Improved glucose recovery from durian peel by alkaline-catalyzed steam pretreatment

Schematic overview of experimental program

A general flow diagram of the alkaline-catalyzed steam pretreatment process to recover glucose from durian peel is depicted in Fig. 2. The process mainly includes sample collection and processing, pretreatment, compositional analysis of raw and pretreated durian peel, and enzymatic hydrolysis. Morphological changes after pretreatment were also studied using X-ray diffraction and scanning electron microscopy.

Collection and processing of durian peel

Collection and processing of durian peel were carried out as previously described in Obeng, Premjet & Premjet (2019). Monthong and chanee peels (refer to Fig. 1) were sampled from Phitsanulok Province, Thailand. The durian peel was cut into smaller fragments, open dried for 10 days, and powdered using a wood miller (SM 100; Rtsch, Rheinis-che StraBe 36-D-42781, Haan, North Rhine-Westphalia, Germany). The powder was subjected to sieving through a mesh with a pore size of 150–300 µm and subsequently put into airtight plastic bags at room temperature prior to further study.

Alkaline-catalyzed steam pretreatment of durian peel

A pretreatment experiment was conducted by varying only one factor at a time while keeping the other fixed. Firstly, the effect of NaOH concentration (1%, 2%, 3%, and 4% w/v) was investigated at an autoclave (TOMY SX-500; Tomy Digital Biology Co., Ltd., Tagara, Nerima-ku, Tokyo, Japan) temperature of 120 °C and 1.0 g/8.0 mL for solid/liquid ratio. Further, durian peel was pretreated by autoclaving at 110 °C, 120 °C, and 130 °C with the best NaOH concentration and 1.0 g/8.0 mL for solid/liquid ratio. The pretreatment process was carried out in a 125 mL Erlenmeyer flask for 60 min. The contents of each flask were thoroughly mixed and tightly covered with aluminum foil before autoclaving. After pretreatment, the pretreated material was quickly placed on ice to cool and subsequently filtered. The solid phase was thoroughly washed with distilled water till a pH of 7.0 was attained and used for further analysis.

Compositional analysis of durian peel

The structural carbohydrates and lignin (Sluiter et al., 2012) contents of durian peel were analyzed according to the National Renewable Energy Laboratory (NREL, Golden, CO, USA) analytical procedures. The contents of monosaccharides were determined as described in Obeng, Premjet & Premjet (2019) using the high-performance liquid chromatography (HPLC) system (Agilent 1100, Agilent Technologies, Waldbronn, Germany). The HPLC system consisted of a G1362A (Agilent Technologies, Waldbronn, Germany) refractive index detector (RID) and Bio-Rad Aminex HPX-87P column (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The HPLC system was run at a temperature of 80 °C and the injection of 20 µL of each sample. Filtered HPLC-grade water was used as an eluent at a flow rate of 0.6 mL/min.

Enzymatic hydrolysis

Enzymatic hydrolysis was carried out following the method described in Obeng, Premjet & Premjet (2018). The untreated and pretreated durian peel was hydrolyzed by suspending 0.1 g of peel (dry weight) in 10 mL of digestion solution. The solution was made up of 0.05 M sodium citrate buffer (pH 4.8, w/v), 2% (w/v) sodium azide, and an enzyme cocktail of 30 filter paper units (FPU) of cellulase (celluclast 1.5 L, Sigma-Aldrich, St. Louis, MO, USA) together with 60 U β-glucosidase (Oriental Yeast Co., Ltd., Tokyo, Japan) per gram of dry biomass. The reaction was conducted in a 50 mL Erlenmeyer flask at a temperature of 50 °C and fixed rotation of 150 rpm (revolution per minute) with a rotary shaker (Innova 4340; New Brunswick Scientific Company, Edison, NJ, USA) for 72 h. Hydrolysates were collected periodically (12 h, 24 h, 48 h, and 72 h) and assayed for glucose concentration using the HPLC. The enzymatic hydrolysis efficiency and glucose recovery were calculated as follows: ${\displaystyle \mathrm{HE}\left(\text{\%}\right)=\frac{\text{Glucose released}\left(\mathrm{g}\right)}{1.11\times \text{Glucan in initial biomass}\left(\mathrm{g}\right)}\times 100}$ ${\displaystyle \mathrm{GR}\left(\text{\%}\right)=\left[\mathrm{SR}\left(\text{\%}\right)\times \text{Glucan}\left(\text{\%}\right)\times 1.11\times \mathrm{HE}\left(\text{\%}\right)\right]\times 100}$ where HE is hydrolysis efficiency, GR is glucose recovery, and SR is solid recovery. The glucan to glucose conversion factor is 1.11.

Scanning electron microscopy (SEM)

Scanning electron microscopy (SEM) analysis was performed with an LEO 1455VP microscope (Zeiss, Gottingen, Germany). The untreated and pretreated durian peel was freeze-dried and mounted on aluminum stubs using double-sided tape for visualization. The surface of the biomass was then sputter-coated with a thin gold layer and visualize under the microscope at a magnification of 500 × and beam accelerating voltage of 20 kV.

X-ray diffraction (XRD)

The untreated and pretreated durian peel were washed with acetone three times, dried at 32 °C, and powdered to the size of 150 µm. The crystallinity of the biomass was then investigated with PANalytical X’pert Pro, PW 3040/60 diffractometer (Almelo, The Netherlands) by scanning from 2 θ = 10° to 40° at the rate of 0.02° s⁻¹. The crystallinity index (CrI) was calculated as follows: ${\displaystyle \mathrm{CrI}\left(\text{\%}\right)=\frac{{\mathrm{I}}_{002}-{\mathrm{I}}_{\mathrm{am}}}{{\mathrm{I}}_{002}}\times 100}$ where I₀₀₂ is the peak intensity of the crystalline peak and I_am is the peak for the amorphous cellulose.

