Sulfite Pretreatment Research Articles

Sulfite (SPORL) pretreatment of switchgrass for enzymatic saccharification

SPORL (Sulfite Pretreatment to Overcome Recalcitrance of Lignocellulose) pretreatment was applied to switchgrass and optimized through an experimental design using Response Surface Methodology within the range of temperature (163–197°C), time (3–37min), sulfuric acid dosage (0.8–4.2% on switchgrass), and sodium sulfite dosage (0.6–7.4% on switchgrass). Performance of SPORL was compared with that of dilute acid (DA) and alkali (AL) in switchgrass pretreatment. Results indicated that SPORL pretreatment improved the digestibility of switchgrass through sufficiently removing hemicellulose, partially dissolving lignin, and reducing hydrophobicity of lignin by sulfonation. The removal of hemicellulose was more critical to substrate digestibility than the removal of lignin during SPORL pretreatment. SPORL pretreated switchgrass had better enzymatic digestibility than DA and AL pretreated ones. The SPORL pretreated switchgrass could be hydrolyzed by 83% within 48h with 15FPU (filter paper unit) cellulase and 30CBU (cellobiose unit) β-glucosidase/g cellulose.

Enzymatic Saccharification of Lignocelluloses Should be Conducted at Elevated pH 5.2–6.2

This study revealed that cellulose enzymatic saccharification response curves of lignocellulosic substrates were very different from those of pure cellulosic substrates in terms of optimal pH and pH operating window. The maximal enzymatic cellulose saccharification of lignocellulosic substrates occurs at substrate suspension pH 5.2–6.2, not between pH 4.8 and 5.0 as exclusively used in literature using T. reesi cellulase. Two commercial cellulase enzyme cocktails, Celluclast 1.5L and CTec2 both from Novozymes, were evaluated over a wide range of pH. The optimal ranges of measured suspension pH of 5.2–5.7 for Celluclast 1.5L and 5.5–6.2 for CTec2 were obtained using six lignocellulosic substrates produced by dilute acid, alkaline, and two sulfite pretreatments to overcome recalcitrance of lignocelluloses (SPORL) pretreatments using both a softwood and a hardwood. Furthermore, cellulose saccharification efficiency of a SPORL-pretreated lodgepole pine substrate showed a very steep increase between pH 4.7 and 5.2. Saccharification efficiency can be increased by 80 % at cellulase loading of 11.3 FPU/g glucan, i.e., from approximately 43 to 78 % simply by increasing the substrate suspension pH from 4.7 to 5.2 (buffer solution pH from 4.8 to 5.5) using Celluclast 1.5L, or by 70 % from approximately 51 to 87 % when substrate suspension pH is increased from 4.9 to 6.2 (buffer solution pH from 5.0 to 6.5) using CTec2. The enzymatic cellulose saccharification response to pH is correlated to the degree of substrate lignin sulfonation. The difference in pH-induced lignin surface charge, and therefore surface hydrophilicity and lignin–cellulase electrostatic interactions, among different substrates with different lignin content and structure is responsible for the reported different enhancements in lignocellulose saccharification at elevated pH.

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 20min with sulfuric acid and sodium bisulfite charges of 2.2 and 8wt/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 0.06mL/g untreated wood, a maximum ethanol titer of 47.4g/L was achieved, resulting in a calculated yield of 285L/tonne of wood using Saccharomyces cerevisiae YRH400 at 35°C and pH 5.5.

Sodium carbonate–sodium sulfite pretreatment for improving the enzymatic hydrolysis of rice straw

The performance of sodium carbonate–sodium sulfite (Na2CO3–Na2SO3) pretreatment on improving the enzymatic hydrolysis of rice straw was investigated. The results indicated that both Na2CO3 and Na2SO3 pretreatments are effective in improving the enzymatic digestibility of rice straw. A higher ratio of Na2SO3 in pretreatment liquor performed better at softening the rice straw through degradation of hemicellulose, delignification, and lignin sulfonation, while it was conserving more polysaccharides during the pretreatment than Na2CO3. The highest sugar recovery of pretreated rice straw, 74.5%, 82.7%, and 59.5% for total sugar, glucan, and xylan, respectively, was obtained at cellulase loading of 20FPU/g-cellulose after the pretreatment at 140°C, chemical charge of 12%, Na2CO3–Na2SO3 ratio of 0:1 (i.e., only Na2SO3 pretreatment). The corresponding delignification ratio of pretreated solid was 53.4%, and 91.4% of the polysaccharides were conserved. The sugar recovery of only Na2CO3 pretreatment at the ratio of 1:0 was 67.1%, 74.4%, and 53.7% for total sugar, glucan, and xylan, respectively, at 140°C, chemical charge of 12%, and enzyme loading of 20FPU/g-cellulose. Its corresponding delignification ratio was 40.3%, and polysaccharides conservation ratio was 88.9%.

