Dataset Information

Enzymatic lignocellulose hydrolysis: Improved cellulase productivity by insoluble solids recycling.

ABSTRACT:

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Background

It is necessary to develop efficient methods to produce renewable fuels from lignocellulosic biomass. One of the main challenges to the industrialization of lignocellulose conversion processes is the large amount of cellulase enzymes used for the hydrolysis of cellulose. One method for decreasing the amount of enzyme used is to recycle the enzymes. In this study, the recycle of enzymes associated with the insoluble solid fraction after the enzymatic hydrolysis of cellulose was investigated for pretreated corn stover under a variety of recycling conditions.

Results

It was found that a significant amount of cellulase activity could be recovered by recycling the insoluble biomass fraction, and the enzyme dosage could be decreased by 30% to achieve the same glucose yields under the most favorable conditions. Enzyme productivity (g glucose produced/g enzyme applied) increased between 30 and 50% by the recycling, depending on the reaction conditions. While increasing the amount of solids recycled increased process performance, the methods applicability was limited by its positive correlation with increasing total solids concentrations, reaction volumes, and lignin content of the insoluble residue. However, increasing amounts of lignin rich residue during the recycle did not negatively impact glucose yields.

Conclusions

To take advantage of this effect, the amount of solids recycled should be maximized, based on a given processes ability to deal with higher solids concentrations and volumes. Recycling of enzymes by recycling the insoluble solids fraction was thus shown to be an effective method to decrease enzyme usage, and research should be continued for its industrial application.

SUBMITTER: Weiss N

PROVIDER: S-EPMC3560254 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Publications

Enzymatic lignocellulose hydrolysis: Improved cellulase productivity by insoluble solids recycling.

Weiss Noah N Börjesson Johan J Pedersen Lars Saaby LS Meyer Anne S AS

Biotechnology for biofuels 20130121 1

<h4>Unlabelled</h4><h4>Background</h4>It is necessary to develop efficient methods to produce renewable fuels from lignocellulosic biomass. One of the main challenges to the industrialization of lignocellulose conversion processes is the large amount of cellulase enzymes used for the hydrolysis of cellulose. One method for decreasing the amount of enzyme used is to recycle the enzymes. In this study, the recycle of enzymes associated with the insoluble solid fraction after the enzymatic hydrolys ...[more]

PMID: 23336604

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Project description:BackgroundTo improve process economics for production of fuels and chemicals from lignocellulosic biomass, high solids concentrations are applied in enzymatic hydrolysis, to increase product concentration and reduce energy input. However, increasing solids concentrations decrease cellulose conversion yields, the so called 'high-solids effect.' Previous work suggests that product inhibition and mixing contribute, but an understanding of how biomass properties influence the high-solids effect, is lacking.ResultsCellulose hydrolysis yields with an industrial cellulase (Ctec2) were measured on pretreated wheat straw and spruce from 5 to 30% dry matter (DM), and compared to yields of an older industrial cellulase mixture (Celluclast 1.5L/Novozym188). For Ctec2, yield was independent of DM below 15-18% DM, while yields decreased with increasing DM above this range, but at different rates for each biomass. For Celluclast 1.5L/Novozym188, yields decreased already from the lowest DM, suggesting that the high-solids effect was more a function of product inhibition, while the yields of the newer Ctec2 mixture were driven more by biomass-water interactions. LF-NMR relaxometry showed that the onset of the high-solids effect for Ctec2 corresponded to the disappearance of free water from the system, and a decrease in water self-diffusion rates. While the spruce had higher yields at low-solids, the wheat straw had higher yields at high-solids conditions, exhibiting that relative yields at low and high-solids are not related. Higher yields corresponded to increased water constraint by the biomass at high-solids conditions. Modifications to the pretreated wheat straw resulted in improved yields, and changes to the inflection point and intensity of the high-solids effect, showing that this effect can be reduced.ConclusionsThe high-solids effect is both enzyme and substrate dependent, and can be reduced by modifying the pretreated biomass, suggesting that pretreatment processes can be designed to achieve similar effects. Yields at low and high-solids concentrations do not correlate for a given biomass, and thus industrial evaluation of biomass recalcitrance should be carried out at high-solids conditions.

Project description:BackgroundAccumulation of recalcitrant oligosaccharides during high-solids loading enzymatic hydrolysis of cellulosic biomass reduces biofuel yields and increases processing costs for a cellulosic biorefinery. Recalcitrant oligosaccharides in AFEX-pretreated corn stover hydrolysate accumulate to the extent of about 18-25 % of the total soluble sugars in the hydrolysate and 12-18 % of the total polysaccharides in the inlet biomass (untreated), equivalent to a yield loss of about 7-9 kg of monomeric sugars per 100 kg of inlet dry biomass (untreated). These oligosaccharides represent a yield loss and also inhibit commercial hydrolytic enzymes, with both being serious bottlenecks for economical biofuel production from cellulosic biomass. Very little is understood about the nature of these oligomers and why they are recalcitrant to commercial enzymes. This work presents a robust method for separating recalcitrant oligosaccharides from high solid loading hydrolysate in gramme quantities. Composition analysis, recalcitrance study and enzyme inhibition study were performed to understand their chemical nature.ResultsOligosaccharide accumulation occurs during high solid loading enzymatic hydrolysis of corn stover (CS) irrespective of using different pretreated corn stover (dilute acid: DA, ionic liquids: IL, and ammonia fibre expansion: AFEX). The methodology for large-scale separation of recalcitrant oligosaccharides from 25 % solids-loading AFEX-corn stover hydrolysate using charcoal fractionation and size exclusion chromatography is reported for the first time. Oligosaccharides with higher degree of polymerization (DP) were recalcitrant towards commercial enzyme mixtures [Ctec2, Htec2 and Multifect pectinase (MP)] compared to lower DP oligosaccharides. Enzyme inhibition studies using processed substrates (Avicel and xylan) showed that low DP oligosaccharides also inhibit commercial enzymes. Addition of monomeric sugars to oligosaccharides increases the inhibitory effects of oligosaccharides on commercial enzymes.ConclusionThe carbohydrate composition of the recalcitrant oligosaccharides, ratios of different DP oligomers and their distribution profiles were determined. Recalcitrance and enzyme inhibition studies help determine whether the commercial enzyme mixtures lack the enzyme activities required to completely de-polymerize the plant cell wall. Such studies clarify the reasons for oligosaccharide accumulation and contribute to strategies by which oligosaccharides can be converted into fermentable sugars and provide higher biofuel yields with less enzyme.

