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Understanding the Relative Importance of the Effects of Soluble and Insoluble Inhibitors on the Enzyme Digestibility of Steam Pretreated Radiata Pine

Lignocellulosic biomass use is one of the keys to growth and development of future biofuels and biochemicals. Amongst the lignocellulosic feedstocks, softwoods are particularly attractive as they have the potential to produce high sugar yields and are prevalent in many forests globally, currently they make up 95% of New Zealand’s forest estate. One of the most promising approaches to the production of lignocellulosic biofuels is the sugar pathway. This pathway involves using enzymes to hydrolyse the carbohydrate polymers in the biomass to monomeric sugars, and then converting these sugars to ethanol. High sugar yields during enzymatic hydrolysis requires an effective pretreatment to disrupt and/or remove some of the lignin and hemicelluloses, so that the cellulose is more accessible to enzymes. Softwoods, such as Pinus radiata, are amongst the most recalcitrant substrates towards enzymatic hydrolysis, typically, they require more severe pretreatment conditions and higher enzyme doses than hardwood or agricultural residues. Steam pretreatment is one simple and cost-effective pretreatment. Steam pretreatment involves heating the biomass in steam or water (150ºC to 250ºC), often in the presence of added acid catalysts. One disadvantage of steam pretreatment is that it can produce compounds that inhibit the subsequent enzymatic hydrolysis. During enzymatic hydrolysis, these inhibitors, particularly lignin, lower the digestibility of the cellulose by non-productively binding the hydrolysis enzymes. This means that high enzyme doses are required increasing the conversion costs. How these inhibitors are formed, how they work and what types of inhibitors are most deleterious during enzymatic hydrolysis is not well understood. The main objective of this thesis was to understand the relative roles of soluble and insoluble fibre components as inhibitors of enzyme hydrolysis of steam pretreated P. radiata as a function of pretreatment severity. Samples: P. radiata sawdust was pretreated using steam explosion under six different pretreatment conditions and separated to produce both water-soluble (filtrate) and insoluble (substrate) samples. These samples were used for the rest of the research. Insoluble inhibition: The digestibility of the insoluble substrates was examined using a commercial enzyme cocktail (Novozymes Cellic® CTec2). Results showed the digestibility of these substrates increased with increasing pretreatment severity due to increases in accessibility. However, when the substrates were tested after ball-milling to a common cellulose accessibility, as determined by Simons’ stain measurements, the digestibility decreased with increasing pretreatment severity. This showed that while increasing pretreatment severity led to greater enzyme inhibition, the inhibition was more than compensated for by increases in the accessibility. As part of this work, modifications were made to the Simons’ stain method to ensure a robust and reliable method was available for measuring cellulose accessibility. Soluble inhibition: The inhibitory effects of filtrates were investigated by comparing changes in the digestibility of both bleached kraft pulp and the insoluble steam pretreated substrates in the presence and absence of filtrates. The results showed that the inhibitory effects of the filtrates were minimal or non-existent under most conditions. It was therefore concluded that the insoluble components of steam pretreated P. radiata were more inhibitory than soluble components. With the commercial enzyme cocktail CTec2, adding the filtrates back during hydrolysis led to small enhancements in digestibility. The results of subsequent experiments were consistent with the enhancements being due to components in the filtrates acting as reductants for the oxidative cellulase enzymes present in this cocktail. Inhibition by lignin: Lignins were isolated from three insoluble substrates of differing pretreatment severity and their inhibitory effects on the digestibility of bleached kraft pulp were determined. Results showed: (i) that the inhibitory effects of added lignins increased with the amount of lignin added; (ii) that more severe pretreatments led to more inhibitory lignins; (iii) that adding the surfactant polyethylene glycol could overcome the inhibitory effects of these lignins. Additionally, the more severe pretreatments produced lignins with greater condensation and more phenolic groups, these in turn negatively correlated to digestibility, indicating the structural composition of lignin plays a role in the extent to which lignin is inhibitory.
Type of thesis
MacAskill, J. J. (2018). Understanding the Relative Importance of the Effects of Soluble and Insoluble Inhibitors on the Enzyme Digestibility of Steam Pretreated Radiata Pine (Thesis, Doctor of Philosophy (PhD)). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/11670
The University of Waikato
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