Biohydrogen and Methane Production from Sugarcane Leaves Pretreated by Deep Eutectic Solvents and Enzymatic Hydrolysis by Cellulolytic Consortia

Abstract

This study determined the optimal conditions for the deep eutectic solvent (DES) pretreatment of sugarcane leaves and the best fermentation mode for hydrogen and methane production from DES-pretreated sugarcane leaves. Choline chloride (ChCl):monoethanolamine (MEA) is the most effective solvent for removing lignin from sugarcane leaves. The optimum conditions were a ChCl: MEA molar ratio of 1:6, 120 °C, 3 h, and substrate-to-DES solution ratio of 1:12. Under these conditions, 86.37 ± 0.36% lignin removal and 73.98 ± 0.42% hemicellulose removal were achieved, whereas 84.13 ± 0.77% cellulose was recovered. At a substrate loading of 4 g volatile solids (VS), the simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) processes yielded maximum hydrogen productions of 3187 ± 202 and 2135 ± 315 mL H₂/L, respectively. In the second stage, methane was produced using the hydrogenic effluent. SSF produced 5923 ± 251 mL CH₄/L, whereas SHF produced 3583 ± 128 mL CH₄/L. In a one-stage methane production process, a maximum methane production of 4067 ± 320 mL CH₄/L with a substrate loading of 4 g VS was achieved from the SSF process. SSF proved to be more efficient than SHF for producing hydrogen from DES-pretreated sugarcane leaves in a two-stage hydrogen and methane production process as well as a one-stage methane production process.

1. Introduction

Biofuels are any liquid, gas, or solid fuel produced from renewable biomass. Examples of biofuels include ethanol, methanol, synthetic gas (syngas), biodiesel, biogas (methane), biochar, bio-oil, and biohydrogen [1]. Biogas, typically a mixture of methane (CH₄) and carbon dioxide (CO₂), is produced worldwide from agricultural, municipal, and industrial wastes via anaerobic digestion (AD) [2].

AD is a well-established process for converting different organic waste into renewable energy, such as methane, with limited environmental impact [3]. AD involves the biological degradation of organic matter. Digestion is driven by anaerobic microorganisms and involves a series of steps, including hydrolysis, acidogenesis, acetogenesis, and methanogenesis [4]. At the beginning of the process, in the hydrolysis step, complex organic polymers are decomposed into monomers such as amino acids, fatty acids, and monosaccharides. Next, acidogenic bacteria convert these monomers into a mixture of short-chain volatile fatty acids during acidogenesis. Acidogenic bacteria are either facultative or strictly anaerobic bacteria belonging to the family Enterobacteriaceae [5]. Next, acetogenic bacteria or acetogens convert volatile fatty acids to acetate, carbon dioxide, and hydrogen in the acetogenesis step which are further used as substrates to produce methane in the methanogenesis step [6]. Therefore, a feedstock used for methane production by AD should be readily biodegradable and free of toxic components that would cause adverse effects on bacteria [7]. AD feedstocks are divided into three main categories: edible food crop resources (first-generation feedstocks), lignocellulosic materials and different organic waste materials (second-generation feedstocks), and algal biomass (third-generation feedstocks) [8]. Despite their high biogas production rates, first-generation feedstocks compete with food production, making them an undesirable biomass source. Therefore, lignocellulosic materials have received attention as alternative feedstocks for biogas production in recent decades owing to their abundance and cost. In this study, sugarcane leaves were used as lignocellulosic material for methane production.

Sugarcane leaves are usually either burnt to enable manual harvest, adding to environmental pollution and greenhouse gases, or left in the field as part of fertilizer, providing soil nutrients. [9]. It is usually left in abundance for up to 600–800 g/m² of sugarcane crops [10]. Dry leaves possess the energy equivalent of 1000 g/m², which is a significant advantage of this feedstock [11]. Sugarcane leaves are primarily composed of lignin (15–20%), hemicellulose (20–35%), and cellulose (35–50%) [12]. Biochemical or thermal methods can convert them into different bioenergy forms and other marketable co-products. Thus, using sugarcane leaves as feedstock to produce methane is necessary to obtain biofuel and mitigate environmental problems. However, the complexity of the structure of lignocellulosic biomasses, such as sugarcane leaves, is a major challenge, making them highly recalcitrant to AD and ultimately resulting in low methane yield [6]. Lignin is a physical barrier to lignocellulosic biomass [13,14,15], preventing enzymes from accessing cellulose [16,17,18,19]. Therefore, a pretreatment step is required to overcome this problem.

Pretreatment steps are essential to make cellulose more accessible for enzymatic hydrolysis by changing the physical and chemical structure of the lignocellulosic biomass and facilitating the conversion of polysaccharides into fermentable sugars [20]. Consequently, methane production from the lignocellulosic biomass is enhanced. One of the pretreatment methods that has received attention in the last decade is ionic liquid (IL) pretreatment. Lignocellulosic biomass pretreatment using ILs offers several attractive features compared to conventional methods [21]. As a molten salt, ILs are composed only of ions, usually a combination of larger organic cations and smaller anions. These features enable ILs ion properties and chemistry to be designed and tailored for desired applications. The dissolution of cellulose in ILs can be increased by introducing higher hydrogen bond (H-bond) basicity and polarity on the anion side and reducing the alkyl chain length on the cation side. This shows that the anion and cation sides of ionic liquids can be changed and tuned to enhance lignocellulosic biomass pretreatment [21]. ILs are considered green solvents due to their characteristics, such as nonvolatility, which makes the application of ILs reduce the air pollution caused by solvent evaporation, having a low toxicity to human health and the environment, biodegradability, being easy to recycle and reuse, and non-corrosive [21]. However, ILs have some disadvantages, including that they are not always “green” and are generally costly. In addition, they can absorb water from the air and evaporate at moderate temperatures. IL synthesis, separation, and purification require many solvents and energy [22,23,24,25]. Recently, a solvent with similar qualities and fewer limitations than IL has been proposed. They are referred to as deep eutectic solvents (DESs). DESs have a high dissolving capacity and a low melting point but are simpler to prepare and less expensive in terms of raw materials [25]. DESs are produced by combining multiple H-bond donors (HBD) and acceptors (HBA) [21]. The number of DES components can be increased to three or more. A DES has a substantially lower melting point than its components (HBD and HBA) due to the strong hydrogen-bonding interaction between HBD and HBA [21]. The capacity of a DES to preferentially cleave the ether bonds between phenylpropane units in lignin heteropolymers [26] is the mechanism by which it delignifies biomass. Although a DES can remove lignin from lignocellulosic biomass, its use as a pretreatment method for feedstock for biogas production remains limited. In this work, DES conditions were optimized to pretreat sugarcane leaves before their usage as an AD feedstock for biogas production. Despite the fact that lignocellulosic biomass pretreatment can improve biogas production, lignocellulosic biomass hydrolysis in AD is still a bottleneck because of the low rate of hydrolysis products slows down the entire AD process. To solve these problems, an enzymatic process was introduced to aid hydrolysis. This can be achieved using enzymes or cellulolytic microorganisms. Cellulolytic microorganisms are effective in recalcitrant cellulosic biomass hydrolysis. These microorganisms have been enriched and isolated from various ecological niches. Consortia are more robust to environmental fluctuations because they are naturally occurring. In this study, cellulolytic microorganisms enriched from rice straw compost (RSC), termite intestines (TI), and the soil around goat and sheep stalls (SGS) were used to construct a cellulolytic consortium. The consortium was further used to hydrolyze the cellulose fraction of sugarcane leaves via separate hydrolysis fermentation (SHF) and simultaneous saccharification and fermentation (SSF). SHF is a method in which enzymatic hydrolysis and fermentation are performed sequentially. This process begins with the enzymatic hydrolysis of biomass or pretreated lignocellulosic biomass at the appropriate temperature for the hydrolyzing enzyme. Subsequently, the fermentation conditions were optimized. However, these two separate processes increase the capital cost of the SHF process. Unlike SHF, SSF involves simultaneous enzymatic hydrolysis and fermentation in the same reactor. SSF can eliminate substrate inhibition because the sugars from the hydrolyzed biomass are directly transformed into biogas by AD [26]. Hence, this study employed SHF and SSF as fermentation modes to hydrolyze sugarcane leaves and produce methane.

