
==== Front
Curr Res Microb Sci
Curr Res Microb Sci
Current Research in Microbial Sciences
2666-5174
Elsevier

S2666-5174(24)00049-X
10.1016/j.crmicr.2024.100267
100267
Articles from the special issue: Emerging Frontiers in Microbial-Mediated Utilization of Crop Residues for Economically Valuable Biomaterials, edited by Debasis Mitra,Periyasamy Panneerselvam, Govindan Selvakumar and Marika Pellegrini
A novel experimental approach for the catalytic conversion of lignocellulosic Bambusa bambos to bioethanol
Paul Souvik Kumar paul.souvikkumar@dubai.bits-pilani.ac.in
a⁎
Das Amar Jyoti b
a Department of Chemical Engineering, BITS Pilani, Dubai campus, International Academic city, Dubai, United Arab Emirates
b Department of Microbiology, Graphic era Deemed University Clement Town, Dehradun, Uttarakhand 248002, India
⁎ Corresponding author. paul.souvikkumar@dubai.bits-pilani.ac.in
18 8 2024
2024
18 8 2024
7 100267© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• Bambusa bambos is a potential source of biofuel precursor.

• Catalytic conversion of lignocellulosic biomass to reducing sugar.

• Conversion of reducing to ethanol through yeast mediated fermentation.

• Ionic liquid recycling and product separation for cost effectiveness.

Bambusa bambos (B.B) biomass is cellulose rich lignocellulosic material, containing 47.49% cellulose, 17.49% hemicellulose, 23.56% lignin was used as a potential substrate for bioethanol production. The research paper investigates the use of B.B biomass as a substrate for bio-ethanol production through a two-phase catalytic conversion process. Four water-regulated regimes were identified to optimize the conversion of lignocellulosic biomass to biofuel precursors. The catalytic hydrolysis of B.B using CuCl2 was conducted for 10 hours at 110˚C, in aprotic ionic liquid (1-Butyl-3-methylimidazolium chloride) medium. The concentrations of glucose and 5-hydroxymethylfurfural (5-HMF) were measured while varying the amount of water addition. Water played a crucial role in the conversion of cellulose to glucose and 5-HMF by influencing product yields through the interplay of transport properties like heat conduction and viscosity. The highest glucose yield was achieved at 60.82% when operating at a water inclusion rate of 115.72 µL water/h for a duration of 6 hours at 110˚C. On the other hand, the maximum HMF yield was observed as 5.84% at water inclusion rate of 77.15 µL water/h for 5 hours at 110˚C. Yeast mediated glucose fermentation resulted in a bioethanol concentration of 5.5 mg/mL utilizing 15 mg/mL of catalytically produced glucose at a temperature of 30°C. After catalytic hydrolysis, the ionic liquid was also efficiently recycled for a sustainable economy.

Graphical abstract

Image, graphical abstract

Keywords

Lignocellulose hydrolysis
Regulatory effects
Aprotic ionic liquid
Product separation
Microbial fermentation
==== Body
pmc1 Introduction

In the pursuit of mitigating climate change and reducing dependency on fossil fuels, the exploration of sustainable energy alternatives has become paramount. Biofuels, derived from renewable sources, offer a promising path towards achieving these goals. However initial excitement about biofuels made from food crops, which were first generation, is now less important than worries for global food security (Armstrong et al, 2016). As a response to these concerns, a paradigm shift towards second-generation biofuels has emerged, focusing on utilizing non-edible lignocellulosic biomass as feedstock (Armstrong et al, 2016). This transition underscores the imperative to identify abundant lignocellulosic materials rich in cellulose content while ensuring minimal disruption to global food supplies.

B.B belongs to grass family and it is widely cultivated in the countries like India, Indonesia, China, Sri Lanka, Malaysia, Central America, Philippines, West Indies etc. (Zhan et al., 2022; Deshmukh and Pathan, 2024). The species is characterized by its tall height and bright-green color. It forms dense thickets with several branched columns. It can grow to heights of 10-35 m and it is commonly found in dry forest zone (Hossain et al., 2022; Chin et al., 2017). The biomass has huge cellulose but low lignin and hemicellulose contents, so it can be regarded as a possible bioethanol source of lignocellulosic material.

However, the primary challenge in the hydrolysis of lignocellulosic biomass is that the plants are inherently resistant to the breakdown of their cell walls (Paul and Chakraborty, 2018). Photosynthesis involves the storage of carbohydrates in the plant cell walls, which are composed of three polymers - lignin, hemicellulose, and cellulose - arranged in layered, interconnected matrices that are resistant to decomposition (Armstrong et al., 2016). Lignin, a polymeric compound forms covalent bonds with hemicellulose, an irregular polysaccharide consisting of xylose linked by β-(1 → 4)-glycosidic bonds, along with side chains of galactose, glucose, mannose, and arabinose (Harmsen et al., 2010). These elements, when combined, reside in the microfibrils that are found inside the secondary cell walls of plants, forming a hydrophobic structure (Silveira et al., 2013). By serving as a barrier, this structure prevents enzymes and solvents from accessing to the crystalline, hydrophilic cellulose chains (Yang et al., 2013). In this way, it protects the glycosidic connections that hold each individual glucose unit together and keeps them from breaking. Cellulose, the most prevalent natural polymer on Earth, is additionally shielded from enzymatic (Dutta and Chakraborty, 2015) and catalytic breakdown (Roy and Chakraborty, 2023) into glucose due to the presence of hydrogen bonds that connect it to the hemicellulose matrix (Dutta and Chakraborty, 2016).

