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

Highlights

• Cellic is more efficient catalyst than Celluclast, but is more sensitive to deactivation.

• Efficient hydrolysis leads to improved recovery of soluble cellulases.

• Alkaline treatment is an efficient method to remove enzymes lignin-adsorbed.

• Enzyme recycling strategies must be customized for each enzyme formulation.

Abstract

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 β-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 β-glucosidase supplementation, respectively. The decrease of Cellic^® CTec2 activity observed along the process was related to deactivation of Cel7A rather than of Cel7B and β-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 β-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.

Introduction

Ethanol is a renewable alternative to petroleum-derived fuels. It can be produced from lignocellulosic materials in various ways, overall consisting of the pretreatment, hydrolysis of cellulose and hemicellulose to monomeric sugars, fermentation and product recovery. Although a significant effort has been made to improve the pretreatment step, an efficient enzymatic hydrolysis still requires high enzyme loadings which contribute to the high costs of the biomass-derived ethanol [1], [2]. Researchers attempted to reduce the cost of enzymatic hydrolysis [3], [4] by (i) minimizing the costs of enzyme production, (ii) increasing the enzymes specific activity, (iii) reducing the required cellulase loading by pretreating the biomass [5], (iv) or recycling the enzymes for multiple rounds of hydrolysis [2], [4], [6], [7], [8], [9], [10], [11], [12], [13].

Cellulase formulations contain a broad spectrum of enzymes that work synergistically to convert cellulose to simpler sugars, namely endoglucanases, cellobiohydrolases, and β-glucosidase. The adsorption of cellulases onto the substrate is the first step in cellulose hydrolysis. Henceforward, a portion of the adsorbed cellulases is gradually release into the supernatant [1], [11], [12], [14]. At the end of the process, for the cellulases present in the supernatant, the simplest recovery method is re-adsorption onto fresh substrate [15] or collection by ultrafiltration [11], [16]. More challenging is the recovery of the portion of cellulases adsorbed on the residual substrate, which accounts for 30–90% of the total enzyme load, depending on the process configuration [14].

Cellulase adsorption onto cellulose is reportedly reversible and often described by the Langmuir isotherm. However, according to various authors, adsorption onto lignin-rich substrates is not strictly reversible [4], [17]. The addition of a desorbent is a traditional approach to detach the enzymes from a lignocellulosic residue [7]. A range of chemicals can be used for that purpose [12], such as surfactants, urea, alkali, glycerol [18], [7], [19], and polyethylene glycol [20]. However, most are not effective enough and additionally some of them deactivate the enzymes. Remarkably, alkaline solutions proved to be among the most efficient ones [4], [7], [9], [10], [21]. In addition, adjusting the pH is operationally easy and cost-effective.

The successful development of recycling enzyme strategies is generally recognized as highly promising and desirable. However, it requires enzymes to be stable enough to endure several cycles of recycling. Stability critically depend on the temperature used in the process [10], [11], [22], but also on the pH, shear stress (from agitators, flow and pumps) and on the contact with the air–liquid interface [23], [24]. Though recycling cellulases can be regarded nowadays as technically feasible, the process must be adapted to each particular enzyme, substrate and operational conditions. We have demonstrated in previous work [10], [11] that recycling Celluclast can be easily achieved as long as a temperature below 50 °C is used and a complete cellulose conversion is achieved. This is possible because after thorough cellulose conversion the enzymes can be recovered from the liquid phase. In this manuscript, we demonstrate that the same is not possible for the highly effective Cellic enzymes, which remain bound to the final residue even after complete hydrolysis, thus making more challenging the recycling process.