Oxidative cleavage of polysaccharides by a termite-derived superoxide dismutase boosts the degradation of biomass by glycoside hydrolases.

Wood-feeding termites effectively degrade plant biomass through enzymatic degradation. Despite their high efficiencies, however, individual glycoside hydrolases isolated from termites and their symbionts exhibit anomalously low effectiveness in lignocellulose degradation, suggesting hereto unknown enzymatic activities in their digestome. Herein, we demonstrate that an ancient redox-active enzyme encoded by the lower termite Coptotermes gestroi, a Cu/Zn superoxide dismutase (CgSOD-1), plays a previously unknown role in plant biomass degradation. We show that CgSOD-1 transcripts and peptides are up-regulated in response to an increased level of lignocellulose recalcitrance and that CgSOD-1 localizes in the lumen of the fore- and midguts of C. gestroi together with termite main cellulase, CgEG-1-GH9. CgSOD-1 boosts the saccharification of polysaccharides by CgEG-1-GH9. We show that the boosting effect of CgSOD-1 involves an oxidative mechanism of action in which CgSOD-1 generates reactive oxygen species that subsequently cleave the polysaccharide. SOD-type enzymes constitute a new addition to the growing family of oxidases, ones which are up-regulated when exposed to recalcitrant polysaccharides, and that are used by Nature for biomass degradation.

Improving cellulases hydrolytic action: An expanded role for electron donors of lytic polysaccharide monooxygenases in cellulose saccharification.

Ascorbic acid (AscA) and gallic acid (GalA) are common electron donors and their boosting effect on lytic polysaccharide monooxygenases (LPMO) has been studied extensively. However, their influence on cellulase hydrolytic action has been ignored. In this work, the effect of AscA and GalA on cellulases hydrolytic action was evaluated. It was found that AscA could increase the hydrolysis of cellulose by cellulases, while GalA showed no effect on cellulases' hydrolytic action. The effect of AscA differed for the monocomponent cellulases: it showed a special boosting effect on cellobiohydrolase, rather than endoglucanase and ß-glucosidase. This promoting effect could be another mechanism behind the boosting effect of the AscA-driven LPMO system on cellulose saccharification. These findings thus advance the understanding of the role of electron donors on cellulose saccharification and offer important clues on how to evaluate the feasibility of electron donors from a new perspective.

Removal of hard COD from acidic eucalyptus kraft pulp bleach plant effluent streams using oxidoreductases.

The bleach plant of a pulp and paper (P&P) mill presents a major source of wastewater containing toxic organic matter characterized as chemical oxygen demand (COD). Due to their high oxidizing power, oxidoreductases hold promise to be a key solution for the removal of dissolved organic material. Here, four oxidoreductases from different enzyme families were selected to treat bleach plant effluents. Haloperoxidase treatment of the final effluent resulted in the highest levels of decolorization (71%) and reduction of aromatic compounds (36%). Using single compound analysis, 27 low molecular weight compounds were found to be persistent throughout the wastewater treatment process and, therefore, classified as hard COD. The tested enzymes efficiently removed several of the identified COD compounds. Hence, this study suggests that the application of oxidoreductases will serve as an environmental-friendly solution for reducing waste from P&P production.

Chemistry of lignin and hemicellulose structures interacts with hydrothermal pretreatment severity and affects cellulose conversion.