Overcoming such barrier is pivotal for unlocking the full potential of lignocellulosic biomass as a sustainable energy source. Recent advancements in biofuel production have focused on innovative approaches to circumvent the challenges associated with lignocellulose hydrolysis (Roy and Chakraborty, 2023; Gaikwad and Chakraborty, 2014). One of the most promising of these is the direct synthesis of 5-hydroxymethylfurfural (HMF) from lignocellulose in an ionic liquid media. Ionic liquids, characterized by their low volatility and excellent solvency for lignocellulose (Wang et al., 2014), serve as efficient pretreatment agents, disrupting the lignin-hemicellulose barrier and enhancing cellulose accessibility (Asghari et al., 2006; Roy and Chakraborty, 2023; Guan et al., 2011). Additionally, recent research has shown that using metal chloride catalysts in combination with water can depolymerize cellulose into glucose (Carraher et al., 2015).

Ionic liquids are useful not only for pretreatment but also for the hydrolysis of cellulose and its subsequent conversion to HMF in the reaction medium (Paul and Chakraborty, 2018; Gaikwad and Chakraborty, 2014). However, optimizing the hydrolysis process presents a nuanced challenge, requiring a delicate balance between maximizing glucose and HMF yield and minimizing undesirable byproducts such as Humin (Roy and Chakraborty, 2023). The tradeoff between hydrolysis and dehydration underscores the importance of strategic water management to achieve optimal biofuel yields (Roy and Chakraborty, 2023). This tradeoff arises due to the requirement of water for cellulose to glucose hydrolysis and the need for limited water during the dehydration process to produce HMF (Xiang et al., 2003).

Within this framework, our work aims at explaining the fundamental mechanisms underlying cellulose hydrolysis in ionic liquid environments and optimize reaction conditions for enhanced biofuel production. While the regulatory effect of water on protic ionic liquid based catalytic hydrolysis of June grass was studied, (Roy and Chakraborty, 2023), we focus on the aprotic ionic liquid mediated hydrolysis of glucose and HMF derived from cellulose extracted from B.B, a widely available lignocellulosic feedstock. Through a systematic investigation of water addition strategies, we seek to (i) determine the optimal quantity and timing of water addition to maximize glucose and HMF yields, (ii) develop robust strategies for enhancing biofuel production efficiency while minimizing environmental impact by leveraging experimental data and theoretical modeling, (iii) recover products while recycling of aprotic ionic liquid after catalytic hydrolysis for cost effectiveness, (iv) conduct microbial fermentation of catalytically produced glucose to ethanol.

2 Materials and methods

2.1 Materials used

B.B was collected from the plant nursery in Warsan, Green Glades Landscaping LLC, Dubai, UAE (5F35+FR Dubai - United Arab Emirates). Ionic liquid, 1-Butyl-3-methyl-imidazolium chloride ([Bmim]Cl), and copper chloride (CuCl2) were purchased from HIMEDIA Labs and SRL, Dubai, UAE, respectively. The GOD-POD test kit was obtained from Accurex Biomedical Pvt. Ltd., Mumbai, India. Sulphuric acid, sodium hydroxide, hydrogen peroxide, 1-Butanol, acetone, and iso-butyl-methyl-ketone (MIBK) were purchased from Merck, Dubai, UAE. Saccharomyces Cerevisiae used in the present study was procured from the departmental pool. Malt, yeast extract, and peptone were purchased from SRL, Dubai, UAE. All chemicals used in this study were of analytical grade.

2.2 Methods

2.2.1 Substrate characterization

The technique described in the existing literature was used to determine the compositional analysis of dry B.B (Toribio-Cuaya et al., 2014; Yan et al., 2016; Paul and Chakraborty, 2018).

2.2.1.1 Determination of acid soluble lignin content

A 500 mg sample of substrate was combined with 10 mL of a 0.275 M H2SO4 solution and agitated at 150 rpm for 24 hours at a temperature of 25°C. The solution was then autoclaved at 118°C for 30 minutes. After cooling, the solid substance was obtained through filtration, while the liquid portion was centrifuged at 3000 revolutions per minute for 15 minutes. The concentration of acid-soluble lignin was measured using a UV–Vis Spectrophotometer (Agilent Technologies) at a wavelength of 280 nm.