There are two ADs for producing methane. The first was a one-stage methane production process. This process is the simplest and most conventional system to perform AD, one in which all AD steps occur in one reactor. One-stage AD has the advantages of low installation and operating costs and a short processing time [27]. The second step is two-stage AD, which separates the first step (acidogenesis and acetogenesis) and the second step (methanogenesis) in two different reactors, allowing the recovery of both hydrogen and methane from this process. By implementing two-stage AD, the system can achieve a more stable operation, higher organic loading capacity, and higher resistance to toxicants and inhibiting substances [28]. To produce methane from DES-treated sugarcane leaves, this study used a one-stage methane production process and a two-stage hydrogen and methane production process. This study aimed to find the best conditions for the DES pretreatment of sugarcane leaves and the best fermentation mode for hydrogen and methane production from DES-pretreated sugarcane leaves.

2. Materials and Methods

2.1. Sugarcane Leaves Preparation

Sugarcane leaves were collected from sugarcane plantations in the Khon Kaen Province, Thailand. First, the sugarcane leaves were dried under the sunlight until less than 10% of the moisture content remained. Next, the dried sugarcane leaves were cut, ground, and sieved using sieve no. 18 (mesh size 1.0 mm) (Central World Intertrade Co., Ltd., Ladkrabang, Bangkok, Thailand) to obtain particles smaller than 1.0 mm. Finally, the sugarcane leaves were stored in a dry plastic box at room temperature (32 ± 2 °C) for further use. The compositions of the sugarcane leaves (all in % (w/w) dry weight) are 36.18 cellulose, 25.23 hemicellulose, 27.68 lignin, and 10.91 ash.

2.2. Deep Eutectic Solvents Preparations

DESs were prepared using different types and molar ratios of HBA and HBD. The DESs used in this study were ChCl:glycerol (G), ChCl:G:aluminum chloride (AlCl₃), and ChCl:MEA. ChCl acts as an HBD, whereas G, a mixture of G, aluminum chloride (AlCl₃), and MEA, acts as HBA. DESs were prepared by varying the molar ratio of each mixture. The ChCl:G mixtures were at 1:2, 1:4, and 1:6 molar ratios, the ChCl:G:AlCl₃ mixtures were at 1:2:0.33, 1:4:0.33, and 1:6:0.33 molar ratios, and the ChCl:MEA mixtures were at 1:6, 1:8, and 1:10, respectively. All analytical grade chemicals were purchased from Elago Enterprises Pty., Ltd. (Elago Enterprises, 5 The Cloisters, Cherrybrook, Sydney, NSW, Australia)

2.3. Cellulolytic Consortium

RSC, TI, and SGS from the Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Thailand, were used as cellulolytic consortium sources. Peptone-cellulose solution (PCS) medium (all in g/L; yeast extract 1, peptones 5, CaCO₃ 2, and NaCl 5) was used to enrich the cellulolytic consortium from RSC, TI, and SGS. Initially, one gram each of RSC, TI, and SGS was added to 30 mL of PCS medium containing 0.15 g of filter paper (Whatman No.1) as the carbon source and an indicator for cellulase activity. Enrichment was performed at 37 °C. Once the filter paper strip was completely degraded, 10% (v/v) of the culture was transferred into the fresh PCS media containing 0.15 g of filter paper Whatman No.1 with 3.0 × 5.5 cm. This process was repeated at least 10 times or until the filter paper weight loss was stable (relative standard deviation < 0.05). Finally, the enriched culture was collected to analyze enzyme activity, including carboxymethyl cellulose degradation (CMCase), filter paper degradation (FPUase), and xylanase, before hydrolysis. The enriched cellulolytic consortium showed CMCase, FPUase, and xylanase activities of 0.38, 8.05, and 1.21 IU/mL. For long-term storage, the cellulolytic consortium was kept in a PCS medium containing 20% glycerol without cellulosic substrates at −80 °C.

2.4. Clostridium butyricum TISTR 1032 Prepartions

The hydrogen producer in this study was C. butyricum TISTR1032. It was purchased from the Thailand Institute of Scientific and Technological Research (TISTR). Strain TISTR1032 was regenerated in a serum bottle containing a cooked meat medium (CMM) (Himedia, Analytical grade, Thane, India). The serum bottle was flushed with nitrogen gas to create anaerobic conditions and incubated at 37 °C for 10 h. Then, 1 mL of the stock culture was added to a serum bottle containing 10 mL of tryptone sucrose yeast extract (TSY) (Himedia, Analytical grade, Thane, India) medium under anaerobic conditions at 150 rpm for 10 h at 37 °C. This process was repeated until an initial cell concentration of 10⁷ cells/mL was obtained. The composition of TSY (all in g/L) was tryptone 5, sucrose 3, yeast extract 5, and K₂HPO₄ 1 [29].

2.5. Anaerobic Sludge Preparations

Anaerobic sludge from the anaerobic digester of SF Khon Kaen Co., Ltd., (SF Khon Kaen, Nai Mueang, Thailand) was used as the inoculum source for the methane production process. The anaerobic digester produced biogas through the Napier silage and chicken manure co-digestion. To prepare methane producers, 5 L of anaerobic sludge was cultivated in a 10 L closed container containing 10 g/L of sugarcane leaves as the carbon source. The container was purged with nitrogen gas for 15 min to create anaerobic conditions and incubated for 2 weeks at room temperature (30 ± 5 °C) for degassing. The acclimatized inoculum was filtered through a filter cloth to remove non-degradable materials before being used for methane production. The anaerobic sludge compositions were 5.62 ± 0.27% of total solids (TS), 3.35 ± 0.08% of VS, 1.46 ± 0.12% of total suspended solids (TSS), and 2.11 ± 0.11% of volatile suspended solids (VSS.)

2.6. The Optimization Factors Affecting the Pretreatment of Sugarcane Leaves

The optimization of the different DESs, the molar ratio of HBA and HBD (ChCl: MEA (1:6, 1:8, and 1:10 molar ratio), ChCl:G (1:2, 1:4, and 1:6 molar ratio), and ChCl:G:AlCl₃ (1:2:0.33, 1:4:0.33, and 1:6:0.33 molar ratio), pretreatment temperature (80 °C, 100 °C, and 120 °C) and time (3 h, 6 h, and 9 h), and substrate-to-DES solution ratio for the pretreatment of sugarcane leaves were examined in the batch test. Next, the substrate-to-DES solution ratios were varied at 1:8, 1:12, 1:16, 1:20, and 1:24 (w/v). All treatments were performed in triplicate. At the end of the heating process, the pretreated solids were washed with hot distilled water several times until a pH of 7.0 was obtained. The pretreated solids were then dried at 60 °C in a hot-air oven until less than 10% of the moisture content remained. Finally, the pretreated solids were stored in sealed plastic bags at 31 ± 2 °C before analysis. The pretreated solids were used as substrates in the SHF and SSF processes for the two-stage hydrogen and methane production and one-stage methane production.

2.7. SHF for Two-Stage Hydrogen and Methane Production and One-Stage Methane Production from Pretreated Sugarcane Leaves

The SHF for the two-stage hydrogen and methane production and one-stage methane production was carried out in batch experiments. The hydrolysis step was investigated separately from that of the fermentation process. The two-stage hydrogen and methane production and one-stage methane production processes were initiated once hydrolysis was completed.

In the hydrolysis step, the experiment was conducted in 120 mL serum bottles with variations of pretreated sugarcane leaf loading of 1, 2, 3, and 4 g VS, respectively. Non-pretreated sugarcane leaves at loadings of 1, 2, 3, and 4 g VS were used as controls. The pretreated and untreated sugarcane leaves at the various loadings were added to the serum bottles containing 50 mL of fermentation media and 10% (v/v) of cellulolytic consortium. The enzyme activities of the cellulolytic consortium, including CMCase, FPUase, and xylanase, were analyzed. The fermentation media was comprised of (all in mg/L) K₂HPO₄ 125, MgCl₂.6H₂O 15, FeSO₄.7H₂O 25, CuSO₄.5H₂O 5, CoCl₂.5H₂O 0.125, NH₄HCO₃ 5240, and NaHCO₃ 6720 [30]. The initial fermentation broth pH was adjusted to 6.5 using either 5 M NaOH or 5 M HCl. The serum bottles were closed tightly using a rubber stopper and aluminum caps and incubated at 37 °C for 7 d [30]. Every 24 h, 1 mL of the hydrolysate was collected from each serum bottle to measure the pH and reduce sugar concentration. At the end of the hydrolysis step, the hydrolysate and solid residue in each serum bottle were used as substrates for the two-stage hydrogen and methane production and one-stage methane production processes.

