Investigation of alkaline hydrogen peroxide pretreatment to enhance enzymatic hydrolysis and phenolic compounds of oil palm trunk

Abstract

Alkaline hydrogen peroxide (AHP) as a pretreatment effectively enhances the increasing enzymatic digestibility of oil palm trunk (OPT) for conversion to biofuels and bioproducts in the biorefinery processes. The effect of hydrogen peroxide concentration (1–5%), temperature (50–90 °C), and time (30–90 min) were studied to find out the optimum condition for the removal of lignin. The optimum condition attained at 70 °C, 30 min, and 3% H₂O₂ g /g of biomass not only increased the cellulose content from 38.67% in raw material to 73.96% but also removed lignin and hemicellulose up to 50% and 57.12%, respectively. The AHP-treated fibers subjected to enzyme hydrolysis showed significant improvement in glucose concentration that increased from 11.77 (± 0.84) g/L (raw material) to 46.15 (± 0.32) g/L with 59.82% enzyme digestibility at 96 h. Scanning electron microscopy (SEM) and Fourier transformation infrared (FT-IR) were employed to analyze the morphology and structural changes of untreated and AHP-treated fibers. SEM results showed disruption of the intact OPT structure resulting in increase of enzyme accessibility to cellulose. The FT-IR identified changes in peaks which indicated structural transformation and dissolution of both lignin and hemicellulose molecules caused by AHP treatment. The black liquor obtained from AHP treatment contained about 5.13 mg gallic acid equivalent (GAE)/g of dry sample of total phenolic content (TPC) and an antioxidant activity of 59.80% and 65.51% inhibitions of DPPH and ABTS assays, respectively. Hence, it is a sustainable approach to utilize waste for the recovery of multiple value-added products during pretreatment process.

Introduction

Upsurging demand of energy and the diminishing natural resources of fossil fuels have gathered the attention of scientists to renewable energy resources as a substitute. Lignocellulosic biomass has the potential to serve as an abundant low-cost agricultural feedstock for the production of bioethanol.

Oil palm trunk (OPT), a valuable lignocellulosic biomass, comprise interpenetrated polymeric components, including cellulose, hemicellulose, and lignin, as potential source of fermentable sugars which can be converted to bio-based fuels (Yetti et al. 2012). Cellulose, the main component of oil palm trunk (OPT), consists of glucose monomeric units linked by β-1,4 glycosidic bonds to form high-molecular weight polymer. Glucose conversion yield of OPT is comparatively higher than rubber wood sawdust and mixed hardwood sawdust (Chin et al. 2011). In addition, hemicellulose, a shorter polymer, contains some sugar monomers, while lignin is a three-dimensional polymer with propyl-phenol units that provide structural support to plant cell wall. The economic life span of OPT begins deteriorating at the age of 25–30 years. Thereby, it is essential to cut the old palm trees and to replant new ones that causes an immense OPT waste generation at plantation sites, which is mostly utilized in making plywood and treated as agricultural waste or left as heap in the fields (Shahirah et al. 2015).

Lignocellulose degradation by anaerobic bacteria is challenging due to its heterogeneous complexity (Reddy and Yang 2005). An effective and specific pretreatment is essential to break down the robust physical, chemical association and disrupt the crystalline structure of cellulose to escalate the access of cellulose enzyme (Saha 2004). There are numerous pretreatments, classified as physical, chemical, and biological methods (Hendriks and Zeeman 2009; Beukes and Pletschke 2011) of lignocellulosic pretreatment in which some methods use acid or alkali at high temperature for breakdown of hemicellulose, removal of lignin, and providing improved accessibility for cellulose hydrolysis (Saha 2003). However, these approaches have major shortcoming of producing sugar degrading products, i.e., hydroxymethylfurfural and furfural which are highly toxic for fermentative micro-organisms. Among these methods, alkaline hydrogen pretreatment (AHP) is one of the utmost favorable chemical pretreatments for a wide range of lignocellulosic biomass that can provide a high degree of enzymatic hydrolysis efficiency. The alkali in AHP is responsible for saponification reactions that cleave acetate bonds on the hemicelluloses, which then increases their solubility (Pedersen and Meyer 2010). AHP pretreatment for delignification is pH dependent with an optimum pH of 11.5–11.6 which is the pKa for hydrogen peroxide decomposition reaction (Gould 1985; Palamae et al. 2014). Hydrogen peroxide participates in natural delignification process by reacting with both aliphatic and aromatic structures of lignin for depolymerization (Yang et al. 2002) and formation of reactive oxygen species (ROS), i.e., hydroxyl radicals and superoxide ions which are responsible for oxidation of phenolic compounds (Pan et al. 1998). During AHP pretreatment, the degraded or solubilized lignin, a natural polymer and rich source of aromatic rings, possesses high antioxidant properties and can be used as an effective free radical scavenger (Sun et al. 2014; Chen et al. 2015).

Lignocellulosic delignification prior to enzymatic hydrolysis and fermentation is one of the key processes that produce appropriate fermentable substrate. Apart from that, cellulose crystallinity, available surface area, porosity, presence of hemicellulose, and lignin are important factors that can affect the enzymatic hydrolysis step, such as patterns of releasing sugar and enzyme activity (Alvira et al. 2010). Enzymatic hydrolysis helps understand the significant effect of AHP as a pretreatment for delignification of OPT by increasing enzyme digestibility.

