Industrial Crops & Products 159 (2021) 113122 Available online 23 November 2020 0926-6690/© 2020 Elsevier B.V. All rights reserved. Co-fermentation of immobilized yeasts boosted bioethanol production from pretreated cotton stalk lignocellulosic biomass: Long-term investigation Kamran Malik a, b , El-Sayed Salama b, *, Marwa M. El-Dalatony a , Mohammed Jalalah c, d , Farid A. Harraz c, e , M.S. Al-Assiri c , Yuanzhang Zheng f , Priyanka Sharma a, b , Xiangkai Li a, ** a MOE, Key Laboratory of Cell Activities and Stress Adaptations, Lanzhou University, Lanzhou, 730000, Gansu Province, PR China b Department of Occupational and Environmental Health, School of Public Health, Lanzhou University, Lanzhou, 730000, Gansu Province, PR China c Promising Centre for Sensors and Electronic Devices (PCSED), Advanced Materials and Nano-Research Centre, Najran University, P.O. Box: 1988, Najran, 11001, Saudi Arabia d Department of Electrical Engineering, Faculty of Engineering, Najran University, P.O. Box: 1988, Najran, 11001, Saudi Arabia e Nanomaterials and Nanotechnology Department, Central Metallurgical Research and Development Institute (CMRDI), P.O. 87 Helwan, Cairo, 11421, Egypt f Department of Molecular Biology, School of Medicine Biochemistry, Indiana University, Indianapolis, 46202, USA A R T I C L E INFO Keywords: Lignocellulosic biomass Pretreatment Yeast immobilization Fermentation Bioethanol ABSTRACT The main concern of lignocellulosic biomass utilization for biofuel production is the presence of lignin which hinder the hemicellulose and cellulose accessibility. In this study, chemical and biological pretreatments have been used for decomposition of the lignocellulosic cotton stalk (CS) into monosaccharides. Long-term fermen- tation/co-fermentation (upto 5 cycles) of pretreated CS by immobilized yeasts (Saccharomyces cerevisiae YPH499 and Pachysolen tannophilus 32691) for bioethanol was investigated. Spectroscopic analysis (including FTIR, XRD, SEM, and TGA) showed the disintegration and abrasion in CS structure after application of both the pre- treatments. The maximum sugar utilization effciency in 1st cycle of co-fermentation by immobilized yeasts was 94.1 and 90.4% with 0.46 and 0.44 g/g bioethanol production in chemical and biological pretreatment, respectively. Moreover, bioethanol yield was slightly sustained till 2nd cycle (0.380.40 g/g). However, bio- ethanol production steadily declined at 3rd cycle and reached to the lowest value at 5th cycle. These results demonstrated that co-fermentation with immobilization approach might signifcantly improve the bioethanol production from pretreated lignocellulosic biomass (including CS). 1. Introduction The major challenge to convert lignocellulosic biomass into biofuel feedstock is its recalcitrant nature, which hinders the lignin removal and accessibility of cellulose and hemicellulose (Zeng et al., 2020). To overcome this issue, multiple pretreatments (including chemical, phys- ical, physicochemical, and biological) have been adopted on the basis of effectiveness and environment safety (Abraham et al., 2020; Rezania et al., 2020). Biological pretreatment emerged as a potential technique, which has an advantage of a low energy input, environment safety, and less inhibitors formation (Yoo et al., 2020). Fungi has been extensively employed over a few decades to pretreat lignocellulosic biomass, as it secretes ligninolytic enzymes for the lignin degradation and improves the enzymatic hydrolysis effciency (Singh, 2020). The ligninolytic en- zymes such as, Manganese peroxidase (MnP), lignin peroxidase (LiP), versatile peroxidase (VP), and laccase (Lac) play a key role in oxidation of the lignin polymers and in the synthesis of aromatic radicals (Kumar et al., 2020), after which the cleavage of aromatic ring, C4-ether breakdown, and demethoxylation happens, which results in lignin depolymerization (Singh, 2020). However, fungal pretreatment exhibi- ted low rate of lignin delignifcation, and is also a time consuming method (Vu et al., 2020). Compared with fungal pretreatments, many cellulolytic, and sulfate reducing bacteria have been used to enhance the biofuel yield (Ali et al., 2020; Di Donato et al., 2019). Some bacteria produces extracellular peroxidases such as LiP, dye-decolorizing peroxidase, Lac, and MnP, * Corresponding author at: Department of Occupational and Environmental Health, School of Public Health, Lanzhou University Lanzhou, 730000, Gansu, PR China. ** Corresponding author: MOE, Key Laboratory of Cell Activities and Stress Adaptations, Lanzhou University, Lanzhou, 730000, Gansu Province, PR China. E-mail addresses: salama@lzu.edu.cn, sayed14@hanyang.ac.kr (E.-S. Salama), xkli@lzu.edu.cn (X. Li). Contents lists available at ScienceDirect Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop https://doi.org/10.1016/j.indcrop.2020.113122 Received 2 October 2020; Received in revised form 5 November 2020; Accepted 10 November 2020