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Quantification of morphochemical changes during in situ enzymatic hydrolysis of individual biomass particles based on autofluorescence imaging.

ABSTRACT: Enzymatic hydrolysis of biomass is an established method for producing biofuels. Lignocellulosic biomass such as corn stover is very inhomogeneous material with big variation on conversion rates between individual particles therefore leading to variable recalcitrance results. In this study, we used noninvasive optical microscopy techniques, such as two-photon microscopy and fluorescence lifetime imaging microscopy, to visualize and analyze morphological and chemical changes of individual corn stover particles pretreated with sulfuric acid during hydrolysis. Morphochemical changes were interpreted based on the fluorescence properties of isolated building blocks of plant cell wall, such as cellulose, hemicellulose, and lignin. Enzymatic hydrolysis resulted in particle size reduction, side wall collapse, decrease of second harmonic signal from cellulose, redshifting of autofluorescence emission, and lifetime decrease attributed to the relative increase of lignin. Based on these observations, tracking compositional change after hydrolysis of individual particles was accomplished. The methodologies developed offer a paradigm for imaging and analyzing enzymatic hydrolysis in vitro and in situ, which could be used for screening enzymes cocktails targeting specific recalcitrant structures or investigating locally enzyme anti-inhibitory agents.

SUBMITTER: Kapsokalyvas D

PROVIDER: S-EPMC7154748 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Quantification of morphochemical changes during in situ enzymatic hydrolysis of individual biomass particles based on autofluorescence imaging.

Kapsokalyvas Dimitrios D Loos Joachim J Boogers Ilco A L A IALA Appeldoorn Maaike M MM Kabel Mirjam A MA Van Zandvoort Marc M

Biopolymers 20191223 3

Enzymatic hydrolysis of biomass is an established method for producing biofuels. Lignocellulosic biomass such as corn stover is very inhomogeneous material with big variation on conversion rates between individual particles therefore leading to variable recalcitrance results. In this study, we used noninvasive optical microscopy techniques, such as two-photon microscopy and fluorescence lifetime imaging microscopy, to visualize and analyze morphological and chemical changes of individual corn st ...[more]

PMID: 31868924

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Project description:Background:Liquid hot water (LHW) pretreatment has been considered as one of the most industrially viable and environment-friendly methods for facilitating the transformation of lignocelluloses into biofuels through biological conversion. However, lignin fragments in pretreatment hydrolysates are preferential to condense with each other and then deposit back onto cellulose surface under severe conditions. Particularly, lignin tends to relocate or redistribute under high-temperature LHW pretreatment conditions. The lignin residues on the cellulose surface would result in significant nonproductive binding of cellulolytic enzymes, and therefore negatively affect the enzymatic conversion (EC) of glucan in pretreated substrates. Although additives such as bovine serum albumin (BSA) and Tween series have been used to reduce nonproductive binding of enzymes through blocking the lignin, the high cost or non-biocompatibility of these additives limits their potential in industrial applications. Results:Here, we firstly report that a soluble soy protein (SP) extracted from inexpensive defatted soy powder (DSP) showed excellent performance in promoting the EC of glucan in LHW-pretreated lignocellulosic substrates. The addition of the SP (80 mg/g glucan) could readily reduce the cellulase (Celluclast 1.5 L®) loading by 8 times from 96.7 to 12.1 mg protein/g glucan and achieve a glucan EC of 80% at a hydrolysis time of 72 h. With the same cellulase (Celluclast 1.5 L®) loading (24.2 mg protein/g glucan), the ECs of glucan in LHW-pretreated bamboo, eucalyptus, and Masson pine substrates increased from 57%, 54% and 45% (without SP) to 87%, 94% and 86% (with 80 mg SP/g glucan), respectively. Similar effects were also observed when Cellic CTec2, a newer-generation cellulase preparation, was used. Mechanistic studies indicated that the adsorption of soluble SP onto the surface of lignin residues could reduce the nonproductive binding of cellulolytic enzymes to lignin. The cost of the SP required for effective promotion would be equivalent to the cost of 2.9 mg cellulase (Celluclast 1.5 L®) protein (or 1.2 FPU/g glucan), if a proposed semi-simultaneous saccharification and fermentation (semi-SSF) model was used. Conclusions:Near-complete saccharification of glucan in LHW-pretreated lignocellulosic substrates could be achieved with the addition of the inexpensive and biocompatible SP additive extracted from DSP. This simple but remarkably effective technique could readily contribute to improving the economics of the cellulosic biorefinery industry.

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:BackgroundValorization of lignocellulosic biomass to obtain clean fuels and high-value chemicals is attractive and essential for sustainable energy and chemical production, but the complex structure of biomass is recalcitrant to catalytic processing. This recalcitrance can be overcome by pretreating biomass into deconstructable components, which involves altering the structural complexities and physicochemical properties. However, the impact of these alterations on biomass deconstruction varies considerably, depending on the pretreatment and subsequent conversion type. Here, we systematically describe the changes in structure and properties of corn stover after ball milling as well as their influence on the following enzymatic saccharification and acid-catalyzed alcoholysis, with the aim of elucidating the relationships between structures, properties and deconstructable potential of lignocellulosic biomass.ResultsBall milling causes dramatic structural changes, since the resistant plant cell walls are destroyed with size reduction to a cellular scale, leading to the increase in surface area and reducing ends, and decrease in crystallinity and thermal stability. As a result, ball-milled corn stover is more susceptible to enzymatic saccharification to fermentable sugars and provides more industrially viable processing approaches, as it is effective at high solids loading and minor enzyme loading, without any other pretreatment. Acid-catalyzed alcoholysis of corn stover to biofuels, on the other hand, is also enhanced by ball milling, but additional processing parameters should be tailored to the needs of efficient conversion. Further, a detailed examination of process variables coupled with a kinetic study indicates that acid-catalyzed alcoholysis is limited by the process variables rather than by the substrate parameters, whereas ball milling facilitates this reaction to some extent, especially under mild conditions, by lowering the activation energy of corn stover decomposition.ConclusionsThe efficient catalytic conversion of biomass is closely related to its structure and properties, an understanding of which offers prospects for the rational improvement of methods aimed at more economic commercial biorefineries.

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Quantification of morphochemical changes during in situ enzymatic hydrolysis of individual biomass particles based on autofluorescence imaging.

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Quantification of morphochemical changes during in situ enzymatic hydrolysis of individual biomass particles based on autofluorescence imaging.

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