The aim of this study is to investigate the use of alkaline hydrogen peroxide (AHP) with the influence of time, temperature, and concentration of hydrogen peroxide (H₂O₂) to identify the optimum AHP condition that can remove lignin and hemicellulose efficiently to help improve the efficiency of enzymatic hydrolysis. To the best of author’s knowledge, such effect of AHP pretreatment at low or moderate temperature, particularly on oil palm trunk, is very limited in the literature. This study examines the potential of recovering both cellulose and phenolic compounds along with antioxidant activity from OPT during the pretreatment process. Production of value-added bio-products, i.e., phytochemicals from OPT and black liquor during pretreatment process for bioethanol production, may not only be environment friendly but also add economic value toward sustainable development for the oil palm industry.

Materials and methods

Materials

Oil palm trunk (Elaeis guineensis Jacq.) was purchased from local agriculturist in Plai Phraya District, Krabi province, Thailand. All chemical reagents were analytical grade and purchased from Sigma-Aldrich. Cellobiose (99%), glucose (99%), sulfuric acid (98%), hydrogen peroxide (30%), sodium hydrogen peroxide (97%), Folin--Ciocalteu reagent (FCR), sodium carbonate, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and DI water were used for the solutions preparation. Steam explosion machine (Kumakai Nitto, Japan), UV–Vis spectrophotometer, water bath, scanning electron microscope (SEM) (Hitachi TM3000, USA), and Fourier transform infrared spectroscopy (FT-IR) (Bruker Co., Karlsruhe, Germany) were used in this experiment.

Methods

Pretreatment

Steam explosion and hot water washing

Initially, the oil palm trunk (OPT) (Elaeis guineensis Jacq.) samples were chopped by wood chipper into 20 × 20 × 5 mm³. A dry sample of 150 g OPT chips was placed in a 2.5-L tank for steam explosion at 210 °C for 4 min (Songprom et al. 2011). The combined pulp slurry was collected and squeezed to get the steam-exploded fibers. Later, the steam-exploded fibers were treated with hot water washing at 80 °C for 30 min with a total solid:liquid ratio of 1:8 (g/mL).

Effect of alkaline hydrogen peroxide pretreatment

The steam-exploded fibers were pretreated with alkaline hydrogen peroxide at varied temperatures of 50 °C, 70 °C, and 90 °C; reaction times were 30 min, 60 min, and 90 min, and hydrogen peroxide loading of 1%, 3%, and 5% (g H₂O₂/g fibers) with the solid loading of 10 wt% and the pH 11.5 (± 0.2) was adjusted with 5 M NaOH solution. Reaction time was started after the sample reached the desired temperature. Overall, 27 different conditions were applied with 3 replicates and the chemical composition of the treated fibers were analyzed after every step of pretreatment by the given standard analysis of the technical association of the pulp and paper industry (TAPPI).

Scanning electron microscopy (SEM) analysis

The morphology of the biomass before and after pretreatment was observed using scanning electron microscope (SEM) (Hitachi TM3000, USA). The acceleration voltage was 5.0 kV. The sample was mounted on aluminum sample stubs, and before analysis, the sputter coated the sample with a thin layer of gold (Azmi et al. 2018).

Fourier transformation infrared (FT-IR) analysis

The presence of any changes in functional groups during pretreatment in the samples was scanned by FT-IR spectroscopy (Bruker Co., Karlsruhe, Germany) with KBr pellets made of 5% sample and pressed into a thin pellet followed by spectra produced as transmittance ranging from 4000 and 500 cm⁻¹ (Serrano et al. 2019).

Enzyme hydrolysis untreated and AHP treated fibers

The enzyme hydrolysis was conducted using Cellic ® CTec2: 10 FPU/g with 10% substrate loading. The CTec2 activity was obtained through following the NERL’s protocol and was 178.5 FPU (Filter paper unit). 10% dry weight of fibers from all three stages (1.raw material; 2. steam-exploded; 3. AHP-treated fibers) was taken separately in a 500-mL Erlenmeyer flask with addition of 270-mL citrate buffer (0.05N, pH 4.8) in it. Enzyme hydrolysis was conducted at 50 °C and 150 rpm. The samples were collected at 0, 2, 4, 6, 8, 10, 12, 15, 18, 24, 36, 48, 60, 72, 84, and 96 h (Kumneadklang et al. 2015). All enzymatic hydrolysates were analyzed for glucose content by high-performance liquid chromatography (HPLC). The enzyme hydrolysis were performed in triplicates and average results are stated. The enzyme digestibility was calculated according to Eq. (1):

$$\text{Enzyme digestibility}\%=\frac{(\text{Glucose})+1.053(\text{Cellobiose})}{1.111f(\text{Biomass})}\times 100$$ 1

where (Glucose) represents glucose concentration (g/L) released during enzyme hydrolysis; (Cellobiose) represents cellobiose concentration (g/L) released during enzyme hydrolysis; f is cellulose fraction in dry biomass (g/g); 1.053 is correction factor of cellobiose to equivalents of glucose; and 1.111 is the factor that converts cellulose to glucose.

