Surfactant-mediated hydrothermal pretreatment of Ryegrass followed by enzymatic saccharification for polyhydroxyalkanoate production

Highlights

• Ryegrass biomass, a non-food crop, due to high carbohydrate (62%) contents is good source of extracting sugars for bio refinery purpose.

• Surfactant pretreatment is an attractive method for improved saccharification of ryegrass biomass.

• High PHA accumulation in Pseudomonas strain using sugars obtained after hydrolysis of pre-treated biomass.

Abstract

Lignocellulosic biomass from non-food crops is an attractive renewable source of sugars. These sugars can be used as feedstock for the microbial production of different industrially relevant compounds. Delignification of lignocellulosic biomass is a major challenge in sugar extraction. In the present study, ryegrass biomass (non-food), which is high in carbohydrates, was pre-treated with the anionic surfactant sodium dodecyl sulphate (SDS) for effective delignification. Enzymatic hydrolysis of the pre-treated biomass led to the extraction of high sugar levels compared with the raw biomass. Furthermore, these sugars were used for the production of poly-hydroxyalkanoates (PHA), which is an alternative to synthetic polymers. Ryegrass treated with 1% SDS solution at 180 °C showed the maximum delignification. This pre-treated biomass was characterised by TGA, FTIR and SEM and showed successful lignin removal. Enzymatic hydrolysis of the resultant pre-treated biomass showed 87% conversion into sugars and was used as a carbon source for the production of medium chain length (mcl) PHAs using two Pseudomonas strains. Both strains accumulated approximately 30% of their cell dry weight in mcl-PHAs. Hence, this study shows for the first time that surfactant pretreatment is effective for lignin solubilisation; this process increased the biomass surface area and improved enzyme actions, resulting in higher sugar extraction.

Introduction

The global production of lignocellulosic plant biomass, which includes forests, crop residues, grasses, municipal and woods, is 200 × 10⁹ tons per year, though only 8–20 tons per year is utilised (Ragauskas et al., 2006). Lignocellulosic biomass is an organic material derived from a biological origin and composed of cellulose (beta-1,4-glucan fibres), hemicellulose (non-cellulosic polysaccharides such as xylans, mannans and glucans) and lignin (complex poly-phenolic structure). Cellulose and hemicellulose are polymers that can be used as carbon sources for microbial growth. Grass biomass is an attractive feedstock, as it is globally abundant throughout the year and has a low maintenance cost (Kataria et al., 2009). In Ireland, the high abundance of ryegrass allows it to be a promising lignocellulose feedstock for biofuel and biochemical production (Sharma et al., 2012). Ryegrass is the most common bunch type of grass that can survive for several growing seasons and has a 9655.6 kg DM/ha forage production (Oliveira and Gonzalez, 1991). Lignin is one of the most important constituents of this biomass. It is present in the plant cell wall and difficult for microorganisms to use during fermentation, as it is resistant to degradation. The conversion of lignocellulosic biomass to biochemicals is a multi-step process that includes pretreatment, hydrolysis and fermentation. Pretreatment is one of the most important and critical processes in bio-refineries for the production of biofuels and bio-based chemicals (Sun and Che, 2002, Kataria et al., 2013). Pretreatment helps to remove lignin and promote the release of cellulose, which promotes effective hydrolysis by cellulolytic enzymes and makes bio-refinery processes more economical. Several pretreatment methods, including acid, alkali, wet oxidation, ammonia fibre explosion, ozonolysis, microwave-induced hydrothermal, and ultrasonic methods, have been employed to destroy lignin (Sun and Che, 2002, Yan et al., 2017, Li et al., 2016). The major challenge with biomass pretreatment is that the decomposition of lignin generates phenolic compounds. Furthermore, the saccharification step, which involves the enzymatic hydrolysis of the pre-treated biomass, can be limited due to the presence of lignin. Lignin absorb a large portion of cellulase and make it unavailable for the cellulosic hydrolysis that results from the extended enzymatic reaction time to achieve a high liberation of sugars (Kumar and Whyman 2009). During pretreatment and enzymatic hydrolysis, surfactants enhance the hydrolysis of lignocellulosic biomass (Eriksson et al., 2002, Lin et al., 2016). When a surfactant is added during pretreatment, it may cause the lignin to be emulsified in the liquid phase and prevent the re-deposition of lignin on the biomass (Qing et al., 2010). Moreover, surfactants have both hydrophobic as well as hydrophilic properties, making them good candidates for lignin removal. Polyhydroxyalkanoates (PHAs) are biodegradable biopolymers with the same properties as conventional synthetic polymers used for plastics. PHAs are intracellularly biosynthesised by different bacterial strains including Pseudomonas sp., Bacillus sp. and Methylobacterium sp (Reddy et al., 2003, Suriyamongkol et al., 2007). The properties of PHA change depending on the monomer composition of the polymer, polymer chain length and length of the side chains. PHAs can be composed of short chain lengths (scl) with 3–5 carbon atoms or medium chain lengths (mcl) with 6–14 carbon atom hydroxyalkanoic acid monomers. The chain length depends on the microbial strain used for synthesis, the carbon source used as the substrate, and the bacterial culture conditions). PHAs are industrially important polymers with various applications including being used in hygiene products, adhesives, films, flavours in foods, dairy products, fabrics and materials and several medical applications (Keshavarz and Roy, 2010). We have already demonstrated that pretreatment and delignification are important for the production of highly digestible biomass and biopolymers (Davis et al., 2013). To the best of our knowledge, few studies have reported on SDS (sodium dodecyl sulphate) pretreatment of ryegrass and its conversion to PHAs. In the present study, SDS, an anionic surfactant, was used for ryegrass pretreatment, and the effectiveness of the pretreatment was determined in terms of the sugars released from the pre-treated biomass by enzymatic hydrolysis. The resulting sugars were evaluated for the production of mcl-PHA using two strains of Pseudomonas.