Understanding of how the plant cell walls of different plant species respond to pretreatment can help improve saccharification in bioconversion processes. Here, we studied the chemical and structural modifications in lignin and hemicellulose in hydrothermally pretreated poplar and wheat straw using wet chemistry and 2D heteronuclear single quantum coherence nuclear magnetic resonance (NMR) and their effects on cellulose conversion. Increased pretreatment severity reduced the levels of ß─O─4 linkages with concomitant relatively increased levels of ß─5 and ßâ”€ß structures in the NMR spectra. ß─5 structures appeared at medium and high severities for wheat straw while only ßâ”€ß structures were observed at all pretreatment severities for poplar. These structural differences accounted for the differences in cellulose conversion for these biomasses at different severities. Changes in the hemicellulose component include a complete removal of arabinosyl and 4-O-methyl glucuronosyl substituents at low and medium pretreatment severities while acetyl groups were found to be relatively resistant toward hydrothermal pretreatment. This illustrates the importance of these groups, rather than xylan content, in the detrimental role of xylan in cellulose saccharification and helps explain the higher poplar recalcitrance compared to wheat straw. The results point toward the need for both enzyme preparation development and pretreatment technologies to target specific plant species.

On the roles of AA15 lytic polysaccharide monooxygenases derived from the termite Coptotermes gestroi.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes which catalyze the oxidative cleavage of polysaccharides. LPMOs belonging to family 15 in the Auxiliary Activity (AA) class from the Carbohydrate-Active Enzyme database are found widespread across the Tree of Life, including viruses, algae, oomycetes and animals. Recently, two AA15s from the firebrat Thermobia domestica were reported to have oxidative activity, one towards cellulose or chitin and the other towards chitin, signalling that AA15 LPMOs from insects potentially have different biochemical functions. Herein, we report the identification and characterization of two family AA15 members from the lower termite Coptotermes gestroi. Addition of Cu(II) to CgAA15a or CgAA15b had a thermostabilizing effect on both. Using ascorbate and O2 as co-substrates, CgAA15a and CgAA15b were able to oxidize chitin, but showed no activity on celluloses, xylan, xyloglucan and starch. Structural models indicate that the LPMOs from C. gestroi (CgAA15a/CgAA15b) have a similar fold but exhibit key differences in the catalytic site residues when compared to the cellulose/chitin-active LPMO from T. domestica (TdAA15a), especially the presence of a non-coordinating phenylalanine nearby the Cu ion in CgAA15a/b, which appears as a tyrosine in the active site of TdAA15a. Despite the overall similarity in protein folds, however, mutation of the active site phenylalanine in CgAA15a to a tyrosine did not expanded the enzymatic specificity from chitin to cellulose. Our data show that CgAA15a/b enzymes are likely not involved in lignocellulose digestion but might play a role in termite developmental processes as well as on chitin and nitrogen metabolisms.

Detection and Characterization of a Novel Copper-Dependent Intermediate in a Lytic Polysaccharide Monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes capable of oxidizing crystalline cellulose which have large practical application in the process of refining biomass. The catalytic mechanism of LPMOs still remains debated despite several proposed reaction mechanisms. Here, we report a long-lived intermediate (t1/2 =6-8 minutes) observed in an LPMO from Thermoascus aurantiacus (TaLPMO9A). The intermediate with a strong absorption around 420 nm is formed when reduced LPMO-CuI reacts with sub-equimolar amounts of H2 O2 . UV/Vis absorption spectroscopy, electron paramagnetic resonance, resonance Raman and stopped-flow spectroscopy suggest that the observed long-lived intermediate involves the copper center and a nearby tyrosine (Tyr175). Additionally, activity assays in the presence of sub-equimolar amounts of H2 O2 showed an increase in the LPMO oxidation of phosphoric acid swollen cellulose. Accordingly, this suggests that the long-lived copper-dependent intermediate could be part of the catalytic mechanism for LPMOs. The observed intermediate offers a new perspective into the oxidative reaction mechanism of TaLPMO9A and hence for the biomass oxidation and the reactivity of copper in biological systems.

A Perspective on the Transition to Plant-Based Diets: a Diet Change May Attenuate Climate Change, but Can It Also Attenuate Obesity and Chronic Disease Risk?