2.2.1.2 Determination of cellulose content

The solid obtained during 0.275 M H2SO4 treatment was dried in an air oven at 80˚C for 10 h and the dried solid was mixed with 10 mL of 0.4 M sodium hydroxide and 100 µL of 14 M hydrogen peroxide at a temperature of 50°C while stirring at 180 rpm for 4 hours. After the reaction, the solid underwent filtration, drying, and subsequent weighing for determing the cellulose content.

2.2.1.3 Determination of alkaline lignin content

Acetic acid (50% v/v) was added to the filtered liquid to acidify it and precipitate alkaline lignin. The solution then underwent centrifugation, and the resulting precipitate containing alkaline lignin was subsequently dried and weighed.

2.2.1.4 Determination of hemicellulose content

500 mg sample of extractive-free biomass was reacted with 10 mL of a 1 M NaOH solution for 24 hours at a temperature of 25°C and a mixing speed of 100 rpm. The insoluble material was filtered and washed. The pH of the filtrate was lowered from 9.0 to 4.0 by adding 50% v/v of acetic acid. The resulting suspension was then centrifuged at 400 rpm and then filtered through a Whatman filter paper. After the removal of the supernatant, the solid remained was dried and weighted to obtain hemicellulose A while, through the stepwise precipitation of the supernatant, hemicellulose B was precipitated with methanol having a v/v ratio of 1:3. Following, the methanol supernatant was filtered and the resulting mixture was centrifuged to obtain the solid hemicellulose B. At this point the solid was weighed and dried at 90°C.

2.2.1.5 Determination of ash and moisture content

The ash content in the lignocellulosic material was analysed by burning 1 g of the biomass at 600°C, for 6 h in a furnace (Thermo Fisher Scientific, USA). Subsequently, the material was weighed after it had been burned. The weight of ash was then divided by the initial dry lignocellulosic material weight to determine the weight percentage of ash. The moisture content of 1 g of lignocellulosic material was measured by drying it at 105°C for 5 hours. The percentage of moisture was calculated by weighing both the original and oven-dried lignocellulosic biomass.

2.2.2 Catalytic hydrolysis

The hydrolysis was carried out in 15 mL glass vials by mixing B.B with the ionic liquid [Bmim]Cl, water, and a metal chloride catalyst (CuCl2). The vials were heated in a temperature-controlled oil bath while the reactants were mixed with a magnetic stirrer. Silicon oil was used as the oil bath medium. Pre-treatment processes were carried out by mixing the substrate and ionic liquid with desired quantities and process conditions for 6 hours at 500 rpm for each experiment before hydrolysis. After pretreatment, the catalyst and water were added to the reaction mixture to initiate the hydrolysis of cellulose. During the experiments, water was added carefully in varying amounts to identify the water-regulated regimes for maximizing product yields. In the case of lower amounts of water addition, a total of 270 µL of water was added, divided into different intervals of time. The water addition rates were selected as 45 µL water/h for 6 h, 38.58 µL water/h for 7 h, 33.75 µL water/h for 8 h, and 30 µL water/h for 9 h. For moderate amounts of water addition, a total of 540 µL of water was added, divided into different time intervals with rates of 90 µL water/h for 6 h, 77.15 µL water/h for 7 h, 67.5 µL water/h for 8 h, and 60 µL water/h for 9 h. In the case of sufficient water addition, a total of 675 µL of water was added, with rates of 112.5 µL water/h for 6 h, 96.43 µL water/h for 7 h, 84.38 µL water/h for 8 h, and 75 µL water/h for 9 h. For optimum/higher amounts of water addition, a total of 810 µL of water was added, with rates of 135 µL water/h for 6 h, 115.72 µL water/h for 7 h, 101.25 µL water/h for 8 h, and 90 µL water/h for 9 h. The reaction temperature was varied from 100˚C to 130˚C with a 10˚C increase each time. The catalyst to substrate ratio was varied from 6% to 14%, with a 2% increase each time. For example, a 6% ratio means 6 mg of catalyst per 100 mg of substrate. The substrate to ionic liquid ratio was varied as 1.67%, 3.33%, 5%, and 5.83%, with 1.67% meaning 0.0167 mg of substrate per mg of ionic liquid. Water amount, reaction time, catalyst to substrate ratio, reaction temperature, and substrate to ionic liquid ratio were varied to optimize different process conditions and to maximize product yields. All the experiments were conducted in triplicates. The average values and standard deviations were calculated among three data points using Microsoft excel 365. The average values and error bars were plotted while representing the data points. The statistical results obtained from Excel is verified in OriginPro (Origin Lab). Small error bars corresponding to all experimental data points suggested the accuracy of our study.

The Water addition strategy is summarized in below Table 1.Table 1 Different water addition strategies (lower, moderate, sufficient, optimum and high) maintained during metal chloride based catalytic hydrolysis of B.B in ionic liquid medium.

