Cellulase stability, adsorption/desorption profiles and recycling during successive cycles of hydrolysis and fermentation of wheat straw

The potential of enzymes recycling after hydrolysis and fermentation of wheat straw under a variety of conditions was investigated, monitoring the activity of the enzymes in the solid and liquid fractions, using low molecular weight substrates. A significant amount of active enzymes could be recovered by recycling the liquid phase. In the early stage of the process, enzyme adsorb to the substrate, then gradually returning to the solution as the saccharification proceeds. At 50 °C, normally regarded as an acceptable operational temperature for saccharification, the enzymes (Celluclast) significantly undergo thermal deactivation. The hydrolysis yield and enzyme recycling efficiency in consecutive recycling rounds can be increased by using high enzyme loadings and moderate temperatures. Indeed, the amount of enzymes in the liquid phase increased with its thermostability and hydrolytic efficiency. This study contributes towards developing effective enzymes recycling strategies and helping to reduce the enzyme costs on bioethanol production.The authors acknowledge funding through FP7 KACELLE (Kalundborg Cellulosic Ethanol) project for supporting this work. We also thank Dra. Lucilia Domingues for supplying the yeast Saccharomyces cerevisiae CEN PK 113 wild type

Biomass saccharification : development of strategies for enzyme recycling

In the present work the recycling of free enzymes after prehydrolysis and simultaneous saccharification and fermentation of pretreated wheat straw under a variety of conditions was investigated. It was found that a significant amount of active cellulase and glucosidase could be recovered by recycling the free cellulases the amount of free enzymes increase with its thermostability and hydrolytic efficiency. At 50° C normally regarded as an acceptable operational temperature for saccharification processes the enzymes significantly loses its activity and this thermal deactivation was independent of initial enzyme concentration used. The degree of cellulose conversion through a series of consecutive hydrolytic/recycling rounds dropped more substantially when low concentrations of cellulases were used. The hydrolysis yield and enzyme recycling efficiency in consecutive recycling rounds can be increased by using high enzyme loadings and moderate temperatures. Furthermore the recovery of cellulases from lignin lignocellulosic hydrolysates and cellulose by alkaline wash at pH 9 and 10 has been analysed

Recycling of cellulases in lignocellulosic hydrolysates using alkaline elution

The recovery of cellulases from lignin, lignocellulosic hydrolysates and cellulose by alkaline washes at pH 9 and 10 was examined. The effect of the pH on the structural stability of purified Cel7A was analyzed by circular dichroism. Purified Cel7A showed conformational changes at pH 9 and 10 that were reversible at pH 4.8. Temperature influenced the enzymatic hydrolysis of wheat straw and may be critical for the efficiency of cellulase recycling from wheat straw hydrolysates. Operation at moderate temperatures (37 °C) resulted in a rate of saccharification 19% higher than that obtained at 50° C, improving cellulase recycling by 49%. Over 60% of the enzyme activity on the synthetic substrate 4-methylumbelliferyl-β-d-cellobioside (MUC) may be recovered by using a simple alkaline wash. This is thus a promising strategy for enzyme recycling that is simple to implement at industrial scale, economical and effective.The authors acknowledge funding through FP7 KACELLE (Kalundborg Cellulosic Ethanol) project for supporting his work. We also thank Dr. Mai Ostergaard Haven for critical reviewing of the manuscript and Dr. Jane Lindedam for supplying the lignin

Enzymatic cellulose oxidation is linked to lignin by long-range electron transfer

Enzymatic oxidation of cell wall polysaccharides by lytic polysaccharide monooxygenases (LPMOs) plays a pivotal role in the degradation of plant biomass. While experiments have shown that LPMOs are copper dependent enzymes requiring an electron donor, the mechanism and origin of the electron supply in biological systems are only partly understood. We show here that insoluble high molecular weight lignin functions as a reservoir of electrons facilitating LPMO activity. The electrons are donated to the enzyme by long-range electron transfer involving soluble low molecular weight lignins present in plant cell walls. Electron transfer was confirmed by electron paramagnetic resonance spectroscopy showing that LPMO activity on cellulose changes the level of unpaired electrons in the lignin. The discovery of a long-range electron transfer mechanism links the biodegradation of cellulose and lignin and sheds new light on how oxidative enzymes present in plant degraders may act in concert.info:eu-repo/semantics/publishe