Current dietary guidelines advocate more plant-based, sustainable diets on the basis of scientific evidence about diet-health relations but also to address environmental concerns. Here, we critically review the effects of plant-based diets on the prevalence of obesity and other health outcomes. Plant-based diets per se have limited efficacy for the prevention and treatment of obesity, but most have beneficial effects in terms of chronic disease risk. However, with the considerable possibilities of translating plant-based diets into various types of dietary patterns, our analysis suggests that potential adverse health effects should also be considered in relation to vulnerable groups of the population. A transition to more plant-based diets may exert beneficial effects on the environment, but is unlikely to affect obesity, and may also have adverse health effects if this change is made without careful consideration of the nutritional needs of the individual relative to the adequacy of the dietary intake.

The Environmental Foodprint of Obesity.

Emissions of greenhouse gases (GHG) are linked to global warming and adverse climate changes. Meeting the needs of the increasing number of people on the planet presents a challenge for reducing total GHG burden. A further challenge may be the size of the average person on the planet and the increasing number of people with excess body weight. We used data on GHG emissions from various sources and estimated that obesity is associated with ~20% greater GHG emissions compared with the normal-weight state. On a global scale, obesity contributes to an extra GHG emissions of ~49 megatons per year of CO2 equivalent (CO2 eq) from oxidative metabolism due to greater metabolic demands, ~361 megatons per year of CO2 eq from food production processes due to increased food intake, and ~290 megatons per year of CO2 eq from automobile and air transportation due to greater body weight. Therefore, the total impact of obesity may be extra emissions of ~700 megatons per year of CO2 eq, which is about 1.6% of worldwide GHG emissions. Inasmuch as obesity is an important contributor to global GHG burden, strategies to reduce its prevalence should prioritize efforts to reduce GHG emissions. Accordingly, reducing obesity may have considerable benefits for both public health and the environment.

Novel redox-active enzymes for ligninolytic applications revealed from multiomics analyses of Peniophora sp. CBMAI 1063, a laccase hyper-producer strain.

The repertoire of redox-active enzymes produced by the marine fungus Peniophora sp. CBMAI 1063, a laccase hyper-producer strain, was characterized by omics analyses. The genome revealed 309 Carbohydrate-Active Enzymes (CAZymes) genes, including 48 predicted genes related to the modification and degradation of lignin, whith 303 being transcribed under cultivation in optimized saline conditions for laccase production. The secretome confirmed that the fungus can produce a versatile ligninolytic enzyme cocktail. It secretes 56 CAZymes, including 11 oxidative enzymes classified as members of auxiliary activity families (AAs), comprising two laccases, Pnh_Lac1 and Pnh_Lac2, the first is the major secretory protein of the fungi. The Pnh_Lac1-mediator system was able to promote the depolymerization of lignin fragments and polymeric lignin removal from pretreated sugarcane bagasse, confirming viability of this fungus enzymatic system for lignocellulose-based bioproducts applications.

Effects of preheating on briquetting and subsequent hydrothermal pretreatment for enzymatic saccharification of wheat straw.

Briquetting of plant biomass with low bulk density is an advantage for handling, transport, and storage of the material, and heating of the biomass prior to the briquetting facilitates the densification process and improves the physical properties of the briquettes. This study investigates the effects of preheating prior to briquetting of wheat straw (WS) on subsequent hydrothermal pretreatment and enzymatic conversion to fermentable sugars. WS (11% moisture content) was densified to briquettes under different conditions; without preheating or with preheating at 75 or 125°C for either 5 or 10 min. Subsequent hydrothermal pretreatment was done for both un-briquetted WS and for briquettes. Enzymatic saccharification was afterwards performed for all samples. The results showed that as expected, nonpretreated WS briquettes gave very low sugar yields (22-29% of the cellulose content), even though preheating at 125°C prior to briquetting (without pretreatment) improved sugar yields somewhat. When combined with pretreatment, briquetting with preheating showed neutral or negative effects on sugar yield. This result suggests that moderate preheating (75°C for 5 min) before briquetting improved bulk density and compressive resistance of briquettes without impeding subsequent enzymatic conversion. However, excessive preheating (75 or 125°C for 10 min) before briquetting may result in irreversible structural modifications that hinder the interaction between biomass and water during pretreatment, thereby decreasing the accessibility of cellulose to enzymatic saccharification.

