The enzymatic hydrolysis and fermentation of pretreated wood substrates

Aspenwood chips were pretreated by steam explosion. The various wood fractions obtained were assayed for their ability to act as substrates for growth and cellulase production of different Trichoderma and Clostridium thermocellum species. Steam exploded aspenwood was as efficiently utilized as solka floc and correspondingly high cellulase activities were detected in the various culture filtrates. When T. harzianum E58 was grown on increasing concentrations of solka floc, highest cellulase and xylanase activities were detected at 1% substrate concentrations while high substrate concentrations (10-20%) inhibited growth and enzyme production. When the cellulosic substrates were supplemented with increasing amounts of glucose, cellulase and xylanase production were inhibited when the glucose concentration exceeded 0.1%. Highest xylanase activities were detected after growth of T. reesei C30 and T. harianum E58 on xylan and solka floc respectively. All of the steam exploded fractions were at least partially hydrolyzed by the T. harzianum E58 cellulase system. The extent of the pretreatment also influenced the ability of Zymomonas mobilis and Saccharomyces cerevisiae to ferment the liberated sugars to ethanol. About 85% of the theoretical yield of ethanol from cellulose could be obtained from the combined hydrolysis and fermentation of pretreated aspenwood.     

The laccase-catalyzed modification of lignin for enzymatic hydrolysis

The efficient use of cellulases in the hydrolysis of pretreated lignocellulosic biomass is limited due to the presence of lignin. Lignin is known to bind hydrolytic enzymes nonspecifically, thereby reducing their action on carbohydrate substrates. The composition and location of residual lignin therefore seem to be important for optimizing the enzymatic hydrolysis of lignocellulosic substrates. The use of lignin-modifying enzymes such as laccase may have potential in the modification or partial removal of lignin from the biomass. In this study, the effect of lignin modification by laccase on the hydrolysis of pretreated spruce (Picea abies) and giant reed (Arundo donax) was evaluated. The substrates were first treated with laccase and then hydrolyzed with commercial cellulases. Laccase modification improved the hydrolysis yield of spruce by 12%, but surprisingly had an adverse effect on giant reed, reducing the hydrolysis yield by 17%. The binding properties of cellulases on the untreated and laccase-treated lignins were further studied using isolated lignins. The laccase treatment reduced the binding of enzymes on modified spruce lignin, whereas with giant reed, the amount of bound proteins increased after laccase treatment. Further understanding of the reactions of laccase on lignin will help to control the unspecific-binding of cellulases on lignocellulosic substrates.     

The laccase-catalyzed modification of lignin for enzymatic hydrolysis

The efficient use of cellulases in the hydrolysis of pretreated lignocellulosic biomass is limited due to the presence of lignin. Lignin is known to bind hydrolytic enzymes nonspecifically, thereby reducing their action on carbohydrate substrates. The composition and location of residual lignin therefore seem to be important for optimizing the enzymatic hydrolysis of lignocellulosic substrates. The use of lignin-modifying enzymes such as laccase may have potential in the modification or partial removal of lignin from the biomass. In this study, the effect of lignin modification by laccase on the hydrolysis of pretreated spruce (Picea abies) and giant reed (Arundo donax) was evaluated. The substrates were first treated with laccase and then hydrolyzed with commercial cellulases. Laccase modification improved the hydrolysis yield of spruce by 12%, but surprisingly had an adverse effect on giant reed, reducing the hydrolysis yield by 17%. The binding properties of cellulases on the untreated and laccase-treated lignins were further studied using isolated lignins. The laccase treatment reduced the binding of enzymes on modified spruce lignin, whereas with giant reed, the amount of bound proteins increased after laccase treatment. Further understanding of the reactions of laccase on lignin will help to control the unspecific-binding of cellulases on lignocellulosic substrates.     

The HCH-1 model of enzymatic cellulose hydrolysis

Solka Floc BW200 was enzymatically hydrolyzed in a batch reactor using a commercial cellulase preparation. A total of 50 different hydrolysis conditions were run within a 10-fold range in enzyme concentration and a 30-fold range in cellulose concentration. The data were evaluated in three ways using five different models. Previous literature models were not as successful in correlating the data as the HCH-1 Model derived in this work.     

