Efficient Biovalorization of Oil Palm Trunk Waste as a Low-Cost Nutrient Source for Bioethanol Production

Abstract

This study aimed to efficiently utilize felled oil palm trunk (OPT) for bioethanol and lactic acid production. OPT was separated into two fractions: oil palm sap (OPS) and OPT fiber. OPS contained substantial amounts of sugars (38–40 g/L) and nitrogen (0.60–0.70 g/L), which can serve as a base medium for bioethanol production. As bioethanol production requires high sugar concentrations, OPS was concentrated, supplemented with OPT fiber, and used for bioethanol production through simultaneous saccharification and fermentation (SSF) by Saccharomyces cerevisiae. Repeated-batch SSF for five cycles efficiently utilized OPT fiber and achieved an average ethanol production of 35–42 g/L in each cycle. To increase the accessibility of the enzyme, OPT fiber was acid-pretreated prior to the SSF process. The combined use of acid-pretreated OPT slurry and concentrated OPS provided the maximum ethanol production of 49.63 ± 1.05 g/L. The fermented broth after ethanol recovery, containing mainly xylose, was used to produce lactic acid at a concentration of 18.85 ± 0.55 g/L. These strategies can greatly contribute to the zero-waste biorefinery of OPT and may also be applicable for the efficient biovalorization of other similar agricultural wastes.

1. Introduction

The depletion of fossil fuels has accelerated the global shift toward utilizing bioethanol as an alternative fuel source. The bioethanol production industry presently relies on first-generation feedstocks such as sugar and starch, competing with the food industry that uses them as food sources and raising significant concerns regarding socio-economic and environmental impacts. Second-generation feedstocks, such as lignocellulosic biomass, offer advantages over first-generation feedstocks, as they hold great promise as low-cost, non-edible, and abundant sources of carbohydrates [1,2]. Lignocellulosic biomass sourced from agricultural materials like wheat straw, rice husk, rice straw, fruit peels, food waste, sugarcane bagasse, corn cobs, coconut shells, and groundnut shells holds promise as valuable feedstock for enhancing value through various processes [3]. Old palm trees have gained much attention in recent years due to the abundant lignocellulosic waste in Thailand. These trees have an average productive lifespan of approximately 25 to 30 years [4]. Approximately 1.44 million tons of felled old oil palm trunks (OPTs) are generated, making them significant sources of biomass [5]. OPT can be separated into two main portions: liquid and solid. Oil palm sap (OPS) is liquid squeezed from OPT. It contains a large amount of fermentable sugars, nitrogen, minerals, and vitamins. It could be considered a low-cost nutrient source for microorganisms [6,7]. The solid portion obtained after OPS squeezing, i.e., OPT fiber, is lignocellulosic biomass, mainly composed of cellulose, hemicellulose, and lignin. Due to the complex structure of lignocellulose hindering its conversion, researchers are exploring various pretreatment techniques followed by hydrolysis to break down this complex substance and convert it into simple sugar units [3,8]. Therefore, OPT fiber needs to be pretreated and hydrolyzed into fermentable sugars and supplemented with nitrogen sources prior to fermentation by microorganisms.

Simultaneous saccharification and fermentation (SSF) stands out as an effective approach for lignocellulosic bioethanol production, as it can reduce the end-product inhibitory effect on enzymatic hydrolysis and decrease overall production time and investment costs. However, attaining a substantial ethanol yield requires high sugar concentrations [9]. Therefore, the lignocellulosic biomass needs to be pretreated to disrupt its rigid structure and improve the accessibility of cellulolytic enzymes toward cellulose fibers to increase the generation of fermentable sugars from biomass [5,10]. An appropriate pretreatment method for lignocellulosic biomass depends on factors such as biomass characteristics, desired end product, and economic viability. Recently, a green in situ lignin protection and fractionation strategy was proposed to upgrade and use all components in lignocellulosic biomass [8,11]. Among the commonly used pretreatment methods, dilute acid pretreatment is regarded as particularly well-suited for large-scale applications [12]. Dilute acid pretreatment employs hydronium ions to accelerate the hemicellulose extraction process. When the hydronium ion is released due to the high reaction temperature, O-glycosidic bonds and acetyl groups break down, resulting in partial depolymerization [13]. As a considerable amount of fermentable sugars from cellulose and hemicellulose are typically released into the liquid fraction during acid pretreatment, it is more desirable to utilize the whole acid-pretreated slurry (solid and liquid fractions together) [1].