For the two-stage hydrogen and methane production process, all serum bottles from the hydrolysis step were uncapped, and the pH was adjusted to 6.5 using 5 M NaOH or 5 M HCl. Then, 10% of C. butyricum TISTR 1032 (10⁷ cells/mL) was added to the serum bottles as the inoculum for hydrogen production. The serum bottles were recapped using a rubber stopper and aluminum caps and then purged with nitrogen gas for 15 min to ensure anaerobic conditions. The serum bottles were then incubated at 37 °C. The hydrogen production stage continued until biogas production ceased. After the first hydrogen fermentation stage, the hydrogenic effluent and solid residues were used as substrates for methane production in the second stage. The serum bottles were uncapped, and 20 g VS/L of acclimatized anaerobic sludge was added to produce methane in the methane stage. The initial fermentation broth pH was adjusted to 7.0 using 5 M NaOH or 5 M HCl.

The hydrolysate and solid residue from the hydrolysis step were used directly to produce methane for the one-stage methane production. The experiment was conducted in a serum bottle containing 20 g VS/L anaerobic sludge as the inoculum. The fermentation broth pH was adjusted to 7.0 using 5M NaOH or 5M HCl. The biogas volume was measured using a wetted glass syringe [31], and the biogas content was analyzed using gas chromatography (GC). The measurement of hydrogen and methane production continued until biogas production ceased. All treatments were performed in triplicate.

2.8. SSF for Two-Stage Hydrogen and Methane Production and One-Stage Methane Production from Pretreated Sugarcane Leaves

SSF for the two-stage hydrogen and methane production and one-stage methane production of pretreated sugarcane leaves was carried out in batch tests. Hydrolysis and fermentation occur simultaneously in this process.

The SSF for the two-stage hydrogen and methane production process was conducted using various loadings of pretreated sugarcane leaves (1, 2, 3, and 4 g VS). The control comprised untreated sugarcane leaves at different loadings as the DES-pretreated sugarcane leaves. The pretreated and untreated sugarcane leaves at the different substrate loadings were added to the serum bottles containing 50 mL of fermentation media, 10% (v/v) of the cellulolytic consortium, and 10% (v/v) of C. butyricum TISTR1032. The pH of the fermentation broth was adjusted to 6.5. The hydrogen production stage continued until biogas production ceased. After the first hydrogen fermentation stage, the hydrogenic effluent and solid residues were used as substrates for methane production in the second stage. At the end of the hydrogen production stage, the serum bottles were uncapped and 20 g VS/L of anaerobic sludge was added to produce methane. The fermentation broth pH was adjusted to 7.0.

Each serum bottle contained 50 mL of fermentation medium, 10% v/v of the cellulolytic consortium, 20 g-VS/L of anaerobic sludge as the inoculum for the one-stage methane production process, and different pretreated sugarcane leaves at various loadings of 1, 2, 3, and 4 g-VS. An experimental setup using untreated sugarcane leaves was used as a control. The solution pH was adjusted to 7.0 by 5 M NaOH or 5 M HCl.

Fermentation was performed in 120 mL serum bottles with a 50 mL working volume. Each serum bottle was capped using a rubber stopper and aluminum caps and closed tightly using a rubber stopper and aluminum caps. The serum bottles were incubated at 37 °C after being purged with nitrogen gas for 15 min to create anaerobic conditions. All procedures were carried out in triplicate. The measurement of hydrogen and methane production continued until biogas production ceased.

2.9. Enzyme acTIVITY Assay

The enriched culture from Section 2.3 was centrifuged at 10,000 rpm for 5 min to remove the cells and residues of the filter paper. The supernatant was used to analyze the enzyme activity. The activity of CMCase, FPUase, and xylanase was determined according to the IUPAC Commission of Biotechnology [32]. Enzyme activity assays were performed using different substrates for each enzyme. As substrates, the CMCase used 0.5 mL of 2% of carboxymethyl cellulose in sodium citrate buffer (0.05 M, pH 4.8). The FPUase used Whatman No.1 filter paper strip (1.0 × 6.0 cm) as the substrate, and xylanase used 0.9 mL of 1% (w/v) birchwood xylan in citrate-phosphate buffer (pH 5.0) as the substrate. The enzymatic assays were performed as described below.

CMCase and FPUase were determined by adding 0.5 mL supernatant to 1.0 mL sodium citrate buffer (0.05 M, pH 4.8). The substrate for each enzyme was then added to the reaction mixture. The mixture was then incubated at 50 °C for 60 min. To stop the reaction, 3.0 mL 3.5-dinitrosalicylic acid (DNS) reagent solution was added to the reaction mixture. The mixture was added to 20 mL of deionized water (DI water) and then left at room temperature for 20 min before measuring the absorbance at 540 nm using a spectrophotometer. Glucose was used as the standard.

The xylanase was determined by adding 1.0 mL of the supernatant to the 0.9 mL of 1% (w/v) birchwood xylan in citrate-phosphate buffer (pH 5.0). The mixture was incubated at 50 °C for 10 min and then cooled to room temperature. Subsequently, 20 mL of DI water was added to the cooled mixture before measuring the absorbance at 540 nm. Xylose was used as the standard.

A blank containing only water and the reaction mixtures was included to compensate for the effects of enzymes and substrates on the enzyme reaction. The control was the reaction mixture without an enzyme or substrate. CMCase, FPUase, and xylanase were measured at 540 nm using an EMC-11D-V spectrophotometer (EMCLab, Duisburg, Germany).

One enzyme unit (IU) was defined as the amount of enzyme hydrolyzing cellulose to release one microgram of glucose (FPUase and CMCase) or xylose (xylanase) per minute under assay conditions.

2.10. Analytical Methods

According to standard methods, the TS, TSS, VS, and VSS were determined [33]. The pH was measured using a pH meter (pH-500, Queen, New York, USA). The chemical compositions of untreated and pretreated sugarcane leaves, including cellulose, hemicellulose, and lignin, were analyzed according to the laboratory analytical procedures (LAP) of the National Renewable Energy Laboratory (NREL) to determine the structural carbohydrates and lignin in biomass [34]. The reducing sugars were analyzed using the 3,5-dinitrosalicylic acid (DNS) method [35]. The biogas composition was analyzed using a GC (GC-104, Shimadzu, Kyoto, Japan) with a thermal conductivity detector and a 2-m stainless steel column packed with Shin carbon (50/80) mesh. The operating conditions were as described previously [36]. The soluble metabolite products in the fermentation broth were measured using HPLC according to the method described in [37].

Compositional analysis was conducted following the standard methods of the NREL analytical procedure [34]. First, the sugarcane leaves were treated with 72% sulfuric acid at 30 °C for 1 h, followed by 4% sulfuric acid-treated samples at 121 °C for 1 h, and then the liquid and solid were separated by vacuum filtration. Next, the cellulose and hemicellulose contents were calculated from the corresponding sugar concentrations obtained from the measurement of liquid fractions by HPLC with conversion factors of 0.90 for glucose and 0.88, respectively, for glucose and xylose. Next, the acid-insoluble lignin was gravimetrically determined from the solid fractions using acid-insoluble lignin and ash. In contrast, the acid-soluble lignin content was determined by measuring the absorbance of the liquid fractions at 205 nm using a spectrophotometer.

2.11. Statistical Analysis

Statistical analysis was conducted with the IBM SPSS statistics program version 21. The means were compared using one-way ANOVA analysis with the Duncan test as a post hoc test. In addition, the means difference of the three samples were compared using an independent-samples t-test. All statistical tests were performed with a 95% confidence level.

2.12. Calculations

The hydrogen and methane volumes were calculated using the mass balance equation [38] and the, hydrogen and methane yields were expressed in mL H₂ or CH₄/g-VS_added. Next, the modified Gompertz equation (Equation (1)) was used to fit the cumulative hydrogen and methane yield curves [39]. Finally, a modified Gompertz equation was used to fit the cumulative hydrogen production curves and obtain the hydrogen production P, the hydrogen production rate R, and λ.

$$H=Pexp\left\{-exp\left[\frac{Rm\times e}{P}\right]\left(\lambda -t\right)+1\right\}$$

(1)

where H is the cumulative hydrogen or methane production (mL), λ is the lag phase time (h), P is the hydrogen or methane production potential (mL), and Rm is the hydrogen or methane production rate (mL H₂/L d or mL CH₄/L d). The incubation time (t) is reported in hours (h) or days. e is an exponential constant equal to 2.718. The equation was plotted using a nonlinear curve fitting in SigmaPlot 11 (Systat Software Inc., Palo Alto, CA, USA).

3. Results

3.1. Effects of Molar Ratio and Type of Deep Eutectic Solvent (DES) on Sugarcane Leaves

The untreated sugarcane leaves had a lower cellulose, hemicellulose, and lignin percentage than the pretreated ones (

Table 1. Effects of DES types and molar ratio on the pretreatment of sugarcane leaves at a 1:16 substrate to DES solution ratio, 6 h, and 80 °C.

