Pretreated Lignocellulosic Substrates Research Articles

Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates

A range of lignocellulosic feedstocks (including agricultural, softwood and hardwood substrates) were pretreated with either sulfur dioxide-catalyzed steam or an ethanol organosolv procedure to try to establish a reliable assessment of the factors governing the minimum protein loading that could be used to achieve efficient hydrolysis. A statistical design approach was first used to define what might constitute the minimum protein loading (cellulases and β-glucosidase) that could be used to achieve efficient saccharification (defined as at least 70% glucan conversion) of the pretreated substrates after 72 hours of hydrolysis. The likely substrate factors that limit cellulose availability/accessibility were assessed, and then compared with the optimized minimum amounts of protein used to obtain effective hydrolysis. The optimized minimum protein loadings to achieve efficient hydrolysis of seven pretreated substrates ranged between 18 and 63 mg protein per gram of glucan. Within the similarly pretreated group of lignocellulosic feedstocks, the agricultural residues (corn stover and corn fiber) required significantly lower protein loadings to achieve efficient hydrolysis than did the pretreated woody biomass (poplar, douglas fir and lodgepole pine). Regardless of the substantial differences in the source, structure and chemical composition of the feedstocks, and the difference in the pretreatment technology used, the protein loading required to achieve efficient hydrolysis of lignocellulosic substrates was strongly dependent on the accessibility of the cellulosic component of each of the substrates. We found that cellulose-rich substrates with highly accessible cellulose, as assessed by the Simons' stain method, required a lower protein loading per gram of glucan to obtain efficient hydrolysis compared with substrates containing less accessible cellulose. These results suggest that the rate-limiting step during hydrolysis is not the catalytic cleavage of the cellulose chains per se, but rather the limited accessibility of the enzymes to the cellulose chains due to the physical structure of the cellulosic substrate.

Enhancing the enzymatic hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content of the associated lignin

To assess the effects that the physical and chemical properties of lignin might have on the enzymatic hydrolysis of pretreated lignocellulosic substrates, protease treated lignin (PTL) and cellulolytic enzyme lignin (CEL) fractions, isolated from steam and organosolv pretreated corn stover, poplar, and lodgepole pine, were prepared and characterized. The adsorption of cellulases to the isolated lignin preparations corresponded to a Langmuir adsorption isotherm. It was apparent that, rather than the physical properties of the isolated lignin, the carboxylic acid functionality of the isolated lignin, as determined by FTIR and NMR spectroscopy, had much more of an influence when lignin was added to typical hydrolysis of pure cellulose (Avicel). An increase in the carboxylic content of the lignin preparation resulted in an increased hydrolysis yield. These results suggested that the carboxylic acids within the lignin partially alleviate non-productive binding of cellulases to lignin. To try to confirm this possible mechanism, dehydrogenative polymers (DHP) of monolignols were synthesized from coniferyl alcohol (CA) and ferulic acid (FA), and these model compounds were added to a typical enzymatic hydrolysis of Avicel. The DHP from FA, which was enriched in carboxylic acid groups compared with the DHP from CA, adsorbed a lower mount of cellulases and did not decrease hydrolysis yields when compared to the DHP from CA, which decreased the hydrolysis of Avicel by 8.4%. Thus, increasing the carboxylic acid content of the lignin seemed to significantly decrease the non-productive binding of cellulases and consequently increased the enzymatic hydrolysis of the cellulose.

High-throughput enzymatic hydrolysis of lignocellulosic biomass via in-situ regeneration

The high cost of lignocellulolytic enzymes is one of the main barriers towards the development of economically competitive biorefineries. Enzyme engineering can be used to significantly increase the production rate as well as specific activity of enzymes. However, the success of enzyme optimization efforts is currently limited by a lack of robust high-throughput (HTP) cellulase screening platforms for insoluble pretreated lignocellulosic substrates. We have developed a cost-effective microplate based HTP enzyme-screening platform for ionic liquid (IL) pretreated lignocellulose. By performing in-situ biomass regeneration in micro-volumes, we can volumetrically meter biomass (sub-mg loading) and also precisely control the amount of residual IL for engineering novel IL-tolerant cellulases. Our platform only requires straightforward liquid-handling steps and allows the integration of biomass regeneration, washing, saccharification, and imaging steps in a single microtiter plate. The proposed method can be used to screen individual cellulases as well as to develop novel cellulase cocktails.

