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Title: Nanobiocatalytic Conversion of Agri-Food Waste into Biochemical Products
Authors: Sillu, Devendra
Supervisor: Agnihotri, Shekhar
Reddy, M. S.
Keywords: Nanobiocatalyst;Biomass valorization;Circular economy;Pretreatment;Bioprocessing
Issue Date: 7-Feb-2023
Abstract: Agri-food waste reduction is a huge priority for many nations including India to develop a resilient food ecosystem. The unavoidable agri-food waste is becoming a major threat to our efforts to alleviate global hunger along with safeguarding socio-economic and environmental interests. On contrary, such waste exhibits immense potential to be repurposed for industrial bioprocessing that could contribute to circular bioeconomy. It is critical to understand ways how we can utilize non-edible waste in generating value-added products via advanced transformation strategies that lead to a more sustainable and prosperous future. The present work thus demonstrates a viable ‘waste-to-feed’ strategy for valorizing agri-food waste using ‘nanobiocatalyst’ with potential benefits. Sugarcane bagasse and potato-peel waste were considered the model representatives of waste from agriculture and food–processing sectors, respectively. The work is broadly classified into three major sections; (i) developing a facile pre-treatment approach for agri-food waste, (ii) fabrication of individual nanobiocatalytic systems involving cellulase and α-amylase enzymes onto halloysite nanotubes (HNTs) as immobilizing template, and (iii) nanobiocatalyst-assisted valorization of agri-food waste to value-added products such as glucose, fructose syrup, and other hydrolysates. These products were employed as medium-alternative for industrial bioprocessing, fermentation and bio-cementation applications. Developing an eco-friendly and facile pre-treatment process is essential for effective bioprocessing of lignocellulosic biomass. A two-step pretreatment approach was developed in this study, employing a solid acid catalyst, i.e., sulfonic acid functionalized magnetic halloysite nanotubes. Acid-functionalized magnetic halloysite nanotubes (MHNTs) were fabricated via mercaptosilane grafting and subsequent oxidation. The solid acid catalyst treatment followed by dilute alkaline treatment of sugarcane bagasse resulted in a more effective removal of hemicellulose (75.27%) and lignin (60.02%) contents from the biomass. The catalyst was easily recoverable using a magnet and could be reused over five consecutive cycles. The presence of high xylose (18.76 g/L) and low concentrations of inhibitory products in wash liquids indicated the effectiveness of the pretreatment approach employed. Sugarcane bagasse after solid-acid catalyst pre-treatments was more amenable towards enzymatic hydrolysis resulting in 60.32% cellulose saccharification. A 2.63-fold higher ethanol production was achieved using pre-treated sugarcane bagasse than its raw form, validating the effectiveness of the pre-treatment approach. A quest for efficient biotransformation of cellulosic material into sustainable biochemical products for recent biotechnological interventions is currently under way. Herein, the fabrication of nanobiocatalyst (NBC) employing halloysite nanotubes (HNTs) as a template for immobilizing cellulase enzyme was attempted, which catalyzed the hydrolysis of cellulose into glucose. Magnetic character was imported to HNTs by in situ anchoring of iron oxide nanoparticles, onto which cellulase was immobilized using aminosilane surface functional chemistry. Characterization studies revealed nanobiocatalyst to be extremely stable during heterogeneous catalysis without compromising their catalytic activity. The optimization of process parameters yielded ∼93.5% activity of cellulase with high enzyme loading (111.6 mg/g support) after immobilization. Immobilized cellulase displayed superior stability at elevated temperatures (≥60°C) and storage capability compared with their free forms. The NBC even retained ∼68.2% of its original activity after seven consecutive uses with a minimum yield of 25.4 mg glucose/g cellulose and was 100% recoverable using a magnet. Displaying a high ionic-liquid tolerance ability is concurrent with superior catalytic potential against CMC and extracted cellulose (pretreated sugarcane bagasse), and achieving ∼50.2% saccharification and 0.56 g glucose/g cellulose within 48 h of continuous operation establishes the commercial viability of using cellulase-immobilized HNTs for efficient cellulose hydrolysis. The glucose stream produced using pretreated sugarcane bagasse was further converted into fructose syrup employing a packed bed reactor with immobilized glucose isomerase. The parameters involved in isomerization reaction were optimized and a high glucose conversion (48.9%) into fructose was achieved. Overall, the nanobiocatalyst-assisted valorization of pretreated sugarcane bagasse for producing a clean glucose stream and fructose syrup was established. The concept of ‘nanobiocatalysis’ creates exciting opportunities for improving enzyme performance via immobilization onto nanomaterials. A nanobiocatalyst consisting of magnetic halloysite nanotube (MHNT)/α-amylase was evaluated to transform food processing waste into an active fermentation medium formulation for low-cost bioprocessing. A high loading of α-amylase (185.5 mg (g of support)−1) was achieved on the surface of MHNTs through polydopamine functionalization. We validated the establishment of an enzyme-support system retaining >89% catalytic activity (27332 IU (g of support)−1) with improved enzyme handling (>99.1% recovery) and reusability (>56% activity, 10 cycles). MHNTs remarkably improved the enzyme kinetics and thermodynamic characteristics along with operational and storage stabilities and mitigated the likely inhibitory effects of cellulose/metal ions as contaminants. In addition to facilitating a continuous production of reducing sugars from the extracted starch over 72 h, the nanobiocatalyst was equally effective in preparing a nutritive food waste hydrolysate as a fermentable medium substitute for batch culturing of E. coli and a single-cell protein (A. niger). The commercial relevance of waste hydrolysate was also investigated to promote calcite precipitation via Bacillus sp. induced biocementation. We evidenced that nanobiocatalyst-assisted “starch depolymerization” released more nutritional components into the hydrolysate suspension, easily accessible to growing microbial cultures. Summarizing all, the natural abundance and low cost of support material (halloysite nanotubes), simple functionalization procedures, and excellent immobilization capability of our system warrants huge potential to be adopted for immobilizing other bio macromolecules beyond enzymes. The successful implementation of the current strategy at an industrial scale will also open new dimensions for transforming other agri-food wastes into biochemical products of commercial relevance.
Description: Ph.D. thesis
Appears in Collections:Doctoral Theses@DBT

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