Total phenolic contents of AHP black liquors

The black liquor that was collected after AHP treatments were used to study its total phenolic contents. A 2.5 mL of Folin--Ciocalteu reagent (0.2 mol/L) and 5 mL of 20% Na₂CO₃ solution were mixed with 0.5 mL of AHP black liquor. The mixture was incubated at room temperature in dark place for 120 min, and the absorbance was measured at 750 nm. The intensity of blue color was measured at 750 nm using UV–Vis spectrophotometer. A calibration curve was obtained using 50, 100, 150, 200, 250, and 300 ppm to calculate the total phenolic content of AHP black liquor as gallic acid equivalent (mg_GAE)/mL of extracts.

Antioxidant analysis of AHP black liquors

DPPH method

The antioxidant activity of black liquor was determined based on the use of the free radical scavenging effect on the DPPH radical by spectrophotometry method. 0.1 mL of AHP black liquor was mixed with methanolic solution of DPPH (3.9 mL of 6 × 10^–5 M) at room temperature for 30 min. The mixture was thoroughly vortex mixed, and the absorbance was measured at 518 nm against the blank. The results expressed as percentage inhibition of the DPPH radical were calculated according to Eq. (2). Trolox (1 g/L) was used as a standard (Alzagameem et al. 2018):

$$\%\text{Inhibition of DPPH}=\left\{\frac{\left(\text{Abs control}-\text{Abs sample}\right)}{\text{Abs control}}\right\}\times 100$$ 2

where Abs control represents absorbance of the DPPH solution without sample and Abs sample represents absorbance of DPPH solution with sample.

ABTS method

The ABTS free radical cation inhibition of antioxidant assay was performed by following the protocol of Okoh et al. (2014) with slight changes because the AHP black liquor was used to determine antioxidant activity of lignin. The preformed radical monocation of ABTS was generated by oxidation of 7 mM ABTS solution with 2.45 mM potassium persulfate solution in equal amount and was kept for 16 h at room temperature in dark. This solution was later diluted with methanol to achieve an absorbance of less than 0.800 at 734 nm. Three milliliters of the ABTS radical cation solution was added to 0.5 mL of AHP black liquor. The reaction mixture was vortexed thoroughly, left in the dark at room temperature for 20 min and measured at 734 nm. The percentage inhibition of ABTS radical from the AHP black liquor was calculated according to Eq. (2). Trolox (1 g/L) was used as a standard.

Analysis of cellobiose and glucose by HPLC

Cellobiose, glucose, and ethanol concentrations were analyzed using high-performance liquid chromatography (HPLC) (Agilent Technologies, Germany) equipped with a Bio-Rad Aminex HPX-87H column, automatic sampler, column heater, and refractive index detector (RID). The column was maintained at 50 °C and eluted with 5 mM sulfuric acid in water at 0.6 mL/min flow rate (Xue et al. 2015).

Statistical analysis

The data were expressed as mean values ± standard deviation (SD). A factorial design was performed to study the effect of factors on one another. Statistically significant differences between chemical compositional analyses of OPT were determined by the Duncan’s New multiple range test, using IBM SPSS Statistics version 23 (IBM Corp, USA). Level of statistical significance was set at 95% (p < 0.05).

Results and discussion

Effect of alkaline hydrogen peroxide pretreatment

To investigate the effect of alkaline hydrogen peroxide pretreatment (AHP) and study the optimum condition for delignification, 27 different conditions were used. Different variables, i.e., pretreatment time, H₂O₂ concentration, and pretreatment temperature, were studied, keeping pH and solid loading constant at 11.5 and 10% dry weight, respectively. The mechanism where alkaline hydrogen peroxide pretreatment enhances enzymatic saccharification appears to involve delignification of lignocellulosic material and increases the degree of hydration of the cellulose polymer (Gould 1984). Alkaline hydrogen peroxide treatment with pH at 11.5 caused a significant lignin degradation of 50%. A summary of these results is presented in Table 1, which represents the % dry weight of cellulose, hemicellulose, lignin, and ash after the AHP pretreatment. The AHP pretreatment increases the surface area, breaking the structural intermolecular bonds between carbohydrates and lignin (Song et al. 2016), disordering the lignin structure, and isolating lignin from the biomass (Mesquita et al. 2016). AHP conditions of 70 °C with 3% and 5% H₂O₂ concentrations for 30 min gave highest celluloses of 73.96% and 73.66%, respectively, which were comparatively higher to that of raw material (38.67%) (Table 2) and in comparison with AHP pretreatment study of Yilmaz et al. (2016) on sunflower for fermentable sugars. Furthermore, at the same optimum condition (70 °C, 30 min, 3% H₂O₂ concentration), the percentage of dry weight of hemicellulose, i.e., 12.9%, was found lower as compared to that of raw material, i.e., 30.22% (Table 2). A good pretreatment method should provide more hydrolyzed hemicelluloses fraction than the hydrolysis of the cellulose fraction. Hemicelluloses are easily hydrolyzed in alkaline medium than cellulose fibrils. The amorphous structure of hemicelluloses helps in hydrolysis, while fibrils being in crystalline form inhibit hydrolysis in alkaline medium (Scheller and Ulvskov 2010). The lowest dry weight percentage of pentosan observed at the same condition was 1.17 ± 0.1, although no significant difference was observed among the rest of the conditions. At the optimum condition of 70 °C, 30 min, 3% H₂O₂ concentration, lignin content showed appreciable decrease to the lowest 11.68%, and a 50% of lignin degradation was observed as compared to that of raw material, i.e., 23.76% dry weight. The results were found comparatively better than the findings of Li et al. (2013, 2016) who studied AHP pretreatment for improving delignification from 36.6 to 50.2% and 37.5 to 40.3%. On the other hand, Alvarez-Vasco and Zhang (2013) studied AHP pretreatment at relatively high temperature which resulted in less delignification of only 22%. The factors, i.e., temperature, time, and H₂O₂ concentration, exhibited significant effects on the percent lignin reduction during pretreatment (Williams 2014). Moreover, the results depicted that the same H₂O₂ concentration at higher temperature resulted in a slight decrease of the composition of both cellulose and lignin which could be attributed to exothermic reaction with froth formation. Other possibility could be the reason that high temperature decomposes H₂O₂ by reducing its oxidative delignification potential, and longer pretreatment time could result in recondensation and repolymerization of solubilized lignin in AHP black liquor (Silverstein et al. 2007). At higher concentration of H₂O₂, the rate of evolution of O₂ increases at rapid rate that reduces the oxygen incorporation at lignin sites, resulting in decreased delignification efficiency (Rojith and Singh 2013). Generally, various concentrations of H₂O₂ are used for lignocellulosic biomass depending upon its lignin content (Ross et al. 2008). Alvarez-Vasco and Zhang (2013) reported the optimum condition of hydrogen peroxide dosages of 4% for softwood, whereas Rabelo et al. (2013) successfully pretreated sugarcane bagasse with optimum H₂O₂ dosage of 7.36%.

