alfa Aesar (Heysham, England). Thiamine was purchased from Alfa Aesar (Ward Hill, MA). Dowex 1X4 resin (chloride form) and biotin were purchased from Sigma-Aldrich (St. Louis, MO). H2SO4 (98%) and NaCl were purchased from VWR (West Radnor, PA). K2HPO4, KH2PO4, MgSO4×7H2O, MnSO4×H2O, FeSO4×7H2O were purchased from Fisher Scientific (Fair Lawn, NJ). Citric acid was purchased from Mallinckrodt Chemicals (Phillipsburg, NJ). CaCO3 was purchased from EMD Chemicals (Gibbstown, NJ). Reinforced Clostridial Broth medium (RCM) was purchased from HIMEDIA laboratories (Mumbai, India). Cellic CTec 2 was obtained from Novozymes North America, Inc (Franklinton, UC). DI-water was produced by the Barnstead Nanopure UV Ultrapure Water System (Thermo Fisher Scientific, Marietta, OH).
Clostridium acetobutylicum ATCC 824 was used for butanol production. It was routinely stored as spores at 4 °C and treated by heat shock at 75 °C for 10 min followed by cooling down in an ice bath prior to cultivation. The RCM medium was sparged with nitrogen and then autoclaved at 121 °C for 15 min. The heat-shocked cells were grown until the optical density (OD) reached 1.30 determined by an UV-vis spectrometer (Thermo Scientific, Madison, WI) at 600 nm.
Organosolv Pretreatment
Loblolly pine wood chips were collected by the Forest Products Laboratory at Auburn University and those free of barks and size of 1.0 ´ 1.0 cm (L × W) were selected for organosolv pretreatment. Wood chips (80 g, oven-dry weight) were soaked in 65% (v/v) ethanol solution with 1.1 % (w/w) sulfuric acid (on the basis of biomass dry weight) overnight (7:1 liquor/solid ratio) and then loaded into a 1 L Parr reactor (Parr Instrument Co., Moline, IL) to be treated at 170 °C for 60 min. The spent liquor (aqueous phase) was separated from solid by vacuum filtration upon the completion of pretreatment. Afterward, if ethanol washing was needed, the solid fraction was washed by 700 mL warm ethanol solution (65 % (v/v), 50 °C) three times to dissolve the ethanol extractable lignin and followed by washing by 700 mL DI water four times to remove the residual ethanol. The cellulose-rich solid fraction was homogenized in a blender for 15 s and then used for fermentation and the aqueous phase was subject to detoxification. They were both stored at 4 °C until use.
The EOL was collected from the spent liquor and the ethanol washes. 3-fold DI water was added to precipitate lignin and then the lignin fraction was separated by filtration and then washed thoroughly with DI water, dried in air and then in the oven (105 °C). Sample from the mixture of filtrate and water washes was taken to determine the water-solubles. The collected materials include 39.0 g wood pulp, 14.2 g EOL and 14.2 g water solubles after pretreatment of 80 g oven-dry wood. The water solubles contained 9.50 g carbohydrates, 2.31 g acid-soluble lignin, 0.35 g HMF, 0.71 g furfural, and 1.37 g acetic acid.
Chemical Analysis of Raw Biomass and Pretreated OPLP
The extractives content in raw biomass, organosolv pretreated OPLP-UW and OPLP-W (unwashed OPLP and washed OPLP) was determined as previously described [34]. The composition analysis of carbohydrate and lignin before and after ethanol organosolv pretreatment was carried out using extractives-free samples as previously described [35]. The sugar content of prehydrolysate was determined according to NREL standard method, NREL/TP-510-42623 [36]. The chemical composition of untreated and ethanol organosolv pretreated loblolly pine is shown in Table 1.
Table 1 Chemical composition of untreated and ethanol organosolv pretreated loblolly pine
|
Untreated (%)
|
Organosolv Treated
|
OPLP-UW (%)
|
OPLP-W (%)
|
Glucan
|
42.30±0.38
|
72.74±0.20
|
82.14±0.03
|
Xylan
|
7.51±0.05
|
2.17±0.01
|
1.69±0.08
|
Galactan
|
2.96±0.05
|
0.36±0.03
|
0.40±0.02
|
Arabinan
|
1.78±0.03
|
0.63±.02
|
0.69±0.05
|
Mannan
|
11.17±0.08
|
1.36±0.00
|
0.99±0.02
|
Ethanol Extractives
|
1.18±0.05
|
9.64±0.12
|
0.79±0.04
|
Acid Insoluble Lignin (AIL)
|
29.45±0.27
|
12.11±0.15
|
13.52±0.10
|
Acid Soluble Lignin (ASL)
|
0.56±0.05
|
0.28±0.00
|
0.35±0.01
|
Ash
|
0.36±0.02
|
0.03±0.00
|
0.04±0.00
|
Total
|
97.27
|
99.31
|
100.61
|
Enzymatic Hydrolysis
Cellic CTec 2 was used in enzymatic hydrolysis of pretreated biomass and its filter paper enzyme activity was 126 FPU/mL. Enzymatic hydrolysis of OPLP-W and OPLP-UW (moisture content, ~70%) was carried out in 125 mL serum bottle with a working volume of 50 mL with glucan loading of 5.8% (w/v). Two different conditions were performed with both OPLP-W and OPLP-UW for SHF and SSF conditions respectively: (1) pH 4.8 controlled by 50 mM citrate buffer, 50 °C and 150 rpm; (2) pH 6.0 controlled by adding 0.25 g CaCO3, 35 °C and 80 rpm. To study the effect of lignin on enzymatic hydrolysis, 0.3 g EOL (equivalent to the amount of lignin removed by washing) was added into the ethanol-washed substrate prior to enzymatic hydrolysis. The mixture was autoclaved at 121°C for 15 min, and then after cooling to room temperature, the enzyme was added to initiate the hydrolysis. Enzyme loading was 25 FPU/g glucan. Samples were taken aseptically to prevent contamination. The enzymatic hydrolysis yield was calculated as glucose released during hydrolysis divided by theoretical total glucose in the substrate.
