Contents lists available at ScienceDirect Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop Improved high solid loading enzymatic hydrolysis of low-temperature aqueous ammonia soaked sugarcane bagasse using laccase-mediator system and high concentration ethanol production Kanak Raj, Chandraraj Krishnan ⁎ Department of Biotechnology, Indian Institute of Technology Madras, Chennai, 600036, India ARTICLE INFO Keywords: High solids loading cellulose hydrolysis High concentration ethanol Aqueous ammonia soaking Low-temperature pretreatment Lignocellulosic biomass Laccase-mediator assisted hydrolysis ABSTRACT High solids loading hydrolysis (HSLEH) of sugarcane bagasse (SCB) pretreated by low temperature aqueous ammonia soaking (AAS) was performed to obtain high concentrations of glucose and further fermentation to high concentration of ethanol. HSLEH was performed by fed batch addition of substrate and one-time enzyme feed at starting point. When the solids loading at 40% was tried in fed-batch hydrolysis, the observed con- centrations of glucose and ethanol were 119 g/l and 58.4 g/l respectively. When laccase (200 U/g solids) with 1- HBT (25 mg/g solids) as a mediator was added to cellulase in HSLEH, the glucose and ethanol concentrations were further enhanced to 157 g/l and 72.4 g/l. The present glucan conversion of 74% in HSLEH was higher when compared with the reported values for biomass pretreated by high temperature steam explosion, acid and alkali pretreatments. The present ethanol concentration (72.4 g/l) was also higher than the reported levels of ethanol in the second-generation bioethanol production. As HSLEH of biomass by low temperature AAS has not been reported so far, the present results clearly demonstrates that LMS assisted HSLEH of biomass pretreated by low temperature AAS is promising for cellulosic ethanol production. 1. Introduction The enzymatic hydrolysis of lignocellulosic biomass to fermentable sugars is an important step in the conversion of biomass to ethanol. Production of high concentration of sugars during hydrolysis is required to achieve high ethanol concentration in subsequent fermentation (Paulova et al., 2015). Production of > 5% ethanol during fermentation is desirable for economical process, which requires a minimum of ≥10% fermentable sugars in the hydrolysate. Formation of 10% fer- mentable sugars, mostly glucose and xylose from lignocellulosic bio- mass has been reported for high solids loading enzymatic hydrolysis (HSLEH). The other advantages of HSLEH are low capital cost of the process as fewer reactors are required, less energy is consumed for heating and cooling and reduced disposal costs because of less water usage (Koppram et al., 2014). Therefore HSLEH can improve the eco- nomics of lignocellulose to ethanol conversion process. However, there are certain challenges involved with HSLEH. It has very little to no free water when the solids loading is above 15%. At HSLEH, yield stress and apparent viscosity of the biomass slurry in- crease and thus the rheology of biomass becomes non-newtonian (Modenbach and Nokes, 2013). This results in high stirring resistance, limitation of mass and heat transfer and low efficiency of biomass hy- drolysis. In additional, the high concentration of sugars released at initial stage inhibits the action of enzymes, which restricts the industrial application of HSLEH (Modenbach and Nokes, 2013). The efficiency of HSLEH depends upon pretreatment method, hy- drolysis conditions, reactor design and enzyme formulation. Pretreatment method has a large effect on sugar yield in enzymatic hydrolysis. Alkaline pretreatments using alkali and ammonia remove lignin selectively and retain both hexose and pentose (Kim et al., 2016; Rekha et al., 2014; Prabakar et al., 2018a, 2018b). Therefore alkali pretreated biomass on enzymatic hydrolysis forms glucose and xylose (Kamalini et al., 2018). Use of ammonia in alkali pretreatment is de- sirable since it can be recovered and recycled. Ammonia based pre- treatment like AFEX (ammonia fiber expansion), ARP (ammonia recycle percolation) have been reported for enzymatic conversion of biomass at high solids loading (Aita et al., 2011). These methods require high temperature and pressure and therefore are energy intensive. Whereas, aqueous ammonia soaking (AAS) at low temperature and atmospheric pressure is a low energy pretreatment method. Since mild pretreatment by AAS results in lower efficiency of hydrolysis when compared with AFEX and ARP methods, AAS pretreated biomass has not been studied https://doi.org/10.1016/j.indcrop.2019.01.032 Received 21 August 2018; Received in revised form 7 January 2019; Accepted 16 January 2019 ⁎ Corresponding author at: Department of Biotechnology, Indian Institute of Technology Madras, Chennai, 600036, India. E-mail address: kcraj@iitm.ac.in (C. Krishnan). Industrial Crops & Products 131 (2019) 32–40 0926-6690/ © 2019 Elsevier B.V. All rights reserved. T