Although OPS and OPT fiber have been separately utilized as nutrient sources for microorganisms [5,7,10], there has been no attempt to efficiently utilize both portions. Moreover, strategies to increase the availability of fermentable sugar during the SSF of OPT fiber for attaining high ethanol yields should also be developed. This study aimed to efficiently valorize both OPS and OPT fiber for bioethanol production following the zero-waste concept. Several strategies to improve bioethanol production were attempted. These include (i) the concentration of OPS to increase the sugar concentration; (ii) the combined use of OPS and OPT fiber through SSF; (iii) repeated-batch SSF of OPT fiber; (iv) the combined use of OPS and pretreated OPT fiber through SSF. Subsequently, the fermented broth after ethanol recovery, which was rich in xylose, was employed for lactic acid production.

2. Materials and Methods

2.1. Microorganisms

Saccharomyces cerevisiae FAI used for bioethanol production was obtained from the culture collection of the Faculty of Agro-Industry, Prince of Songkla University, Thailand. Active yeast cells were pre-cultured in a growth medium consisting of yeast extract 10 g; peptone 20 g; and glucose 20 g per 1 L at 30 °C and 150 rpm for 18 h and used as inoculum with a yeast cell concentration of about 10⁹ cells/mL.

Lactobacillus pentosus TISTR 920 used for lactic acid production was obtained from the Thailand Institute of Scientific and Technological Research (TISTR, Pathum Thani, Thailand). The bacterial inoculum was prepared by pre-culturing in MRS medium consisting of proteose peptone 10 g; beef extract 10 g; yeast extract 5 g; glucose 20 g; polysorbate 80 1 g; ammonium citrate 2 g; sodium acetate 5 g; magnesium sulfate 0.1 g; manganese sulfate 0.05 g; and dipotassium phosphate 2 g per 1 L. The pre-culture was incubated under anaerobic conditions at 37 °C for 18–24 h and used as inoculum with a cell concentration of about 10⁸ colony-forming units/mL.

2.2. Preparation of OPS and OPT Fiber

Oil palm trees, approximately 25 years old, were obtained from Krabi Province, Thailand. The trunks were sectioned into pieces and squeezed for OPS production using a laboratory-scale pressing machine [6]. OPS was subsequently processed by centrifugation at 6000 rpm for 15 min and stored at −20 °C until use. The remaining solid residues (OPT fiber) were dried at 105 °C until constant weight. Subsequently, they were milled using a laboratory mill and sieved through 20- and 30-mesh screens to a particle size of 0.75–2.0 mm, which mainly constituted the vascular bundle fraction. OPT fiber was analyzed for cellulose, hemicellulose, and lignin contents.

2.3. Ethanol Production from OPS and OPT Fiber through SSF and Repeated-Batch SSF

Original OPS with an initial sugar concentration of 40 g/L and concentrated OPS with an initial sugar concentration of 80 g/L were used as base medium instead of a synthetic medium for ethanol production. Different OPT fiber loadings of 5%, 10%, and 15% w/v were supplemented with cellulase (iKnowzyme, Reach Biotechnology, Pathum Thani, Thailand) at 15 filter paper units (FPU) per gram of OPT fiber for ethanol production through SSF. The yeast inoculum (10%) was introduced and incubated at 30 °C and 150 rpm. Samples were collected for the analysis of cell growth, ethanol production, and sugar consumption. Repeated-batch SSF was carried out using appropriate OPS concentrations and appropriate OPT fiber loadings. After the first batch of SSF, the fermented broth was replaced with fresh OPS, and the residual OPT fiber was repeatedly used for five cycles. Cellulase was also added at 15 FPU per gram of OPT fiber in each cycle.

