Pretreated Oil Palm Empty Fruit Research Articles

Rehabilitation of Paddy Fields using Subcritical Water Pretreated Oil Palm Empty Fruit Bunch

Rehabilitation of Paddy Fields using Subcritical Water Pretreated Oil Palm Empty Fruit Bunch

Utilization of pretreated oil palm empty fruit bunches and their hydrolysate for ethanol production by Indonesian ethanologenic yeast

Abstract. Anita SH, Oktaviani M, Hermiati E. 2023. Utilization of pretreated oil palm empty fruit bunches and their hydrolysate for ethanol production by Indonesian ethanologenic yeast. Biodiversitas 24: 5243-5252. Oil Palm Empty Fruit Bunches (OPEFB) represent a polysaccharide-rich raw material with promising potential for ethanol production. This study aimed to investigate the ethanologenic yeasts, specifically Saccharomyces cerevisiae InaCC Y93 and Kluyveromyces marxianus InaCC Y119, affect bioethanol production in three different systems: Separate Hydrolysis and Fermentation (SHF), Simultaneous Saccharification and Fermentation (SSF), and Prehydrolysis-Simultaneous Saccharification and Fermentation (PSSF). This work is distinguished by the use of indigenous Indonesian yeast strains, including a thermotolerant strain. In the pretreatment process, 1.13% oxalic acid was added to OPEFB and subjected to microwave treatment at 190°C for 3.01 min. Subsequently, cellulase enzymes (40 FPU/g) and a 10% (w/v) yeast inoculum were introduced into 5.27 g dry weight of pretreated OPEFB pulp. The OPEFB acid hydrolysate was also subjected to fermentation. Ethanol content was monitored at 24 h intervals for 72 h. The PSSF system employs K. marxianus InaCC Y119 at 48 h exhibited the highest ethanol concentration, yielding 0.290 g/g, equivalent to approximately 51.20% of the theoretical yield. Additionally, K. marxianus InaCC Y119 demonstrated its capability to ferment the OPEFB acid hydrolysate into ethanol. These findings underscore the considerable potential of K. marxianus for applications in fermenting both hexose and pentose sugars to produce ethanol within higher-temperature systems.

Lactic Acid Production from Sequential Inorganic Salt Pretreated Oil Palm Empty Fruit Bunch via Simultaneous Saccharification and Fermentation

Lactic acid is produced from inorganic salt pretreated oil palm empty fruit bunch (OPEFB) for the first time through simultaneous saccharification and fermentation (SSF). OPEFB is an agricultural waste that can be turned into lactic acid (LA), a highly desired chemical product. An inorganic salt (Na3PO4.12H2O-FeCl3) pretreatment precedes SSF with Bacillus coagulans DSM2314. The effect of solid loading, concentration CaCO3 and enzyme loading on LA generation is studied using design of experiments. The results show that solid loading and concentration CaCO3 affect LA yield. 50g/L biomass, 50g/L CaCO3 concentration, and 50 FPU/g cellulase enzyme yield maximal LA (46.66/L) and yield (0.93/g OPEFB). The model created to predict LA production was then validated.

Large scale production of succinic acid by fermentation of sequential inorganic salt pretreated oil palm empty fruit bunch

The abundance of accumulated oil palm wastes has led to their usage as substrate in the production of organic acid such as succinic acid (SA). For the first time, oil palm empty fruit bunch (OPEFB) which was pretreated using inorganic salts was utilized for SA production in a large-scale fermenter. OPEFB was pretreated sequentially using 15% (w/v) Na3PO4.12H2O and 5% (w/v) ZnCl2 solution before undergoing fermentation process in a 16 L bioreactor in the presence of Actinobacillus succinogenes ATCC 55618. The optimum fermentation conditions were adopted from small-scale setting and implemented in the large-scale setting. Two different fermentation approaches were studied; simultaneous saccharification and fermentation (SSF) and modified simultaneous saccharification and fermentation (mSSF). This research revealed that mSSF produced higher concentration of total reducing sugars (TRS) yield and SA compared to the SSF method. SSF of inorganic salts pretreated OPEFB produced maximum SA concentration (38.85 gL-1) and yield (0.39 gg-1). Meanwhile highest TRS yield obtained from pre-hydrolysis was 0.79 gg-1 at 72 h and SA (50.5 gL-1) was acquired under the mSSF at 132 h. Therefore, mSSF is a novel process that has propitious potential for SA production in a large-scale setting which can be further executed in the industrial scale.