Table 1.

Effect of alkaline hydrogen peroxide (AHP) pretreatment of oil palm trunk at different temperatures, times, and hydrogen peroxide concentrations

Temperature (°C)	Time (min)	% Concentration (g H2O2/g biomass)	% Dry weight of hemicellulose	% Dry weight of cellulose	% Dry weight of lignin	% Dry weight of ash
50	30	1	11.05 ± 0.17ef	66.56 ± 0.02	18.02 ± 0.95fghijk	0.89 ± 0.04
50	30	3	11.02 ± 0.31gh	67.61 ± 0.03	17.96 ± 0.16fghij	0.81 ± 0.01
50	30	5	9.93 ± 0.19e	69.77 ± 0.05	17.11 ± 0.56cdegf	0.77 ± 0.09
50	60	1	5.8 ± 0.98pq	67.86 ± 0.02	19.22 ± 1.14kl	1.12 ± 0.01
50	60	3	10.73 ± 0.78kl	64.92 ± 0.06	17.51 ± 0.91defgh	1.11 ± 0.08
50	60	5	9.57 ± 0.33q	63.39 ± 0.04	15.71 ± 0.72b	1.11 ± 0.03
50	90	1	3.64 ± 0.11k	62.54 ± 0.09	19.11 ± 0.68jkl	1.08 ± 0.07
50	90	3	9.17 ± 0.44pq	64.38 ± 0.07	18.91 ± 0.80ijkl	1.11 ± 0.19
50	90	5	9.88 ± 0.12j	67.23 ± 0.05	18.31 ± 0.75ghijk	0.91 ± 0.11
70	30	1	12.85 ± 0.34a	69.83 ± 0.04	16.11 ± 0.42bc	0.96 ± 0.14
70	30	3	12.9 ± 0.21a	73.96 ± 0.03	11.68 ± 0.24a	0.95 ± 0.06
70	30	5	12.45 ± 0.46b	73.66 ± 0.01	12.11 ± 0.63a	0.94 ± 0.04
70	60	1	12.24 ± 0.11c	69.91 ± 0.013	17.75 ± 0.31efghi	0.90 ± 0.07
70	60	3	11.62 ± 0.51i	66.24 ± 0.03	17.21 ± 0.21cdfeg	0.91 ± 0.12
70	60	5	7.27 ± 0.67hi	70.74 ± 0.03	17.12 ± 0.94cdefg	0.90 ± 0.16
70	90	1	10.05 ± 0.33kl	65.60 ± 0.04	16.87 ± 0.53cdef	1.07 ± 0.09
70	90	3	9.91 ± 0.61klm	68.18 ± 0.001	16.66 ± 0.52bcde	1.05 ± 0.07
70	90	5	4.8 ± 0.23pq	68.74 ± 0.05	16.32 ± 0.33bcd	1.02 ± 0.03
90	30	1	10.83 ± 0.7lmn	64.37 ± 0.07	19.21 ± 0.88kl	1.02 ± 0.09
90	30	3	4.21 ± 0.91op	69.59 ± 0.003	19.65 ± 0.60l	1.01 ± 0.87
90	30	5	8.65 ± 0.43g	70.13 ± 0.018	19.54 ± 0.36l	0.94 ± 0.06
90	60	1	9.9 ± 0.99pq	63.33 ± 0.022	19.63 ± 0.48l	1.09 ± 0.09
90	60	3	7.57 ± 0.11ij	70.22 ± 0.008	19.00 ± 0.12jkl	1.08 ± 0.06
90	60	5	5.7 ± 0.45k	70.41 ± 0.01	18.87 ± 0.97ijkl	1.08 ± 0.05
90	90	1	10.01 ± 0.65no	64.48 ± 0.06	18.44 ± 0.42hijkl	1.10 ± 0.01
90	90	3	7.73 ± 0.44mn	67.14 ± 0.011	18.09 ± 0.28fghijk	1.04 ± 0.01
90	90	5	8.61 ± 0.1g	70.17 ± 0.11	17.89 ± 0.64fghij	1.01 ± 0.02

^a−qMean values within the same column with different letters are significantly different (p < 0.05)

Table 2.