2.4. Ethanol Production from Acid-Pretreated OPT Fiber through SSF

A dilute acid pretreatment of the OPT fiber was performed following the modified method of Wischral et al. [14]. Briefly, the OPT fiber was added to 100 mL of 1% H₂SO₄ aqueous solution in a 100 mL capped bottle, and the solid-to-liquid ratio was set at 10% (w/v). The bottles were sealed and autoclaved. The pretreatment was conducted at 121 °C for 30 min. The acid-pretreated OPT fiber (AOPT) slurry was used for ethanol production. The pH was adjusted to 6, and the cellulase enzyme was added at 15 FPU/g of AOPT. When AOPT was used alone, yeast extract was added at 3 g/L as a nitrogen source. The combined use of OPS and AOPT was also performed. The fermentation process and sampling were carried out as mentioned above.

2.5. Utilization of Residual Liquid from the SSF of AOPT for Lactic Acid Production

Ethanol in the fermented broth from the SSF of AOPT was recovered by rotary evaporation at 70 °C, and the residual liquid was used for lactic acid production by L. pentosus TISTR 920. The culture pH was adjusted to 6. After 10% (v/v) inoculation, the culture was incubated at 37 °C and stirred at 100 rpm under anaerobic conditions for 72 h.

2.6. Analytical Methods

The concentration of yeast cells was measured using a hemocytometer (counting chamber, BOE-14V, Boeco, Hamburg, Germany). The ethanol concentration was determined using a gas chromatographer (Shimadzu, Kyoto, Japan) equipped with a Stabilwax column (Restek Corp., Bellefonte, PA, USA). The reducing sugar and total sugar concentrations were analyzed using the dinitrosalicylic (DNS) method and the phenol sulfuric method, respectively.

In lactic acid production, total acid was measured by the titration method. The determination of the sugar and acid compositions was performed using a high-pressure liquid chromatography (HPLC) system (Agilent 1260, Santa Clara, MA, USA) equipped with an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) [15]. The chemical compositions of both untreated and pretreated OPT fiber were analyzed for cellulose, hemicellulose, and lignin contents by the AOAC standard method. Briefly, the lignin and acid detergent fiber (ADF) content was measured using the AOAC standard method 973.18 [16], and the neutral detergent fiber (NDF) content was measured using the AOAC standard method 992.16 [17]. The cellulose content was calculated indirectly from the difference between the ADF and the lignin contents, and the hemicellulose content was calculated from the difference between the NDF and the ADF contents. The statistical analysis was performed by one-way analysis of variance and by means of Duncan’s multiple range tests at a 95% confidence level.

3. Results and Discussion

3.1. Ethanol Production from OPS and OPT Fiber through SSF

OPS with a total sugar concentration of 40 g/L, was used as base medium for ethanol production without adding any other nutrients. OPT fiber was added to OPS as an additional carbon source for ethanol production through the SSF process. In a traditional SSF process, a prehydrolysis step is typically required to provide an adequate initial sugar amount for the initial growth of yeast cells [18]. In this study, as OPS initially contained a high amount of sugars, OPT fiber was used in the SSF process without the prehydrolysis step. Figure 1a shows the ethanol production using OPT fiber loadings at 5–15% in OPS. The cellulase loading was 15 FPU/g OPT fiber. The yeast consumed sugar rapidly and produced higher amounts of ethanol with increasing OPT fiber loadings. Using OPS (40 g/L) alone without any OPT fiber loading resulted in the lowest ethanol production of 11.49 ± 0.41 g/L at 24 h of fermentation. When OPT fiber was added to OPS at 5%, 10%, and 15% loading, ethanol production increased up to 15.02 ± 1.61 g/L, 16.45 ± 0.21 g/L, and 17.20 ± 0.04 g/L, respectively. It should be noted that ethanol production using OPT fiber loading at 10% was not significantly different from that using 15% OPT fiber loading. This could be due to mass transfer limitations when using too high solid loading [19]. Figure 1b shows that the total sugar concentration when using only OPS rapidly decreased during 24 h of fermentation, while when using OPS supplemented with OPT, it decreased more slowly, likely due to the more available sugars released from the saccharification of OPT (Figure 1c). Although a certain amount of sugar remained in the culture broth after 24 h, there was no significant increase in ethanol production after this period. The pH decreased from 5–5.5 to lower than 4, possibly due to the production of organic acids, including acetic acid and succinic acid. These acids were reported as byproducts during batch alcoholic fermentation by yeast but at very low levels of 0.005–0.015 g/g of glucose consumed [19,20]. It was reported that this yeast strain could produce ethanol similarly in this pH range [21,22].

