Xuejun Pan

Substrate Pretreatment: The Key to Effective Enzymatic Hydrolysis of Lignocellulosics?

Although the structure and function of cellulase systems continue to be the subject of intense research, it is widely acknowledged that the rate and extent of the cellulolytic hydrolysis of lignocellulosic substrates is influenced not only by the effectiveness of the enzymes but also by the chemical, physical and morphological characteristics of the heterogeneous lignocellulosic substrates. Although strategies such as site-directed mutagenesis or directed evolution have been successfully employed to improve cellulase properties such as binding affinity, catalytic activity and thermostability, complementary goals that we and other groups have studied have been the determination of which substrate characteristics are responsible for limiting hydrolysis and the development of pretreatment methods that maximize substrate accessibility to the cellulase complex. Over the last few years we have looked at the various lignocellulosic substrate characteristics at the fiber, fibril and microfibril level that have been modified during pretreatment and subsequent hydrolysis. The initial characteristics of the woody biomass and the effect of subsequent pretreatment play a significant role on the development of substrate properties, which in turn govern the efficacy of enzymatic hydrolysis. Focusing particularly on steam pretreatment, this review examines the influence that pretreatment conditions have on substrate characteristics such as lignin and hemicellulose content, crystallinity, degree of polymerization and specific surface, and the resulting implications for effective hydrolysis by cellulases.

Woody biomass pretreatment for cellulosic ethanol production: technology and energy consumption evaluation.

This review presents a comprehensive discussion of the key technical issues in woody biomass pretreatment: barriers to efficient cellulose saccharification, pretreatment energy consumption, in particular energy consumed for wood-size reduction, and criteria to evaluate the performance of a pretreatment. A post-chemical pretreatment size-reduction approach is proposed to significantly reduce mechanical energy consumption. Because the ultimate goal of biofuel production is net energy output, a concept of pretreatment energy efficiency (kg/MJ) based on the total sugar recovery (kg/kg wood) divided by the energy consumption in pretreatment (MJ/kg wood) is defined. It is then used to evaluate the performances of three of the most promising pretreatment technologies: steam explosion, organosolv, and sulfite pretreatment to overcome lignocelluloses recalcitrance (SPORL) for softwood pretreatment. The present study found that SPORL is the most efficient process and produced highest sugar yield. Other important issues, such as the effects of lignin on substrate saccharification and the effects of pretreatment on high-value lignin utilization in woody biomass pretreatment, are also discussed.

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.

Strategies to Enhance the Enzymatic Hydrolysis of Pretreated Softwood with High Residual Lignin Content

Pretreatment of Douglas-fir by steam explosion produces a substrate containing approx 43% lignin. Two strategies were investigated for reducing the effect of this residual lignin on enzymatic hydrolysis of cellulose: mild alkali extraction and protein addition. Extraction with cold 1% NaOH reduced the lignin content by only approx 7%, but cellulose to glucose conversion was enhanced by about 30%. Before alkali extraction, addition of exogenous protein resulted in a significant improvement in cellulose hydrolysis, but this protein effect was substantially diminished after alkali treatment. Lignin appears to reduce cellulose hydrolysis by two distinct mechanisms: by forming a physical barrier that prevents enzyme access and by non-productively binding cellulolytic enzymes. Cold alkali appears to selectively remove a fraction of lignin from steam-exploded Douglas-fir with high affinity for protein. Corresponding data for mixed softwood pretreated by organosolv extraction indicates that the relative importance of the two mechanisms by which residual lignin affects hydrolysis is different according to the pre- and post-treatment method used.

Pretreatment of woody biomass for biofuel production: energy efficiency, technologies, and recalcitrance

This mini review discusses several key technical issues associated with cellulosic ethanol production from woody biomass: energy consumption for woody biomass pretreatment, pretreatment energy efficiency, woody biomass pretreatment technologies, and quantification of woody biomass recalcitrance. Both total sugar yield and pretreatment energy efficiency, defined as the total sugar recovery divided by total energy consumption for pretreatment, should be used to evaluate the performance of a pretreatment process. A post-chemical pretreatment wood size-reduction approach was proposed to significantly reduce energy consumption. The review also emphasizes using a low liquid-to-wood ratio (L/W) to reduce thermal energy consumption for any thermochemical/physical pretreatment in addition to reducing pretreatment temperature.