Table 1Total amount of water addition (µL)	Range	Hourly (µL water/h) water addition	
270	Lower	(a) 45 µL/h for 6 h	
(b) 38.58 µL/h for 7 h	
(c) 33.75 µL/h for 8 h	
(d) 30 µL/h for 9 h	
540	Moderate	(a) 90 µL/h for 6 h	
(b) 77.15 µL/h for 7 h	
(c) 67.5 µL/h for 8 h	
(d) 60 µL/h for 9 h	
675	Sufficient	(a) 112.5 µL/h for 6 h	
(b) 96.43 µL/h for 7 h	
(c) 84.38 µL/h for 8 h	
(d) 75 µL/h for 9 h	
810	Optimum and high	(a) 135 µL/h for 6 h	
(b) 115.72 µL/h for 7 h	
(c) 101.25 µL/h for 8 h	
(d) 90 µL/h for 9 h	

2.2.3 Product separation and Ionic liquid recycling

After hydrolysis, the products (glucose, HMF) were separated, and the ionic liquid was recycled following procedures outlined in the literature (Paul and Chakraborty, 2018; Roy and Chakraborty, 2023). An immiscible organic solvent composed of Butanol and MIBK ((V1-Butanol: VMIBK = 3:7, Morganicsolvent: MIL = 3:1) was introduced into the reaction mixture. Subsequently, the ionic liquid phase and the organic phase were isolated from each other. HMF concentrations were determined in both the organic and ionic liquid phases (Saha and Abu-Omar, 2014; Dashtban et al., 2014; Paul and Chakraborty, 2018). Glucose, which is insoluble in the organic phase, remained in the ionic liquid phase where its concentration was quantified. To regenerate the ionic liquid, the reaction mixture was supplemented with 5 mL of a 40 wt% potassium phosphate tribasic (K3PO4) solution, followed by stirring at 100 rpm for 15 minutes at a temperature of 70°C (Shill et al., 2011). After cooling and subsequent centrifugation, the solution underwent phase separation. The first layer was located at the top and it was identified as composed of the ionic liquid in excess. The middle layer was the solid phase which include the lignocellulosic substrate. Lastly, the lower layer formed by K3PO4 salt rich phase (Paul and Chakraborty, 2018). The phase with a high amount of ionic liquid was separated and into a flask, a mixture of water and acetone in equal volume was added to avoid gel formation. The precipitated lignin from the ionic liquid was separately isolated by evaporating the acetone-water mixture at 120°C which led to the formation of lignin particulate. The glucose, which should have been diluted in the medium for the subsequent microbial fermentation, was measured in the medium with the ionic liquid. Lastly, the ionic liquid without lignin was recycled to repeat the treatment (Sun et al., 2009). It was found that nanofiltration membranes can effectively separate glucose from the ionic-liquid-based medium with good selectivity – although glucose and ionic liquid [Bmim]Cl displayed some miscibility – with selectivity values of up to 36.6 (Avram et al., 2017).

2.2.4 Yeast mediated fermentation for bio-ethanol production

For fermentation of glucose obtained from catalytic hydrolysis, a 20 mL solution of MGYP media was prepared (Paul and Chakraborty, 2018). This media was created by combining 15 mL of glucose, with a concentration of 15 mg/mL derived from the hydrolysis process, with malt and yeast extract (80 mg each), as well as 120 mg of peptone, in 5 mL of distilled water. Consequently, a total of 20 mL of fermentation media was prepared. The microorganism Saccharomyces cerevisiae was utilized for the fermentation process. The autoclaved media were filled with the yeast, which was then incubated for 60 hours at 25°C, 30°C, and 35°C while spinning at 100 rpm. It is important to note that all experiments were conducted twice to ensure accuracy and reliability of the results. The average values and standard deviations were calculated among three data points using Microsoft excel 365. The average values and error bars were plotted while representing the data points.

2.2.5 Analytical techniques use for analysis

2.2.5.1 Glucose measurement with high performance liquid chromatography (HPLC)

The concentration of glucose was measured using an HPLC system from Agilent Technologies, USA. A ZORBAX Carbohydrate Analysis Column (5 μm, 4.6 × 150 mm^2) was utilized in the HPLC setup to quantify the glucose concentration, coupled with a refractive index (RI) detector. The mobile phase consisted of a solution of 75:25 (v/v) acetonitrile: water, flowing at a rate of 1.5 mL/min, while maintaining an operational temperature of 25°C with injection volume of 10 µL and retention time of 3.8 minutes. The calibration curve was prepared by measuring the area of standard glucose samples of concentration varying from 1 mg/mL to 10 mg/mL at the same operating conditions. The linear equation obtained from the calibration curve is used to estimate the concentration of glucose in the samples taken from the reaction mixture.

2.2.5.2 HMF estimation

The level of HMF was quantified using a high-performance liquid chromatography (HPLC) system with model Perkin Elmer Series from USA and the Wakosil II 5C18 column (5 μm, 4.6 × 150 mm2). A mobile phase consisting of a 90:10 (v/v) mixture of methanol and water was employed at a temperature of 30°C, with a flow rate of 1 mL/min, injection volume of 10 µL, retention time of 2.5 minutes, and a wavelength of 282 nm to quantify the concentration of HMF (Yan et al., 2008). The yield of each sample was calculated using the formula: yield (%) = (product concentration (mg/mL) / cellulose concentration in raw substrate (mg/mL)) × 100 (Paul and Chakraborty, 2018). The calibration curve was prepared by measuring the area of standard HMF samples of concentration varying from 0.001 mg/mL to 0.01 mg/mL at the same operating conditions. The linear equation obtained from the calibration curve is used to estimate the concentration of HMF in the samples taken from the reaction mixture.