Whole sugar 2,3-butanediol fermentation for oil palm empty fruit bunches biorefinery by a newly isolated Klebsiella pneumoniae PM2

Effective utilization of cellulose and hemicelluloses is essential to sustainable bioconversion of lignocellulose. A newly isolated xylose-utilizing strain, Klebsiella pneumoniae PM2, was introduced to convert the biomass “whole sugars” into high value 2,3-butanediol (2,3-BDO) in a biorefinery process. The fermentation conditions were optimized (30°C, pH 7, and 150 rpm agitation) using glucose for maximum 2,3-BDO production in batch systems. A sulfite pretreated oil palm empty fruit bunches (EFB) whole slurry (substrate hydrolysate 119.5 g/L total glucose mixed with pretreatment spent liquor 80 g/L xylose) was fed to strain PM2 for fermentation. The optimized biorefinery process resulted in 75.03 ± 3.17 g/L of 2,3-BDO with 0.78 ± 0.33 g/L/h productivity and 0.43 g/g yield (87% of theoretical value) via a modified staged separate hydrolysis and fermentation process. This result is equivalent to approximately 135 kg 2,3-BDO and 14.5 kg acetoin precursors from 1 ton of EFB biomass without any wastage of both C6 and C5 sugars.

High-titer bio-succinic acid production from sequential alkalic and metal salt pretreated empty fruit bunch via simultaneous saccharification and fermentation

Bio-succinic acid is produced from inorganic salt pretreated oil palm empty fruit bunch (OPEFB) for the first time through simultaneous saccharification and fermentation (SSF). OPEFB is an agriculture waste that can be converted into succinic acid (SA) which is a high value added product sought after in the chemical industry. In this study OPEFB is pretreated using sequential inorganic salt (sodium phosphate dodecahydrate and zinc chloride) before undergoing SSF using Actinobacillus succinogenes ATCC 55618. Parameters that influenced the SA production such as substrate loading, inoculum size and enzyme loading are investigated using design of experiments. The results revealed that biomass loading and enzyme loading have significant impact in SA yield. Under condition of 100 g/L biomass, 5% inoculum and cellulase enzyme 40 FPU/g OPEFB, maximum concentration of SA (65.2 g/L) and yield (0.65 g/g OPEFB) are obtained. Subsequently the model developed for predicting SA production has been successfully validated.

Naganishia cerealis IN1S2.5 oil production from the hydrolysate of NaOH-impregnated and catalyst steam explosion pretreated oil palm empty fruit bunch

NaOH-impregnation with catalyst steam explosion was found to be an efficient pretreatment method for oil palm empty fruit bunch (OPEFB) as a substrate for oil production by Naganishia cerealis IN1S2.5. Cellulase hydrolysis of the pretreated OPEFB yielded glucose at 0.364 g/g. Investigation of N. cerealis IN1S2.5 oil production in the OPEFB hydrolysate revealed a maximum oil yield (2.46 g/L) when the C/P molar ratio of the OPEFB hydrolysate was adjusted to 25.71, supplemented with Ca2+ and Zn2+, and set to pH 4. The N. cerealis IN1S2.5 oil was comprised of oleic (37.6%), palmitic (36.2%), and steric (17.9%) acids, all (w/w), as the major fatty acids. Predicted properties of the produced biodiesel indicated the potential of N. cerealis IN1S2.5 oil as a biodiesel feedstock.

