Roland Gleisner

Sulfite pretreatment (SPORL) for robust enzymatic saccharification of spruce and red pine

This study established a novel process using sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust and efficient bioconversion of softwoods. The process consists of sulfite treatment of wood chips under acidic conditions followed by mechanical size reduction using disk refining. The results indicated that after the SPORL pretreatment of spruce chips with 8-10% bisulfite and 1.8-3.7% sulfuric acid on oven dry (od) wood at 180 degrees C for 30 min, more than 90% cellulose conversion of substrate was achieved with enzyme loading of about 14.6 FPU cellulase plus 22.5 CBU beta-glucosidase per gram of od substrate after 48 h hydrolysis. Glucose yield from enzymatic hydrolysis of the substrate per 100 g of untreated od spruce wood (glucan content 43%) was about 37 g (excluding the dissolved glucose during pretreatment). Hemicellulose removal was found to be as critical as lignin sulfonation for cellulose conversion in the SPORL process. Pretreatment altered the wood chips, which reduced electric energy consumption for size reduction to about 19 Wh/kg od untreated wood, or about 19 g glucose/Wh electricity. Furthermore, the SPORL produced low amounts of fermentation inhibitors, hydroxymethyl furfural (HMF) and furfural, of about 5 and 1 mg/g of untreated od wood, respectively. In addition, similar results were achieved when the SPORL was applied to red pine. By building on the mature sulfite pulping and disk refining technologies already practiced in the pulp and paper industry, the SPORL has very few technological barriers and risks for commercialization.

On energy consumption for size-reduction and yields from subsequent enzymatic saccharification of pretreated lodgepole pine

This study investigated the effects of chemical pretreatment and disk-milling conditions on energy consumption for size-reduction and the efficiency of enzymatic cellulose saccharification of a softwood. Lodgepole pine wood chips produced from thinnings of a 100-year-old unmanaged forest were pretreated by hot-water, dilute-acid, and two SPORL processes (Sulfite Pretreatment to Overcome Recalcitrance of Lignocellulose) at acid charge on oven dry (od) wood of 0% and 2.21%. The pretreated wood chips were then milled using a laboratory disk mill under various solids-loadings and disk-plate gaps to produce substrates for enzymatic hydrolysis. We found that post-chemical-pretreatment size-reduction of forest biomass can decrease size-reduction energy consumption by 20-80% depending on the pretreatment applied under 20% solids-loading and a disk-plate gap of 0.76 mm in milling. SPORL with a sodium bisulfite charge of 8% and sulfuric acid charge of 2.21% on wood was the most effective in decreasing size-reduction energy consumption. Solids-loading had the most significant effect on disk-milling energy. When solids-loading was reduced from 30% to 3%, disk-milling energy could be decreased by more than a factor of 10 for wood chips pretreated by both SPORL and dilute-acid at an acid charge of 2.21%. The enzymatic hydrolysis glucose yields (EHGY) from the substrates produced by all pretreatments were independent of the solids-loading in milling, indicating that these energy savings in size-reduction can be realized without affecting EHGY. When wood chips were pretreated by SPORL with 2.21% acid charge, size-reduction energy consumption was decreased to less than 50 Wh/kg od wood at a practical solids-loading of approximately 10-20%, equivalent to that used in size-reduction of agriculture biomass, with excellent EHGY of about 370 g per kg od wood. Similar effects on size-reduction energy savings and excellent EHGY were also achieved when large disk-plate gaps (up to 1.52 mm studied) were applied in disk-milling of wood chips pretreated by SPORL with acid.

Sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust enzymatic saccharification of hardwoods.

This study demonstrates sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust bioconversion of hardwoods. With only about 4% sodium bisulfite charge on aspen and 30‐min pretreatment at temperature 180°C, SPORL can achieve near‐complete cellulose conversion to glucose in a wide range of pretreatment liquor of pH 2.0–4.5 in only about 10 h enzymatic hydrolysis. The enzyme loading was about 20 FPU cellulase plus 30 CBU β‐glucosidase per gram of cellulose. The production of fermentation inhibitor furfural was less than 20 mg/g of aspen wood at pH 4.5. With pH 4.5, SPORL avoided reactor corrosion problem and eliminated the need for substrate neutralization prior to enzymatic hydrolysis. Similar results were obtained from maple and eucalyptus.

High titer ethanol production from simultaneous enzymatic saccharification and fermentation of aspen at high solids: a comparison between SPORL and dilute acid pretreatments.