Statistical analysis

All experiments in this study were conducted on a dry weight basis and included a minimum of three replicates. Data reported are the mean values with their standard deviations. The data were statistically analyzed using one-way analysis of variance (ANOVA) at a 5% significance level. Tukey’s test was used for multiply comparisons. Microsoft Excel 2010 was used for drawing graphs.

Compositional analysis

Compositional analysis (dry weight) of the untreated durian peel in the current study revealed the presence of mainly glucan, xylan, and lignin (refer to Tables 1 and 2). These components represent approximately 70% and 69% of the total content of monthong and chanee peels, respectively.

Impact of sodium hydroxide concentration

Pretreatment by different concentrations of NaOH had a significant effect on the chemical composition of both monthong and chanee peels (refer to Table 1). The glucan content of both cultivars significantly (p = 0.0001) increased with a concomitant decrease (p = 0.0001) in both xylan and lignin as NaOH concentration increased at the set temperature of 120 °C. When compared to untreated durian peel, a maximum of 1.5-fold increase in glucan content was observed after pretreatment with 4% (w/v) NaOH for both cultivars. A decrease in lignin content was more obvious in peels from both cultivars. Delignification in the range of approximately 62% to 89% for pretreated monthong peel and 60% to 88% for pretreated chanee peel were observed.

Hydrolysis efficiency of pretreated monthong and chanee was significantly (p = 0.0001) improved when NaOH concentration was increased (refer to Figs. 3 and 4). Maximum hydrolysis efficiency (90.89%) was observed by pretreating monthong peel with 4% (w/v) NaOH concentration, which was similar (p = 1.000) to that (90.85%) observed by pretreatment with 3% (w/v) NaOH concentration (refer to Fig. 3). However, maximum (p = 0.027) hydrolysis efficiency (90.09%) was observed by pretreating chanee peel with 4% (w/v) NaOH concentration (refer to Fig. 4). Glucose recovery in pretreated peels from both cultivars was significantly (p = 0.0001) improved following enzymatic hydrolysis. Maximum glucose from pretreated monthong (34.92%) and chanee (32.92%) were significantly (p = 0.0001) recovered when peels were exposed to 2% (w/v) NaOH concentration. However, reduction in the glucose recovered from both cultivars was observed when pretreatment of peels was conducted at higher (3% and 4% w/v) NaOH concentrations. Considering substantial recovery of glucose and maximum delignification, 2% NaOH (w/v) is observed as an optimum dosage with 60 min of reaction time for pretreatment of durian peel from both cultivars.

Impact of autoclave temperature

Autoclave heating in this study contributed to durian peel with significantly (p = 0.0001) lower lignin, xylan, and higher glucan contents than those of their respective untreated peel biomass (refer to Table 2). Increasing autoclave temperature resulted in decreasing lignin and xylan contents in monthong and chanee peels. In contrast, the relative amount of glucan in durian peel increased when autoclave temperature was increased. Durian peel subjected to 130 °C presented the lowest lignin content of approximately 86% and 85% delignification for monthong and chanee peels, respectively.

Increasing autoclave temperature resulted in a significant (p = 0.0001) increase in enzymatic hydrolysis efficiency in both pretreated monthong (refer to Fig. 5) and chanee (refer to Fig. 6) peels. The highest enzymatic hydrolysis efficiency of approximately 91% and 90% were achieved in monthong and chanee peels pretreated at an autoclave temperature of 130 °C, respectively. In contrast, increasing autoclave temperature resulted in a significant decrease (p = 0.0001) in glucose recovery of both pretreated monthong and chanee peels. Maximum glucose of about 36% and 35% in monthong (refer to Fig. 5) and chanee (refer to Fig. 6) peels pretreated at 110 °C, respectively, were recovered.

Ultrastructural morphology

The impact of alkaline-catalyzed steam pretreatment on structural changes in monthong and chanee peel was analyzed. The ultrastructural morphology of the untreated peel from monthong and chanee revealed structural uniformity with less surface destruction (refer to Figs. 7–10). However, pretreated monthong and chanee peels showed more destructive surface morphology (refer to Figs. 7B–7E, 8B–8E, 9B–9D, and 10B–10D). Generally, after pretreatment by different NaOH concentrations, the fibers of both monthong and chanee peels were disconnected. This resulted in the creation of long cracks on the surface of the peel biomass. The cracks became more extensive and deep as NaOH concentration was increased.

The surface of monthong and chanee peels pretreated at the various autoclave temperatures was also rougher and more disordered. There was a fragmentation of the surface structure of peel biomass by increasing autoclave temperature. At 130 °C, the complete collapse of the peel biomass surface structure was observed.

Crystalline structure

Modifications to cellulose crystallinity (refer to Figs. S1–S4) during alkaline-catalyzed steam pretreatment of durian peel were also investigated. The crystallinity index (CrI) for both cultivars (refer to Tables 1 and 2) increased after the pretreatment process. Increasing NaOH concentration during pretreatment led to an increase in CrI for monthong and chanee (refer to Table 1). The CrI of untreated monthong peel increased from 28.01% to 44.62% after pretreatment with 4% NaOH concentration. For untreated chanee peel, CrI increased from 20.72% to 37.71% following pretreatment with a 4% NaOH. Similarly, CrI for monthong and chanee increased as autoclave temperature was increased. High CrI values of 43.03% and 35.63% for monthong and chanee, respectively, were attained at the highest autoclave temperature of 130 °C (refer to Table 2).