Effect of hydrothermal pretreatment severity on lignin inhibition in enzymatic hydrolysis.

Hydrothermal pretreatment is commonly used for enhancing enzymatic hydrolysis of lignocellulosics. Spruce and wheat straw were pretreated with increasing severity and lignin characteristics were analysed. The effect of enzymatically isolated lignin on the hydrolysis of Avicel and the adsorption of a cellobiohydrolase onto lignin was measured. Non-pretreated lignins had only a minor effect on Avicel hydrolysis. The structural changes in lignin accompanying hydrothermal pretreatment were associated with increased binding and inactivation of the cellulase on the lignin surface. The inhibitory effect was more pronounced in spruce than in wheat straw lignin. However, similar pretreatment severities caused similar levels of inhibition in Avicel hydrolysis for both biomass sources. The combined severity factor of the pretreatment correlated well with the inhibitory effect of lignin.

Enzymatic hydrolysis is limited by biomass-water interactions at high-solids: improved performance through substrate modifications.

BACKGROUND: To 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. RESULTS: Cellulose 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. CONCLUSIONS: The 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.

The impact of silicon on cell wall composition and enzymatic saccharification of Brachypodium distachyon.

BACKGROUND: Plants and in particular grasses benefit from a high uptake of silicon (Si) which improves their growth and productivity by alleviating adverse effects of biotic and abiotic stress. However, the silicon present in plant tissues may have a negative impact on the processing and degradation of lignocellulosic biomass. Solutions to reduce the silicon content either by biomass engineering or development of downstream separation methods are therefore targeted. Different cell wall components have been proposed to interact with the silica pool in plant shoots, but the understanding of the underlying processes is still limited. RESULTS: In the present study, we have characterized silicon deposition and cell wall composition in Brachypodium distachyon wild-type and low-silicon 1 (Bdlsi1-1) mutant plants. Our analyses included different organs and plant developmental stages. In the mutant defective in silicon uptake, low silicon availability favoured deposition of this element in the amorphous form or bound to cell wall polymers rather than as silicified structures. Several alterations in non-cellulosic polysaccharides and lignin were recorded in the mutant plants, indicating differences in the types of linkages and in the three-dimensional organization of the cell wall network. Enzymatic saccharification assays showed that straw from mutant plants was marginally more degradable following a 190 °C hydrothermal pretreatment, while there were no differences without or after a 120 °C hydrothermal pretreatment. CONCLUSIONS: We conclude that silicon affects the composition of plant cell walls, mostly by altering linkages of non-cellulosic polymers and lignin. The modifications of the cell wall network and the reduced silicon concentration appear to have little or no implications on biomass recalcitrance to enzymatic saccharification.

Properties important for solid-liquid separations change during the enzymatic hydrolysis of pretreated wheat straw.

OBJECTIVES: The biochemical conversion of lignocellulosic biomass into renewable fuels and chemicals provides new challenges for industrial scale processes. One such process, which has received little attention, but is of great importance for efficient product recovery, is solid-liquid separations, which may occur both after pretreatment and after the enzymatic hydrolysis steps. Due to the changing nature of the solid biomass during processing, the solid-liquid separation properties of the biomass can also change. The objective of this study was to show the effect of enzymatic hydrolysis of cellulose upon the water retention properties of pretreated biomass over the course of the hydrolysis reaction. RESULTS: Water retention value measurements, coupled with 1H NMR T2 relaxometry data, showed an increase in water retention and constraint of water by the biomass with increasing levels of cellulose hydrolysis. This correlated with an increase in the fines fraction and a decrease in particle size, suggesting that structural decomposition rather than changes in chemical composition was the most dominant characteristic. CONCLUSIONS: With increased water retained by the insoluble fraction as cellulose hydrolysis proceeds, it may prove more difficult to efficiently separate hydrolysis residues from the liquid fraction with improved hydrolysis.