Native aspen (Populus tremuloides) was pretreated using sulfuric acid and sodium bisulfite (SPORL) and dilute sulfuric acid alone (DA). Simultaneous enzymatic saccharification and fermentation (SSF) was conducted at 18% solids using commercial enzymes with cellulase loadings ranging from 6 to 15 FPU/g glucan and Saccharomyces cerevisiae Y5. Compared with DA pretreatment, the SPORL pretreatment reduced the energy required for wood chip size-reduction, and reduced mixing energy of the resultant substrate for solid liquefaction. Approximately 60% more ethanol was produced from the solid SPORL substrate (211 L/ton wood at 59 g/L with SSF efficiency of 76%) than from the solid DA substrate (133 L/ton wood at 35 g/L with SSF efficiency 47%) at a cellulase loading of 10 FPU/g glucan after 120 h. When the cellulase loading was increased to 15 FPU/g glucan on the DA substrate, the ethanol yield still remained lower than the SPORL substrate at 10 FPU/g glucan.

High titer ethanol production from SPORL-pretreated lodgepole pine by simultaneous enzymatic saccharification and combined fermentation.

Lodgepole wood chips were pretreated by sulfite pretreatment to overcome recalcitrance of lignocelluloses (SPORL) at 25% solids loading and 180 °C for 20 min with sulfuric acid and sodium bisulfite charges of 2.2 and 8 wt/wt% on an oven-dry wood basis, respectively. The pretreated wood chips were disk-milled with pretreatment spent liquor and water, and the solid fraction was separated from the liquor stream. The liquor was neutralized and concentrated through vacuum evaporation. Quasi-simultaneous enzymatic saccharification of the cellulosic solids and combined fermentation with the concentrated liquor was conducted at up to 20% total solids loading. Fed-batching of the solids facilitated liquefaction and saccharification, as well as managing instantaneous inhibitor concentrations. At a commercial cellulase (CTec2) loading of only 9 FPU or 0.06 mL/g untreated wood, a maximum ethanol titer of 47.4 g/L was achieved, resulting in a calculated yield of 285 L/tonne of wood using Saccharomyces cerevisiae YRH400 at 35 °C and pH 5.5.

Using sulfite chemistry for robust bioconversion of Douglas-fir forest residue to bioethanol at high titer and lignosulfonate: a pilot-scale evaluation.

This study demonstrated at the pilot-scale (50 kg) use of Douglas-fir forest harvest residue, an underutilized forest biomass, for the production of high titer and high yield bioethanol using sulfite chemistry without solid-liquor separation and detoxification. Sulfite Pretreatment to Overcome the Recalcitrance of Lignocelluloses (SPORL) was directly applied to the ground forest harvest residue with no further mechanical size reduction, at a low temperature of 145°C and calcium bisulfite or total SO2 loadings of only 6.5 or 6.6 wt% on oven dry forest residue, respectively. The low temperature pretreatment facilitated high solids fermentation of the un-detoxified pretreated whole slurry. An ethanol yield of 282 L/tonne, equivalent to 70% theoretical, with a titer of 42 g/L was achieved. SPORL solubilized approximately 45% of the wood lignin as directly marketable lignosulfonate with properties equivalent to or better than a commercial lignosulfonate, important to improve the economics of biofuel production.

Comparisons of SPORL and Dilute Acid Pretreatments for Sugar and Ethanol Productions from Aspen

This study reports comparative evaluations of sugar and ethanol production from a native aspen (Populus tremuloides) between sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) and dilute acid (DA) pretreatments. All aqueous pretreatments were carried out in a laboratory wood pulping digester using wood chips at 170°C with a liquid to oven dry (od) wood ratio (L/W) of 3:1 at two levels of acid charge on wood of 0.56 and 1.11%. Sodium bisulfite charge on od wood was 0 for DA and 1.5 or 3.0% for SPORL. All substrates produced by both pretreatments (except DA with pretreatment duration of 0) had good enzymatic digestibility of over 80%. However, SPORL produced higher enzymatic digestibility than its corresponding DA pretreatment for all the experiments conducted. As a result, SPORL produced higher ethanol yield from simultaneous saccharification and fermentation of cellulosic substrate than its corresponding DA pretreatment. SPORL was more effective than its corresponding DA pretreatment in reducing energy consumption for postpretreatment wood chip size‐reduction. SPORL, with lower energy input and higher sugar and ethanol yield, produced higher sugar and ethanol production energy efficiencies than the corresponding DA pretreatment. Published 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011

Pilot-scale demonstration of SPORL for bioconversion of lodgepole pine to bioethanol and lignosulfonate.