Anaerobic digestion of fungally pre-treated oil palm empty fruit bunches: energy and carbon emission footprint

Oil palm empty fruit bunches (OPEFB) is one of the abundant lignocellulosic biomass in Indonesia. However, high lignin content in EFOB has becoming one of the obstacles in valorising it as bioenergy resources, as it is hardly broken down by the microorganisms. Therefore, pre-treatment is often needed to enhance the biodegradation process of organic content in OPEFB. The biological pre-treatment is besieged to enhance the utilisation of OPEFB as bioenergy resources. This study was aimed to investigate the impact of fungal pre-treatment on the OPEFB characteristics and its methane potential, and to estimate the energy and carbon emission saving from anaerobic digestion (AD) of OPEFB. In this study, OPEFB was treated using Phanerocheate chrysosporium under the standard condition and incubated for 7 and 14 days. The energy and carbon emission footprint were calculated using AD Assessment Tool (ADAT) software. The results showed that fungal pre-treatment was able to increase organic matter and enhance methane potential. Estimation on energy (i.e. electrical and heat), however, indicated a lower energy balance and carbon emissions saving for fungal pre-treated OPEFB. The higher moisture content (MC) on treated OPEFB may reduce the biogas and methane production. Yet, further research is still required to have an in-depth understanding of improving the efficacy of biogas production from OPEFB by incorporating other green pre-treatment approaches.

Enhanced cellulase and reducing sugar production by a new mutant strain Trichoderma harzianum EUA20

Trichoderma harzianum EU2-77 was a mutant strain of the wild-type strain T. harzianum NP13a isolated in Singapore. A multi-mutagenesis one-screening (MMOS) method was developed to further improve strain EU2-77 and a new mutant EUA20 was obtained. It exhibited filter paper cellulase (FPase) activity up to 14.79IU/mL within 6 days shake flask cultivation. Activities of FPase, endoglucanase, β-glucosidase, and xylanase, and protein content by EUA20 were respectively increased to 5.73, 4.35, 7.34, 1.80 and 2.70 folds. Using pretreated oil palm empty fruit bunch (OPEFB) and corncob powder as the substrates, strain EUA20 presented approximate 6.52 and 8.80IU/ml FPase activity. Reducing sugar yield of 615.8 and 636.8mg/g biomass were respectively obtained for OPEFB and corncob powder using cellulolytic enzymes of strain EUA20. Our results demonstrated that mutant strain EUA20 had great potential in on-site cellulase production for effective biomass bioconversion.

Optimization of Cellulase and Xylanase Productions by Streptomyces thermocoprophilus TC13W Using Low Cost Pretreated Oil Palm Empty Fruit Bunch

The cellulase and xylanase productions from the pretreated empty fruit bunch as the low-cost substrate in a submerged fermentation were investigated. The objectives of this study were selected the local strain that produced high cellulase and xylanase and enhanced the enzymes production by the selected strain. Ten Streptomyces strains were cultivated in the complex medium [(g/l); CaCl2·2H2O 0.1, MgSO4·7H2O 0.1, KH2PO4 0.5, K2HPO4 1.0, NaCl 0.2, NH4NO3 1.0, yeast extract 5.0 and Tween 80 0.5 with alkaline peroxide pretreated empty fruit bunch (APEFB) 20 in distilled water, pH 7.0] in an incubator shaker at 150 rpm and 45 °C for 120 h. The strain TC13W gave the highest cellulase and xylanase activities (280 and 878 U/g APEFB, respectively). This strain was identified by 16S rDNA method as Streptomyces thermocoprophilus (96% similarity). Cellulase and xylanase productions by S. thermocoprophilus TC13W in the optimized medium with 1% (w/v) APEFB and 0.5% (w/v) yeast extract, pH 6.5 at 150 rpm and 40 °C for 120 h gave the maximum cellulase and xylanase activities of 925 and 1796 U/g APEFB, respectively. The increasing of cellulase and xylanase activities in the optimized medium was 3.30 and 2.04 folds, respectively in comparison to the original medium. These in-house enzymes could be used as the promising candidate enzyme for produce various value-added products via an enzymatic hydrolysis of the lignocellulosic waste.

Optimisation of Simultaneous Saccharification and Fermentation (SSF) for Biobutanol Production Using Pretreated Oil Palm Empty Fruit Bunch.

