Utilization of methyltrioctylammonium chloride as new ionic liquid in pretreatment of sugarcane bagasse for production of cellulase by novel thermophilic bacteria

Highlights

• Thermophilic bacteria were isolated from less explored mesophilic environments.

• Ammonium based ionic liquid was used for the pretreatment of sugarcane bagasse.

• Pretreated sugarcane bagasse was used for cellulase production from thermophiles.

• The promising strains belonged to A. thermoaerophilus and B. borstelensis.

Abstract

Fermentation of carbohydrates present in lignocellulosic (LC) biomass is facilitated by lignin removal, which is usually achieved by adopting various pretreatment methods to provide the enzymes proper access to their respective substrates. Pretreatment using ionic liquid (IL) is relatively recent advancement and considered as mild and green process. ILs can dissolve extensive quantities of biomass and depolymerize the cellulose. In this context, an abundantly available LC biomass, sugarcane bagasse (SB), was pretreated using alkali or with an IL, methyltrioctylammonium chloride, and was used for cellulase production from thermophilic bacteria. In all, 26 indigenously isolated thermophilic bacterial strains were quantitatively screened for cellulase production. 16S rDNA sequences of the promising isolates UE10 and UE27 revealed relatedness with Brevibacillus borstelensis, while the strain UE1 belonged to Aneurinibacillus thermoaerophilus. Cellulase production was compared by utilizing alkali pretreated and IL pretreated SB and the later was found more appropriate. UE1, UE10 and UE27 yielded 22.2, 22.18 and 33.3 IU mL⁻¹ of endoglucanase, respectively, by fermenting IL pretreated SB. The changes in SB structure after pretreatment were evaluated by scanning electron microscopy. This study demonstrated the potential of novel thermophilic bacterial strains to utilize IL pretreated SB for production of industrially important enzyme, cellulase.

Introduction

Sugarcane bagasse (SB), is an abundantly available industrial residue in Pakistan which is one of the largest sugarcane producing countries. SB is having a considerable complex structure with 25 % lignin, 25 % hemicellulose and 50 % cellulose (Qadir et al., 2018). Cellulose present in SB can be hydrolyzed to glucose and other soluble sugars by using microbial cellulases and hence it has potential to be used as fermentation raw material. SB, however, needs specialized treatment prior to its conversion into fermentable sugars so that cellulose become amenable to enzymatic hydrolysis (Arshad and Ahmed, 2016).

Pretreatment of LC breaks the carbohydrate-lignin complex that can be achieved by adopting various methods. However, every method has its own limitation, for instance, mechanical methods require intensive energy; physicochemical methods require specialized equipment that can withstand high temperatures and pressures; alkali pretreatment has environmental safety issues; and biological methods have excessive residence time (Canilha et al., 2012). Therefore, the development of alternative pretreatment processes is needed. The growing research towards pretreatment methods led to the use of ionic liquids (ILs) in this regard that opened a new horizon towards green solutions for the modern world’s chemical and energy requirements.

ILs exhibit promising physical and chemical characteristics including less toxicity, low volatility, thermal stability and good recyclability (Reddy, 2015), therefore, favored over other chemicals to disrupt hydrogen bonding between cellulose chains and to decrease crystallinity (Haghighi Mood et al., 2013). There are a number of advantages offer by IL compared to alkali including recyclability (Yoon et al., 2011). In the current study, an ammonium based IL was chosen in contrast to the widely reported imidazolium based ILs. Although, imidazolium based ILs efficiently delignify LC substrates, however their toxicity to fermenting bacteria is also well cited (Docherty et al., 2007). Moreover, these are not readily biodegradable and hence studies are needed to assess the potential environmental hazard (Ejaz et al., 2019). Methyltrioctylammonium chloride is a water insoluble quaternary ammonium salt. In comparison to traditional method of oxidizing cyclohexanol or cyclohexanone with potassium permanganate or nitric acid, the use of methyltrioctylammonium chloride is a more environmentally friendly (Pourreza and Zavvar Mousavi, 2004). Nonetheless, the production of cellulase from thermophilic bacteria using IL pretreated SB has not frequently been reported. Thermophiles can be isolated from various environments including mesophilic niches (Tariq et al., 2018). Considering the advantages of carrying out chemical processes at elevated temperatures, thermophiles get due attention for biomass utilization and saccharification, as well. The reactions carried out at higher temperature offer various advantages such as reduced chances of microbial contamination, reduced and enhanced rate of reaction and solubility of substrate (Golan, 2011). Cellulases exhibit considerable diversity in its structure and hence are classified into different types. Indeed, primary structure of a protein determined the thermostability of an enzyme (Rigoldi et al., 2018). A novel protein fold, which has shaped like an α-barrel of 12 helices connected by loops that form the active site, was reported to be present in thermostable cellulase (Juy et al., 1992). Moreover, according to Chang et al. (2016), thermostable chimeric cellulase attributed thermostability towards the presence of a 3₁₀ helix. The aim of the present study was to establish a continuous research line for the isolation of new thermophilic bacterial strain which can ferment SB with promising biotechnological and environmental potential and to compare alkali and ionic liquid pretreatment of SB.