Application of a microassay method to study enzymatic hydrolysis of pretreated wheat straw

AbstractBACKGROUND: The conversion of lignocellulosic biomass to ethanol includes a disruptive pretreatment process followed by enzyme‐catalyzed hydrolysis of the cellulose and hemicellulose components to fermentable sugars. As the cost and hydrolytic efficiency of enzymes are major factors that restrict the commercialization of biomass conversion processes, significant efforts are made nowadays to improve the enzymatic mixtures and make the process cost‐effective.RESULTS: In this work, enzymatic microassays have been developed and validated to test new different enzymatic formulations on real lignocellulosic substrates. Homogeneous handsheets from steam pretreated wheat straw were elaborated to be used as substrate. The microassay was adapted to test both water‐insoluble solids and the whole slurry as substrates. Results in hydrolysis microassays were comparable with those obtained in standard flask assays using pretreated wheat straw. Moreover, using the enzymatic microassays, two novel preparations have been evaluated, demonstrating the ability of microassays to discriminate between different enzymatic mixtures.CONCLUSIONS: This enzymatic microassay represents a rapid method to test the performance of new selected cellulase enzymes on real pretreated lignocellulosic substrates. This microassay will enable evaluation of enzyme components separately, or optimized mixtures. Copyright © 2010 Society of Chemical Industry

The effect of isolated lignins, obtained from a range of pretreated lignocellulosic substrates, on enzymatic hydrolysis

The influence of the residual lignin remaining in the cellulosic rich component of pretreated lignocellulosic substrates on subsequent enzymatic hydrolysis was assessed. Twelve lignin preparations were isolated by two isolation methods (protease treated lignin (PTL) and cellulolytic enzymatic lignin (CEL)) from three types of biomass (corn stover, poplar, and lodgepole pine) that had been pretreated by two processes (steam and organosolv pretreatments). Comparative analysis of the isolated lignin showed that the CEL contained lower amounts of carbohydrates and protein than did the PTL and that the isolated lignin from corn stover contained more carbohydrates than did the lignin derived from the poplar and lodgepole pine. The lower yields of acid insoluble lignin (AIL) obtained from the corn stover when using the PTL method indicated that the lignin from the corn stover had a higher hydrophilicity than did the lignin from the poplar and lodgepole pine. The isolated lignin preparations were added to the reaction mixture containing crystalline cellulose (Avicel) and their possible effects on enzymatic hydrolysis were assessed. It was apparent that the lignin isolated from lodgepole pine and steam pretreated poplar decreased the hydrolysis yields of Avicel, whereas the other isolated lignins did not appear to decrease the hydrolysis yields significantly. The hydrolysis yields of the pretreated lignocellulose and those of Avicel containing the PTL showed good correlation, indicating that the nature of the residual lignin obtained after pretreatment significantly influenced hydrolysis.

Enzymatic hydrolysis of steam-pretreated lignocellulosic materials with Trichoderma atroviride enzymes produced in-house

BackgroundImprovement of the process of cellulase production and development of more efficient lignocellulose-degrading enzymes are necessary in order to reduce the cost of enzymes required in the biomass-to-bioethanol process.ResultsLignocellulolytic enzyme complexes were produced by the mutant Trichoderma atroviride TUB F-1663 on three different steam-pretreated lignocellulosic substrates, namely spruce, wheat straw and sugarcane bagasse. Filter paper activities of the enzymes produced on the three materials were very similar, while β-glucosidase and hemicellulase activities were more dependent on the nature of the substrate. Hydrolysis of the enzyme preparations investigated produced similar glucose yields. However, the enzymes produced in-house proved to degrade the xylan and the xylose oligomers less efficiently than a commercial mixture of cellulase and β-glucosidase. Furthermore, accumulation of xylose oligomers was observed when the TUB F-1663 supernatants were applied to xylan-containing substrates, probably due to the low β-xylosidase activity of the enzymes. The efficiency of the enzymes produced in-house was enhanced by supplementation with extra commercial β-glucosidase and β-xylosidase. When the hydrolytic capacities of various mixtures of a commercial cellulase and a T. atroviride supernatant produced in the lab were investigated at the same enzyme loading, the glucose yield appeared to be correlated with the β-glucosidase activity, while the xylose yield seemed to be correlated with the β-xylosidase level in the mixtures.ConclusionEnzyme supernatants produced by the mutant T. atroviride TUB F-1663 on various pretreated lignocellulosic substrates have good filter paper activity values combined with high levels of β-glucosidase activities, leading to cellulose conversion in the enzymatic hydrolysis that is as efficient as with a commercial cellulase mixture. On the other hand, in order to achieve good xylan conversion, the supernatants produced by the mutant have to be supplemented with additional β-xylosidase activity.