DES	Molar Ratio	pH	Pretreatment			Composition of Residues
			Cellulose Recovery (%)	Hemicellulose Removal (%)	Lignin Removal (%)	Cellulose (%)	Hemicellulose (%)	Lignin (%)
Untreated	-	-	-	-	-	36.18 ± 0.73 f	25.23 ± 0.02 a	27.68 ± 0.35 a
ChCl/MEA	1:6	14.08	71.86 ± 0.37 c,d	69.88 ± 2.93 b,c	77.62 ± 0.79 a	57.62 ± 0.30 b,c	16.84 ± 1.64 d	13.73 ± 0.48 e
	1:8	14.12	73.49 ± 0.53 c	71.07 ± 0.54 b	77.50 ± 0.33 a	58.92 ± 0.42 a	16.21 ± 0.30 d	13.80 ± 0.30 e
	1:10	14.18	70.96 ± 2.10 d	68.21 ± 1.45 c	76.41 ± 0.85 a	55.33 ± 1.64 d	17.28 ± 0.79 d	14.07 ± 0.51 e
ChCl/G	1:2	7.93	82.37 ± 0.71 b	33.60 ± 1.16 e	28.98 ± 1.23 d	39.40 ± 0.34 e	22.15 ± 0.45 b	25.99 ± 0.45 b
	1:4	6.40	85.08 ± 0.81 a	33.57 ± 1.67 e	25.11 ± 0.61 e	38.72 ± 0.53 e	21.09 ± 0.53 b,c	26.08 ± 0.21 b
	1:6	6.60	82.14 ± 1.49 b	37.35 ± 2.39 d	29.41 ± 0.80 d	39.12 ± 0.71 e	20.81 ± 0.79 c	25.72 ± 0.29 b
ChCl/G/AlCl3	1:2:0.33	0.40	65.65 ± 0.23 f	87.00 ± 0.07 a	68.57 ± 0.26 c	59.28 ± 0.20 a	8.19 ± 0.05 e	21.72 ± 0.18 c
	1:4:0.33	0.38	66.61 ± 0.70 e,f	88.54 ± 0.48 a	70.27 ± 0.54 b	58.74 ± 0.62 a,b	7.05 ± 0.29 f	20.06 ± 0.37 d
	1:6:0.33	0.36	68.11 ± 0.81 e	88.21 ± 0.22 a	68.39 ± 0.19 c	57.37 ± 0.68 c	6.92 ± 0.13 f	20.37 ± 0.13 d

Untreated, untreated sugarcane leaves; ChCl, choline chloride; MEA, monoethanolamine; G, glycerol; AlCl₃, aluminum chloride; and DES, deep eutectic solvent. Values marked with the same letters are not significantly different (p < 0.05).

). These results implied that the DES pretreatment efficiently removed the lignin content in the lignocellulosic biomass.

The results demonstrated that the molar ratio of ChCl to MEA in sugarcane leaf pretreatment did not affect lignin removal, hemicellulose removal, or cellulose recovery (Table 1). Based on these data, the optimal molar ratio for the pretreatment of sugarcane leaves with ChCl/MEA at ratios of 1:6, 1:8, and 1:10 was determined to be 1:6 because the MEA concentration was the lowest. The highest lignin removal efficiency was observed at molar ratios of 1:2 and 1:6 when sugarcane leaves were pretreated with ChCl/glycerol (G). In contrast, the molar ratio variations of 1:6 and 1:4 resulted in the highest hemicellulose removal and cellulose recovery. Because this study focuses on the ability of DESs to remove lignin from sugarcane leaves, a molar ratio that can remove the majority of lignin was chosen for the pretreatment of sugarcane leaves. Additionally, a low molar ratio leads to a smaller amount of each HBA and HBD being considered. Therefore, the optimum ChCl/G molar ratio was determined to be 1:2. When ChCl/G/AlCl₃ was employed as the pretreatment solvent, ChCl/G/AlCl₃, at a molar ratio of 1:4:0.33, achieved maximum lignin removal. However, the molar ratio had no significant influence on hemicellulose removal and cellulose recovery. Thus, the optimum molar ratio of ChCl/G/AlCl₃ is 1:4:0.33. The molar ratio affects the DES’s physical properties, such as freezing point, density, and viscosity.

Lignin removal efficiency ranged from 76.41–77.62% at various molar ratios in DESs with strong bases (pH 14.08–14.18), i.e., ChCl/MEA (Table 1). This is not surprising because lignin is a base-soluble biopolymer [40]; therefore, basic solvents are advantageous for its removal. Furthermore, it was discovered that the more basic the solvent, the more lignin was extracted [40]. Under basic conditions, the breakage of ether links in lignin and ester bonds between lignin and hemicellulose leads to lignin removal [40] and dissolution [41].

At the optimum ChCl/G ratio of 1:2, the lignin removal was only 28.98 ± 1.23%, which is the lowest compared to other DES types (Table 1), indicating that ChCl/G is less effective in removing lignin from sugarcane leaves. This is because ChCl/G has a neutral pH (6.4–7.93), making the ChCl/G solution less efficient at eliminating lignin [42].

DESs with a strong acid i.e., ChCl/G/AlCl₃ (pH of 0.8–0.9), removed lignin in the moderate range of 68.39–70.27% but showed the highest hemicellulose removal (Table 1). In general, pretreatment solutions with low pH affect hemicellulose hydrolysis but have an intense effect on lignin dissolution and cellulose hydrolysis [41].

In the pretreatment processes, pH and viscosity were considered the main factors when determining the DES performance. The pH-influenced lignin removal from the lignocellulosic biomass occurs because lignin is a base-soluble polymer. Therefore, a DES with a high pH is considered for use as a pretreatment agent [40]. In contrast, DESs possess a comparatively higher viscosity (>100 cP) at room temperature which significantly limits their extraction applications. Additionally, their high viscosity reduces the mass transfer rate between the sample and extraction phase owing to the formation of extensive H-bond networks between the HBA and HBD components [43].

ChCl: MEA had the highest pH compared to ChCl/G and ChCl/G/AlCl₃. Because lignin is a base-soluble polymer, ChCl/MEA basicity makes it more effective for lowering the lignin concentration in the biomass while maintaining high cellulose recovery; hence, it is more advantageous for lignin removal. However, the results showed that ChCl/MEA had an insignificant effect on lignin removal at different molar ratios (Table 1). A higher molar ratio leads to more solvents, which increases the production costs when applied on an industrial scale. Therefore, the selected DESs in subsequent experiments were ChCl/MEA at a 1:6 molar ratio.

3.2. Effects of Pretreatment Time and Pretreatment Temperature

This study was conducted by varying the pretreatment time to 3, 6, and 9 h, while the pretreatment temperatures were varied at 80, 100, and 120 °C. ChCl/MEA at a 1:6 molar ratio was used as the solvent to pretreat the sugarcane leaves. At a pretreatment time of 3 h, the pretreatment temperature of 120 °C shows the lowest lignin content of 8.95 ± 0.15%, while still recovering the highest cellulose (70.17 ± 0.20%) compared to the other pretreatment temperatures (

Table 2. Effects of the temperatures and time on the pretreatment of sugarcane leaves at a 1:16 substrate to DES solution ratio using 1:6 molar ratio of ChCls/MEA as DES.

