(111b) Advancements in Biochemical Conversion of Different Lignocellulosic Feedstocks for Bioethanol Production: A Pilot Scale Approach

The conversion of lignocellulosic biomass into bioethanol and its blending with gasoline for the transportation sector can reduce the dependency on fossil fuels. However, several factors are affecting the commercialization of lignocellulosic biorefinery, including the enzyme costs, incomplete conversion of polymeric carbohydrates into monomeric sugars, and the formation of fermentative inhibitors. Fermentative inhibitors deter the fermentation efficiency of hydrolysates which leads to reduced yield and productivity [1]. Generally, fermentative inhibitors can be classified into three categories: process-derived, inherent, and supplemented. Process-derived fermentative inhibitors include Furfural, 5-Hydroxymethylfurfural (5-HMF), levulinic acid, and formic acid. These are formed during the pretreatment of lignocellulosic biomass under acidic conditions with moderate to high temperatures [2]. Furfural is a decomposed product of xylose and arabinose, and 5-HMF is a decomposed product of glucose and fructose. Further decomposition of 5-HMF forms levulinic acid and formic acid, whereas furfural forms only formic acid. Apart from these, acetic acid, glucuronic acid, ferulic acid, vanillic acid, coumaric acid, benzoic acid, hydroxybenzoic acid, and syringaldehyde are the inherent inhibitors derived during the hydrolysis of lignocellulosic biomass [3–5]. Acetic acid, glucuronic acid, and ferulic acid are the structural constituents of hemicellulose, which is linked with the xylan backbone [6]. Moreover, vanillic acid, coumaric acid, syringaldehyde, etc., are the structural components of lignin [7]. Finally, citrate buffer and sulfate ions are the unavoidable major supplement inhibitors used during the enzymatic hydrolysis of pretreated lignocellulosic biomass. Citrate buffer plays a vital role during the enzymatic hydrolysis biomass, which restricts the pH change of the hydrolysis medium [8]. Generally, 50 mM citrate buffer strength is used to maintain the enzymatic hydrolysis medium pH between 4.8 and 5.5; it deters microbial metabolic growth in the subsequent fermentation of hydrolysates [9, 10]. Therefore, in addition to the process-derived and inherent inhibitors, 50 mM citrate buffer strength also acts as an inhibitor during hydrolysate fermentation.

Several pretreatment technologies were investigated to deconstruct the complex network of lignocellulosic biomass. Dilute sulfuric acid and alkali are the most widely used inorganic catalytic agents for the pretreatment of lignocellulosic biomass. Dilute sulfuric acid pretreatment hydrolyzes most of the hemicellulose fraction but forms sugar decomposition products [11, 12]. In contrast, alkaline pretreatment delignifies the lignocellulosic biomass, which enhances enzymatic digestibility but eliminates most of the hemicellulose fraction along with the minor fraction of cellulose, which eventually affects the total ethanol yield per dry ton of lignocellulosic biomass [13]. Moreover, washing pretreated biomass required considerable water to remove residual acid or base before the enzymatic hydrolysis [14]. Several studies also performed enzymatic hydrolysis of dilute sulfuric acid pretreated biomass by directly adjusting the pH at 4.8 to 5.5 with ammonium hydroxide instead of washing [15]. However, it forms sulfate ions that deter microbial metabolic growth, leading to low ethanol yield and productivity [16]. In addition, high levels of ammonium salts in the stillage required a specialized wastewater treatment section in the downstream processing [15].

Hydrothermal pretreatment is a leading approach for the possible commercialization of 2G biorefinery because they avoid the addition of chemical catalysts that add operating costs and complicate downstream processing. Hydrothermal pretreatment deconstructs the conglomerate structure of lignocellulosic biomass and makes accessibility to the enzymes to hydrolyze hemicellulose and cellulose into monomeric sugars (glucose, xylose, and arabinose), and subsequent microbial conversion of sugars into bioethanol [17]. In this regard, we developed a two-stage pretreatment process whereby biomass is treated at 190 °C for 10 min at 50% (w/w) moisture, followed by disc milling [18]. Disc milling further opens the cellulose fibers and increases sugar yields in the subsequent enzymatic hydrolysis. This process has since been scaled up with a continuous steam explosion reactor using bioenergy sorghum as the feedstock [19]. Fed-batch enzymatic hydrolysis of pretreated biomass at high solids loading (50% w/v) yields 230 g/L sugar concentration by the cellulose and hemicellulose hydrolysis efficiency of 65% and 85%, respectively [20]. Another significant advantage of the hydrothermal pretreatment followed by mechanical refining is that it minimizes process-derived fermentative inhibitors like furfural, 5-Hydroxymethylfurfural (5-HMF), levulinic acid, and formic acid. Our approach also reduced the usage of citrate buffer strength to 0.5 mM during the enzymatic hydrolysis process because the hydrothermal pretreatment was conducted at a lower severity without using acid catalysts and limited the formation of sugar decomposition products [21]. However, inherent inhibitors are still released from the lignin and hemicellulose fractions [3–6].

Hydrothermal pretreatment followed by a mechanical refining approach has been investigated on different lignocellulosic feedstocks such as oilcane 1566 (a genetically modified sugarcane that produces lipids along with sugars), energy cane, and miscanthus × giganteus. In this study, we made more advancements in the enzymatic hydrolysis step, which was carried out in distilled water without controlling the pH by acids or bases, and the sugar yields were compared with standard citrate buffer strength (50 mM). Fed-batch enzymatic hydrolysis was conducted with 50% (w/v) solid loading at 50 °C for 96 h using cellulase (NS 22257) and hemicellulase (NS 22254). In the 50 mM citrate buffer containing enzymatic hydrolysis medium, 215.28±0.69 g/L, 249.2±0.44 g/L, 238.52±0.56 g/L, and 192.78±0.99 g/L sugar concentrations were obtained from bioenergy sorghum, oilcane 1566, energy cane and miscanthus × giganteus, respectively. Whereas 215.05±0.62 g/L, 239.61±0.98 g/L, 231.85±1.45 g/L, and 184.65±0.44 g/L sugar concentrations were attained from bioenergy sorghum, oilcane 1566, energy cane and miscanthus × giganteus, respectively without using citrate buffer. Relatively similar sugar concentrations were obtained either using 50 mM citrate buffer or without citrate buffer. Further, enzymatic hydrolysates derived without citrate buffer were fermented by commercial genetically modified xylose-fermenting Saccharomyces cerevisiae for bioethanol production. Fermentation of bioenergy sorghum, oilcane 1566, energy cane, and miscanthus × giganteus hydrolysates produced 78.56±1.44 g/L, 59.23±0.66 g/L, 68.28±3.03 g/L, and 51.18 g/L bioethanol, respectively.

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