Comparison of methods to assess the enzyme accessibility and hydrolysis of pretreated lignocellulosic substrates

Fiber size analysis, water retention value, and Simons' stain measurements were assessed for their potential to predict the susceptibility of a given substrate to enzymatic hydrolysis. Slight modifications were made to the fiber size analysis and water retention protocols to adapt these measurements to evaluate substrates for cellulolytic hydrolysis rather than pulps for papermaking. Lodgepole pine was pretreated by the steam and ethanol-organosolv processes under varying conditions. The Simons' stain procedure proved to be an effective method for indicating the potential ease of enzymatic hydrolysis of substrates pretreated by either process or when the pretreatment conditions were altered.

The characterization of pretreated lignocellulosic substrates prior to enzymatic hydrolysis, part 1: A modified Simons' staining technique

To date, there is limited knowledge available regarding the key features of pretreated lignocellulosic substrates that promote the effective enzymatic hydrolysis of the cellulose component to glucose during bioconversion processes to produce ethanol. Fundamentally, cellulase enzymes require access to the cellulose to carry out effective hydrolysis. Porosity and the overall surface area of substrates have major structural features influencing the hydrolysis of pretreated substrates by cellulases. Simons' Stain (SS) is a potentially useful semiquantitative method for estimating the available surface area of lignocellulosic substrates. In this study, a modified, rapid SS method was developed, where the processing time was decreased from >50 to 6 h and the maximum dye adsorbed on the substrate was calculated using the adsorption isotherm for the orange and blue components of the dye mixture. The modified SS test readily measures the decrease in accessibility and hydrolyzability of a steam pretreated substrate that had been dried under three different drying regimes. For each of the lignocellulosic substrates, the total dye adsorption correlated well with the hydrolysis yields resulting in a correlation coefficient of r(2) = 0.95. The modified SS procedure is an effective tool for assessing how lignocellulosic substrates might be potentially hydrolyzed by cellulases.

Heterologous Expression of Aspergillus niger β-d-Xylosidase (XlnD): Characterization on Lignocellulosic Substrates

The gene encoding a glycosyl hydrolase family 3 xylan 1,4-beta-xylosidase, xlnD, was successfully cloned from Aspergillus niger strain ATCC 10864. The recombinant product was expressed in Aspergillus awamori, purified by column chromatography, and verified by matrix-assisted laser desorption ionization, tandem time of flight (MALDI-TOF/TOF) mass spectroscopy of tryptic digests. The T (max) was determined using differential scanning microcalorimetry (DSC) to be 78.2 degrees C; the K (m) and k (cat) were found to be 255 microM and 13.7 s(-1), respectively, using pNP-beta-D-xylopyranoside as substrate. End-product inhibition by D-xylose was also verified and shown to be competitive; the K (i) for this inhibition was estimated to be 3.3 mM. XlnD was shown to efficiently hydrolyze small xylo-oligomers to monomeric xylose, making it a critical hydrolytic activity in cases where xylose is to be recovered from biomass conversion processes. In addition, the presence of the XlnD was shown to synergistically enhance the ability of an endoxylanase, XynA from Thermomyces lanuginosus, to convert xylan present in selected pretreated lignocellulosic substrates. Furthermore, the addition of the XynA/XlnD complex was effective in enhancing the ability of a simplified cellulase complex to convert glucan present in the substrates.