DES	Temp (°C)/Time (h)	Pretreatment				Composition of Residues
		Cellulose Recovery (%)	Hemicellulose Removal (%)	Lignin Removal (%)	Cellulose (%)	Hemicellulose (%)	Lignin (%)
Untreated					36.56 ± 0.77 k	27.31 ± 0.03 a	27.68 ± 0.35 a
ChCl/MEA (1:6)	80/3	69.25 ± 0.97 g	57.76 ± 0.72 h	73.49 ± 0.86 i	53.37 ± 0.75 j	24.32 ± 0.41 b	15.47 ± 0.50 b
	80/6	68.69 ± 1.51 g	66.60 ± 1.90 g	78.67 ± 0.52 h	57.61 ± 1.27 h	20.93 ± 1.19 c	13.55 ± 0.33 c
	80/9	68.37 ± 1.13 g	70.23 ± 1.40 f	80.81 ± 0.61 g	60.93 ± 1.01 g	19.82 ± 0.93 d	12.95 ± 0.41 d
	100/3	71.54 ± 0.30 f	70.29 ± 0.30 f	83.45 ± 0.02 e,f	64.25 ± 0.27 f	19.93 ± 0.20 d	11.25 ± 0.01 e
	100/6	72.81 ± 0.42 e	75.05 ± 0.96 a,b	85.96 ± 0.28 d	67.52 ± 0.39 d	17.28 ± 0.66 f,g	9.86 ± 0.20 f
	100/9	72.04 ± 0.40 e,f	76.57 ± 0.19 a	87.06 ± 0.01 c	69.64 ± 0.39 b,c	16.92 ± 0.14 g,h	9.47 ± 0.01 f,g
	120/3	78.75 ± 0.22 a	70.64 ± 0.51 e,f	86.73 ± 0.22 c	70.17 ± 0.20 b	19.54 ± 0.34 d	8.95 ± 0.15 h,i
	120/6	78.32 ± 0.38 a	72.57 ± 0.37 d	87.95 ± 0.53 b	71.91 ± 0.34 a	18.12 ± 0.24 e,f	8.07 ± 0.35 j
	120/9	78.80 ± 0.89 a	74.54 ± 0.79 b,c	89.18 ± 0.29 a	71.33 ± 0.81 a	17.22 ± 0.54 f,gh	7.42 ± 0.20 k

Untreated: untreated sugarcane leaves; ChCl: choline chloride; and MEA: monoethanolamine. Values marked with the same letters are not significantly different (p < 0.05).

). Similar results were obtained with pretreatment times of 6 h and 9 h at a pretreatment temperature of 120 °C. At 120 °C, the lowest lignin content (i.e., the highest lignin removal) was attained when the pretreatment time was 9 h. However, the cellulose recovery was not significantly different from the other pretreatment conditions. At 120 °C, with various pretreatment times of 3 h, 6 h, and 9 h, the system energy inputs were 622, 1198, and 1774 kJ/g-biomass, respectively. Therefore, the lowest energy occurred with the 3 h pretreatment time. Thus, the optimum pretreatment time and temperature for the pretreatment of sugarcane leaves with ChCl/MEA were 3 h at 120 °C.

3.3. Effect of the Substrate to DES Solution Ratio

The effect of the substrate to DES solution ratio was determined at the optimum conditions using ChCl/MEA at a 1:6 molar ratio, 120 °C, and 3 h. The substrate to DES solution ratios were 1:8, 1:12, 1:16, 1:20, and 1:24. The lowest lignin contents of 8.13 ± 0.57% and 8.15 ± 0.33%, respectively, were achieved at the substrate to DES solution ratios of 1:16 and 1:20, with lignin removal of 88.23 ± 0.83% and 88.06 ± 0.48% (

Table 3. Effects of the substrate to DES solution ratio using a 1:6 molar ratio of ChCls/MEA to DES at a temperature of 120 °C and time of 3 h on the pretreatment of sugarcane leaves.

The Substrate to DES Solution Ratio	Pretreatment			Composition of Residues
	Cellulose Recovery (%)	Hemicellulose Removal (%)	Lignin Removal (%)	Cellulose (%)	Hemicellulose (%)	Lignin (%)
Untreated				36.56 ± 0.77 d	27.31 ± 0.03 a	27.68 ± 0.35 a
1:8	81.84 ± 2.23 b	72.40 ± 0.68 c	86.86 ± 0.16 b	69.81 ± 0.62 c	17.59 ± 0.43 b	8.49 ± 0.10 b,c
1:12	84.13 ± 0.77 a	73.98 ± 0.42 b	86.37 ± 0.36 b	72.54 ± 0.67 b	16.76 ± 0.27 c	8.90 ± 0.23 b
1:16	81.44 ± 1.21 b	74.62 ± 0.41 b	88.23 ± 0.83 a	74.27 ± 1.11 a	17.29 ± 0.28 b	8.13 ± 0.57 c
1:20	81.31 ± 1.11 b	75.78 ± 0.45 a	88.06 ± 0.48 a	73.34 ± 1.00 a,b	16.32 ± 0.30 c	8.15 ± 0.33 c
1:24	84.38 ± 0.15 a	76.29 ± 0.10 a	86.42 ± 0.09 b	74.27 ± 0.13 a	15.59 ± 0.07 d	9.05 ± 0.06 b

Untreated: untreated sugarcane leaves; values marked with the same letters are not significantly different (p < 0.05).

). The energy inputs at 1:16 and 1:20 substrate to DES solution under the optimum conditions of 1:6 molar ratio ChCl/MEA, 120 °C, and 3 h, were 622 and 776 kJ/g-biomass, respectively. Therefore, the ratio of 1:16 substrates to DES solution is the optimal ratio when the lignin content after pretreatment is considered the criterion.

At ratios of 1:12 and 1:24 substrate to DES solution, the cellulose recovery was 84.13 ± 0.77% and 84.38 ± 0.02%, respectively (Table 3). However, at a ratio of 1:24 substrate to DES solution, the results indicate the highest lignin content compared to other variations in the substrate to DES solution ratio. Because a ratio of 1:12 substrate (467 kJ/g-biomass) to DES solution had a lower energy input than at 1:24 (930 kJ/g-biomass), a 1:12 substrate to DES solution ratio was considered the optimum condition for cellulose recovery.

The ratio of 1:8 substrate to DES solution yielded the lowest cellulose concentration of 69.81 ± 0.62%. This could be because the solvent cannot break more ether bonds in lignin-carbohydrate complexes because of the high substrate concentrations used at this ratio, resulting in minimal lignin removal (Table 3). A high ratio of substrates to the DES solution may also lead to the accumulation of solid particles and an increase in system viscosity [44]. Additionally, the viscous system limits the mass transfer between the samples and the extraction phase. Consequently, the 1:8 substrate to DES solution ratio is not optimal because it diminishes lignin removal and cellulose recovery capabilities.

The substrate to DES solution ratio of 1:16 resulted in greater lignin removal. In contrast, at a substrate to DES solution ratio of 1:12, the cellulose recovery was higher with a low hemicellulose content of 16.76 ± 0.2%. At a substrate to DES solution ratio of 1:12, the energy input was lower than that at 1:16, resulting in energy inputs of 467 kJ/g-biomass and 622 kJ/g-biomass, respectively. The results indicated that the optimum substrate to DES solution ratio was 1:12, resulting in low lignin content and high cellulose content.

3.4. Enzymatic Hydrolysis of DES Pretreated Sugarcane Leaves by the Cellulolytic Consortium

Sugarcane leaves were pretreated using DESs at a molar ratio of 1:6 and 120 °C for 3 h, and a substrate to DES solution ratio of 1:12 before being subjected to enzymatic hydrolysis. Untreated sugarcane leaves were used as the controls. The highest reducing sugar concentrations of 1.49 ± 0.28 g/L and 2.36 ± 0.02 g/L were obtained from untreated and DES-pretreated sugarcane leaves on day 7 (

Table 4. The effects of substrate loading on the enzymatic hydrolysis of untreated and DES pretreated sugarcane leaves by the cellulolytic consortium.

Substrate Loading (g-VSadded)	Reducing Sugar Concentration (g/L)
	Day 0	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7
U-1	0.15 ± 0.02 a	0.40 ± 0.01 e	0.28 ± 0.01 e	0.40 ± 0.04 d	0.36 ± 0.01 e	0.44 ± 0.01 d	0.37 ± 0.01 f	0.26 ± 0.24 f
U-2	0.16 ± 0.02 a	0.21 ± 0.01 g	0.43 ± 0.00 e	0.50 ± 0.00 d	0.78 ± 0.05 d	0.88 ± 0.05 c	0.96 ± 0.02 d	0.85 ± 0.04 e
U-3	0.17 ± 0.02 a	0.81 ± 0.03 c	0.50 ± 0.02 d	0.72 ± 0.02 c	0.90 ± 0.02 c,d	0.98 ± 0.00 c	1.70 ± 0.01 d	1.13 ± 0.01 d
U-4	0.17 ± 0.02 a	0.35 ± 0.02 f	0.36 ± 0.00 f	0.74 ± 0.02 c	1.12 ± 0.20 b	1.66 ± 0.33 a	1.64 ± 0.29 c	1.49 ± 0.28 c
P-1	0.17 ± 0.02 a	0.41 ± 0.02 e	0.36 ± 0.03 f	0.49 ± 0.02 d	0.47 ± 0.09 e	0.60 ± 0.14 d	0.61 ± 0.11 e	0.35 ± 0.07 f
P-2	0.17 ± 0.02 a	0.70 ± 0.01 d	0.57 ± 0.02 c	0.86 ± 0.15 b	0.95 ± 0.11 c	1.43 ± 0.02 b	1.65 ± 0.06 c	1.85 ± 0.15 b
P-3	0.16 ± 0.00 a	1.18 ± 0.03 a	1.52 ± 0.01 a	1.56 ± 0.02 a	1.71 ± 0.02 a	1.78 ± 0.02 a	1.97 ± 0.08 b	1.95 ± 0.07 b
P-4	0.17 ± 0.02 a	0.95 ± 0.03 b	1.10 ± 0.03 b	0.92 ± 0.03 b	1.25 ± 0.03 b	1.88 ± 0.01 a	2.18 ± 0.02 a	2.35 ± 0.02 a

Untreated: untreated sugarcane leaves; P: pretreated sugarcane leaves; values marked with the same letters are not significantly different (p < 0.05).