2.2.5.3 Microbial cell growth

0.5 mL of sample was collected from the growth media and then centrifugation was carried out at 5000 rpm for a duration of 20 minutes. The cells were then washed with fresh media, dried in a hot air oven at 50°C for 30 minutes, and subsequently weighed.

2.2.5.4 Ethanol estimation

The bioethanol assays of the samples were determined using Perkin Elmer, USA gas chromatography system. This system used the flame ionization detector; and nitrogen was employed as the carrier gas. In this research, the analysis was done on a 10% OV–17 column under the isothermal condition where the temperature was set to 90°C. The injector and detector temperatures were maintained at 110°C and 150°C, respectively. To establish the calibration curve for ethanol estimation, standard solutions of ethanol within the concentration range of 2–10 μl/mL were employed.

3 Results and discussion

3.1 Reaction mechanism for Ionic liquid mediated catalytic conversion of lignocellulosic biomass

Ionic liquids exhibited superior solvent properties when it came to lignocellulosic raw materials, as their enhanced thermal and electrochemical stabilities allowed for customization through the selection of various cation-anion pairs to cater to specific reactions (Gusain et al., 2017). Ionic liquids could be categorized into two main groups: protic ionic liquids (PILs) and aprotic ionic liquids (APILs) (Thawarkar et al., 2015). PILs were generated by the transference of H+ ions from a Bronsted acid or base, whereas APILs were produced by the incorporation of R+ ions (commonly an alkyl group) to a heteroatom lone pair (Mirjafari et al., 2013). Currently, APILs were the primary catalysts used in the conversion of lignocelluloses, but there was an increasing focus on PILs. APILs utilized the one-electron reduction mechanism in the Oxygen Reduction Reaction (ORR), resulting in the production of superoxide anion (Ejigu and Walsh, 2014). The anion species exhibited strong nucleophilic characteristics and played an active role in cleaving the glycosidic bonds within the cellulose chain by forming a supramolecular structure with assistance from the alkyl group found in the APILs. Ionic liquids had the capability to facilitate various intermolecular reactions, including hydrogen bonding, ionic interactions, dispersion forces, and dipolar interactions. Anions present in ionic liquids showed a strong preference for taking up hydrogen bonds. Many chemicals that were usually insoluble in water could be dissolved by these anions. An ionic liquid's ability to form hydrogen bonds between an insoluble chemical and the anion in the ionic liquid determined how well it would dissolve the substance (Matthews et al., 2015).

Glucose was formed when β-(1-4)-glycosidic links in the cellulose chain were broken during ionic liquid-mediated catalytic hydrolysis. Water, an ionic liquid (Bmim)Cl, and a metal chloride catalyst (CuCl2) were present during this process (Goswami et al., 2016; Su et al., 2009; Zhou et al., 2011). As a Lewis acid catalyst, the mixture of catalysts, ionic liquid, and solvents forms a supramolecular complex with cellulose, which is represented as [Cellulosem(Bmim)+(CuCl3(H2O)n)−]. After that, a water molecule degraded the supramolecule, producing α-glucose, which then converted into β-glucose.

Cellulose to α- glucose: (1) (Bmim)Cl+CuCl2+(H2O)nkf⇌kb(Bmim)++(CuCl3(H2O)n)−,

(2) Cellulosem+Bmim++[CuCl3(H2O)n]−→k1Cellulosem(Bmim)+(CuCl3(H2O)n)−.

(3) Cellulosem(Bmim)+(CuCl3(H2O)n)−+H2O→k2Glucoseα+Bmim++(CuCl3(H2O)n)−+Cellulosep+Cellulosem−p−1.

α- glucose to β- glucose : (4) Glucoseα+Bmim++CuCl3−kf1⇌kb1Glucoseα(Bmim)+(CuCl3)−,

The β-glucose converted to fructose through a reversible reaction (Zhang and Zhao, 2010; Guan et al., 2011; Peng et al., 2010). Fructose underwent dehydration to yield HMF along with the formation of three water molecules (Desir et al., 2019). Eventually, HMF underwent rehydration to produce LA and FA in a 1:1 molar ratio (Gaikwad and Chakraborty, 2014; Shen and Wyman, 2012).

β- glucose to HMF, LA and FA: (5) Glucoseα(Bmim)+(CuCl3)−kf2⇌kb2Glucoseβ+Bmim++CuCl3−.

(6) Glucoseβ+Bmim++CuCl3−kf3⇌kb3Glucoseβ(Bmim)+(CuCl3)−,

(7) Glucoseβ(Bmim)+(CuCl3)−+H2Okf4⇌kb4Fructose+Bmim++CuCl3−+H2O,

(8) Fructosekf5⇌kb5HMF+3H2O,

(9) HMF+2H2O→k6LA+FA.