Abstract The process sulfite pretreatment to overcome recalcitrance of lignocelluloses (SPORL) has been the focus of this study. Pilot-scale (50 kg) pretreatment of wood chips of lodgepole pine (Pinus contorta Douglas ex Loudon) killed by mountain pine beetle (Dendroctonus ponderosae Hopkins) were conducted at 165°C with a dilute sulfite solution of pH 2 for bioconversion to ethanol and lignosulfonate (LS). The pretreatment duration was optimized in laboratory bench scale experiments with a certain severity based on a combined hydrolysis factor (CHF). The sodium bisulfite loading was 8% and the liquor to wood ratio 3. The pretreated solids were disk milled together with the spent liquid and the resultant slurry with a 25% solids content was directly (without detoxification) submitted to a simultaneous enzymatic saccharification and fermentation (SSF) with Saccharomyces cerevisiae YRH400 at cellulase loading of 35 ml kg-1 of untreated wood. At solids loading of 20%, the alcohol yield was 288 l t-1 wood (with a final concentration of 52.2 g l-1), which corresponds to a 72.0% theoretical yield based on total glucan, mannan, and xylan. The LS from SPORL was highly sulfonated and its molecular weight was lower than that of a purified commercial softwood LS, and therefore it has a high potential as a directly marketable co-product.

Comparisons of high titer ethanol production and lignosulfonate properties by SPORL pretreatment of lodgepole pine at two temperatures

Mountain pine beetle killed lodgepole pine wood chips were pretreated by SPORL (Sulfite Pretreatment to Overcome the Recalcitrance of Lignocelluloses) at 180 °C for 25 min and 165 °C for 75 min using the same chemical loadings, which represent the same pretreatment severity. The pretreated whole slurries were used to produce lignosulfonate and ethanol through simultaneous enzymatic saccharification and combined fermentation (SSCombF) up to solid loadings of 18% without detoxification. Low temperature pretreatment reduced furan formation, which facilitated ethanol production as measured by ethanol productivity and sugar consumption. The improved carbohydrate yields at 165 °C also produced high ethanol yields (liter per tonne wood) at all SSCombF solids loadings. An ethanol yield and titer of 306 L per tonne wood, or approximately 72% theoretical, and 47.1 g L−1, respectively, were achieved without detoxification at 165 °C. Lignosulfonates (LS) produced from the two SPORL runs are highly sulfonated but have lower molecular weight than a commercial high purity softwood LS. Both infrared and NMR spectra of LS from SPORL treated wood chips were compared with those of the commercial LS. The LSs from SPORL treated wood chips were found to have better dispersion properties than the commercial LS.

Conversion of SPORL pretreated Douglas fir forest residues into microbial lipids with oleaginous yeasts

Douglas fir is the dominant commercial tree grown in the United States. In this study Douglas fir residue was converted to single cell oils (SCO) using oleaginous yeasts. Monosaccharides were extracted from the woody biomass by pretreating with sulfite and dilute sulfuric acid (SPORL process) and hydrolyzing using commercial cellulases. A new SPORL process that uses pH profiling was compared to the traditional method. Both processes yielded 77 g l−1 concentration of sugars. The SPORL generated sugars were evaluated for conversion to SCO using yeasts Lipomyces tetrasporus and Yarrowia lipolytica in batch cultures containing SPORL sugars diluted to 60% v/v supplemented with nitrogen at an appropriate C:N ratio of 75:1. An extended lag phase was observed for both yeasts, which was eliminated by including SPORL sugars diluted to 40% v/v in the seed cultures for acclimation. The maximum lipid concentrations were 3.18–5.13 g l−1. This corresponded to yields of 0.06–0.17 g lipid per g beginning sugars and productivities of 0.99–1.42 g l−1 d−1. Lipid concentrations for L. tetrasporus were further amplified by using two schemes incorporating multiple batch cultures. In the first, the yeast was grown in 40% v/v SPORL sugars and the entire contents of this fermentation transferred to undiluted SPORL sugars not supplemented with nitrogen. The result was the production of 13.4 g l−1 lipids within 3 days. This corresponds to a yield of 0.174 g g−1 and a productivity of 4.47 g l−1 d−1. The second approach was to thrice transfer the yeast cells in 60% v/v SPORL sugars supplemented with limited nitrogen to promote further lipid formation. The end result was 18.1 g l−1 of lipids with a process yield and productivity of 0.104 g g−1 and 1.29 g l−1 d−1, respectively. This is the first report that the authors are aware of demonstrating the feasibility of converting unconditioned woody biomass to single cell oil.