). However, it should be noted that the substrate loading should not be greater than 4 g of volatile solids (VS)_added because solid accumulation can occur, limiting the mass transfer between the substrate and enzyme. Additionally, at the high loading of the pretreated biomass, enzymatic digestibility and fermentation efficiency were dramatically reduced as mixing became increasingly difficult as viscosity increased [44]. Therefore, a 4 g-VS_added was chosen as the optimum substrate loading for enzymatic hydrolysis by the cellulolytic consortium using DES-pretreated sugarcane leaves as substrates.

3.5. The Two-Stages Hydrogen and Methane Production by SHF and SSF

3.5.1. Hydrogen Production

The SHF and SSF processes were carried out to determine an efficient fermentation process to produce hydrogen and methane through a two-stage hydrogen and methane production process. Various substrate loadings, 1–4 g-VS, of DES-pretreated sugarcane leaves under the optimum conditions of a 1:6 molar ratio, 120 °C, 3 h, and a substrate to DES solution ratio of 1:12 were used as the substrate. Additionally, untreated sugarcane leaves were used as a control at various substrate loadings of 1–4 g-VS.

The hydrogen production rate and hydrogen production potential of the DES-pretreated sugarcane leaves by SSF of the two-stage hydrogen and methane production processes were higher than those of untreated sugarcane leaves (

Table 5. Comparison of hydrogen production by SSF and SHF of the two-stage hydrogen production process.

Substrate Loading (g-VSadded)	SSF-Two Stages Hydrogen Production					SHF-Two Stages Hydrogen Production
	Hydrogen Production (mL/L)	Hydrogen Production Rate (mL/L d)	Lag Phase (λ) (d)	Hydrogen Yield (mL/g-VSadded)	R2	Hydrogen Production (mL/L)	Hydrogen Production Rate (mL/L d)	Lag Phase (λ) (d)	Hydrogen Yield (mL/g-VSadded)	R2
U_1	21 ± 4 e	1.8 ± 0.8 h	0.0	1.0 ± 0.1 f	0.9826	17 ± 1 e	1.5 ± 0.2 h	0.5	0.8 ± 0.1 f	0.9844
U_2	66 ± 5 e	10.0 ± 2.0 g,h	2.0	1.7 ± 0.1 f	0.9935	32 ± 15 e	6.2 ± 6.4 g,h	4.0	0.8 ± 0.4 f	0.9814
U_3	133 ± 14 e	22.0 ± 3.3 f,g	2.0	2.2 ± 0.2 f	0.9958	52 ± 10 e	14.2 ± 5.2 g,h	5.0	0.9 ± 0.2 f	0.9892
U_4	229 ± 20 e	34.7 ± 3.9 e,f	2.0	2.9 ± 0.2 f	0.9962	99 ± 81 e	20.2 ± 15.4 f,g,h	5.0	1.2 ± 1.0 f	0.9885
P_1	152 ± 7.0 e	23.0 ± 2.2 f,g	0.5	7.6 ± 0.3 e	0.9947	48 ± 19 e	7.9 ± 3.6 g,h	3.0	2.4 ± 1.0 f	0.9902
P_2	821 ± 48 d	85.9 ± 5.1 d	4.0	20.5 ± 1.2 c	0.9956	682 ± 154 d	40.9 ± 10.7 e	4.0	17.0 ± 3.8 d	0.9916
P_3	1840 ± 201 c	211.2 ± 14.7 b	4.0	30.7 ± 3.3 b	0.9978	1700 ± 136 c	112.4 ± 7.2 c	5.0	28.1 ± 1.9 b	0.9962
P_4	3187 ± 209 a	317.5 ± 20.5 a	5.0	39.8 ± 2.6 a	0.9941	2135 ± 315 b	197.2 ± 20.5 b	7.0	26.7 ± 3.9 a	0.9952

Untreated: untreated sugarcane leaves; P: pretreated sugarcane leaves; values marked with the same letters are not significantly different (p < 0.05).

). The highest hydrogen production and production rates of 3187 ± 202 mL H₂/L and 317.5 ± 20.5 mL H₂/L.d, respectively, were achieved by SSF using DES-pretreated sugarcane leaves at a substrate loading of 4 g VS (Table 5). Due to the presence of lignin in the untreated sugarcane leaves, their hydrogen production was lower than that of the DES-pretreated sugarcane leaves.

Similar findings on hydrogen production were obtained with SHF, and similar findings on hydrogen production were obtained with the SHF process. DES-pretreated sugarcane leaves had higher hydrogen production, a higher hydrogen production rate, and a shorter lag phase than the untreated (Table 5). The highest hydrogen production and hydrogen production rate were achieved at 4 g VS of DES-pretreated sugarcane leaves, resulting in 2135 ± 315 mL H₂/L and 197.2 ± 20.5 mL H₂/L.d, respectively. The untreated sugarcane leaves at 4 g VS produced hydrogen and hydrogen production rates of 99 ± 81 mL H₂/L and 20.2 ± 15.4 mL H₂/L, respectively.

3.5.2. Methane Production

The hydrogenic effluent from the first stage was used to produce methane during the second stage. Methane production (3583 ± 128 mL CH₄/L) from the SHF process using the optimum substrate loading of 4 g VS of DES-pretreated sugarcane leaves was lower than that from the SSF process (5923 ± 251 mL CH₄/L) (

Table 6. Comparison of methane production by SSF and SHF of the two-stage methane production process.

Substrates Loading (g-VSadded)	SSF Two-Stages Methane Production					SHF Two-Stages Methane Production
	Methane Production (mL/L)	Methane Production Rate (mL/L d)	Lag Phase (λ) (d)	Methane Yield (mL/g-VSadded)	R2	Methane Production (mL/L)	Methane Production Rate (mL/L d)	Lag Phase (λ) (d)	Methane Yield (mL/g-VSadded)	R2
U_1	1448 ± 354 f	29.9 ± 10.4 e	14	115.9 ± 28.3 c,d,e,f	0.9931	1468 ± 441 f	33.3 ± 13.9 d,e	13	117.4 ± 35.3 c,d,e,f	0.9921
U_2	2633 ± 390 c,d,e	64.7 ± 9.7 c,d,e	11	105.3 ± 15.6 d,e,f	0.9968	1974 ± 545 e,f	43.1 ± 15.6 d,e	12	91.1 ± 21.4 e,f	0.9972
U_3	3009 ± 516 b,c	63.1 ± 15.6 c,d,e	12	80.3 ± 13.8 e,f	0.9949	2847 ± 530 b,c,d	53.8 ± 3.1 d,e	10	75.9 ± 14.1 e,f	0.9950
U_4	3311 ± 806 b,c	76.8 ± 18.8 b,c,d,e	19	66.2 ± 16.1 e,f	0.9954	3097 ± 244 b,c	70.1 ± 3.6 b,c,d,e	18	61.9 ± 4.9 f	0.9926
P_1	2654 ± 277 c,d,e	101.8 ± 27.7 b,c	6	212.3 ± 22.2 a	0.9950	2077 ± 308 d,e,f	68.4 ± 25.4 b,c,d,e	6	166.1 ± 24.6 a,b,c,d	0.9873
P_2	3402 ± 760 b,c	109.7 ± 68.4 b,c	3	178.5 ± 72.0 a,b,c	0.9660	3644 ± 473 b	77.5 ± 25.3 b,c,d,e	16	201.2 ± 113.8 a,b	0.9795
P_3	5179 ± 291 a	160.1 ± 15.2 a	1	138.1 ± 7.8 b,c,d,e	0.9751	3221 ± 417 b,c	113.8 ± 30.9 b	1	85.9 ± 11.1 e,f	0.9797
P_4	5923 ± 251 a	159.3 ± 19.0 a	1	118.5 ± 5.0 c,d,e,f	0.9828	3583 ± 128 b	79.3 ± 9.8 b,c,d	2	71.7 ± 2.6 e,f	0.9778

Untreated: untreated sugarcane leaves; P: pretreated sugarcane leaves; values marked with the same letters are not significantly different (p < 0.05).