Separate Hydrolysis and Fermentation (SHF)
For the SHF process, the mixture obtained from enzymatic hydrolysis (pH 4.8 controlled by 50 mM citrate buffer, 50 °C and 150 rpm) was applied for fermentation. The volume of the mixture after enzymatic hydrolysis became 45 ml due to the loss in the sample taken. It was brought to 50 ml after inoculation (10% v/v) and glucan loading became 5.2% (w/v). Upon completion of enzymatic hydrolysis, it was supplemented with previous filter-sterilized nutrients stock: 50ml vitamins (p-aminobenzoic acid, 1 g/L, thiamine, 1 g/L, biotin, 0.01 g/L), 0.25 mL minerals (MgSO4⋅7H2O, 40 g/L, MnSO4⋅H2O, 2 g/L, FeSO4⋅7H2O, 2 g/L, NaCl, 2 g/L) and 0.5 mL buffer (K2HPO4, 50 g/L, KH2PO4, 50 g/L, CH3COONH4, 220 g/L). CaCO3 (0.25 g) was added into the broth to control the pH during fermentation. Then the mixture in serum bottle was vacuumed and flushed with nitrogen for 7 cycles to remove oxygen by using a purge valve. The fermentation was initiated by adding 5 mL inoculum (10 % inoculation).
Simultaneous Saccharification and Fermentation (SSF)
The SSF process of both OPLP-W and OPLP-UW was carried out in 125 mL serum bottle with working volume of 50 mL with glucan loading of 5.2% (w/v). They were autoclaved at 121°C for 15 min and then supplemented with previous filter-sterilized nutrients stock the same as SHF process listed above. CaCO3 (0.25 g) was added into the broth. Then the slurry in the serum bottle was vacuumed and flushed with nitrogen for 7 cycles to remove oxygen by using a purge valve. The enzyme loading was 25 FPU/g glucan and the enzyme was sterilized by passing through a 0.2 µm membrane filter. The fermentation was initiated by adding enzyme and 5 mL inoculum (10% inoculation). Both SHF and SSF were carried out at 35 °C and 80 rpm.
SSF Process Supplemented with Detoxified Prehydrolysates
Ethanol in prehydrolysate was evaporated at 40 °C in a rotary evaporator (IKA RV10 basic) and the pH was adjusted to 4.0 with NaOH before evaporation. The concentrated prehydrolysate was then diluted with DI water to make the total volume the same as that before evaporation. Two-step detoxification was carried out as described previously [32]. Briefly, the pH of prehydrolysate was adjusted to 10 by adding Ca(OH)2 and incubated at 90 °C and 100 rpm for 30 min. Afterward, 10 g activated Dowex 1X4 resin was added to 100 mL prehydrolysate and the whole mixture was incubated at 25 °C and 100 rpm for 1 h. The liquid was separated from the resin by centrifuge at 4000 rpm for 10 min and then the pH was adjusted back to 7 with H2SO4. The detoxified prehydrolysate was supplemented into SSF in place of water.
All fermentations were performed in duplicates. Samples were taken periodically for sugar and ABE analysis. Butanol yield was calculated as butanol produced divided by glucose content in the pretreated substrate (and prehydrolysate if applicable) and is expressed as g/g. ABE yield was calculated as the total ABE produced divided by glucose content in the pretreated substrate (and prehydrolysate if applicable) and is expressed as g/g.
Sugars and Products Analysis
The sugar content was quantified by a Shimadzu (LC-20A) HPLC system consisting of a degasser, autosampler, LC-20AD pump, and RID-10A detector, equipped with a 300 mm × 7.8 mm i.d., 9 µm, Aminex HPX-87P column and a 30 mm × 4.6 mm i.d. guard column of the same material (Bio-Rad, Hercules, CA). Nano-pure water was used as mobile phase running at 0.6 mL/min. The column temperature was maintained at 85 ºC. Acetic acid, butyric acid, ethanol, acetone, butanol, HMF, and furfural were quantified by the same HPLC system (Shimadzu LC-20A) equipped with an Aminex HPX-87H column. The mobile phase was composed of 5 mM of sulfuric acid running isocratic at 0.6 mL/min. The column temperature was kept at 45 °C.