This study was conducted in order to optimise simultaneous saccharification and fermentation (SSF) for biobutanol production from a pretreated oil palm empty fruit bunch (OPEFB) by Clostridium acetobutylicum ATCC 824. Temperature, initial pH, cellulase loading and substrate concentration were screened using one factor at a time (OFAT) and further statistically optimised by central composite design (CCD) using the response surface methodology (RSM) approach. Approximately 2.47 g/L of biobutanol concentration and 0.10 g/g of biobutanol yield were obtained after being screened through OFAT with 29.55% increment (1.42 fold). The optimised conditions for SSF after CCD were: temperature of 35 °C, initial pH of 5.5, cellulase loading of 15 FPU/g-substrate and substrate concentration of 5% (w/v). This optimisation study resulted in 55.95% increment (2.14 fold) of biobutanol concentration equivalent to 3.97 g/L and biobutanol yield of 0.16 g/g. The model and optimisation design obtained from this study are important for further improvement of biobutanol production, especially in consolidated bioprocessing technology.

Enzymatic saccharification of liquid hot water and dilute sulfuric acid pretreated oil palm empty fruit bunch and sugarcane bagasse

Oil palm empty fruit bunch (OPEFB) and sugarcane bagasse (SB) are potential feedstocks for the production of bioethanol. In this study OPEFB and SB were pretreated by liquid hot water and dilute sulfuric acid (3% H2SO4), and continued with enzymatic saccharification. Heating treatment for both methods was conducted in an autoclave at 121 °C for 1 hr. The saccharification was performed up to 72 hours with cellulase enzyme loading of 10, 20, and 30 FPU per g biomass. Results showed that OPEFB and SB pretreated with H2SO4 produced higher reducing sugars than those pretreated by liquid hot water. Higher enzyme loading also resulted in higher reducing sugars. Reducing sugars obtained from enzymatic saccharification of OPEFB were higher than those obtained from SB. The highest total reducing sugars (50.48 g/100 g biomass) was obtained from OPEFB pretreated with 3% H2SO4 at enzyme loading of 30 FPU per g biomass.

Thermogravimetric analysis and kinetic modeling of low-transition-temperature mixtures pretreated oil palm empty fruit bunch for possible maximum yield of pyrolysis oil

The impacts of low-transition-temperature mixtures (LTTMs) pretreatment on thermal decomposition and kinetics of empty fruit bunch (EFB) were investigated by thermogravimetric analysis. EFB was pretreated with the LTTMs under different duration of pretreatment which enabled various degrees of alteration to their structure. The TG-DTG curves showed that LTTMs pretreatment on EFB shifted the temperature and rate of decomposition to higher values. The EFB pretreated with sucrose and choline chloride-based LTTMs had attained the highest mass loss of volatile matter (78.69% and 75.71%) after 18 h of pretreatment. For monosodium glutamate-based LTTMs, the 24 h pretreated EFB had achieved the maximum mass loss (76.1%). Based on the Coats-Redfern integral method, the LTTMs pretreatment led to an increase in activation energy of the thermal decomposition of EFB from 80.00 to 82.82–94.80 kJ/mol. The activation energy was mainly affected by the demineralization and alteration in cellulose crystallinity after LTTMs pretreatment.

Anaerobic co-digestion of hydrolysate from alkali pre-treated oil palm empty fruit bunches with biodiesel waste glycerol

With an attempt to utilize bio-waste, oil palm empty fruit bunches (OPEFB) were investigated as feedstock for biogas production. Bench scale experiments were carried out employing a two-stage process for the digestion of pre-treated OPEFB with and without the addition of biodiesel waste glycerol (WG). Two continuous stirred tank reactors (CSTR) were operated for acid and methane production. Alkali pre-treated OPEFB were initially acidified to produce an acid hydrolysate solution. Different proportions of WG, between 0.4 and 0.8% (by volume of feed), were increased stepwise. The results indicated that the acidification of these pre-treated brunches generated approximately 4.25 g/L of volatile fatty acid at an optimum pre-treatment condition with soaking in 2% of NaOH for 48 h. This was an increase of 47% compared to the acidification of raw OPEFB. The maximum methane production was achieved with the addition of 0.4% WG, which yielded a gas production of 0.542 and 0.369 L g CODrem biogas and methane, respectively. These conditions can improve the gas yield of biogas and methane by a factor of 1.67 and 2.84, respectively. However, a higher supplementation of WG seemed to promote more instability, which was reflected by a lower methane concentration and organic removal efficiency.