3.2 Amount of water addition

The crucial determinant of product distribution was the amount of water added every hour within specific intervals during catalytic hydrolysis. Water functioned as a cross-catalyst, initially hydrolyzing solid cellulose into monomeric glucose and then facilitating glucose dehydration to produce HMF. As water was added to the reaction mixture, the yield of glucose increased over time, reaching a peak value before declining. Fig.s 1 (a-d) depicted the temporal dynamics of glucose production for varying amounts of water additions: lower (total 270 µL water addition) (Fig. 1 (a)), moderate (total 540 µL water addition) (Fig. 1 (b)), sufficient (total 675 µL water addition) (Fig. 1 (c)), and higher (total 810 µL water addition) (Fig. 1 (d)). The maximum glucose yield was observed at a water addition rate of 115 µL water/h for 6 h (Fig. 1 (d)), with a total of 695 µL water added to the system. Adding more water beyond this point decreased the glucose yield, indicating the formation of an inhibited water-regulated regime in the system. In contrast, the HMF yield increased continuously with lower water additions (Fig. 2 (a)), reaching its peak at 385.75 µL water addition with a water addition rate of 77.15 µL water/h for 5 h, before declining. The production of HMF maximized at lower water additions because the conversion of glucose to HMF is a dehydration reaction, while the conversion of cellulose to glucose is a hydrolysis reaction. Hence, the highest yield of HMF was achieved with lower water additions compared to glucose (Roy and Chakraborty, 2023). Previous studies also suggested that high water addition during ionic liquid mediated hydrolysis of biomass reduced the solubility of cellulose present in the lignocellulosic biomass (Swatloski et al., 2002). Moreover, high water concentration provided enough water molecules to compete with anions of ionic liquid for the formation of hydrogen bonds with cellulose. This led to partial dissolution of lignocellulosic biomass in ionic liquid (Kilpelainen et al., 2007). The water content in case of chloride-based ionic liquid should be more restrictive (Brandt et al., 2010).Fig. 1 Temporal dynamics of glucose yield for various water addition rates (a) 270 µL (b) 540 µL (c) 675 µL (d) 810 µL of water added every hour up to 6, 7, 8, and 9 hours during ionic liquid mediated catalytic hydrolysis of B.B in oil-bath reactor at the optimum catalyst to substrate loading of 12 wt.%, and the substrate to ionic liquid loading of 5 wt.% at temperature of 110˚C.

Fig 1

Fig. 2 Temporal dynamics of HMF yield for various water addition rates (a) 270 µL (b) 540 µL of water added every hour up to 6, 7, 8, 9 hours during ionic liquid mediated catalytic hydrolysis of B.B in oil-bath reactor at the optimum catalyst to substrate loading of 12 wt.%, and the substrate to ionic liquid loading of 5 wt.% at temperature of 110˚C.

Fig 2

3.3 Effect of reaction time

The effect of optimal reaction time was another crucial process parameter that was determined during catalytic hydrolysis. According to water addition strategy, water was added for 6, 7, 8 and 9 h to produce glucose and HMF during catalytic hydrolysis (Fig. 1, Fig. 2). From the temporal evaluation of glucose and HMF, for the cases of different amount of water addition, it was observed that no glucose and HMF are produced at zeroth hour. As the catalyst CuCl2 was added after zero hour, in the presence of metal chloride catalyst reaction initiated. The glucose yield reached its maximum value till the time of continuous water addition and then the yield was decreased with reaction hours as soon as the water addition was stopped. Fig. 1 (a-b) showed that the glucose yield was maximum at 6, 7, 8 and 9 h according to water addition strategy and decreased thereafter. This was attributed to the fact that after certain interval of time, as the water addition was stopped and the water addition ranges were within the limited and moderate water regulated regime, the system faced scarcity of water which led to production of HMF (Fig. 2 (a-b)). For Fig. 1 (c-d), we observed the same trend of glucose yield as for Fig. 1 (a-b). But in these two cases, as the water addition ranges were within sufficient and higher water regulated regime, after certain interval of time, excess water was present in the system, which, in turn, led to precipitation of cellulose (Paul and Chakraborty, 2018; Medronho et al., 2012) and resulted in reduction of glucose yield.

Fig. 2 (a) showed the increase in HMF yield with reaction time irrespective of water addition strategy as the system was operated under limited water regulated regime which favored dehydration reaction (Zhang and Zhao, 2010; da Silva Lacerda et al., 2015). For further increase of water as showed in Fig. 2 (b), the HMF yield maximized after certain interval of time and then decrease. This was also due to the fact that after certain time, the amount of water presence in the system was more and the condition became nonideal for the production of HMF. Previous study also suggested that prolonged reaction time led to decrease in reducing sugar yield due to the formation of inhibitory complexes in the reaction mixture (Yoon et al., 2011). Thus, reaction time was found to be an important parameter that could be regulated with the amount of water addition to obtain desired product distribution that maximized yields of glucose (115.72 µL water/h for 6 h) and HMF (77.15 µL water/h for 5 h).

