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

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

Precipitation of Trichoderma reesei commercial cellulase preparations under standard enzymatic hydrolysis conditions for lignocelluloses

Comparative studies between commercial Trichoderma reesei cellulase preparations show that, depending on the preparation and loading, total protein precipitation can be as high as 30 % under standard hydrolysis conditions used for lignocellulosic materials. ATR-IR and SDS-PAGE data verify precipitates are protein-based and contain key cell wall hydrolyzing enzymes. Precipitation increased considerably with incubation temperature; roughly 50–150 % increase from 40 to 50 °C and 800 % greater at 60 °C. All of the reported protein losses translated into significant, and often drastic, losses in activity on related 4-nitrophenyl substrates. In addition, supplementation with the non-ionic surfactant PEG 6,000 decreased precipitation up to 80 % in 24 h precipitation levels. Protein precipitation is potentially substantial during enzymatic hydrolysis of lignocelluloses and should be accounted for during lignocellulose conversion process design, particularly when enzyme recycling is considered.This work was supported by the project "Demonstrating Industrial scale second generation bioethaol production-Kalundborg Cellulosic Ethanol Plant" under the EU FP7 framework program and the project "Development of improved second generation (2G) bioethanol technology to prepare for commercialization under the Danish Energy Technology and Demonstration Programme (EUDP)

Celluclast and Cellic® CTec2:Saccharification/fermentation of wheat straw, solid–liquid partition and potential of enzyme recycling by alkaline washing

The hydrolysis/fermentation of wheat straw and the adsorption/desorption/deactivation of cellulases were studied using Cellic® CTec2 (Cellic) and Celluclast mixed with Novozyme 188. The distribution of enzymes cellobiohydrolase I (Cel7A), endoglucanase I (Cel7B) and -glucosidase of the two formulations between the residual substrate and supernatant during the course of enzymatic hydrolysis and fermentation was investigated. The potential of recyclability using alkaline wash was also studied. The efficiency of hydrolysis with an enzyme load of 10 FPU/g cellulose reached >98 % using Cellic® CTec2, while for Celluclast a conversion of 52 % and 81 %, was observed without and with -glucosidase supplementation, respectively. The decrease of Cellic® CTec2 activity observed along the process was related to deactivation of Cel7A rather than of Cel7B and -glucosidase. The adsorption/desorption profiles during hydrolysis/fermentation revealed that a large fraction of active enzymes remained adsorbed to the solid residue throughout the process. Surprisingly, this was the case of Cel7A and -glucosidase from Cellic, which remained adsorbed to the solid fraction along the entire process. Alkaline washing was used to recover the enzymes from the solid residue. This method allowed efficient recovery of Celluclast enzymes; however, this may be achieved only when minor amounts of cellulose remain present. Regarding the Cellic formulation, neither the presence of cellulose nor lignin restricted an efficient desorption of the enzymes at alkaline pH. This work shows that the recycling strategy must be customized for each particular formulation, since the enzymes found e.g.in Cellic and Celluclast bear quite different behaviour regarding the solid-liquid distribution, stability and cellulose and lignin affinity.P7 KACELLE (Kalundborg Cellulosic Ethanol, Grant no. 239379) project for supporting this work. FCT Strategic Project of UID/BIO/ 04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP- 01-0124-FEDER-027462) and the Project “BioEnv—Biotechnology and Bioengineering for a sustainable world”, REF. NORTE-07-0124- FEDER-000048, co-funded by the Programa Operacional Regional do Norte (ON.2—O Novo Norte), QREN, FEDER