). Substrate loading influences VFAs production. The maximum methane content under the optimum condition obtained from the SHF and SSF processes were 51% and 50 %, respectively (data not shown). The results revealed that 4 g-VS of DES-pretreated sugarcane leaves gave the highest VFAs production (Figure 1) and hydrogen (Table 5) and methane production (Table 6) in both SSF and SHF processes.

3.6. One-Stage Methane Production by SHF and SSF

For the SHF process, DES-pretreated sugarcane leaves at 3 g-VS gave the highest methane production of 3723 ± 340 mL CH₄/L (

Table 7. Effects of substrate loading on enzymatic hydrolysis of untreated and DES pretreated sugarcane leaves by the cellulolytic consortium.

Substrates Loading (g-VSadded)	SSF One-Stage Methane Production					SHF One-Stage Methane Production
	Methane Production (mL/L)	Methane Production Rate (mL/L d)	Lag Phase (λ) (d)	Methane Yield (mL/g-VSadded)	R2	Methane Production (mL/L)	Methane Production Rate (ml/L d)	Lag Phase (λ) (d)	Methane Yield (mL/g-VSadded)	R2
U_1	1632 ± 481 f,g	27.4 ± 14.0 e	9	130.5 ± 38.5 a,b,c	0.9848	1597 ± 12 f,g	21.5 ± 6.3 e	10	127.8 ± 33.5 a,b,c	0.9918
U_2	2015 ± 370 e,f,g	30.0 ± 7.0 d,e	9	80.6 ± 14.8	0.9816	2179 ± 405 e,f,g	33.4 ± 5.1 d,e	18	87.2 ± 16.2 d,e,f,g	0.9938
U_3	2735 ± 186 c,d,e	42.6 ± 3.1 c,d,e	16	76.3 ± 5.5 d,e,f,g	0.9907	1941 ± 558 e,f,g	29.2 ± 11.3 e	16	51.7 ± 14.9 e,f,g	0.9629
U_4	2988 ± 112 b,c,d	46.8 ± 4.9 c,d,e	12	59.8 ± 2.2 e,f,g	0.9940	2304 ± 304 d,e,f	35.9 ± 7.5 c,d,e	15	46.1 ± 6.1 g	0.9867
P_1	2029 ± 104 e,f,g	41.8 ± 3.7 c,d,e	4	162.4 ± 8.3 a	0.9948	1853 ± 242 f,g	34.5 ± 7.9 d,e	9	148.3 ± 19.3 a,b	0.9948
P_2	2155 ± 449 e,f,g	59.3 ± 31.2 b,c	2	113.0 ± 44.9 b,c,d	0.9905	1478 ± 796 g	44.6 ± 17.7 c,d,e	2	74.7 ± 34.3 d,e,f,g	0.9600
P_3	3474 ± 613 a,b,c	79.8 ± 15.8 b	1	92.6 ± 16.3 c,d,e,f	0.9875	3723 ± 340 a,b	60.3 ± 13.4 b,c	12	99.3 ± 9.1 c,d,e	0.9965
P_4	4067 ± 319 a	120.5 ± 9.5 a	4	81.3 ± 6.4 d,e,f,g	0.9936	3349 ± 415 a,b,c	54.7 ± 16.6 c,d	3	67.0 ± 8.3 e,f,g	0.9793

Untreated: untreated sugarcane leaves; P: pretreated sugarcane leaves; values marked with the same letters are not significantly different (p < 0.05).

). Under these conditions, the methane content was 53 % (data not shown). Methane production increased with increasing substrate concentrations. However, methane production was reduced when the substrate concentration reached 4-gVS. This may be because of the accumulation of VFAs during AD. The results in Figure 2 show that at 4 g VS, acetic acid, butyric acid, and propionic acid accumulated in the methanogenic effluent.

One-stage methane production by SSF showed that the highest methane production of 4067 ± 319 mL CH₄/L was achieved using 4 g-VS DES-pretreated sugarcane leaves as the substrate (Table 7). Under these conditions, the methane content was approximately 56%. This result indicates that SSF supports a higher concentration of the substrate to produce methane than SHF for one-stage methane production. This might be because the soluble metabolite products produced by SSF were directly converted to methane during the methanogenesis stage, as shown in Figure 2. Therefore, there was no accumulation of VFAs which can inhibit and reduce methane production.

4. Discussion

4.1. The Effect of Molar Ratio and Type of Deep Eutectic Solvents (DESs) on Sugarcane Leaves

An efficient pretreatment can be achieved via the breakage of the lignin structure, which is the main biomass protective barrier, thereby enhancing the accessibility of solvents or enzymes into the biomass for sugar hydrolysis [45]. Furthermore, the DES mechanisms in biomass delignification are due to their ability to selectively cleave ether bonds without affecting C–C linkages [45]. Moreover, the ability of DES to donate and accept protons and electrons could reduce cellulose crystallinity owing to the disruption of H-bonds in the lignocellulosic biomass [45].

A strong basicity solvent is expected to remove lignin via saponification of intermolecular ester linkages, crosslinking xylan, hemicelluloses, and other components, such as lignin and other hemicelluloses. With the elimination of crosslinks, the porosity of lignocellulosic materials rises [46]. The basic solvent used in pretreatment operations causes swelling, which leads to an increase in the internal surface area, a decrease in the degree of polymerization, a reduction in crystallinity, separation of structural links between lignin and carbohydrates, and disruption of the lignin structure [46].

The H-bonds present in ChCl/G were identified as Ch⁺ and glycerol (cationic H-bond), chloride ions (Cl⁻) and glycerol (anionic H-bond), ChCl ion pairs (doubly ionic H-bonds), and glycerol-glycerol (neutral H-bonds) [47]. The close to neutral pH of ChCl/G is due to the Cl⁻ ion being surrounded by glycerol, resulting in a stronger glycerol-glycerol bond than the Cl⁻-glycerol bond. Moreover, the H-bond strength of lignin was stronger than that of ChCl/G lignin (lignin-lignin > Cl⁻/glycerol > Ch⁺/glycerol) [47]. Moreover, the H-bond energy of β-O-4 ether linkages in lignin is greater than that of ChCl/G [47]. Thus, ChCl/G, which has a weak H-bond interaction, cannot break the ether contained in the biomass [47].

The method of using strong acid solvents to disrupt the van der Waals forces, H-bonds, and covalent bonds that hold the cellulose and hemicellulose structures together in the lignocellulosic biomass, results in hemicellulose solubilization and cellulose reduction [48]. Furthermore, the acid catalyst hydronium ions cause long cellulose and hemicellulose chains to break down into sugar monomers [49]. Although using acid in the lignocellulosic pretreatment process removes hemicellulose and increases the lignocellulose pore size, the main disadvantage is the formation of inhibitors such as hydroxymethylfurfural (HMF), furfural, and other by-products, such as phenylic compounds and aliphatic carboxylic acids [41]. Additionally, the downstream process requires pH neutralization and significant biomass size reduction [50].

4.2. The Effect of Pretreatment Time and Pretreatment Temperature

The results of the variations in pretreatment temperature at each pretreatment time indicated that increasing the pretreatment temperature could increase the DES’s effectiveness in removing lignin from the lignocellulosic biomass. During this process, the cellulose content increased as the lignin content in the biomass decreased. This is because the application of heat in the DES pretreatment process can reduce the biomass mechanical strength and break the β-O-4-aryl ether bonds in the lignin-carbohydrate structure. Breaking this bond causes lignin to dissolve in the DES, causing a decrease in the lignin content in the biomass [51]. Additionally, an increase in the pretreatment temperature means that more energy is provided in the pretreatment system, which can increase the ability of the DES to break ether bonds [52]. Therefore, it is necessary to consider the energy used during the pretreatment process to determine the optimum pretreatment time and temperature.

4.3. Effects of the Substrate to DES Solution Ratio

The number of substrates used must be considered when determining the optimal substrate to DES solution ratio. The main benefit of using numerous substrates is that it improves the process efficiency because there is more biomass in the reaction system. Using more substrates can lower the amount of energy used in the pretreatment process [53] and utilizing a lower substrate to DES solution ratio can cause the solvent to break more ether bonds of lignin-carbohydrate complexes because of the high substrate concentrations used at this ratio, resulting in minimal lignin removal. A high ratio of substrates to the DES solution may also lead to the accumulation of solid particles and an increase in system viscosity [44]. In addition, the viscous system limits the mass transfer between the samples and the extraction phase.