Effects of SPORL and Dilute Acid Pretreatment on Substrate Morphology, Cell Physical and Chemical Wall Structures, and Subsequent Enzymatic Hydrolysis of Lodgepole Pine

The effects of pretreatment by dilute acid and sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) on substrate morphology, cell wall physical and chemical structures, along with the subsequent enzymatic hydrolysis of lodgepole pine substrate were investigated. FE-SEM and TEM images of substrate structural morphological changes showed that SPORL pretreatment resulted in fiber separation, where SPORL high pH (4.2) pretreatment exhibited better fiber separation than SPORL low pH (1.9) pretreatment. Dilute acid pretreatment produced very poor fiber separation, consisting mostly of fiber bundles. The removal of almost all hemicelluloses in the dilute acid pretreated substrate did not overcome recalcitrance to achieve a high cellulose conversion when lignin removal was limited. SPORL high pH pretreatment removed more lignin but less hemicellulose, while SPORL low pH pretreatment removed about the same amount of lignin and hemicelluloses in lodgepole pine substrates when compared with dilute acid pretreatment. Substrates pretreated with either SPORL process had a much higher cellulose conversion than those produced with dilute acid pretreatment. Lignin removal in addition to removal of hemicellulose in SPORL pretreatment plays an important role in improving the cellulose hydrolysis of the substrate.

Comparison of different alkali-based pretreatments of corn stover for improving enzymatic saccharification

Corn stover was treated with NaOH, NaOH+anthraquinone (AQ), NaOH+Na2SO3 (alkaline), NaOH+Na2SO3 (neutral), and NaOH+Na2S, respectively. The treated corn stover was subjected to hydrolysis with cellulase (20 FPU/g dry biomass) and β-glucosidase (10IU/g dry biomass). Compared with other pretreatment methods, alkaline sodium sulfite pretreatment (ASSP) at a relatively low temperature of 140°C provided for the best lignin removal of about 92%. After ASSP with 10wt.% of the total alkali charge (Na2SO3:NaOH=1:1) at 140°C for 30min and subsequent enzymatic hydrolysis, a total sugar yield of 78.2% was obtained on the basis of the amount of glucose and xylose released from raw corn stover. This yield was 24.0% higher than that achieved with NaOH only under the same conditions. Therefore, the supplement of Na2SO3 in alkali pretreatment can facilitate delignification and significantly improve the enzymatic saccharification.

Fractionation of Forest Residues of Douglas-fir for Fermentable Sugar Production by SPORL Pretreatment

Douglas-fir (Pseudotsuga menziesii) forest residues were physically fractionated through sieving. The bark and wood were separated for large-sized fractions (>12.7 mm), and their contents were determined. The chemical compositions of the large fractions were calculated based on the contents and chemical compositions of the bark and wood. The chemical compositions of the fine fractions were analyzed. The bark and wood content in the fine fractions was calculated based on the measured glucan and lignin contents in each fraction. It was found that fractionation by particle/chip size can effectively fractionate bark and wood and therefore lignin from carbohydrates. The large-sized fractions (>12.7 mm) represent approximately 60 % of the collected forest residues but only contain approximately 37 % of the total bark and 35 % of the total ash, or a selectivity over bark and ash of 1.6 and 1.7, respectively. Pretreatment of forest residues by sulfite pretreatment to overcome recalcitrance of lignocelluloses and subsequent enzymatic hydrolysis revealed the presence of 14.3 % bark can reduce substrate enzymatic digestibilities (SED) 16 % compared with that from a bark-free sample. The SED of a bark is 41 % compared with 73 % for wood when pretreated under the same conditions. Separating pretreatment of bark from wood is beneficial for producing a more enzymatically digestible substrate. The results from the present study could have significant implications for harvesting forest residues.