Compositional analysis of raw material and pretreated oil palm trunk

Condition	% Yield	% Dry weight of hemicellulose	% Dry weight of cellulose	% Dry weight of lignin	% Dry weight of ash	% Dry weight of pentosan
Raw material	100	30.22	38.67	23.76	1.62	23.30
Steam explosion	68.34	8.12	61.76	20.32	1.16	4.10
AHP pretreatment	39.00	12.9	73.96	11.68	0.95	1.17

In delignification mechanism, hydroxyl radicals produced from the breakdown of H₂O₂ in alkaline condition stimulate the deconstruction of hemicellulose and lignin (Rabetafika et al. 2014). However, H₂O₂ promotes the delignification by oxidative reaction (Lai 2001). The findings indicate that AHP is a promising pretreatment method for the effective retention of cellulose and efficient removal of lignin at low or moderate temperature (Li et al. 2018). The individual factors influenced cellulose content significantly (p < 0.05) (Table 1). Increase in temperature from 50 to 70 °C was directly proportional to cellulose content (66.36–69.65%); however, a decrease in cellulose content from 69.65 to 67.76% was observed when temperature increased from 70 to 90 °C, respectively. A similar trend was observed by Upajak et al. (2018), which may be attributed to higher temperature resulted in an excessive quantity of radicals led to decrease the recovery of cellulose (Bajpai 2012; Li et al. 2014). Thus, a higher temperature during alkaline hydrogen peroxide pretreatment should be avoided (Gupta and lee 2009). Results also depicted that longer pretreatment time significantly (p < 0.05) affected cellulose content. The cellulose content were 69.50%, 67.77%, and 66.49% at 30, 60, and 90 min, respectively. Decrease in cellulose content due to longer pretreatment time may be attributed to increase in solubilization of cellulose from the solid into liquid phase of AHP pretreatment that converts into monomeric sugar to produce inhibitory by-products (Mosier et al. 2005; Upajak et al. 2018). Moreover, at higher pretreatment temperature, a short pretreatment time is required to achieve maximum delignification (Rabelo et al. 2008). There was a direct relationship in increasing H₂O₂ concentration and cellulose content, which statistically varied significantly (p < 0.05). The H₂O₂ at 1%, 3%, and 5% led to 66.05%, 68.35%, and 69.36% of cellulose content, respectively. Generally, the interaction of these factors did not significantly influence cellulose content (p > 0.05). The removal of lignin and hemicellulose before enzymatic hydrolysis of lignocellulose is highly necessary because it increases the digestibility of cellulose. Comparatively, alkaline pretreatment requires higher temperature, greater amount of water for washing fibers, and sticky in nature due to the presence of high concentration of sodium hydroxide which makes it less environmental friendly.

Compositional analysis of raw material and pretreated oil palm trunk (OPT)

Prior to pretreatment, the chemical composition of raw material of oil palm trunk was 11.22% extractive substances, 30.22% hemicellulose, 38.67% cellulose, 23.76% lignin, 1.62% ash, and 23.30% pentosan on dry weight basis. However, after steam explosion and AHP pretreatment, the content of cellulose ascended to 73.96%, whereas the percentage quantity on dry weight basis was found to be descended to 12.9% hemicellulose, 11.68% lignin, 0.95% ash, and 1.17% pentosan. Moreover, after each step of pretreatment, such as steam explosion, 68.34% of fibers were recovered, whereas after AHP pretreatment, the fibers were reduced to 39% (Table 2).

Scanning electron microscopy (SEM) analysis

SEM was employed to observe the cross-sectional areas of the raw OPT and AHP-treated fibers (Fig. 1). The surface of raw OPT was intact and hard surface with well-ordered fiber bundles, which was not easy for the accessibility of enzyme to cellulose (Fig. 1a). The steam-exploded fibers were still compact and showed hardness (Fig. 1b). On the other hand, after AHP treatment, a structural change and physical deformation were evident (Fig. 1c). The fibers turn out to be rough, loose, disrupt with cracks, and pores on the surface (Phitsuwan et al. 2016). These structural changes were attributed to the degradation of lignin and hemicellulose (Zhang et al. 2019), disruption of linkages between lignin and hemicellulose (Cabrera et al. 2014), as well as the radical reaction of OH and lignin (Correia et al. 2013). The fibers were well separated, which could reduce the structural barrier and provide more surface area for enzyme accessibility (Sun et al. 2015).

Fig. 1.