Mechanisms of enzymatic glycoside hydrolysis

The determination of a large number of three-dimensional structures of glycosidases, both free and in complex with ligands, has provided valuable new insights into glycosidase catalysis, especially when coupled with results from studies of specifically labelled glycosidases and kinetic analyses of point mutants.     

Polarized-light-stimulated enzymatic hydrolysis of xylan

The 1- to 2-h illumination of xylanase with visible polarized light (PL) prior to the action of that enzyme upon beechwood xylan significantly increased its activity. The activity only negligibly decreased on 3 months storage. The hydrolysis of xylan proceeded in three well-distinguished stages. In the first and fastest stage the effect of illumination was only slightly positive. The effect of the stimulation was noted in the second, slower stage. Enzyme stimulated with PL, preferably by means of the 2-h illumination, performed better than enzyme stimulated with nonpolarized light and non-stimulated enzyme. In the last, the slowest stage, the rates of the reaction were nearly the same using either stimulated or non-stimulated enzyme.     

Semicontinuous enzymatic hydrolysis of lignocelluloses

Lignocelluloses (steamed hardwood and hardwood kraft pulp) were semicontinuously hydrolyzed on a large scale [2-2. 5 kg of substrate vs. 20, 000 IU filter paperase (FPase)] using a 10-L hydrolysis reactor with an ultrafiltration unit for the recovery and reuse of cellulases. The substrate was added to the reactor at appropriate intervals to keep a concentration of approximately 5% (w/v). All of the enzyme was added at the beginning and no further addition was done. The ultrafiltration unit was operated intermittently rather than continuously due to its enough capacity (dilution rate of 2.5 h(-1)) and making the enzyme durable. The enzyme required to produce one gram of reducing sugar in this reactor was 27.3 FPase IU/g RS for steamed hardwood and 7.4 FPase IU/g RS for hardwood kraft pulp. The sugar composition of hydrolyzate was unaltered virtually from beginning to end of the hydrolysis in spite of the progressive loss of enzyme activities. The analysis of the enzyme composition in the hydrolyzate during hydrolysis revealed that an exo-beta-D-glucanase component was adsorbed selectively at the stages of advanced hydrolysis extent.     

Role of oxidative enzymatic treatments on enzymatic hydrolysis of softwood

The impact of oxidative modification and partial removal of lignin by laccase-mediator treatments on the enzymatic hydrolysis of steam-pretreated softwood (SPS) was evaluated. Two mediators, N-hydroxy-N-phenylacetamide (NHA) and its acetylated precursor, were oxidized by the laccase from Trametes hirsuta, and their effects on the activity of cellulolytic enzymes and on the hydrolysis yield of SPS were examined. Both simultaneous and sequential combinations of laccase-mediator treatments with commercial cellulases increased the sugar yield in the enzymatic hydrolysis of SPS. The maximal increase was 21% when a sequential treatment was applied. Laccase treatment alone was also shown to improve hydrolysis. NHA oxidized by laccase inhibited significantly the cellulases of Trichoderma reesei, but the presence of the solid substrate protected the activities against oxidative inactivation. Surface analysis of the lignocellulosic substrate before and after the laccase and cellulase treatments revealed an enrichment of lignin and an increase of carboxylic groups on the surface of the hydrolysis residue.     

The combined enzymatic hydrolysis and fermentation of hemicellulose to 2,3-butanediol

Summary Hemicellulose-rich fractions from several agricultural residues were converted to 2,3-butanediol by a combined enzymatic hydrolysis and fermentation process. Culture filtrates from Trichoderma harzianum E58 were used to hydrolyze the substrates while Klebsiella pneumoniae fermented the liberated sugars to 2,3-butanediol. Approximately 50–60% of a 5% (w/v) xylan preparation could be hydrolyzed and quantitatively converted to 2,3-butanediol using this procedure. Although enzymatic hydrolysis was optimal at pH 5.0 and 50° C, the combined hydrolysis and fermentation was most efficient at pH 6.5 and 30° C. Combined hydrolysis and fermentation resulted in butanediol levels that were 20–40% higher than could be obtained with a separate hydrolysis and fermentation process. The hemicellulose-rich water-soluble fractions obtained from a variety of steam-exploded agricultural residues could be readily used by the combined hydrolysis and fermentation approach resulting in butanediol yields of 0.4–0.5 g/g of reducing sugar utilized.