Effects of enzyme loading and β-glucosidase supplementation on enzymatic hydrolysis of switchgrass processed by leading pretreatment technologies

Abstract

The objective of this work is to investigate the effects of cellulase loading and β-glucosidase supplementation on enzymatic hydrolysis of pretreated Dacotah switchgrass. To assess the difference among various pretreatment methods, the profiles of sugars and intermediates were determined for differently treated substrates. For all pretreatments, 72 h glucan/xylan digestibilities increased sharply with enzyme loading up to 25 mg protein/g-glucan, after which the response varied depending on the pretreatment method. For a fixed level of enzyme loading, dilute sulfuric acid (DA), SO₂, and Lime pretreatments exhibited higher digestibility than the soaking in aqueous ammonia (SAA) and ammonia fiber expansion (AFEX). Supplementation of Novozyme-188 to Spezyme-CP improved the 72 h glucan digestibility only for the SAA treated samples. The effect of β-glucosidase supplementation was discernible only at the early phase of hydrolysis where accumulation of cellobiose and oligomers is significant. Addition of β-glucosidase increased the xylan digestibility of alkaline treated samples due to the β-xylosidase activity present in Novozyme-188.

Introduction

Lignocellulosic biomass is currently considered the most promising long-term feedstock for production of bioethanol. The recalcitrance of the feedstock is the major hurdle in the bioconversion process. In the plant cell structure, cellulose fibrils are embedded in a matrix of lignin and hemicellulose. This forms a chemical and structural barrier for enzymatic degradation of cell wall sugars and subsequent microbial fermentation to ethanol (Lynd et al., 2002, Kumar and Wyman, 2009). For cellulase enzymes to be able to effectively access the cellulose, the cell wall structure needs to be disrupted. Various pretreatment methods are employed by in order to remove these barriers. The pretreatment mode of action varies depending upon the method applied. Pretreatment often results in a change of composition, alteration of physical properties including cellulose crystallinity. The CAFI team has put forward a collaborative effort to examine some of the promising pretreatment technologies including: soaking in aqueous ammonia (SAA), ammonia fiber expansion (AFEX), calcium hydroxide (Lime), liquid hot water (LHW), dilute sulfuric acid (DA), and SO₂ (Wyman et al., 2005b).

The goal of this research as part of the CAFI project is to assess the enzyme requirement for various pretreatments and seek ways to reduce the enzyme loading. In many cases the required enzyme loading is too high to be economically feasible (Mosier et al., 2005, Kumar and Wyman, 2008a). Since the enzyme is one of the major costs involved in producing cellulosic ethanol, pretreatment advances are still needed to reduce the overall production costs (Wyman et al., 2005a). A typical cellulase enzyme mixture consists of many different enzymes that function synergistically to break down the cell wall polysaccharides. One of the key enzymes in the cellulase system is exo-glucanase which produces cellobiose as an intermediate product (Zhao et al., 2004). It is well known that cellobiose inhibits the action of exo-glucanase (Xiao et al., 2004). If the cellulase complex does not have sufficient β-glucosidase activity, external supplementation is necessary to increase the enzyme efficiency (Lynd et al., 2002). Enzymatic hydrolysis of cellulose and hemicellulose also produces glucose and xylose oligomers as reaction intermediates. Although inhibition of cellulase activity by monomeric sugars and cellobiose is well documented (Kumar and Wyman, 2008a, Quing and Wyman, 2011), limited information is available with regard on the effect of oligomers. More detailed investigations are required to understand how the enzyme loading is influenced by pretreatment method and the type of substrate. The technical objective of this work is to investigate the effects of cellulase loading and β-glucosidase supplementation on production of monomeric and oligomeric sugars. Efforts were put forward to observe data over the entire duration of hydrolysis and to seek explanations for the difference in hydrolysis behavior, if any, among the substrates generated by various CAFI methods.