3.4 Effect of reaction temperature

The variation in reaction temperature was observed to exert a significant effect on the yield of glucose and HMF (Fig. 3) at optimized water concentration and time. The yields of glucose (Fig. 3(a)) and HMF (Fig. 3(b)) increased from 48.27% to 60.82%, and from 3.57% to 5.84%, respectively, as the reaction temperature was increased from 100˚C to 110˚C. However, when the reaction temperature was further increased to 120˚C, the yields of glucose and HMF decreased. No traces of levulenic acid (LA) and formic acid (FA) were observed within the temperature range of 100˚C to 120˚C. To find out if LA and FA were being formed throughout the reaction, the temperature was increased to 130˚C because higher temperatures encouraged the formation of these acids. However, at 130˚C, no traces of LA or FA were detected, and the yields of HMF and glucose further declined. This phenomenon can be attributed to the fact that the conversion of HMF to undesired side products like humin was taking place instead of the production of LA and FA at 130˚C (Peng et al., 2010) and decrease in the yields of glucose and HMF were observed. Past research (Paul and Chakraborty, 2018; Shen and Wyman, 2012) showed that temperatures above 160˚C were advantageous for the formation of LA and FA. The reaction setup did not allow for tests beyond 130˚C due to the evaporation of water in the system at higher temperatures, since all experiments were carried out under semi-batch settings to find the ideal water addition technique. Thus, the reaction temperature was observed to be an important parameter that could be regulated to maximize the yields of glucose and HMF in semi-batch conditions.Fig. 3 Effect of reaction temperature on the yields of (a) glucose at 115.72 µL water/h for a duration of 6 hours (b) HMF at 77.15 µL water/h for 5 hours during ionic liquid mediated catalytic hydrolysis of B.B in oil-bath reactor at the optimum catalyst to substrate loading of 12 wt.%, and the substrate to ionic liquid loading of 5 wt.%.

Fig 3

3.5 Effect of catalyst to substrate loadings

The optimization of metal chloride catalyst loading was conducted alongside lignocellulosic substrate loading. Fig.s 4 (a-b) illustrated the yields of glucose and HMF, respectively, for different catalyst to substrate loadings (w/w) ranging from 6% to 14%, with a 2% increase at each step, at optimum water addition, reaction time, reaction temperature, and substrate to ionic liquid ratio of 5%. Fig. 4 (a) indicated that at the optimum temperature of 110˚C and with an optimum water addition rate of 115.72 µL water/h for 6 h, the yield of glucose increased by 14.53% as the catalyst ratio per unit of lignocellulosic biomass increased from 6% to 12%. Similarly, in Fig. 4(b), the maximum yield of HMF increased by 2.87% at the optimum temperature of 110˚C and with an optimum water addition rate of 77.15 µL water/h for 5 h within the same range (6-12%) of catalyst to substrate loadings. However, as the catalyst to substrate ratio increased from 12% to 14%, the yields of glucose and HMF decreased by 2.55% and 0.72%, respectively. This reduction can be attributed to the fact that a high catalyst ratio led to the production of unwanted side reactions (Eminov et al., 2016). Consequently, a catalyst to substrate loading of 12% was determined to be the optimum for ionic liquid-mediated catalytic hydrolysis of B.B under semi-batch conditions.Fig. 4 Effect of catalyst to substrate loadings on the yields of (a) glucose at 110˚C, with 115.72 µL water/h for a duration of 6 hours (b) HMF at 110˚C, with 77.15 µL water/h for 5 hours during ionic liquid mediated catalytic hydrolysis of B.B in oil-bath reactor. (substrate to ionic liquid loading kept constant at 5 wt.%).

Fig 4

3.6 Effect of substrate to ionic liquid loadings

The substrate to ionic liquid loading was optimized at the optimal amount of water addition, time, temperature, and catalyst loading. The substrate to ionic liquid ratio was varied as 1.67%, 3.33%, 5%, and 5.83% during the catalytic hydrolysis of B.B. Fig. 5(a-b) illustrated the effect of substrate to ionic liquid loading on the yields of glucose and HMF. The yields of glucose and HMF were optimized at a 5% substrate to ionic liquid loading, and the yields decreased with increased substrate to ionic liquid loadings. Low substrate loading favored its dispersion in the solution and increased its regeneration rates. Conversely, a high loading of substrate to ionic liquid resulted in limitations in heat and mass transfer within the reaction mixture, thereby reduced the accessibility of the ionic liquid to the biomass (da Costa Lopes et al., 2013). From existing literature, it was also observed that as the initial loading of biomass increases, a lower amount of biomass was dissolved (Tan and Lee, 2012). In order to produce biofuels in a cost-efficient manner, the ionic liquid was extracted using a potassium sulfate solution while achieving an 80% recovery rate. Subsequently, the recovered ionic liquid was reused in further reactions.Fig. 5 Effect of substrate to ionic loadings on the yields of (a) glucose at 110˚C, with 115.72 µL water/h for a duration of 6 hours (b) HMF at 110˚C, with 77.15 µL water/h for 5 hours during ionic liquid mediated catalytic hydrolysis of B.B in oil-bath reactor. (Catalyst to substrate loading kept constant at 12 wt.%).