Laccase-derived lignin compounds boost cellulose oxidative enzymes AA9.

BACKGROUND: The discovery of lignin as activator for the redox enzyme lytic polysaccharide monooxygenases (LPMOs) for the oxidation of cell-wall polysaccharides opens a new scenario for investigation of the interplay between different lignocellulose-degrading enzymes. The lignin-active enzymes in one hand, and the carbohydrate active in the other, are linked through a variety of electrons carrier molecules either derived from lignin or enzymatically transferred. Likewise, in nature, many lignocellulose-degrading organisms are expressing those enzymes simultaneously, and we wanted to test if a major commercial available lignin oxidase enzyme, i.e., laccase could benefit and synergize the activity of the LPMOs by depolymerizing the insoluble lignin. RESULTS: In this work, two fungal laccases together with a mediator (ABTS) were used to isolate low-molecular-weight lignin from lignocellulosic biomass. The isolated lignins were used as electron donors for activation of LPMOs. A direct correlation between the low-molecular-weight lignin isolated with laccases and an increased activity of a cellulolytic cocktail containing LPMO was found when pure cellulose was hydrolyzed. We then tried to implement existing commercial cellulases cocktail with laccase enzymes, but under the conditions tested, the co-incubation of laccases with LPMOs showed a substrate competition towards oxygen inhibiting the LPMO. In addition, we found that laccase treatment may cause other modifications to pure cellulose, rendering the material more recalcitrant for enzymatic saccharification. CONCLUSIONS: Laccase-mediated system was able to depolymerize lignin from pre-treated and native sugarcane bagasse and wheat straw, and the released phenolic molecules were able to donate electrons to LPMO enzymes boosting the overall enzymatic hydrolysis of cellulose. Likewise, other poly-phenol oxidase, we might have just started showing possible pros or cons in applying several oxidase enzymes for a simultaneous degradation of cellulose and lignin, and we found that the competition towards oxygen and their different consumption rates must be taken into account for any possible co-application.

Undefined cellulase formulations hinder scientific reproducibility.

In the shadow of a burgeoning biomass-to-fuels industry, biological conversion of lignocellulose to fermentable sugars in a cost-effective manner is key to the success of second-generation and advanced biofuel production. For the effective comparison of one cellulase preparation to another, cellulase assays are typically carried out with one or more engineered cellulase formulations or natural exoproteomes of known performance serving as positive controls. When these formulations have unknown composition, as is the case with several widely used commercial products, it becomes impossible to compare or reproduce work done today to work done in the future, where, for example, such preparations may not be available. Therefore, being a critical tenet of science publishing, experimental reproducibility is endangered by the continued use of these undisclosed products. We propose the introduction of standard procedures and materials to produce specific and reproducible cellulase formulations. These formulations are to serve as yardsticks to measure improvements and performance of new cellulase formulations.

Predicting the ethanol potential of wheat straw using near-infrared spectroscopy and chemometrics: The challenge of inherently intercorrelated response functions.

The combination of NIR spectroscopy and chemometrics is a powerful correlation method for predicting the chemical constituents in biological matrices, such as the glucose and xylose content of straw. However, difficulties arise when it comes to predicting enzymatic glucose and xylose release potential, which is matrix dependent. Further complications are caused by xylose and glucose release potential being highly intercorrelated. This study emphasizes the importance of understanding the causal relationship between the model and the constituent of interest. It investigates the possibility of using near-infrared spectroscopy to evaluate the ethanol potential of wheat straw by analyzing more than 1000 samples from different wheat varieties and growth conditions. During the calibration model development, the prime emphasis was to investigate the correlation structure between the two major quality traits for saccharification of wheat straw: glucose and xylose release. The large sample set enabled a versatile and robust calibration model to be developed, showing that the prediction model for xylose release is based on a causal relationship with the NIR spectral data. In contrast, the prediction of glucose release was found to be highly dependent on the intercorrelation with xylose release. If this correlation is broken, the model performance breaks down. A simple method was devised for avoiding this breakdown and can be applied to any large dataset for investigating the causality or lack of causality of a prediction model.