4.4. The Enzymatic Hydrolysis of DES Pretreated Sugarcane Leaves by the Cellulolytic Consortium

The DES-pretreated sugarcane leaves had a higher reducing sugar content than untreated sugarcane leaves in all substrate loadings used (Table 4). This might be due to the complex structure of untreated sugarcane leaves that inhibits the cellulolytic consortium enzyme from accessing the cellulose contained in the lignocellulosic biomass structure of sugarcane leaves [37]. This causes the enzymatic hydrolysis process to be inefficient, as proven by the low sugar content of untreated sugarcane leaves as substrates for enzymatic hydrolysis. Therefore, pretreatment is essential for increasing the efficiency of the enzymatic hydrolysis process. Moreover, solid accumulation can occur due to the utilization of high substrate loading which limits the mass transfer between the substrates and the enzyme. Additionally, at a high loading of pretreated biomass, enzymatic digestibility and fermentation efficiency were dramatically reduced as mixing became increasingly difficult as viscosity increased [44].

4.5. The Two Stages of Hydrogen and Methane Production by SHF and SSF

4.5.1. Hydrogen Production

The untreated sugarcane leaves showed a lower hydrogen production and a longer lag phase than the DES-pretreated sugarcane leaves, which was caused by lignin in untreated sugarcane leaves. Lignin acts as a physical barrier to prevent enzyme access to hydrolyze cellulose and reduces hydrolysis efficiency [37]. Therefore, the cellulolytic consortium cannot access the cellulose in sugarcane leaves, resulting in less availability of fermentable sugars. Consequently, hydrogen production was low.

A comparison of the two-stage process of hydrogen production by SHF and SSF revealed that at high substrate loading, SSF was more efficient in producing hydrogen than SHF (Table 5). This is because of the accumulation of hydrolysis products in the SHF. In contrast, for SSF, the hydrolysis products are simultaneously produced and consumed. Thus, there is no accumulation of hydrolysis products that may inhibit the process.

4.5.2. Methane Production

Our results showed that SHF produced more acetic and butyric acid than SSF (Figure 1), which can cause a decrease in the pH of the SHF reactor system and result in low methane production (Table 6). Low pH adversely affects the activity of methanogens, resulting in low methane production during the AD process [37]. Methanogenic bacteria have been found to prefer a pH range of 6.5 to 7.5 to generate methane [54]. They have modest growth rates and are particularly sensitive to environmental changes. A reactor with a pH less than 6.0 is frequently related to reduced methane generation [55].

Substrate loading influences VFA production. The results revealed that 4 g VS of DES-pretreated sugarcane leaves gave the highest VFA production (Figure 2) and hydrogen (Table 5) and methane production (Table 6) in both SSF and SHF processes. It should be noted that utilizing a higher substrate loading of over 4 g VS could lead to system failure owing to the fast generation of VFAs [56]. Additionally, high substrate loading could drive the process to incomplete organic matter degradation owing to inhibition by overloading [56]. The results showed that increasing the substrate loading increased VFA production (Figure 2). This might be due to the increased availability of organic compounds, that is, fermentable sugars, at high substrate loading.

A literature search reveals insufficient information on two-stage and one-stage methane production from pretreated sugarcane leaves. Therefore, the methane yield obtained from this study (SSF and SHF) was compared with the literature search using a one-stage process (

Table 8. The comparison between the methane yield obtained in this study with the literature searches using pretreated sugarcane leaves as the substrate.

Substrate	Pretreatment Method	Pretreatment Conditions	Fermentation Mode	Methane Yield	References
Sugarcane leaves	Ammonium fiber explosion (AFEX) pretreatment	80–120 °C, 60 min	N/A-One-stage	336 mL/g-VSadded	[58]
	Sodium hydroxide (6% NaOH) pretreatment	25 °C, 3 days	N/A-One-stage	287 mL/g-TSadded	[57]
	Potassium hydroxide (KOH) pretreatment	170 °C, 60 min	N/A-One-stage	205 mL/g-TSadded	[59]
	Liquid hot water (LHW) pretreatment	190 °C, 60 min	N/A-One-stage	162 mL/g-TSadded	[59]
	Dilute acid (DA) pretreatment	170 °C, 15 min	N/A-One-stage	156 mL/g-TSadded	[59]
	DES Pretreatment	120 °C, 3 h	SSF-Two-stage	118 mL/g-VSadded	This study
	DES Pretreatment	120 °C, 3 h	SSF-One-stage	81 mL/g-VSadded	This study

N/A means the data was not applicable.

). The maximum methane yield of 118 and 81 mL/g-VS_added from DES-pretreated sugarcane leaves under optimum conditions for SSF two-stage hydrogen and methane production and one-stage methane production is less than that of sugarcane leaves pretreated with other pretreatment methods (Table 8). The results may have been affected by the disparity between the inoculum source and the initial substrate load. For instance, Luo et al., 2018 [57] utilized a NaOH pretreated sugarcane leaf with an initial concentration of 65 g-TS/L to produce methane, but our study utilized only 4 g-VS/L DES pretreated sugarcane leaf (equal to 4.62 g-TS/L). Pretreatment duration in this study is less than that of the ammonium fiber explosion (AFEX), NaOH, KOH, liquid hot water (LHW), and dilute acid (DA) methods.

Moreover, the pretreatment temperature used in this research was lower than the KOH, LHW, and DA pretreatment. Results revealed the advantages of the DES pretreatment in terms of energy efficiency. Furthermore, DES can be recovered and regenerated for subsequent pretreatment. AFEX pretreatment, on the other hand, utilizes more complicated equipment, which is likely to increase initial production costs, as well as high pressure in explosive systems, which can reduce the efficiency of the pretreatment process.

4.6. One-Stage Methane Production by SHF and SSF

The results in Figure 2 show that at 4 g VS, acetic acid, butyric acid, and propionic acid accumulated in the methanogenic effluent. The accumulation of this product is due to a separate enzymatic hydrolysis process that converts the cellulose in DES-pretreated sugarcane leaves to glucose, which will later be converted in the acidogenesis process to produce VFAs. After the enzymatic hydrolysis process, glucose production increases the VFAs in acidogenesis, which causes the accumulation of VFAs, thereby reducing methane production at the end of AD [60]. Moreover, acetic acid accumulation may be due to the cellulolytic consortium producing such VFAs during cellulose degradation. In contrast, for SSF, the VFAs were directly consumed by methanogenic bacteria to produce methane. Therefore, the VFA products in the SSF were lower than those in the SHF, leading to higher methane production in the SSF.

As shown in Figure 2, the results indicate that SSF supports a higher substrate loading concentration to produce methane than SHF for one-stage methane production. This might be because the soluble metabolite products produced by SSF were directly converted to methane during the methanogenesis stage. Therefore, there was no accumulation of VFAs which can inhibit and reduce methane production. The loading substrates in the AD system are critical factors that can affect the efficiency of the AD system. At low substrate loadings, there is a possibility that the biogas produced will make the overall process inefficient. However, at the same time, if the loading substrate is too high, the products produced during the AD process will accumulate, which will cause AD process inhibition [61].

Lignocellulosic biomass biodegradability increases with decreasing lignin content [62]. Therefore, reducing the lignin content can improve the effectiveness of enzymatic hydrolysis by a cellulolytic consortium as the enzymes can easily access the cellulose in pretreated substrates. In this study, the lignin content of sugarcane leaves pretreated with DES was significantly reduced, resulting in a significant improvement in biogas production. Therefore, it can be concluded that sugarcane leaves pretreated with DES at 4-g-VS under optimal conditions and the addition of a cellulose consortium for enzymatic hydrolysis can increase methane production through one-stage methane production by SSF.

5. Conclusions

A DES pretreatment efficiently removes lignin from lignocellulosic biomass. ChCl: MEA is the most effective at eliminating the lignin in the sugarcane leaves, with a lignin removal efficiency of 76.41–77.62% for every molar ratio. The two-stage hydrogen and methane production from DES-pretreated sugarcane leaves hydrolyzed by a cellulolytic consortium demonstrated increased hydrogen production compared to untreated sugarcane leaves in SSF and SHF processes. Methane production in a single stage by SSF and SHF from DES-treated sugarcane leaves and hydrolysis by a cellulolytic consortium was greater than methane production from untreated sugarcane leaves. SSF is an optimal fermentation method for two-stage hydrogen and methane production and one-stage methane production. This study demonstrates the utilization of sugarcane leaves to produce bioenergy (hydrogen and methane) while mitigating the PM 2.5 issues associated with burning sugarcane leaves.