Citation: Saini, R.; Chen, C.-W.; Patel, A.K.; Saini, J.K.; Dong, C.-D.; Singhania, R.R. Valorization of Pineapple Leaves Waste for the Production of Bioethanol. Bioengineering 2022, 9, 557. https://doi.org/10.3390/ bioengineering9100557 Academic Editors: Giovanni Esposito and Martin Koller Received: 26 August 2022 Accepted: 14 October 2022 Published: 15 October 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). bioengineering Article Valorization of Pineapple Leaves Waste for the Production of Bioethanol Reetu Saini 1,† , Chiu-Wen Chen 1,2,3,† , Anil Kumar Patel 1,4 , Jitendra Kumar Saini 5 , Cheng-Di Dong 1,3,4, * and Reeta Rani Singhania 2,3,4, * 1 Institute of Aquatic Science and Technology, National Kaohsiung University of Science and Technology, Kaohsiung City 81157, Taiwan 2 Sustainable Environment Research Center, National Kaohsiung University of Science and Technology, Kaohsiung City 81157, Taiwan 3 Department of Marine Environmental Engineering, National Kaohsiung University of Science and Technology, Kaohsiung City 81157, Taiwan 4 Centre for Energy and Environmental Sustainability, Lucknow 226029, India 5 Department of Microbiology, Central University of Haryana, Mahendergarh 123031, India * Correspondence: cddong@nkust.edu.tw (C.-D.D.); reetasinghania@nkust.edu.tw (R.R.S.) † These authors contributed equally to this work. Abstract: Being a lignocellulose-rich biomass, pineapple leaves waste (PL) could be a potential raw material for the production of biofuel, biochemicals, and other value-added products. The main aim of this study was to investigate the potential of pineapple leaves in the sustainable production of bioethanol via stepwise saccharification and fermentation. For this purpose, PL was subjected to hydrothermal pretreatment in a high-pressure reactor at 150 ◦ C for 20 min without any catalyst, resulting in a maximum reducing sugar yield of 38.1 g/L in the liquid fraction after solid-liquid separation of the pretreated hydrolysate. Inhibitors (phenolics, furans) and oligomers production were also monitored during the pretreatment in the liquid fraction of pretreated PL. Enzymatic hydrolysis (EH) of both pretreated biomass slurry and cellulose-rich solid fraction maintained at a solid loading (dry basis) of 5% wt. was performed at 50 ◦ C and 150 rpm using commercial cellulase at an enzyme dose of 10 FPU/gds. EH resulted in a glucose yield of 13.7 and 18.4 g/L from pretreated slurry and solid fractions, respectively. Fermentation of the sugar syrup obtained by EH of pretreated slurry and the solid fraction was performed at 30 ◦ C for 72 h using Saccharomyces cerevisiae WLP300, resulting in significant ethanol production with more than 91% fermentation efficiency. This study reveals the potential of pineapple leaves waste for biorefinery application, and the role of inhibitors in the overall efficiency of the process when using whole biomass slurry as a substrate. Keywords: pineapple leaves waste; bioethanol; Saccharomyces; separate hydrolysis and fermentation; enzymatic hydrolysis; hydrothermal pretreatment 1. Introduction Taiwan is the third-largest producer of pineapple, with an annual pineapple produc- tion of nearly 42,000 tons in 2019 [1,2]. Taiwanese pineapples have a delicate texture and balanced flavor that make them among the world’s best. Pineapple fields are primarily located in the central and southern regions of the country [1]. The cultivation of pineapple generates a large amount of plant waste. Approximately 250 metric tons of wet plant residues per hectare are generated, which mainly comprise leaves. The crown of the pineap- ple itself accounts for 10–25% of the total weight of the pineapple [2] The pineapple’s crown is made up of two types of leaves which vary in their length. Long leaves are utilized by many companies for the production of fibers for various applications; in contrast, the short leaves are not suitable for fiber production and are, therefore, left behind causing a disposal problem. These leaves contain a significant amount of cellulose and hemicellulose Bioengineering 2022, 9, 557. https://doi.org/10.3390/bioengineering9100557 https://www.mdpi.com/journal/bioengineering