To increase the ethanol production, the initial sugar concentration of OPS was increased to 80 g/L by evaporation before use for ethanol production (Figure 2). Ethanol production increased up to 40.14 ± 0.52 g/L when using concentrated OPS (Figure 2a). When concentrated OPS was supplemented with OPT fiber, ethanol production slightly increased with increasing OPT loadings. Figure 2b shows that the sugar content decreased rapidly during 24 h of fermentation. It seemed that the saccharification rate was lower than the consumption rate, possibly because the high initial sugar concentration of 80 g/L inhibited cellulase activity [23]. However, it should be noted that the addition of OPT fiber to OPS could increase the viability of yeast, resulting in rapid yeast growth within 24 h of fermentation (Figure 2c).

3.2. Repeated-Batch SSF for Efficient Ethanol Production from OPS and OPT Fiber

To enhance ethanol production and avoid end-product inhibition, repeated-batch SSF was performed on the remaining 10% cell culture broth and residual OPT fiber from the previous cycle. Therefore, there was no need to prepare an inoculum for the next cycle. Moreover, as the OPT fiber was repeatedly used, it could also serve as a support material for yeast cell immobilization. It was then expected that repeated-batch SSF would enhance ethanol productivity and, at the same time, reduce the cost of the inoculum. In each cycle, the fermented culture broth was replaced with fresh OPS and cellulase at 15 FPU/g OPT. Repeated-batch SSF was performed for five cycles. As shown in Figure 3, ethanol production in the first cycle was as high as 42.7 ± 1.70 g/L, likely due to active cell growth. Cell concentration slightly decreased in the subsequent batch. This might be because of the lower availability of sugars from OPT fiber. Figure 3a shows that ethanol production by this strategy remained high for more than 70% of the first cycle and was in the range of 34–36 g/L. This approach has been reported to bring several benefits, including labor and seed preparation time saving, and results in enhanced productivity and cost-effectiveness. This fermentation mode can maintain a consistently high substrate concentration across multiple runs and effectively prolong the high-productivity phase of the microorganisms [24,25].

3.3. Ethanol Production from Acid-Pretreated OPT Fiber through SSF

Acid pretreatment can enhance the availability of sugar for ethanol production, primarily due to the presence of acidic ions, which cleave the cellulose and hemicellulose chains, leading to the formation of sugar fragments [26]. Especially, dilute acid pretreatment is widely used for disrupting the highly resistant and intricate lignocellulosic structure, thus facilitating the release of sugars through enzymatic hydrolysis. While the use of high acid concentrations leads to adverse effects such as sugar losses, the formation of inhibitors (acetic acid, furfural, and 5-hydroxylmethylfurfural) at high levels, and equipment corrosion [12]. In this study, OPT fiber was acid-pretreated using 1% (v/v) H₂SO₄ at 121 °C for 30 min. The composition of the acid hydrolysate characterized using HPLC is shown in

Table 1. Composition of hydrolysate from acid pretreatment of OPT.