Research on Pretreatment Process and Product Detection in Enzymatic Hydrolysis of Lignocellulosic Biomass

Sulfite pretreatment was explored in enzymatic hydrolysis of bagasse for the first time, and was compared with dilute acid pretreatment. The results showed that enzymatic hydrolysis yield of bagasse after sulfite treatment was lower than that of the bagasse pretreated with dilute acid. Meanwhile, complexity of sulfite pretreatment and its high cost made it infeasible for industrialized production. Results also showed positive correlation of bagasse pretreatment weight loss to enzymatic hydrolysis yield in dilute acid pretreatment processes, which made substrate weight loss a plausible parameter in pretreatment evaluation.

Quantitative predictions of bioconversion of aspen by dilute acid and SPORL pretreatments using a unified combined hydrolysis factor (CHF)

A combined hydrolysis factor (CHF) was developed to predict xylan hydrolysis during pretreatments of native aspen (Populus tremuloides) wood chips. A natural extension of previously developed kinetic models allowed us to account for the effect of catalysts by dilute acid and two sulfite pretreatments at different pH values. When xylan is modeled as two fractions with different hydrolysis rates, previously identified as fast and slow xylan, the model closely matches the experimental data. Extent of xylan hydrolysis is strongly correlated with pretreatment solids yield, energy consumption for size reduction, and substrate enzymatic digestibility (SED). Composition of the pretreatment hydrolysate was less correlated with extent of hydrolysis due to carbohydrate decomposition reactions. Substrate cellulose enzymatic conversion and enzymatic hydrolysis glucose yield can be predicted to approximately 10% accuracy using CHF alone.

Precipitation of lignosulphonates from SPORL liquid by calcium hydroxide treatment

Precipitation of lignosulphonates from the liquor for sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) by addition of Ca(OH)2 was investigated in this work. The experiment was conducted in a reaction temperature range of 20 to 75oC for 90 minutes with Ca(OH)2 charge varying from 20 to 90 g/L and a range of liquid enrichment ratio of 1 to 5. It was found that increased Ca(OH)2 charge, duration time, reaction temperature, and liquor concentration each tended to improve lignosulphonates precipitation, but tended to hurt fermentable sugars conservation. Application of Ca(OH)2 20 g/L to SPORL liquid without enrichment at 30oC for 90 minutes could be an optimal condition. Under this condition, 25.95% of the lignosulphonates was precipitated for further utilization, while calculated amounts of 106.46% of glucose and 60.25% of xylose were conserved for further fermentation.

Ethanol production from poplar wood through enzymatic saccharification and fermentation by dilute acid and SPORL pretreatments

Dilute acid (DA) and Sulfite Pretreatment to Overcome Recalcitrance of Lignocelluloses (SPORL) pretreatments were directly applied to wood chips of four poplar wood samples of different genotypes (hereafter referred to as poplars; Populus tremuloides Michx. ‘native aspen collection’; Populus deltoides Bartr. ex Marsh×Populus nigra L. ‘NE222’ and ‘DN5’; P. nigra×Populus maximowiczii A. Henry ‘NM6’) to evaluate their bioconversion potential. Plant biomass recalcitrance (PBR) was defined to quantitatively determine the recalcitrance of the poplars. Using DA pretreatment, NM6 produced the lowest bioconversion efficiency with a total monomeric sugar yield of 18% theoretical and an ethanol yield of 0.07Lkg−1 of wood compared with an aspen sugar yield of 47% theoretical and an ethanol yield of 0.17Lkg−1 of wood. Similar comparisons following SPORL pretreatment were 43% versus 55% and 0.11 versus 0.20Lkg−1 of wood for NM6 and aspen, respectively. Bioconversion performance of NE222 and DN5 fell between that of aspen and NM6. While substrate lignin content and lignin removal by pretreatments did not affect substrate enzymatic digestibility, the wood lignin content was found to negatively affect xylan or hemicellulose removal using both DA and SPORL pretreatments. The ability of lignin protecting hemicellulose removal dictates PBR through affecting disk milling energy for size reduction of pretreated wood chips, substrate enzymatic digestibility. The SPORL pretreatment not only improved sugar and ethanol yields over DA for all four poplars, but also better dealt with the differences among them, suggesting better tolerance to feedstock variability.

