The complete enzymatic saccharification of agarose and its application to simultaneous saccharification and fermentation of agarose for ethanol production

Abstract

A sugar platform equipped with acetic acid, multiple agarases and neoagarobiose hydrolase (NABH) converted recalcitrant agar polysaccharide into monosugars, which was evaluated by simultaneous saccharification and fermentation (SSF). The sugar platform was divided into chemical liquefaction and enzymatic saccharification. The chemical liquefaction was carried out in mild conditions (using a dilute acetic acid at 80 °C for 1–6 h) to avoid the production of fermentation inhibitors and hence the highest degree of liquefaction of 95.6% (w/w) was obtained. We mimicked the natural agarolytic pathway using three microbial agarases (Aga16B, Aga50D and DagA) and NABH, and the enzyme system converted 79.1% of agarose to monosugars. The chemical liquefaction and SSF of 30 g/l agarose resulted in 4.4 g/l ethanol concentration and 49.3% of the theoretical ethanol yield to d-galactose. This is the first report on the complete enzymatic conversion of agarose into its monosugars and the SSF of agarose into ethanol.

Introduction

Bioethanol as a sustainable biofuel has attracted worldwide attention because of the oil crisis and climate change (Sheehan and Himmel, 1999). Production of bioethanol based on crops such as corn or sugar cane has been criticized for driving an increase in world food prices and land overuse (Hahn-Hägerdal et al., 2006). Lignocellulosic bioethanol has been suggested to overcome the limitations of crop-based bioethanol (Alvira et al., 2010). Bioethanol from lignocellulosic biomass also has shortcomings. Even if lignocellulosic biomass can be derived from energy crops, it may not compensate for the opportunity costs caused by sharing land with food crops (Singh et al., 2011). In addition, only a few countries have sufficient arable land capable of cultivating lignocellulosic biomass for commercial bioethanol production.

In contrast to land plant biomass, marine biomass has emerged as the next-generation biomass holding several benefits. Marine biomass (i) has a high productivity per unit area, (ii) does not compete with food crops, (iii) can grow in undemanding cultivation conditions (e.g. anywhere under the proper sunlight or even improper environment in agriculture) and (iv) often possesses high carbohydrate content. Specifically, red algal biomass has appeared as a promising alternative for ethanol production (John et al., 2011).

The major constituent of red algal biomass is a recalcitrant agar polysaccharide that is mainly composed of agarose (Renn, 1997). Agarose is a linear polysaccharide, which has alternative α-1,3- and β-1,4 linkage groups between two monosugars such as d-galactose and 3,6-anhydro-l-galactose (AHG) (Araki, 1956). In the practice of converting red algal biomass into bioenergy or valuable biochemicals, the critical step is the decomposition process of agarose to give fermentable monosugars. The sugar platform converting biomass into fermentable monosugars can be achieved by chemical liquefaction and enzymatic saccharification of the insoluble polymer (e.g. corn starch) (Naik et al., 2010). Despite that chemical and enzymatic methods have been adopted in the sugar platform for lignocellulosic biomass using liquefaction and saccharification processes (Jung et al., 2011, Ko et al., 2009, Kumar et al., 2009), no standard processes for agar polysaccharide, the major constituent of red algal biomass, has been established yet.

In the enzymatic decomposition of agarose, microbial agarolytic enzymes can be utilized by the agarolytic pathway using β-agarases (Michel et al., 2006). As shown in Fig. 1, β-agarases decompose agarose by breaking the β-1,4 glycosidic linkages. The end product of the β-agarase action is converged to a disaccharidic neoagarobiose (l-3,6-anhydrogalactosyl-α-1,3-d-galactose) that should be further hydrolyzed by a key enzyme of neoagarobiose hydrolase (NABH) into two monosugars such as d-galactose and AHG (Ha et al., 2011, Lee et al., 2009). Many agarases including NABH involved in the natural agarolytic pathway have been characterized but only a few agarases are commercially available. Those known agarases never have been applied for the saccharification process of agar polysaccharides. In microbial polysaccharide hydrolysis, enzymatic depolymerization often requires several enzymes in a multiple modular process having a steady and rate-determining step (Warren, 1996).The enzymatic hydrolysis of agarose will not be an efficient process unless proper chemical liquefaction is applied to overcome the enzymatic rate-determining step. Above all, any enzymatic process by agarases will not produce monosugars without NABH.

Compared with enzymatic hydrolysis of agar polysaccharide, chemical decomposition employs acidic catalysts to attack random or specific glycosidic linkages of agarose and then to release agarooligosaccharide (Kazłowski et al., 2008). Such chemical decomposition is relatively simple because it uses only heat and an acidic catalyst. However, the chemical process for agarose has critical disadvantages as in the process for lignocellulose. Because the reaction extent of the chemical catalysis is hard to be controlled, over-decomposition often releases fermentation inhibitors such as furfurals that give rise to problems during ethanol production (Ando et al., 1986). The process is also prone to cause environmental pollution (Olsson and Hahn-Hägerdal, 1996).

In order to resolve these problems in the chemical and enzymatic processes described above, we attempted to develop an efficient sugar platform for agarose using a mild chemical liquefaction followed by enzymatic saccharifiaction mimicking a natural process. The sugar platform combining chemical and enzymatic processes is well known for lignocellulosic biomass (Hamelinck et al., 2005). In the processing of lignocellulosic biomass, a chemical pretreatment using strong acid such as sulfuric acid alters the structure of biomass to help enzymatic attack on the substrates. However, because red algal biomass is mainly composed of agar polysaccharides, strong acid treatment gives rise to considerable amounts of fermentation inhibitor by over-decomposition of sugar components (Delgenes et al., 1996). In agarose, AHG and d-galactose are the monomeric unit sugars having a 1:1 ratio but AHG is acid-labile and quickly decomposes into substances inhibitory to fermentation (Yang et al., 2009). Consequently, we determined to use a weak acid such as acetic acid and relatively mild reaction conditions for the maximum production of monosugars (Roleda et al., 1997). Various sizes of oligosaccharides produced in the mild chemical liquefaction can be further hydrolyzed by an environment-friendly enzymatic process.

Here, we suggest the sugar platform for production of fermentable sugars from agarose by chemical and enzymatic processes. The platform includes chemical liquefaction by an acidic catalyst in a mild condition and the enzymatic saccharification of the pretreated agarose into monomeric sugars using three different types of agarases and NABH. The effectiveness of chemical liquefaction was evaluated by following enzymatic hydrolysis yield. In order to validate the sugar platform, we conducted the simultaneous saccharification and fermentation (SSF) for bioethanol production using the unliquefied and weak acid-pretreated agarose.