Project description:BackgroundXylan, the major constituent of hemicellulose, is composed of β-(1,4)-linked xylopyranosyl units that for the backbone, with side chains formed by other chemical moieties such as arabinose, galactose, mannose, ferulic acid and acetyl groups. Acetyl xylan esterases and α-L-arabinofuranosidases are two important accessory enzymes that remove side chain residues from xylan backbones and may act in synergy with other xylanolytic enzymes. Compared with enzymes possessing a single catalytic activity, multifunctional enzymes can achieve lignocellulosic biomass hydrolysis using a less complex mixture of enzymes.ResultsHere, we cloned an acetyl xylan esterase (PcAxe) from Penicillium chrysogenum P33 and expressed it in Pichia pastoris GS115. The optimal pH and temperature of the recombinant PcAxe (rPcAxe) for 4-nitrophenyl acetate were 7.0 and 40 °C, respectively. rPcAxe is stable across a broad pH range, retaining 100% enzyme activity om pH 6-9 after a 1 h incubation. The enzyme tolerates the presence of a wide range of metal ions. Sequence alignment revealed a GH62 domain exhibiting α-L-arabinofuranosidase activity with pH and temperature optima of pH 7.0 and 50 °C, in addition to the expected esterase domain. rPcAxe displayed significant synergy with a recombinant xylanase, with a degree of synergy of 1.35 for the hydrolysis of delignified corn stover. Release of glucose was increased by 51% from delignified corn stover when 2 mg of a commercial cellulase was replaced by an equivalent amount of rPcAxe, indicating superior hydrolytic efficiency.ConclusionsThe novel bifunctional enzyme PcAxe was identified in P. chrysogenum P33. rPcAxe includes a carbohydrate esterase domain and a glycosyl hydrolase family 62 domain. This is the first detailed report on a novel bifunctional enzyme possessing acetyl xylan esterase and α-L-arabinofuranosidase activities. These findings expand our current knowledge of glycoside hydrolases and pave the way for the discovery of similar novel enzymes.

Project description:Glycoside hydrolases (GH) cleave carbohydrate glycosidic bonds and play pivotal roles in living organisms and in many industrial processes. Unlike acid-catalyzed hydrolysis of carbohydrates in solution, which can occur either via cyclic or acyclic oxocarbenium-like transition states, it is widely accepted that GH-catalyzed hydrolysis proceeds via a general acid mechanism involving a cyclic oxocarbenium-like transition state with protonation of the glycosidic oxygen. The GH45 subfamily C inverting endoglucanase from Phanerochaete chrysosporium (PcCel45A) defies the classical inverting mechanism as its crystal structure conspicuously lacks a general Asp or Glu base residue. Instead, PcCel45A has an Asn residue, a notoriously weak base in solution, as one of its catalytic residues at position 92. Moreover, unlike other inverting GHs, the relative position of the catalytic residues in PcCel45A impairs the proton abstraction from the nucleophilic water that attacks the anomeric carbon, a key step in the classical mechanism. Here, we investigate the viability of an endocyclic mechanism for PcCel45A using hybrid quantum mechanics/molecular mechanics (QM/MM) simulations, with the QM region treated with the self-consistent-charge density-functional tight-binding level of theory. In this mechanism, an acyclic oxocarbenium-like transition state is stabilized leading to the opening of the glucopyranose ring and formation of an unstable acyclic hemiacetal that can be readily decomposed into hydrolysis product. In silico characterization of the Michaelis complex shows that PcCel45A significantly restrains the sugar ring to the 4C1 chair conformation at the -1 subsite of the substrate binding cleft, in contrast to the classical exocyclic mechanism in which ring puckering is critical. We also show that PcCel45A provides an environment where the catalytic Asn92 residue in its standard amide form participates in a cooperative hydrogen bond network resulting in its increased nucleophilicity due to an increased negative charge on the oxygen atom. Our results for PcCel45A suggest that carbohydrate hydrolysis catalyzed by GHs may take an alternative route from the classical mechanism.

Dataset Information

Enzymatic lignocellulose hydrolysis: Improved cellulase productivity by insoluble solids recycling.

Unlabelled

Background

Results

Conclusions

Publications

Enzymatic lignocellulose hydrolysis: Improved cellulase productivity by insoluble solids recycling.

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