Effect Of Lignin Sulfonation On Enzymatic Lignin Modification And Saccharification Of Woody Biomass

Lignocellulose is the most abundant renewable natural resource. Utilization oflignocellulose to produce biofuel is a promising approach to alleviate energy shortage whilereduce human impact on climate. However, current technologies for producing cellulosicbiofuel are not economical. Utilization of lignin for co-product development and improvingbioconversion yield are keys to improve the economics of cellulosic biofuel production forcommercialization. This dissertation studied the potential of lignin sulfonation on ligninmodification for high value utilization of lignin and for improving the efficiency of enzymatichydrolysis of lignocelluloses.Lignin sulfonation is the important approach to improve lignin water solubility, whilecondensation polymerization is the main method to increase the molecular weight of lignin.Both properties are critical for many industrial applications such as dispersants andplasticizers. With the very limited number of sulfite pulp mills worldwide, lignin sulfonationbecomes a dominant production process for producing lignosulfonate. Kraft hardwood ligninwas modified by laccase and laccase/xylanase system, followed by sulfomethylation. It washypothesized that laccase can promote both polymerization and depolymerization. Themolecular weight of sulfomethylated lignin was measured by gel permeation chromatography(GPC), and structural changes of enzymatic modified lignin were characterized by headspacegas chromatography (HS-GC), infrared spectroscopy (IR) and nuclear magnetic resonance(NMR). The results indicate that application of laccase and laccase/xylanase had limitedeffect on lignin molecular weight (Mw), but increased polydispersity. The demethylation bylaccase oxidation increased the content of phenolic group, while the content of syringyl unitdecreased. At the same time, laccase oxidized the phenolic end-groups into stabilized radicalsthat subsequently underwent radical-radical couplings. The charge density of lignin modelcompounds was calculated by molecular simulation. The larger charge density of aromaticring after laccase modification was found to be beneficial for sulfomethylation. Due to thedecrease of steric hindrance, xylanase addition increased the reaction rate of laccasemodification. The sulfomethylation reactivity, measured by the content of sulfonic group, wasincreased by33%after enzymatic modification. In addition, the dispersion efficiency ofsulfomethlated kraft lignin with enzymatic modification was improved significantly.The dissertation also studied the modification of a hardwood sodium lignosulfonate(NaLS) by commercial laccase. It was found laccase did not change the content of sulfonicgroups and surface charge. Laccase was able to promote both polymerization and depolymerization. In the beginning, the cleavage of some linkages and demethylation bylaccase oxidation decreased the Mwof NaLS, while the content of phenolic groups wasincreased. With the increase in incubation time, the phenolic group content decreased and theMwof NaLS increased, suggesting that polymerization reactions dominated. Due to theincrease in Mwand the changes of adsorption conformation, the adsorption amount of NaLSon MgO particles increased. Moreover, the modified NaLS improved the fluidity of slurriesby23%. Among all the modified NaLS, the dispersion ability of the one with laccaseincubated for2h was the best. Electrostatic self-assembly was used to study the changes inadsorption conformation of NaLS by laccase modification. The NaLS macromolecule withlaccase incubated for2h was an ellipsoidal microgel form, beneficial to the adsorptionprocess. However, the NaLS macromolecule shifted to a more spherical form after incubationwith laccase for36h. The stronger steric hindrance between NaLS macromolecules led to lessadsorption amount.The ability of commercial horseradish peroxidase (HRP) for polymerizinglignosulfonates (LSs) was also investigated in aqueous solution at room temperature. GPCresults showed a significant increase in Mwof LSs. With different dosages of HRP, LSs withdifferent Mwcould be obtained. When6g/L HRP was added, the Mwof NaSKL (sulfonatedkraft lignin) and NaLS could increase by8.5-fold and4.7-fold. During the HRP incubation,phenolic groups were oxidized to phenoxyl radicals. These radicals could undergoradical-couplings directly. On the other hand, unbound electron could transfer to the para-andortho-carbon of the phenols and-carbon of the branch, undergoing radical-couplings.Among the radical-couplings,-O-4’ was predominant. Moreover, the sulfonic group contentincreased by22%and13%for NaSKL and NaLS after HRP incubation, respectively. Theadsorption of LSs by HRP incubation was studied by QCM-D. After HRP incubation, theadsorption amount of LSs increased significantly. Furthermore, the increased Mwand sulfonicgroup contents resulted in the stronger steric hindrance and electronic repulsion. Thedispersion ability of HRP incubated LSs slurry was improved. Therefore, the HRP/H2O2incubation is a promising approach for preparing LSs with high molecular weight.The effects of lignin sulfonation on enzymatic hydrolysis of lignocelluloses were alsoevaluated in this dissertation. Lignosulfonate is a natural surfactant. Many studies indicatedthat surfactant can reduce the nonproductive bonding of cellulase to substrate lignin.Furthermore, sulfite pretreatments were found very effective for saccharification oflignocelluloses. Mountain pine beetle killed Lodgepole pine (BD4) wood chips werepretreated using an acidic sulfite solution of approximately pH=2.0at a liquor to wood ratio of3and sodium bisulfite loading of8wt%on wood. The combined hydrolysis factor (CHF)was used to design a scale-up pretreatment on2000g wood chips. BD4wood chips werepretreated at180oC for25min and165oC for75min using the same chemical loadings and atthe same pretreatment severity measured by CHF. The pretreated whole slurries were used toproduce LS and ethanol through simultaneous enzymatic saccharification and combinedfermentation (SSCombF) up to solids loading of18%without detoxification. Lowtemperature pretreatments (165oC) reduced furan formation, which facilitated ethanolproduction as measured by ethanol productivity and sugar consumption. The improvedcarbohydrate yields at165oC also produced high ethanol yields (liter per tonne wood) at allSSCombF solids loadings. An ethanol yield and titer of306L tonne-1wood, or approximately72%theoretical, and47g/L, respectively, were achieved without detoxification from thewood pretreated at165oC. LS produced from the two SPORL runs are highly sulfonated buthave lower molecular weight than a commercial high purity softwood LS. Both infrared andNMR spectra of LS from SPORL treated wood chips were compared with those of thecommercial LS. The LSs from SPORL treated wood chips were found to have betterdispersion ability than the commercial LS.To further study the effect of lignin sulfonation on bioconversion of lignocelluloses, twoLSs from sulfite pulping process and one sulfonated kraft lignin were applied to purecellulose and lignocellulose (dilute acid and SPORL pretreated aspen, and kraft alkaline andsulfite pretreated lodgepole pine). All three lignin samples inhibited enzymatic hydrolysis ofpure cellulose, but enhanced the enzymatic hydrolysis of lignocellulosic substrates. In order tounderstand the intrinsic properties of LS on enzymatic hydrolysis of lignocellulose, threedifferent fractions of LS were obtained by ultrafiltration. The smallest fraction (Mw=1700Da)was found to improve the enzymatic hydrolysis of pure cellulose. Perhaps the high sulfonicgroup content can form complex with cellulase to result in a stable adsorption cellulasebinding to cellulose. However, the largest fraction (Mw=21000Da) with low sulfonic groupcontent reduced cellulose saccharification, perhaps due to precipitation of the largeLS-cellulase aggregates and reduced cellulase binding to cellulose. When applying LS tolignocellulosic substrates the alkaline pretreated lodgepole pine had the most significantimprovement. This might be due to the fact that lignin on this substrate has the highest affinityto cellulase compared with the lignin of the other three lignocellulosic substrates. Theapplication of LS blocked substrate lignin to bound cellulase to improve cellulase activity forhydrolysis.