4-Hydroxybenzoic acid from hydrothermal pretreatment of oil palm empty fruit bunches – Its origin and influence on biomass conversion

An unknown major compound, characteristically occurring during processing of oil palm empty fruit bunches was identified with LC-DAD-ESI-MS/MS to be 4-hydroxybenzoic acid. Lignin from oil palm empty fruit bunches contains 4-hydroxybenzoic acid so a tempting conclusion was that the 4-hydroxybenzoic acid originated from lignin. However, another hypothesis to its origin was also tested. The route considered involves degradation of rhamnose to 5-methylfuran-2-carbaldehyde followed by reaction with formic acid. Experimental hydrothermal pretreatment of pure rhamnose in the presence of formic acid revealed that 5-methylfuran-2-carbaldehyde is in fact a degradation product from rhamnose, analogous to glucose degradation to 5-(hydroxymethyl)-2-furaldehyde. However, the subsequent step of carboxylation with formic acid to form 4-hydroxybenzoic acid was found not to take place in practice at realistic biomass hydrothermal pretreatment conditions. 5-methylfuran-2-carbaldehyde only differs from furfural by having an extra methyl group and the degradation route indicates that it may be a new important degradation compound to consider in other biomass feedstocks rich in deoxysugars such as rhamnose or fucose, e.g. pectin rich biomasses. Assessment of the influence of 4-hydroxybenzoic acid in the enzymatic hydrolysis of pretreated oil palm empty fruit bunches as well as its presence during fermentation showed that 4-hydroxybenzoic acid is not inhibiting or mediating neither on the enzymatic hydrolysis or fermentation in the quantified range from 0.1 g/L to 1 g/L, indicating an option for reaping the 4-hydroxybenzoic acid from the biomass liquor directly after hydrothermal pretreatment for biorefinery value-addition.

Effects of changes in chemical and structural characteristic of ammonia fibre expansion (AFEX) pretreated oil palm empty fruit bunch fibre on enzymatic saccharification and fermentability for biohydrogen

Oil palm empty fruit bunch (OPEFB) fibre is widely available in Southeast Asian countries and found to have 60% (w/w) sugar components. OPEFB was pretreated using the ammonia fibre expansion (AFEX) method and characterised physically by the Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy. The results show that there were significant structural changes in OPEFB after the pretreatment step, and the sugar yield after enzymatic hydrolysis using a cocktail of Cellic Ctec2® and Cellic Htec2® increased from 0.15gg−1 of OPEFB in the raw untreated OPEFB sample to 0.53gg−1 of OPEFB in AFEX-pretreated OPEFB (i.e. almost a fourfold increase in sugar conversion), which enhances the economic value of OPEFB. A biohydrogen fermentability test of this hydrolysate was carried out using a locally isolated bacterium, Enterobacter sp. KBH6958. The biohydrogen yield after 72h of fermentation was 1.68mol H2 per mol sugar. Butyrate, ethanol, and acetate were the major metabolites.

Effect of Acid Hydrolysis and Thermal Hydrolysis on Solubility and Properties of Oil Palm Empty Fruit Bunch Fiber Cellulose Hydrogel

Cellulose hydrogel was produced from pretreated oil palm empty fruit bunch fiber (EFB) that went through acid hydrolysis and thermal hydrolysis. The pretreated EFB was dissolved in LiOH/urea aqueous solution using the rapid dissolution method and was subjected to a crosslinking process with the aid of epichlorohydrin to form hydrogel. The effects of both hydrolyses’ time on average molecular weight (Mŋ), solubility, and properties of EFB hydrogels were evaluated. Both hydrolyses led to lower Mŋ, lower crystallinity index (CrI) and hence, resulted in higher cellulose solubility. X-ray diffraction (XRD) characterization revealed the CrI and transition of crystalline structure of EFB from cellulose I to II. The effects of hydrolysis time on the transparency, degree of swelling (DS), and morphology of the regenerated cellulose hydrogel were also investigated using an ultraviolet-visible (UV-Vis) spectrophotometer and a Field emission scanning electron microscope (FESEM), respectively. These findings provide an efficient method to improve the solubility and properties of regenerated cellulose products.

