Chapter 1
Chapter 1
Integrating high solids enzymatic hydrolysis and co-culture fermentation to improve ethanol production from deep eutectic solvent pretreated rice straw
One. Introduction
The growing concerns over climate change and the rapidly declining fossil fuel resources due to increasing global energy demand have intensified interest in sustainable and renewable biofuels. Ethanol stands out among available renewable liquid fuels because of its compatibility with existing energy infrastructure, without requiring major modifications to vehicles (e.g., E five to ten blends), pipelines, or storage facilities. However, industrial-scale production of ethanol from first-generation feedstocks (e.g., sweet corn, sugarcane, maize,
ABSTRACT
ABSTRACT
Rice straw is an abundant agricultural residue with significant potential for sustainable ethanol production. However, the major bottlenecks in converting rice straw into ethanol are biomass recalcitrance, high enzyme costs, and inefficient utilisation of mixed sugars. In this study, an integrated process combining microwave-assisted ChCl:glycerol pretreatment, optimised high-solids enzymatic hydrolysis at low enzyme loading, and co-culture fermentation was developed to enhance ethanol production from rice straw. High-solids enzymatic hydrolysis conditions were optimised by applying central composite design and response surface methodology. At a solids loading of nine point eight nine percent, the highest total sugar yield of ninety-four point nine two percent was obtained compared to a total sugar of forty-three point two nine percent at thirty percent solids loading. The optimal hydrolysis conditions of seventeen percent solids loading, three FPU per gram cellulose enzyme loading, and seventy-five hours hydrolysis time were predicted by the quadratic model and validated, resulting in seventy-five point seven percent total sugar yield. Fermentation of the resulting hydrolysates demonstrated that co-culture fermentation outperformed mono- and sequential cultures, achieving a maximum ethanol concentration of forty-one point one grams per liter, with corresponding yields and volumetric productivity of zero point four six grams per gram and one point seven one grams per liter per hour, respectively. In comparison, co-culture fermentation of hydrolysates derived from one percent H two SO four pretreatment resulted in lower ethanol yield (zero point three five grams per gram) and productivity (zero point six seven grams per liter per hour). Thus, the ability to attain high ethanol titre and yield at reduced enzyme dosage and high solids loading highlights the effectiveness of microwave-assisted deep eutectic solvent pretreatment and co-culture fermentation using Saccharomyces cerevisiae and Candida tropicalis. This integrated strategy provides an innovative approach to advancing lignocellulosic bioethanol production from agricultural residues.
soybeans, etc.) could negatively impact food security and land use. Thus, there is a need to develop efficient second-generation ethanol processes based on non-food lignocellulosic biomass (agricultural residues, wood, and energy crops). Rice straw is one of the most abundant agricultural residues, with an estimated annual global production of around seven hundred thirty-one million tons, and Asia alone accounts for about fifty percent of that output. Rice straw is composed of thirty-two to thirty-nine percent cellulose, twenty to thirty-six percent hemicellulose, fourteen to twenty-two percent lignin and ten to seventeen percent ash. On the other hand, the presence of silica in lignocellulosic biomass has been reported to decreased conversion of carbohydrates into monomeric sugars during enzymatic hydrolysis.
Despite its high cellulose and hemicellulose content, rice straw remains vastly underutilised and is often improperly disposed of or burned in open fields, a practise that has been banned in major agricultural regions due to its severe environmental consequences. These characteristics make rice straw an attractive and renewable feedstock for ethanol production. However, pretreatment is a prerequisite for deconstructing the recalcitrant lignocellulosic structure of rice straw enabling enhanced enzymatic hydrolysis and release of fermentable sugars. Conventional pretreatment methods typically require high energy inputs, substantial chemical consumption, and the formation of inhibitors, which negatively affect downstream processing. On the other hand, pretreatment methods using deep eutectic solvents offer a better alternative due to their low toxicity, biodegradability, and cost-effectiveness. Microwave assisted-ChCl: glycerol pretreatment has been applied to rice straw leading to high delignification with minimal loss of cellulose/hemicellulose components. The use of microwave heating enables rapid and uniform heating, thereby reducing pretreatment time and improving delignification efficiency. However, to fully realise the industrial-scale potential of microwave pretreatment, future research is required to optimise pretreatment process parameters, improve unit production, and reduce production costs.
However, despite advances in pretreatment technologies, enzymatic hydrolysis represents a major cost bottleneck in ethanol production from lignocellulosic biomass, primarily due to the high enzyme dosages required to achieve acceptable sugar yields for industrial-scale fermentation. Another key challenge associated with ethanol production from rice straw is the efficient fermentation of mixed sugars resulting from enzymatic hydrolysis, as the hydrolysates contain both hexoses and pentoses. Thus, the application of high-solids enzymatic hydrolysis is essential to increase sugar concentrations and improve process economics. This also presents additional challenges, such as poor mass transfer, high viscosity, and end-product inhibition. Therefore, integrated processes that synergistically combine effective pretreatment with tailored enzymatic hydrolysis conditions can significantly enhance cellulose conversion efficiency while reducing enzyme costs. To this end, design of experiments using Central Composite Design and Response Surface Methodology is a valuable tool widely employed to optimise process conditions and achieve high product yields.
Saccharomyces cerevisiae is the most widely used yeast for large-scale ethanol production because of its high ethanol productivity under optimal conditions, however, it cannot utilise pentose sugars. In contrast, Candida tropicalis can utilise pentoses such as xylose, although it generally yields lower ethanol when used alone. Co-culture fermentation has emerged as a promising strategy to enable simultaneous utilisation of mixed sugars, improve ethanol yield, and enhance overall process efficiency. Despite the potential of co-culture fermentation, the performance of S. cerevisiae-C. tropicalis co-culture systems using enzymatic hydrolysates derived from deep eutectic solvent-pretreated rice straw remains poorly understood. In this study, an integrated process is developed to improve bioethanol production from rice straw by optimising high-solids enzymatic hydrolysis at low enzyme loading followed by co-culture fermentation using S. cerevisiae and C. tropicalis. Efficiencies of mono-culture, co-culture, and sequential culture fermentation of hydrolysates of ChCl:glycerol pretreated rice straw were evaluated. In addition, hydrolysates obtained from rice straw pretreated by a conventional method (one percent H two SO four, one hundred twenty-one degrees Celsius, thirty minutes, autoclave) were treated to co-culture fermentation using S. cerevisiae and C. tropicalis and benchmarked against ChCl:glycerol pretreated rice straw. Thus, the findings from this study are expected to provide insights into cost-effective process integration strategies for advancing sustainable lignocellulosic bioethanol production.