Contents lists available at ScienceDirect Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab An investigation on citrus peel as the lignocellulosic feedstock for optimal reducing sugar synthesis with an additional scope for the production of hydrolytic enzymes from the aqueous extract waste Divya Joslin Mathias, Sourav Kumar, Vivek Rangarajan * Department of Chemical Engineering, Birla Institute of Technology and Science-Pilani, K.K. Birla Goa Campus, Zuarinagar, 403726, Goa, India ARTICLE INFO Keywords: Citrus peel Acid hydrolysis Bioethanol production Pectinase production ABSTRACT In the current study, the use citrus peels as the lignocellulosic feedstock for the production of reducing sugars and hydrolytic enzymes was evaluated. Citrus peels were subjected to a two-step pre-treatment procedure to obtain two major fractions-the water soluble pectin rich fraction and the water insoluble solid fraction enriched with cellulose and hemicellulose. Three parameters (solid loading, acid concentration and time) that were found to be critically influencing the acid hydrolysis process were optimized by using Response surface methodology (RSM) - central composite design (CCD) for the improved synthesis of reducing sugars. A maximal total reducing sugar concentration of 13.34 g/L was predicted for the optimal processing conditions of solid loading = 3.87% w/w, acid concentration = 1% w/w and time = 48.4 min and at the processing temperature of 121 °C in a steam autoclave. Experimental validation studies carried out at these optimal conditions showed a slightly higher reducing sugar concentration of 13.65 ± 0.3 g/L than the predicted value. Further studies demonstrating the use of pectin-rich liquid fraction as the nutrient medium for hydrolytic enzymes production resulted in enzyme activities of 5.38 ± 0.2 IU/mL for pectinase and 1.17 ± 0.1 FPU (Filter Paper Units) for cellulase, without supplementation of any salts to the medium. These results suggest that the pre-treated citrus peels can serve as a potent feedstock for bioethanol production, while by-products resulting from pre-treatment can be employed to produce hydrolytic enzymes such as pectinases and cellulases as demonstrated through this work. 1. Introduction Lignocellulosic feedstocks are generated as wastes from food/fruit processing industries. These wastes are rich in carbohydrate content and thus form potential source of raw materials for biofuel production. About 1.3 billion metric tons of solid and liquid fruit biomass wastes are produced every year in the form of peels, stalks, leaves, seeds, etc (Perlack et al., 2005). Although some fraction of these wastes is utilized as cattle feed and composts, major portion of these wastes are discarded directly into landfills where they decompose and promote breeding of several pathogens (Seidl and Goulart, 2016). Some major fruit biomass feedstocks used in bio-refineries includes banana peels, apple pomace, sugarcane bagasse citrus fruits, etc. Citrus peels among the fruit wastes have been extensively studied, owing to their high carbohydrate con- tent accompanied by lower lignin content and they constitute 25–30% by mass the whole fruit (Boluda-Aguilar et al., 2010). Citrus peel wastes are mainly composed of cellulose, hemicellulose, lignin, pectin and trace amounts of tannins, ash, etc., (Ravindran and Jaiswal, 2016). Citrus peel biomass is a rigid structure that cannot be directly processed for bioethanol production due to the presence of lignin, pectin and traces of tannins, etc. Therefore, a pre-treatment step is required to completely remove these components and liberate the carbohydrate fraction for synthesis of bioethanol. Furthermore, the pectin fraction also can be utilized for the production of hydrolytic enzymes. Upon depolymerisation, cellulose and hemicellulose liberate hexose and pentose sugars respectively (Vyas et al., 2018). Presence of lignin during hydrolysis may result in production of phenolic com- pounds, which is highly toxic to microorganisms (Park and Kim, 2012). Therefore, a pre-treatment step is necessary to remove all the toxic compounds and to effectively disrupt the lignocellulose matrix prior to hydrolysis. Apart from removal of lignin, an effective pre-treatment technique must produce maximum amount of carbohydrates and less inhibitory products such as furfurals (Negro et al., 2016). Biomass pre- treatment techniques can be classified into three main categories-phy- sical, chemical and biological treatment. Physical pre-treatment in- cludes mechanical attrition and ultrasonic treatment. Physical pre- https://doi.org/10.1016/j.bcab.2019.101259 Received 27 February 2019; Received in revised form 29 May 2019; Accepted 19 July 2019 * Corresponding author. E-mail address: vivekr@goa.bits-pilani.ac.in (V. Rangarajan). Biocatalysis and Agricultural Biotechnology 20 (2019) 101259 Available online 20 July 2019 1878-8181/ © 2019 Elsevier Ltd. All rights reserved. T