Scanning Electron microscopy (SEM) analysis of a raw oil palm trunk (OPT), b steam-exploded fibers c alkaline hydrogen peroxide (AHP)-treated fibers

Fourier transformation infrared (FT-IR) analysis

The FT-IR spectroscopy was used to investigate the chemical structure changes of raw material after steam-exploded and AHP-treated fibers (Fig. 2). FT-IR analysis helped identify characteristic peaks of lignin and cellulose. Changes in peaks indicate structural transformation induced due to alkaline hydrogen peroxide pretreatment. The broad absorption 3445–3390 cm⁻¹ was related to OH stretching frequencies of cellulose (Jia et al. 2011). The insignificant shift in the spectra of the aliphatic methylene bands at 2924–2917 cm⁻¹ in steam-exploded (Fig. 2b) and AHP-treated fibers (Fig. 2c) was ascribed to C–H stretching which implied that the reaction did not involve any degradation process of the cellulosic fiber (Li et al. 2014; Mokhothu and John 2015). The noticeable peak at 1706 cm⁻¹ in raw material was attributed to the vibrations of carboxylic groups in lignin, which became missing after AHP pretreatment, suggesting that the carboxylic and ester bonds in hemicellulose and lignin were broken (Phitsuwan et al. 2016). In comparison to raw material, the AHP-pretreated fibers displayed significant decrease in band intensities at 1457 (C‒H deformation), 1508 (aromatic skeletal vibrations), and 1636 cm⁻¹ (conjugated C=O stretch), which indicates the breakdown of the aromatic structure of lignin. Band at 1374 cm⁻¹ after AHP treatment is ascribed to C-H deformation of cellulose and hemicellulose (Wang et al. 2013). The intensity of the peak at 1244 cm⁻¹ in steam-exploded fibers sharply decreased and in AHP-treated fibers is attributed to the removal of hemicellulose (Lamaming et al. 2015; Chieng et al. 2017). A sharp band at 897 cm⁻¹ which was missing in raw material was observed in AHP-treated fibers has been attributed the typical structure of cellulose due to β-glycosidic linkage between the sugar units in cellulose (Zhang et al. 2019). Complementing these results, the AHP treatment of OPT fibers successfully showed the presence of a pure cellulose and removed a considerable amount of lignin and hemicellulose.

Fig. 2.

Fourier transformation infrared (FT-IR) analysis of (A) raw oil palm trunk (OPT), (B) steam-exploded fibers (C) alkaline hydrogen peroxide (AHP)-treated fibers

Enzyme hydrolysis of untreated and AHP-treated fibers

In this study, enzyme hydrolysis of raw material, steam-exploded, and AHP-pretreated fibers were extensively studied. The native raw material was relatively resistant to enzymatic hydrolysis, resulting in a low cellobiose concentration of 1.75 ± 0.32 g/L at 18th hour and glucose concentration of 11.77 ± 0.84 g/L at 48th hour (Fig. 3a). However, after 48 hour, glucose concentration decreased significantly. Raw material of OPT exhibited a relatively intact and rigid surface structure which is an obstacle for enzymes to access cellulose (Wang et al. 2018). Other characteristics of substrate that affect enzymatic hydrolysis are lignin content, distribution and structure (Draude et al. 2001), hemicellulose content, cellulose crystallinity, degree of polymerization, and particle size of the substrate (Zhu et al. 2009).

Fig. 3.

The enzyme hydrolysis of a raw oil palm trunk (OPT), b steam-exploded fibers c alkaline hydrogen peroxide (AHP)-treated fibers. (filled circle) Glucose g/L (open circle) Cellobiose g/L

On the other hand, the steam-exploded fibers were used for enzyme hydrolysis and, on comparison with raw material, gave relatively higher cellobiose concentration of 6.71 ± 0.33 g/L at 4th hour and glucose concentration of 27.67 ± 0.22 g/L at 96th hour (Fig. 3b). The results indicated that steam exploded helped remove hemicellulose, due to which the efficiency of enzymatic hydrolysis improved.

After the majority of hemicellulose in OPT removed during the pretreatment, the remaining solid (mainly cellulose and lignin) was more porous and could be more easily hydrolyzed by enzymes into glucose. In order to evaluate the effects of the pretreatment process and conditions on subsequent enzymatic hydrolysis, the fibers after the alkaline hydrogen peroxide pretreatment were hydrolyzed. An increase in cellobiose concentration 7.27 ± 0.35 g/L at 8th hour was observed during enzymatic hydrolysis of the AHP-treated fibers (Fig. 3c). The results clearly indicated that alkaline hydrogen peroxide treatment being an oxidative pretreatment agent improved the efficiency of enzymatic hydrolysis (Shen et al. 2011). It also had significant effect on glucose concentration that increased from 11.77 ± 0.84 g/L (raw material) to 46.15 ± 0.32 g/L of glucose. In addition, enzyme digestibility increased from 16.41% (raw material) to 59.82% (AHP treated fibers) at 96th hour. The Michaelis–Menten equation was used to calculate the initial velocity (V₀) of enzyme hydrolysis, which exhibited (V₀) 0.80 g/h in raw material, 0.91 g/h in steam-exploded fibers, and 1.58 g/h in AHP-treated fibers (Table 3). The results depicted that significant increase in cellobiose and glucose concentration accelerated due to the potential effect of AHP pretreatment which causes an enormous quantity of delignification and hemicellulose removal and led to an increase in the porosity of biomass, increased internal surface area, and decrease in crystallinity (Pezoa et al. 2010). Resultantly, there are more cellulose for the accessibility of enzyme (Song et al. 2016), reducing the nonspecific bonding to enzyme and ultimately enhancing the enzymatic digestibility (Mesquita et al. 2016; Li et al. 2018; Zhang et al. 2019).