Structure and enzymatic accessibility of leaf and stem from wheat straw before and after hydrothermal pretreatment

BACKGROUND: Biomass recalcitrance is affected by a number of chemical, physical and biological factors. In this study we looked into the differences in recalcitrance between two major anatomical fractions of wheat straw biomass, leaf and stem. A set of twenty-one wheat cultivars was fractionated and illustrated the substantial variation in leaf-to-stem ratio between cultivars. The two fractions were compared in terms of chemical composition, enzymatic convertibility, cellulose crystallinity and glucan accessibility. The use of water as a probe for assessing glucan accessibility was explored using low field nuclear magnetic resonance and infrared spectroscopy in combination with hydrogen-deuterium exchange. RESULTS: Leaves were clearly more degradable by lignocellulolytic enzymes than stems, and it was demonstrated that xylose removal was more linked to glucose yield for stems than for leaves. Comparing the locations of water in leaf and stem by low field NMR and FT-IR revealed that the glucan hydroxyl groups in leaves were more accessible to water than glucan hydroxyl groups in stems. No difference in crystallinity between leaf and stem was observed using wide angle x-ray diffraction. Hydrothermal pretreatment increased the accessibility towards water in stems but not in leaves. The results in this study indicate a correlation between the accessibility of glucan to water and to enzymes. CONCLUSIONS: Enzymatic degradability of wheat straw anatomical fractions can be indicated by the accessibility of the hydroxyl groups to water. This suggests that water may be used to assess glucan accessibility in biomass samples

Light-driven oxidation of polysaccharides by photosynthetic pigments and a metalloenzyme

Oxidative processes are essential for the degradation of plant biomass. A class of powerful and widely distributed oxidative enzymes, the lytic polysaccharide monooxygenases (LPMOs), oxidize the most recalcitrant polysaccharides and require extracellular electron donors. Here we investigated the effect of using excited photosynthetic pigments as electron donors. LPMOs combined with pigments and reducing agents were exposed to light, which resulted in a never before seen 100-fold increase in catalytic activity. In addition, LPMO substrate specificity was broadened to include both cellulose and hemicellulose. LPMO enzymes and pigment derivatives common in the environment of plant-degrading organisms thus form a highly reactive and stable light-driven system increasing the turnover rate and versatility of LPMOs. This light-driven system may find applications in biotechnology and chemical processing

Continuous recycling of enzymes during production of lignocellulosic bioethanol in demonstration scale

Recycling of enzymes in production of lignocellulosic bioethanol has been tried for more than 30 years. So far, the successes have been few and the experiments have been carried out at conditions far from those in an industrially feasible process. Here we have tested continuous enzyme recycling at demonstration scale using industrial process conditions (high dry matter content and low enzyme dosage) for a period of eight days. The experiment was performed at the Inbicon demonstration plant (Kalundborg, Denmark) capable of converting four tonnes of wheat straw per hour. 20% of the fermentation broth was recycled to the hydrolysis reactor while enzyme dosage was reduced by 5%. The results demonstrate that recycling enzymes by this method can reduce overall enzyme consumption and may also increase the ethanol concentrations in the fermentation broth. Our results further show that recycling fermentation broth also opens up the possibility of lowering the dry matter content in hydrolysis and fermentation while still maintaining high ethanol concentrations.M.O. Haven wishes to thank the Danish Agency for Science, Technology, and Innovation, grant no. 09-053694 for financial support. The other authors wish to thank the European Seventh Framework Program, grant no. 239379 (the KACELLE project) for financial support