Effect of sulfite pretreatment to overcome the recalcitrance of lignin (SPROL) on enzymatic saccharificaiton of corn stalk

In order to maximize the glucose yield in the subsequent enzymatic hydrolysis, corn stalk was pretreated with sulfite to overcome the recalcitrance of lignocellulose (SPORL) under different operational conditions (pretreatment temperature, bisulfite charge, and pH). The parameters optimized included from 0 to 7% (w/w) on od dry (od) substrate, pretreatment temperature 160 to 190 oC, and pH 2.2 to 6.7 required in the process. The results indicated that after the SPORL pretreatment of corn stalk with 7% bisulfite at 180 oC for 30 min, about 69% and 62% of enzymatic conversion efficiency and glucose yield were achieved, respectively, with emzyme loading of about 5 FPU cellulase per gram of cellulose plus β-glucosidase after 72 h hydrolysis. Temperature had a positive effect on enzymatic hydrolysis. The enzymatic conversion efficiency was reached 81.04% with 7% sodium bisulfite at 190 oC for 30 min. The pH of pretreatment liquor plays a crucial role in enhancing enzymatic digestibility of SPORL substrate.

Efficient ethanol production from beetle-killed lodgepole pine using SPORL technology and Saccharomyces cerevisiae without detoxification

This study applied Sulfite Pretreatment to Overcome Recalcitrance of Lignocelluloses (SPORL) to evaluate the potential of mountain pine beetle-killed lodgepole pine for ethanol production using conventional Saccharomyces cerevisiae without hydrolysate detoxification. The results indicate that the beetle-killed trees are more susceptible to SPORL pretreatment than live trees in addition to having enriched glucan and mannan content as reported in the literature. Ethanol yields of 200 and 250 L/metric ton wood were achieved from a live tree and a dead tree (four years after infestation) without process optimization. Ethanol yield of 220 L/metric ton of wood was obtained from a downed tree with more advanced decomposition, which is approximately 10% more than that from a corresponding live tree. Process mass and energy balance analyses suggest that net ethanol energy output (before distillation, lignin energy excluded) from the decomposing tree was approximately 3.2 GJ/metric ton wood, which is 23% more than that from a corresponding live tree. The study demonstrated the robustness of the SPORL process and the utility of beetle-killed trees for cellulosic ethanol production even after many years post mortality.

Sulfite Pretreatment (SPORL) for Robust Enzymatic Saccharification of Corn Stalks

This study demonstrates sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust bioconversion of corn stalks. With only about 3% sodium bisulfite charge on corn stalks and 30 min pretreatment at temperature 180°C, SPORL can achieve near complete cellulose conversion to glucose in 48h enzymatic hydrolysis. Sulfuric acid charge has great effect on SPORL pretreatment. Enzymatic cellulose conversion of substrate increased with the increase in acid charge under the studied conditions. Bisulfite with different counter-ions can be used in the SPORL. Magnesium as counter ion was found to be more effective on enzymatic hydrolysis of substrate than sodium or ammonium.

Optimizing sulfite pretreatment for saccharification of wheat straw using orthogonal design

An orthogonal designed experiment was used to investigate the effects of sulfite pretreatment on the components separation and saccharification of wheat straw. The process involved sulfite pretreatment of wheat straw under acidic conditions followed by mechanical size reduction using a high consistency refiner. Reaction temperature, retention time, and charges of sodium bisulfite and sulphuric acid were considered as key factors. The results showed the four factors had impact on saccharification of wheat straw. Raising the temperature, increasing the charge of sodium bisulfite or sulphuric acid, or extending the retention time would improve the dissolution of pentosan, lignin, and saccharification efficiency, while causing further conversion of pentose. The separation of lignin and pentosan from wheat straw was the main cause of improvements in saccharification. With an enzyme loading of 5 FPU cellulase plus 4 CBU β-glucosidase per gram of o.d. substrate, a glucose yield 72.45% was achieved using the substrate pretreated under the conditions of temperature 180 oC, sodium bisulfite charge 3%, sulfuric acid charge 1.48%, and retention time 20 min.