Ethanol fermentation of various pretreated and hydrolyzed substrates at low initial pH

Lignocellulosic materials represent an abundant feedstock for bioethanol production. Because of their complex structure pretreatment is necessary to make it accessible for enzymatic attack. Steam pretreatment with or without acid catalysts seems to be one of the most promising techniques, which has already been applied for large variety of lignocellulosics in order to improve enzymatic digestibility. During this process a range of toxic compounds (lignin and sugar degradation products) are formed which inhibit ethanol fermentation. In this study, the toxicity of hemicellulose hydrolysates obtained in the steam pretreatment of spruce, willow, and corn stover were investigated in ethanol fermentation tests using a yeast strain, which has been previously reported to have a resistance to inhibitory compounds generated during steam pretreatment. To overcome bacterial contamination, fermentations were carried out at low initial pH. The fermentability of hemicellulose hydrolysates of pretreated lignocellulosic substrates at low pH gave promising results with the economically profitable final 5 vol% ethanol concentration corresponding to 85% of theoretical. Adaptation experiments have shown that inhibitor tolerance of yeast strain can be improved by subsequent transfer of the yeast to inhibitory medium.

Evaluation of Minimal Trichoderma reesei Cellulase Mixtures on Differently Pretreated Barley Straw Substrates

The commercial cellulase product Celluclast 1.5, derived from Trichoderma reesei (Novozymes A/S, Bagsvaerd, Denmark), is widely employed for hydrolysis of lignocellulosic biomass feedstocks. This enzyme preparation contains a broad spectrum of cellulolytic enzyme activities, most notably cellobiohydrolases (CBHs) and endo-1,4-beta-glucanases (EGs). Since the original T. reesei strain was isolated from decaying canvas, the T. reesei CBH and EG activities might be present in suboptimal ratios for hydrolysis of pretreated lignocellulosic substrates. We employed statistically designed combinations of the four main activities of Celluclast 1.5, CBHI, CBHII, EGI, and EGII, to identify the optimal glucose-releasing combination of these four enzymes to degrade barley straw substrates subjected to three different pretreatments. The data signified that EGII activity is not required for efficient lignocellulose hydrolysis when addition of this activity occurs at the expense of the remaining three activities. The optimal ratios of the remaining three enzymes were similar for the two pretreated barley samples that had been subjeced to different hot water pretreatments, but the relative levels of EGI and CBHII activities required in the enzyme mixture for optimal hydrolysis of the acid-impregnated, steam-exploded barley straw substrate were somewhat different from those required for the other two substrates. The optimal ratios of the cellulolytic activities in all cases differed from that of the cellulases secreted by T. reesei. Hence, the data indicate the feasibility of designing minimal enzyme mixtures for pretreated lignocellulosic biomass by careful combination of monocomponent enzymes. This strategy can promote both a more efficient enzymatic hydrolysis of (ligno)cellulose and a more rational utilization of enzymes.

Bioconversion of lignocellulosics

During the 3 years from 1986 to 1988, two International Energy Agency projects, CPD 2 (Pretreatment of Lignocellulosics) and CPD 5 (Conversion of C5, Sugars to Ethanol), were combined to form Task IV — Bioconversion of Lignocellulosics. Two meetings were held, the first in Graz, Austria in 1986 and the second in Ottawa, Canada in 1988. Proceedings from each of these meetings were distributed among the participants. Round-robin tests on the chemical analysis of pretreated lignocellulosic substrates and the enzymatic hydrolysis of these substrates were carried out by the network participants. Industrial groups from member countries were invited to participate at the Ottawa meeting. Various economic models were presented to determine the technoeconomic factors influencing the commercial viability of a bioconversion process. The success of the network was reflected in the attendance at the meetings, the excellent rapport of the participants and the useful information arising from the round-robin comparisons.

The nature of inhibitory materials present in pretreated lignocellulosic substrates which inhibit the enzymatic hydrolysis of cellulose

Wheat straw and aspen wood chips were pretreated by steam explosion and the various fractions were assayed for the presence of materials which inhibited the enzymatic hydrolysis of the cellulose component of the lignocellulosic substrates to glucose. The inhibitory material could be removed from all of the fractions by simple water extraction. The inhibitory substances were shown to primarily inhibit the B-glucosidase component of the cellulase complex of Trichoderma harzianum E58. The furan derivatives, furfural and hydroxymethyl furfural were not inhibitory at concentrations normally found in steam exploded lignocellulosic substrates.