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Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification.

ABSTRACT: BACKGROUND:Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification. RESULTS:In this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion(a) (AFEX™-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin. CONCLUSIONS:Our findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.

SUBMITTER: Gao D

PROVIDER: S-EPMC4272552 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification.

Gao Dahai D Haarmeyer Carolyn C Balan Venkatesh V Whitehead Timothy A TA Dale Bruce E BE Chundawat Shishir Ps SP

Biotechnology for biofuels 20141213 1

<h4>Background</h4>Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a ...[more]

PMID: 25530803

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Project description:Two strategies leading to enzyme saving during saccharification of pretreated lignocellulo-starch biomass (LCSB) was investigated which included reducing enzyme dosage by varying their levels in enzyme cocktails and enhancing the fermentable sugar yield in enzyme-reduced systems using detoxification chemicals. Time course release of reducing sugars (RS) during 24-120 h was significantly higher when an enzyme cocktail containing full dose of cellulase (16 FPU/g cellulose) along with half dose each of xylanase (1.5 mg protein/g hemicelluloses) and Stargen (12.5 μl/g biomass) was used to saccharify conventional dilute sulphuric acid (DSA) pretreated biomass compared to a parallel system where only one-fourth the dose of the latter two enzymes was used. The reduction in RS content in the 120 h saccharified mash to the extent of 3-4 g/L compared to the system saccharified with full complement of the three enzymes could be overcome considerably by supplementing the system (half dose of two enzymes) with detoxification chemical mix incorporating Tween 20, PEG 4000 and sodium borohydride. Microwave (MW)-assisted DSA pretreated biomass on saccharification with enzyme cocktail having full dose of cellulase and half dose of Stargen along with detoxification chemicals gave significantly higher RS yield than DSA pretreated system saccharified using three enzymes. The study showed that xylanase could be eliminated during saccharification of MW-assisted DSA pretreated biomass without affecting RS yield when detoxification chemicals were also supplemented. The Saccharification Efficiency and Overall Conversion Efficiency were also high for the MW-assisted DSA pretreated biomass. Since whole slurry saccharifcation of pretreated biomass is essential to conserve fermentable sugars in LCSB saccharification, detoxification of soluble inhibitors is equally important as channelling out of insoluble lignin remaining in the residue. As one of the major factors contributing to the cost of ethanol production from LCSB is the cost of enzymes, appropriate modification of enzyme cocktail based on the composition of the pretreated biomass coupled with effective detoxification of the slurry would be a promising approach towards cost reduction.

Project description:Corn stover is the most abundant form of crop residue that can serve as a source of lignocellulosic biomass in biorefinery approaches, for instance for the production of bioethanol. In such biorefinery processes, the constituent polysaccharide biopolymers are typically broken down into simple monomeric sugars by enzymatic saccharification, for further downstream fermentation into bioethanol. However, the recalcitrance of this material to enzymatic saccharification invokes the need for innovative pre-treatment methods to increase sugar conversion yield. Here, we focus on experimental glucose conversion time-courses for corn stover lignocellulose that has been pre-treated with different acid-catalysed processes and intensities. We identify the key parameters that determine enzymatic saccharification dynamics by performing a Sobol's sensitivity analysis on the comparison between the simulation results from our complex stochastic biophysical model, and the experimental data that we accurately reproduce. We find that the parameters relating to cellulose crystallinity and those associated with the cellobiohydrolase activity are predominantly driving the enzymatic saccharification dynamics. We confirm our computational results using mathematical calculations for a purely cellulosic substrate. On the one hand, having identified that only five parameters drastically influence the saccharification dynamics allows us to reduce the dimensionality of the parameter space (from nineteen to five parameters), which we expect will significantly speed up our fitting algorithm for comparison of experimental and simulated saccharification time-courses. On the other hand, these parameters directly highlight key targets for experimental endeavours in the optimisation of pre-treatment and saccharification conditions. Finally, this systematic and two-fold theoretical study, based on both mathematical and computational approaches, provides experimentalists with key insights that will support them in rationalising their complex experimental results.

Project description:BackgroundMultiple analytical methods have been developed to determine the ratios of aromatic lignin units, particularly the syringyl/guaiacyl (S/G) ratio, of lignin biopolymers in plant cell walls. Chemical degradation methods such as thioacidolysis produce aromatic lignin units that are released from certain linkages and may induce chemical changes rendering it difficult to distinguish and determine the source of specific aromatic lignin units released, as is the case with nitrobenzene oxidation methodology. NMR methods provide powerful tools used to analyze cell walls for lignin composition and linkage information. Pyrolysis-mass spectrometry methods are also widely used, particularly as high-throughput methodologies. However, the different techniques used to analyze aromatic lignin unit ratios frequently yield different results within and across particular studies, making it difficult to interpret and compare results. This also makes it difficult to obtain meaningful insights relating these measurements to other characteristics of plant cell walls that may impact biomass sustainability and conversion metrics for the production of bio-derived fuels and chemicals.ResultsThe authors compared the S/G lignin unit ratios obtained from thioacidolysis, pyrolysis-molecular beam mass spectrometry (py-MBMS), HSQC liquid-state NMR and solid-state (ss) NMR methodologies of pine, several genotypes of poplar, and corn stover biomass. An underutilized approach to deconvolute ssNMR spectra was implemented to derive S/G ratios. The S/G ratios obtained for the samples did not agree across the different methods, but trends were similar with the most agreement among the py-MBMS, HSQC NMR and deconvoluted ssNMR methods. The relationship between S/G, thioacidolysis yields, and linkage analysis determined by HSQC is also addressed.ConclusionsThis work demonstrates that different methods using chemical, thermal, and non-destructive NMR techniques to determine native lignin S/G ratios in plant cell walls may yield different results depending on species and linkage abundances. Spectral deconvolution can be applied to many hardwoods with lignin dominated by S and G units, but the results may not be reliable for some woody and grassy species of more diverse lignin composition. HSQC may be a better method for analyzing lignin in those species given the wealth of information provided on additional aromatic moieties and bond linkages. Additionally, trends or correlations in lignin characteristics such as S/G ratios and lignin linkages within the same species such as poplar may not necessarily exhibit the same trends or correlations made across different biomass types. Careful consideration is required when choosing a method to measure S/G ratios and the benefits and shortcomings of each method discussed here are summarized.

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Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification.

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Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification.

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