Fig 5

3.7 Optimization of process parameters

The highest glucose yield was obtained as 60.82% at 110˚C with a water inclusion rate of 115.72 µL water/h for 6 hours with substrate to ionic liquid ratio of 5 wt.% and catalyst to substrate ratio of 12 wt.% (Fig. 1 (d)). Conversely, for HMF, the maximum yield was obtained as 5.84% at 110˚C with a water inclusion rate of 77.15 µL water/h for 5 hours with substrate to ionic liquid ratio of 5 wt.% and catalyst to substrate ratio of 12 wt.% (Fig. 2 (b)).

3.8 Microbial fermentation of glucose to bio-ethanol

The glucose produced during catalytic hydrolysis of cellulose extracted from B.B was microbially (Saccharomyces cerevisiae) fermented to produce bioethanol. The initial concentration of glucose was maintained at 15 mg/ml. The concentration of glucose decreased with time as the yeast utilized glucose as carbon source for its growth. The concentration of ethanol increased gradually with time and with the utilization of glucose during fermentation. The reaction temperature was optimized during fermentation and it was varied as 25˚C, 30˚C and 35˚C. The glucose remained unutilized by the microorganism for the case of 25˚C and 35˚C, whereas, complete utilization of glucose was observed at 30˚C (Fig. 6 (a)). Similarly, the production of ethanol maintained a linear relationship with the depletion of glucose (Fig. 6 (b)). As the glucose was completely utilized by the end of 60 h of fermentation, the highest concentration of ethanol was observed as 5.5 mg/mL with conversion efficiency of 36.66% at 60 h at 30˚C. Hence, optimization of the fermentation process showed that the optimal reaction temperature, and fermentation time for maximum ethanol production were 30˚C, and 60 h, respectively.Fig. 6 Temporal dynamics of (a) glucose concentrations and (b) bioethanol concentrations at various temperatures during yeast mediated fermentation of glucose, produced from catalytic hydrolysis of B. B in ionic liquid medium.

Fig 6

In this work, we used non-edible lignocellulosic biomass B.B for second generation biofuel production. Compositional analysis showed that high cellulose and low lignin and hemicellulose contents, make the substrate a great candidate for the conversion of cellulosic ethanol. However, physicochemical study showed that along with its high cellulose content, B.B had high crystallinity. This made the substrate recalcitrant and resistant to break β-(1-4)-glycosidic bonds in cellulose. Current work showed the ways of overcoming these challenges by exploring catalytic hydrolysis of B.B while varying several process parameters with disparate water regulated regimes (ranging from low to high) for converting long chain cellulose molecules to monomeric sugars in aprotic ionic liquid medium. The ionic liquid was recycled for further use and the glucose obtained from catalytic hydrolysis was fermented microbially to produce ethanol.

4 Conclusion

In this study, we explored the impact of water on the transportation and chemical reactions involved in the catalytic hydrolysis of lignocellulosic B.B into glucose, 5-hydroxymethylfurfural (HMF) with the aid of alkali metal (CuCl2) catalysts within an aprotic ionic liquid environment while investigating the regulatory effects of water on these processes. Water worked as a cross catalyst in the hydrolysis process of cellulose into glucose and it played a significant role in the dehydration of glucose to 5-HMF by eliminating three water molecules. The concentration of water, time, and temperature were recognized as the key process parameters for controlling the distribution of the product. The optimum yield of glucose was observed as 60.82% when the temperature was set at 110˚C and the rate of water addition was 115.72 µL water per hour for a duration of 6 hours with catalyst to substrate ratio of 12% and substrate to ionic liquid ratio of 5%. On the other hand, the maximum yield of HMF was observed as 5.84% when the temperature was also at 110˚C, but the rate of water addition was 77.15 µL water per hour for a duration of 5 hours with catalyst to substrate ratio of 12% and substrate to ionic liquid ratio of 5%. This novel experimental strategy of producing biofuel precursors while conducting oil-bath mediated catalytic hydrolysis of B.B in ionic liquid medium had considerable economic promise. The experimental result showed 1 kg of B.B containing 474.9 g of cellulose can produce 322 g of glucose and 120 g of ethanol could be produced during microbial fermentation.

Declarations

• Ethical Approval: Not applicable

• Consent to Participate: The authors have consent regarding participation in the present work.

• Consent to Publish: Not applicable

• Funding: Not applicable

• Competing Interests: The authors declare that they have no competing or conflict of interest.

• Availability of data and materials: All data generated during the present study are included in the manuscript.

CRediT authorship contribution statement

Souvik Kumar Paul: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization. Amar Jyoti Das: Conceptualization, Methodology, Formal analysis, Investigation, Resources, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.
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