Expression and characterization of a cellobiohydrolase (CBH7B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris

The gene encoding a cellobiohydrolase 7B (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, subcloned, and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kDa. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module. Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55°C with 4-methylumbelliferryl-cellobioside as the substrate and retained 85% of its activity following 24 H incubation at 50°C. Despite the lack of activity toward microcrystalline substrates, this enzyme worked synergistically with the commercial enzyme cocktail Cellic® CTec2 to enhance saccharification by 39% when added to a reaction mixture containing 0.25% alkaline pretreated oil palm empty fruit bunch (OPEFB). Attenuated total reflectance Fourier transform infrared spectroscopy suggested a reduction of lignin and crystalline cellulose in OPEFB samples supplemented with CBH7B. Scanning electron microscopy revealed greater destruction extent of OPEFB strands in samples supplemented with CBH7B as compared with the nonsupplemented control. Therefore, CBH7B has the potential to complement commercial enzymes in hydrolyzing lignocellulosic biomass.

Effect of Buffering System on Acetone-Butanol-Ethanol Fermentation by Clostridium acetobutylicum ATCC 824 using Pretreated Oil Palm Empty Fruit Bunch

Change of pH has been identified as the most significant parameter in modulating the transition between the conversions of acids into solvents in acetone-butanol-ethanol (ABE) fermentation by Clostridia. Thus, ABE fermentation at various phosphate buffer concentrations and initial pH values was conducted using pure glucose and sugars derived from pretreated oil palm empty fruit bunch (OPEFB). A higher solvent concentration (2.93 g/L) was obtained in the fermentation using 20 g/L of glucose with buffer compared with one without buffer that produced 1.34 g/L of solvents. Approximately 8.77 and 9.15 g/L of solvents were produced from fermentation using 40 g/L of glucose with and without buffer, respectively. In the latter conditions, at an initial pH of 5.5, 8.77 g/L of solvents was obtained, which was the highest concentration compared to other initial pH values. Increasing the buffer concentration to 0.2 M at an initial pH of 6.0 resulted in acid accumulation of 16.83 g/L but reduced the solvent production to 1.36 g/L. In addition, ABE fermentation using 20 g/L of sugars from pretreated OPEFB produced 2.25 g/L of solvents with a yield of 0.13 g/g, which was comparable with fermentation using 20 g/L of glucose conducted in a buffering system.

Simultaneous enzymatic saccharification and ABE fermentation using pretreated oil palm empty fruit bunch as substrate to produce butanol and hydrogen as biofuel

Simultaneous saccharification and acetone–ethanol–butanol (ABE) fermentation was conducted in order to reduce the number of steps involved in the conversion of lignocellulosic biomass into butanol. Enzymatic saccharification of pretreated oil palm empty fruit bunch (OPEFB) by cellulase produced 31.58 g/l of fermentable sugar. This saccharification was conducted at conditions similar to the conditions required for ABE fermentation. The simultaneous process by Clostridium acetobutylicum ATCC 824 produced 4.45 g/l of ABE with butanol concentration of 2.75 g/l. The butanol yield of 0.11 g/g and ABE yield of 0.18 g/g were obtained from this simultaneous process as compared to the two-step process (0.10 g/g of butanol yield and 0.14 g/g of ABE yield). In addition, the simultaneous process also produced higher cumulative hydrogen (282.42 ml) than to the two-step process (222.02 ml) after 96 h of fermentation time. This study suggested that the simultaneous process has the potential to be implemented for the integrated production of butanol and hydrogen from lignocellulosic biomass.