Biomass-water interactions correlate to recalcitrance and are intensified by pretreatment: An investigation of water constraint and retention in pretreated spruce using low field NMR and water retention value techniques.

The underlying mechanisms of the recalcitrance of biomass to enzymatic deconstruction are still not fully understood, and this hampers the development of biomass based fuels and chemicals. With water being necessary for most biological processes, it is suggested that interactions between water and biomass may be key to understanding and controlling biomass recalcitrance. This study investigates the correlation between biomass recalcitrance and the constraint and retention of water by the biomass, using SO2 pretreated spruce, a common feedstock for lignocellulosic biofuel production, as a substrate to evaluate this relationship. The water retention value (WRV) of the pretreated materials was measured, and water constraint was assessed using time domain Low Field Nuclear Magnetic Resonance (LFNMR) relaxometry. WRV increased with pretreatment severity, correlating to reduced recalcitrance, as measured by hydrolysis of cellulose using commercial enzyme preparations. Water constraint increased with pretreatment severity, suggesting that a higher level of biomass-water interaction is indicative of reduced recalcitrance in pretreated materials. Both WRV and water constraint increased significantly with reductions in particle size when pretreated materials were further milled, suggesting that particle size plays an important role in biomass water interactions. It is suggested that WRV may be a simple and effective method for measuring and comparing biomass recalcitrance. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:146-153, 2017.

The effect of harvest time, dry matter content and mechanical pretreatments on anaerobic digestion and enzymatic hydrolysis of miscanthus.

Miscanthus x giganteus was harvested as both green and mature biomass and the dry matter content of the driest harvest was artificially decreased by adding water in two subsamples, giving a total of five dry matter contents. All five biomass types were mechanically pretreated by roller-milling, extrusion or grinding and accumulated methane production and enzymatically-accessible sugars were measured. Accumulated methane production was studied using sigmoid curves that allowed comparison among the treatments of the rate of the methane production and ultimate methane yield. The green biomass gave the highest methane yield and highest levels of enzymatically-accessible cellulose. The driest biomass gave the best effect from extrusion but with the highest energy consumption, whereas roller-milling was most efficient on wet biomass. The addition of water to the last harvest improved the effect of roller-milling and equalled extrusion of the samples in efficiency.

The binding of cellulase variants to dislocations: a semi-quantitative analysis based on CLSM (confocal laser scanning microscopy) images.

Binding of enzymes to the substrate is the first step in enzymatic hydrolysis of lignocellulose, a key process within biorefining. During this process elongated plant cells such as fibers and tracheids have been found to break into segments at irregular cell wall regions known as dislocations or slip planes. Here we study whether cellulases bind to dislocations to a higher extent than to the surrounding cell wall. The binding of fluorescently labelled cellobiohydrolases and endoglucanases to filter paper fibers was investigated using confocal laser scanning microscopy and a ratiometric method was developed to assess and quantify the abundance of the binding of cellulases to dislocations as compared to the surrounding cell wall. Only Humicola insolens EGV was found to have stronger binding preference to dislocations than to the surrounding cell wall, while no difference in binding affinity was seen for any of the other cellulose variants included in the study (H. insolens EGV variants, Trichoderma reesei CBHI, CBHII and EGII). This result favours the hypothesis that fibers break at dislocations during the initial phase of hydrolysis mostly due to mechanical failure rather than as a result of faster degradation at these locations.