Table 3.

Effect of pretreatment on enzyme hydrolysis of oil palm trunk

S#	Parameter	Oil palm trunk (OPT)	Steam exploded (fibers)	AHP treated (fibers)
01	Glucose concentration (g/L)	11.77	27.67	46.15
02	Enzyme digestibility in %	16.41	37.18	59.82
03	Initial velocity of enzyme (V0) (g/h)	0.80	0.91	1.58

Total phenolic contents of AHP black liquor

Lignin-derived phenolic compounds are one of the factors significantly reducing the production of lignocellulosic bio-ethanol. Several phenolic compounds, i.e., phenolic acids, tannins, and gallic acid has been observed to be released from lignocellulosic biomass during different pretreatment processes (Chen et al. 2007; Panagiotou and Olsson 2007). In AHP treatment, hydrogen peroxide is able to breakdown the phenolic compounds when it is under alkaline conditions and temperature that exposes the phenolic ring and causes carboxylic groups to be added to the macromolecular structure (Diaz et al. 2014). Black liquors of all 27 AHP treatments were used to study the total phenolic contents. The highest total phenolic content was 9.98 ± 1.14 mg GAE/g_sample, whereas the lowest total phenolic content was of 1.84 ± 0.49 mg GAE/g_sample (Table 4). In addition, the optimum AHP condition that led to a significant lignin removal of 50% had the total phenolic contents of 5.13 ± 0.16 mg GAE/g_sample. The AHP black liquor had relatively higher TPC than the ethanolic extract of palm oil residues, i.e., empty fruit brunch, 1.79 mg GAE/g_sample; palm press fibers, 3.59 mg GAE/g_sample; palm kernel cake, 5.19 mg GAE/g_sample (Souko et al. 2019); and oil palm fronds, 4.691 mg GAE/g_sample (Boateng et al. 2014). The results of this study found higher when compared with sunflower meal, a by-product from the biodiesel industry that showed a TPC value of 4.4 mg GAE/g (Kachrimanidou et al. 2015). It is reasonable that phenolic compounds present in black liquor are responsible for H₂O₂ scavenging (Sroka and Cisowski 2003). Phenolic compounds which can act as antioxidants and free radical scavengers display a wide range of physiological and biological properties (Hagerman et al. 1998). The structure of phenolic compounds, location, and degree of hydroxylation are important in determining their antioxidant activity (Decker 1998).

Table 4.

Total phenolic content and antioxidant activity of black liquor derived from alkaline hydrogen peroxide (AHP) pretreatment of oil palm trunk at different temperatures, times, and hydrogen peroxide concentrations

Temperature (°C)	Time (min)	% Concentration (gH2O2/g biomass)	TPC (mg GAE/g dry sample)	Percentage inhibition (%)
				DPPH	ABTS
50	30	1	3.13 ± 0.24q	53.30 ± 0.77gh	51.79 ± 0.87j
50	30	3	7.22 ± 0.42de	54.19 ± 1.84efgh	57.58 ± 1.13fghi
50	30	5	1.84 ± 0.49r	52.45 ± 2.35g	50.42 ± 1.58j
50	60	1	2.97 ± 0.47pq	53.82 ± 0.23fgh	55.10 ± 2.08gh
50	60	3	5.93 ± 0.74ghi	55.70 ± 1.61defgh	58.33 ± 2.51efg
50	60	5	9.98 ± 1.14a	58.47 ± 1.90abcd	61.23 ± 0.74cde
50	90	1	4.76 ± 0.35lmn	56.48 ± 0.83cdefg	58.12 ± 1.64fgh
50	90	3	8.27 ± 0.26b	52.49 ± 1.34g	59.57 ± 0.47cdef
50	90	5	8.95 ± 0.96a	61.06 ± 0.23a	68.65 ± 1.21a
70	30	1	4.36 ± 0.34mno	56.44 ± 2.20cdefg	61.39 ± 0.53cd
70	30	3	5.13 ± 0.16kl	59.80 ± 0.46abc	65.51 ± 1.32b
70	30	5	7.03 ± 0.53def	60.28 ± 1.42ab	67.18 ± 0.74ab
70	60	1	8.01 ± 0.37cd	60.32 ± 0.48ab	64.71 ± 0.56b
70	60	3	6.62 ± 0.64ghi	57.48 ± 1.12bcde	59.92 ± 0.78cdef
70	60	5	6.93 ± 0.32fgh	57.33 ± 0.83bcdef	57.92 ± 0.35fgh
70	90	1	4.68 ± 0.13lmn	59.89 ± 1.56abc	58.34 ± 3.45efg
70	90	3	5.68 ± 0.18jkl	57.55 ± 1.82bcde	57.03 ± 0.51fghi
70	90	5	5.69 ± 0.30ijk	56.85 ± 0.61bcdefg	58.10 ± 1.69fgh
90	30	1	2.95 ± 0.09q	55.19 ± 1.81defgh	54.67 ± 3.14i
90	30	3	4.73 ± 0.14lmn	55.08 ± 2.89defgh	57.09 ± 1.85fghi
90	30	5	6.01 ± 0.71fghi	58.25 ± 1.82abcd	61.75 ± 1.61c
90	60	1	3.49 ± 0.43opq	38.34 ± 2.81i	43.52 ± 0.80k
90	60	3	7.61 ± 0.71cde	56.70 ± 0.42de	59.35 ± 2.38cdef
90	60	5	8.39 ± 1.10c	57.03 ± 1.47bcdefg	58.58 ± 0.43defg
90	90	1	4.07 ± 0.80nop	56.66 ± 4.77cdefg	56.17 ± 1.22ghi
90	90	3	5.57 ± 0.62jkl	54.37 ± 1.70efgh	57.43 ± 2.22fghi
90	90	5	7.27 ± 0.73de	55.85 ± 1.51cdefg	58.12 ± 1.42fgh