On Polydispersity of Plant Biomass Recalcitrance and Its Effects on Pretreatment Optimization for Sugar Production

This paper discusses a property associated with plant biomass recalcitrance to enzyme and microbial deconstructions in sugar production from cellulose and hemicelluloses. The hemicelluloses are more readily hydrolyzed to sugars than is cellulose. As a result, optimization to maximize individual glucose and hemicellulose sugar recovery is not possible. This property is an inherent feature of plant biomass and is named polydispersity of plant biomass recalcitrance (PPBR) in this study. A set of pretreatment experiments using eucalyptus and sulfite pretreatment to overcome recalcitrance of lignocelluloses was conducted. The results were used to predict the conditions for individually maximizing enzymatic glucose and xylose yields. The predicted maximal yields were used to quantitatively illustrate the PPBR concept. The effect of PPBR on pretreatment optimization and strategies for maximal sugar recovery using two-stage pretreatment are discussed.

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.

Statistical optimization of sulfite pretreatment of corncob residues for high concentration ethanol production

In this study, a central composite design of response surface method was used to optimize sulfite pretreatment of corncob residues, in respect to sulfite charge (5-10%), treatment time (1-2h), liquid/solid (l/s) ratio (6:1-10:1) and temperature (150-180°C) for maximizing glucose production in enzymatic hydrolysis process. The relative optimum condition was obtained as follows: sulfite charge 7.1%, l/s ratio 7.6:1, temperature 156°C for 1.4h, corresponding to 79.3% total glucan converted to glucose+cellobiose. In the subsequent simultaneous saccharification and fermentation (SSF) experiments using 15% glucan substrates pretreated under this kind of conditions, 60.8 g ethanol l(-1) with 72.2% theoretical yield was obtained.

Evaluation of Mountain Beetle-Infested Lodgepole Pine for Cellulosic Ethanol Production by Sulfite Pretreatment to Overcome Recalcitrance of Lignocellulose

The potentials of deteriorated mountain pine beetle (Dendroctonus ponderosae)-killed lodgepole pine (Pinus contorta) trees for cellulosic ethanol production were evaluated using the sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) process. The trees were harvested from two sites in the United States Arapaho-Roosevelt National Forest, Colorado. The infestation age of the trees varied from zero to about 8 years. Mild (170 °C) and harsh (180 °C) SPORL pretreatments were conducted. The chemical charges were sulfuric acid of 2.21% and sodium bisulfite of 8% on oven dry wood for the harsh and half of those for the mild pretreatment. The results suggest that beetle-caused mortality enriched glucan content by as much as 3 percentage points (or 7.5%) in wood. The glucan enrichment seems to increase with infestation age. The enriched glucan can be captured after SPORL pretreatment followed by enzymatic hydrolysis. The killed trees are more susceptible to SPORL pretreatment, which enhanced substrate enzymatic digestibility (SED). Enzymatic hydrolysis glucose yields (EHGY) from killed trees were about 5-20% higher than those from their corresponding live trees. Total fermentable sugar productions from dead trees (including a tree laying on the ground) were 4-14% higher than corresponding production from live trees, depending on pretreatment conditions and infestation age. An ethanol yield of 267 L/metric ton of wood or 69% theoretical value was achieved from a tree infested 4 years, 7% higher than the 250 L/metric ton of wood from the corresponding live tree. The results also demonstrated the robustness of SPORL pretreatment for lodgepole pine.

Eliminating inhibition of enzymatic hydrolysis by lignosulfonate in unwashed sulfite-pretreated aspen using metal salts

This study demonstrated the efficiency of Ca(II) and Mg(II) in removing inhibition of enzymatic hydrolysis by lignosulfonate through non-productive adsorption of enzymes. Adding 1 mmol/g cellulose of either metal salt restores approximately 65% of the activity lost when a pure cellulose/cellulase solution is spiked with lignosulfonate. Addition of either Ca(II) or Mg(II) is also effective in counteracting soluble inhibitors of cellulase present in unwashed aspen solid substrate produced by SPORL (sulfite pretreatment to overcome recalcitrance of lignocelluloses). Soluble inhibitors are often removed by thoroughly washing the lignocellulosic solid substrate following pretreatment. It was determined that adding 1 mmol of MgSO(4)/g substrate (oven dry) to the unwashed aspen substrate gave enzymatic substrate digestibility (SED) equivalent to that of washing for a range of enzyme loadings. These results demonstrate that applying divalent metal salts eliminates the need for washing, thereby saving considerable process water and cost for production of chemicals and biofuels from lignocellulose.