Comparative study of SPORL and dilute-acid pretreatments of spruce for cellulosic ethanol production

The performance of two pretreatment methods, sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) and dilute acid (DA), was compared in pretreating softwood (spruce) for fuel ethanol production at 180 degrees Celsius for 30 min with a sulfuric acid loading of 5% on oven-dry wood and a 5:1 liquor-to-wood ratio. SPORL was supplemented with 9% sodium sulfite (w/w of wood). The recoveries of total saccharides (hexoses and pentoses) were 87.9% (SPORL) and 56.7% (DA), while those of cellulose were 92.5% (SPORL) and 77.7% (DA). The total of known inhibitors (furfural, 5-hydroxymethylfurfural, and formic, acetic and levulinic acids) formed in SPORL were only 35% of those formed in DA pretreatment. SPORL pretreatment dissolved approximately 32% of the lignin as lignosulfonate, which is a potential high-value co-product. With an enzyme loading of 15 FPU (filter paper units) per gram of cellulose, the cellulose-to-glucose conversion yields were 91% at 24h for the SPORL substrate and 55% at 48 h for the DA substrate, respectively.

The bioconversion of mountain pine beetle-killed lodgepole pine to fuel ethanol using the organosolv process.

Lodgepole pine (Pinus contorta) killed by mountain pine beetle (Dendroctonus ponderosae) (BLP) was compared with healthy lodgepole pine (HLP) for bioconversion to ethanol and high‐value co‐products. The BLP and HLP chips were pretreated using an ethanol organosolv process at a variety of severities. It was shown that the BLP was easier to pretreat and delignify than were the HLP chips. The resulting pretreated BLP substrate had a lower residual lignin, lower degree of polymerization of cellulose, lower cellulose crystallinity, smaller fiber size and thereby a better enzymatic hydrolysability than did the HLP substrates. However, under the same conditions, the BLP showed lower substrate yield and cellulose recovery than did the HLP, which likely resulted from the excessive hydrolysis and subsequent decomposition of the cellulose and hemicellulose during the pretreatment. The BLP wood yielded more ethanol organosolv lignin than was obtained with the HLP material. The HLP lignin had a lower molecular weight and narrower distribution than did the BLP lignin. It appears that the beetle killed LP is more receptive to organosolv pretreatment other than a slightly lower recovery of carbohydrates. Biotechnol. Biotechnol. Bioeng. 2008;101: 39–48.

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.

Adsorption of Cellulase on Cellulolytic Enzyme Lignin from Lodgepole Pine

Enzymatic hydrolysis of lignocellulosic materials is significantly affected by cellulase adsorption onto the lignocellulosic substrates and lignin. The presence of lignin plays an important role in lignocellulosic hydrolysis and enzyme recycling. Three cellulase preparations (Celluclast, Spezyme CP, and MSUBC) were evaluated to determine their adsorption onto cellulolytic enzyme lignin (CEL) from steam-exploded Lodgepole pine (SELP) and ethanol (organosolv)-pretreated Lodgepole pine (EPLP). The adsorption affinity of cellulase (Celluclast) onto isolated lignin (CEL-EPLP and CEL-SELP) was slightly higher than that from corresponding EPLP and SELP substrates on the basis of the Langmuir constants. Effects of temperature, ionic strength, and surfactant on cellulase adsorption onto isolated lignin were also explored in this study. Thermodynamic analysis of enzyme adsorption onto isolated lignin (Gibbs free energy change DeltaG(0) approximately -30 kJ/mol) indicated this adsorption was a spontaneous process. The addition of surfactant (0.2% w/v) could reduce the adsorption of cellulase onto CEL-SELP by 60%. Two types of adsorption isotherm were compared for cellulase adsorption onto isolated lignin. A Langmuir adsorption isotherm showed better fit for the experimental data than a Freundlich adsorption isotherm.

Updates on softwood-to-ethanol process development.

Softwoods are generally considered to be one of the most difficult lignocellulosic feedstocks to hydrolyze to sugars for fermentation, primarily owing to the nature and amount of lignin. If the inhibitory effect of lignin can be significantly reduced, softwoods may become a more useful feedstock for the bioconversion processes. Moreover, strategies developed to reduce problems with softwood lignin may also provide a means to enhance the processing of other lignocellulosic substrates. The Forest Products Biotechnology Group at the University of British Columbia has been developing softwood-to-ethanol processes with SO2-catalyzed steam explosion and ethanol organosolv pretreatments. Lignin from the steam explosion process has relatively low reactivity and, consequently, low product value, compared with the high-value coproduct that can be obtained through organosolv. The technical and economic challenges of both processes are presented, together with suggestions for future process development.