TPC total phenolic contents, GAE gallic acid equivalent, DPPH 2,2-diphenyl-1-picrylhydrazyl, ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid

^a−qMean values within the same column with different letters are significantly different (p < 0.05)

Antioxidant activity of AHP black liquors

The percentage inhibitions of DPPH and ABTS were employed to evaluate the antioxidant activity of all 27 black liquors collected after alkaline hydrogen peroxide pretreatment. As free radicals are very reactive and can stay for short period of time; thereby, DPPH and ABTS (free radicals) were placed in dark at room temperature.

Black liquor, also known as spent liquor in paper and pulp industry, comprises lignin fractions that hold antioxidant activity (Niemi et al. 2011) and used in thermoplastic industry (Gosselink et al. 2004). In this study, the AHP condition, which gave the highest total phenolic content, exhibited high percentage inhibitions of DPPH and ABTS of 58.47 ± 1.90 and 61.06 ± 0.23, respectively (Table 4), whereas the AHP condition with low total phenolic content had lowest DPPH and ABTS with percentage inhibitions of 38.34 ± 2.81 and 43.52 ± 0.80, respectively.

The optimum AHP condition (70 °C, 30 min, and 3% H₂O₂ concentration) that caused significant removal of lignin exhibited higher percentage inhibitions of DPPH and ABTS, i.e., 59.80 ± 0.46 and 68.65 ± 1.21, respectively (Table 4), which indicated the presence of high percentage of degraded lignin in black liquor (Niemi et al. 2011). The Pearson coefficients (R*) were used to study the multiple correlation between total phenolic contents (TPCs) and antioxidant activity (DPPH and ABTS assays). The antioxidant activity showed positive correlation with the total phenolic content. The correlation found for the DPPH and ABTS assays was R² = 0.7204. The results displayed higher DPPH percentage inhibition when compared with those of Santos et al. (2014) and Meliana and Setiawan (2016), exhibiting the values of 54.76 and 54.91% inhibitions of DPPH, respectively. Moreover, the results were observed approximately closer to the findings of Alzagameem et al. (2018), i.e., 62.21% inhibition of DPPH. In contrast, Boateng et al. (2014) depicted higher percentage inhibition of DPPH in black liquor ranging from 70 to 97% compared to BHT (50–80%) as a standard. The degraded lignin in AHP black liquor showed high DPPH and ABTS inhibitions but lower than trolox (98–99.5%) as a standard. The capacity inhibition of DPPH radicals was lower than the capacity inhibition of ABTS radicals that may be attributed to the mechanism of radicals catching for the lignin (Huang et al. 2005; Arshanitsa et al. 2013).

The findings of this study revealed that the pretreatment liquor, mostly discarded as waste in bioethanol refineries, comprises antioxidants activity and phenolic compounds which could efficiently be recovered for sustainable bioethanol and phytochemicals production.

Conclusion

The results of this study revealed that increase in the pretreatment temperature and time may decrease the delignification process due to unstable nature of H₂O₂ at high temperature and production of inhibitory by-products, such as hydroxymethylfurfural and furfural. The optimum condition (70 °C, 30 min, 3% H₂O₂) removed significant amount of lignin (50%) and hemicellulose (57.31%). Moreover, it increased 35% of cellulose content when compared to the raw material. The findings were supported by SEM and FT-IR analysis of untreated and treated biomass samples showing change in physical deformation of outer surface of biomass, and also the enzymatic hydrolysis efficiency of OPT was dramatically enhanced when treated with the optimum condition of AHP pretreatment. During the pretreatment process, a large amount of black liquor generates which is mostly discarded. In the current study, the black liquor from the optimum condition of AHP pretreatment showed promising amount of total phenolic contents and exhibited high antioxidant activity. However, it can be utilized in biorefinery process to recover antioxidants and phenolic compounds that may help in sustainable bioethanol production. Overall, it is also concluded that AHP treatment comparatively more environmental friendly than alkaline pretreatment which requires high temperature and more water for washing fibers after pretreatment.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no potential conflict of interest regarding submission and publication of this manuscript.