Composition	Concentration (g/L)
Glucose	19.84 ± 0.27
Xylose	11.33 ± 0.23
Cellobiose	2.32 ± 0.21
Arabinose	2.50 ± 0.03
Acetic acid	5.08 ± 0.18
Furfural	0.76 ± 0.09
5-Hydroxymethylfurfural	0.42 ± 0.00

. The main sugars solubilized during the acid pretreatment were glucose and xylose. The generated acetic acid, furfural, and 5-hydroxymethylfurfural were 5.08 ± 0.18 g/L, 0.76 ± 0.09 g/L, and 0.42 ± 0.00 g/L, respectively. It was reported that a dilute acid pretreatment of lignocellulosic biomass generated 3.1–10.5 g/L of acetic acid, 0.0498–1.479 g/L of furfural, and 0.0148–0.104 g/L of 5-hydroxymethylfurfural depending on the acid concentration and the pretreatment time [15]. The chemical composition of the untreated and acid-pretreated OPT fiber was analyzed. After acid pretreatment, the acid-insoluble lignin content decreased from 13.7% to 7.50%. The cellulose content increased from 56.6 % to 70.9%, while the hemicellulose content decreased from 23.4% to 5.1%. The increase in cellulose content was because lignin and hemicellulose were removed [8,11]. Similarly, Zhao et al. [27] reported that pretreating sugarcane bagasse with 2% sulfuric acid at 121 °C for 2 h led to 85% solubilization of hemicelluloses and a 16% reduction in lignin content. However, the high-temperature acid-catalyzed pretreatment of sugarcane bagasse also led to an improved recovery of dissolved sugars from hemicellulose. This process also enhanced the enzymatic digestibility of the solid cellulose component, facilitating the conversion of cellulose into sugar monomers [28].

Figure 4 shows the sugar yields from the enzymatic hydrolysis of un-pretreated OPT fiber and acid-pretreated OPT fiber (AOPT). The initial sugar concentration of AOPT was as high as 46.33 ± 1.63 g/L. After the enzymatic hydrolysis of AOPT, the sugar concentration was increased up to 57.00 ± 0.72 g/L. Meanwhile, the enzymatic hydrolysis of un-pretreated OPT fiber provided a lower sugar concentration of 30.97 ± 2.36 g/L. Figure 5 shows ethanol production using the liquid fraction of AOPT, namely, hemicellulose hydrolysate, and the whole slurry of AOPT through the SSF process. The results show that the maximum ethanol production of 23.17 ± 0.75 g/L was achieved through SSF of the whole slurry of AOPT. The use of only the hemicellulose hydrolysate gave a low ethanol production of 12.8 ± 0.24 g/L and the SSF of only the AOPT fiber gave the lowest ethanol production of 9.09 ± 0.09 g/L due to the lower available carbon source left after acid pretreatment (Figure 5a). Acid pretreatment also dissolves hemicellulose and a fraction of cellulose [10]. It should be noted that yeast cell growth when using the whole slurry of AOPT was also higher than when using only the solid or liquid fraction of AOPT (Figure 5d), possibly due to the presence of a higher sugar concentration (Figure 5c). Similarly, Lee et al. [29] found that a dilute acid pretreatment of rice straw using 1% H₂SO₄ at 160 °C significantly improved the yield of reducing sugars and ethanol production through SSF due to the abundance of glucose, xylose, arabinose, and other solubilized sugars such as galactose and mannose within the hydrolysates.

3.4. Ethanol Production from Whole Slurry of AOPT and Concentrated OPS

To provide a higher carbon source for ethanol production, concentrated OPS was supplemented with the whole slurry of pretreated OPT. OPT at the different solid loadings of 10% and 15% (w/v) was acid-pretreated using 1% H₂SO₄ at 121 °C for 30 min. The pH of the whole slurry of pretreated OPT was adjusted to 6. Subsequently, OPS with an initial sugar concentration of 80 g/L was added, and ethanol production was performed through SSF. As shown in Figure 6a, when concentrated OPS was supplemented with the whole slurry of pretreated OPT, ethanol production was higher than when using concentrated OPS supplemented with un-pretreated OPT. However, ethanol production using 10% and 15% AOPT loadings was not significantly different. This observation might be attributed to the mass transfer limitation when using too high solid loading. The higher sugar concentration at 10% AOPT loading also confirmed the better mixing efficiency. Figure 6c shows that the total sugar concentration increased during 12 h likely because the saccharification rate by cellulase was higher than the consumption rate by the yeast, and the rapid decrease after 12 h indicated a faster consumption rate due to the higher yeast cell concentration. Figure 6d shows that there was no significant difference in yeast cell growth when using 10% and 15% AOPT loadings. Similarly, Sharma et al. [30] also found that the maximum ethanol yield was achieved at 10% solid loading, whereas a higher solid loading (20%) resulted in reduced ethanol efficiency. This is because the use of high solid loadings can negatively affect processes due to mass transfer limitations, enzyme activity losses, and water availability constraints [31,32]. Figure 7 shows the photographs and microscopic photographs of OPT fiber before and after acid pretreatment and AOPT residue after SSF. After acid pretreatment, the OPT bundles broke down and released holocellulose fiber. The AOPT residue after SSF became a fine powder.

3.5. Valorization of Stillage after Ethanol Fermentation for Lactic Acid Production

The hydrolysis residues and stillage after ethanol fermentation are typically considered waste materials, requiring a series of post-treatments to mitigate their environmental impact before disposal. However, these post-treatments add extra costs and reduce the economic efficiency of lignocellulosic bioethanol production. Fortunately, recycling techniques for the efficient utilization of these wastes have been recognized as viable solutions to address this issue [33]. In this study, the fermentation broth after ethanol separation contained a certain amount of xylose (

Table 2. Sugar composition of fermented broth after SSF and ethanol recovery.

Substrate loading	Glucose (g/L)	Xylose (g/L)	Arabinose (g/L)	Cellobiose (g/L)
OPS 80 g/L + 10% AOPT	3.5 ± 0.04	11.9 ± 0.35	1.70 ± 0.06	1.84 ± 0.06
OPS 80 g/L + 15% AOPT	4.0 ± 0.09	13.7 ± 0.02	1.90 ± 0.11	1.40 ± 0.13

), which is worthy of being recycled. To valorize these residual sugars, which may be an effective way to reuse them, the fermented broth was used as a nutrient source for lactic acid (LA) production by xylose-assimilating L. pentosus TISTR 920.

Figure 8 shows that L. pentosus TISTR 920 utilized sugars in the fermented broth and produced total acid at the concentration of 20.7 g/L. This included lactic acid production at 18.9 ± 0.55 g/L and acetic acid production at 2.06 ± 0.16 g/L. It has been noted that stillage, the main waste product after ethanol distillation, consists of soluble substances (including residual sugars and ethanol), residual cellulases, and yeasts [33]. Cesaro and Belgiorno [34] reported that stillage typically has high chemical oxygen demand (COD) values. It was suggested that anaerobic digestion could be an effective method for reusing stillage to produce biomethane. Another study, conducted by Zhang et al. [35], showed that the wastewater after pervaporation of acetone–butanol–ethanol (ABE) could be used as a buffer for saccharification. After four cycles of fermentation, approximately 86% of wastewater discharge was reduced. This study showed that it is possible to valorize non-fermented sugars for lactic acid production, also contributing to a reduction in wastewater discharge. The photocatalytic reaction to convert sugars to lactic acid is also another strategy for the valorization of non-fermented sugars [36].

4. Conclusions

This study showed the efficient valorization of OPT for bioethanol and lactic acid production. OPT was separated into the two fractions of OPS and OPT fiber. As OPS contains sugars and nitrogen, it could be used as a sole nutrient source for ethanol production. Concentrated OPS supplemented with 10% OPT loading was used for ethanol production through the SSF process. Repeated-batch fermentation was performed to use the OPT fiber efficiently. Through this strategy, the ethanol production obtained in each cycle was 35–42 g/L. In addition, the acid pretreatment of the OPT fiber enhanced the availability of sugars, and ethanol production was improved up to 49.63 ± 1.05 g/L. Furthermore, the fermented broth after ethanol recovery, which was xylose-rich, could be used to produce lactic acid at a concentration of 18.85 ± 0.55 g/L. These strategies can contribute greatly to the zero-waste biorefinery of OPT and may also be applicable for the efficient biovalorization of other similar agricultural wastes.