Industrial Crops & Products 196 (2023) 116503

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Industrial Crops & Products

journal homepage: www.elsevier.com/locate/indcrop

Fungal pretreatment and acid post-treatment for fractionation and

biovalorization of palm biomass wastes into fungal oil, bioethanol, and
lactic acid
Benjamas Cheirsilp a, *, Asma Billateh a, Rawitsara Intasit a, Apichat Upaichit a,
Piyarat Boonsawang a, Yasmi Louhasakul b
a
International Program of Biotechnology, Center of Excellence in Innovative Biotechnology for Sustainable Utilization of Bioresources, Faculty of Agro-Industry, Prince of
Songkla University, Hat Yai, Songkhla 90110, Thailand
b
Faculty of Science Technology and Agriculture, Yala Rajabhat University, Yala 95000, Thailand

A R T I C L E I N F O A B S T R A C T

Keywords: This research aimed to develop prototype for fully utilizing palm empty fruit brunches (EFB) for production of
Chemical pretreatment sugars and biofuels. EFB was biologically pretreated using oleaginous fungus and post-treated using mild acid.
Ethanol This process can remove lignin and fractionate hemicellulose from cellulose pulp, which was confirmed by
Fungal pretreatment
chemical composition, functional groups, and crystallinity changes. The cellulose pulp was used for ethanol
Palm biomass wastes
production via simultaneous saccharification and fermentation (SSF). To increase initial sugar concentration
Simultaneous saccharification and
fermentation prior to ethanol production, repeated-prehydrolysis (RPH) was attempted. The RPH for four cycles gave the
maximum sugars of 60.9 ± 1.9 g/L, and the subsequent SSF gave the highest ethanol of 35.2 ± 2.7 g/L with an
ethanol yield of 0.40 g/g-sugar. Moreover, during fungal pretreatment the fungus also accumulated lipids with
high potential as biodiesel feedstocks, and the hemicellulose hydrolysate obtained after acid post-treatment
could be used for lactic acid fermentation. This study has shown a practical biorefinery roadmap for bio­
valorization of palm biomass wastes into multiple bioproducts, which may greatly contribute to Bio-Circular-
Green (BCG) economy.

1. Introduction Biological pretreatment of biomass is more advantageous than

chemical and thermal pretreatment because it not only improves the
Palm oil is one of the most important agro-industries in Thailand. enzymatic digestibility of the biomass but also produces other valuable
Thailand is also the world’s third largest crude palm oil producer, with byproducts. A variety of microorganisms can produce lignocellulolytic
an average yield of approximately 2 million tons per year, or 1.2 % of the enzymes and biologically pretreat lignocellulosic biomass. These
world’s crude palm oil production (Beaudry et al., 2018). In general, the include bacteria, yeast, and filamentous fungi. Among them, the fila­
extraction of palm oil from one ton of fresh fruit bunches by the wet mentous fungi are superior choice because of their ability to secrete
process generates several wastes, including 234 kg of empty fruit multi-extracellular enzymes to hydrolyze biomass. Industrially, two
bunches (EFB), 180 kg of palm fibers, 73 kg of palm kernel shells, 180 kg filamentous fungi, i.e., Trichoderma spp. and Aspergillus spp. have been
of decanter sludge, and 0.75 m3 of wastewater (Liew et al., 2015). EFB recognized as effective lignocellulolytic enzyme producers (Hemansi
has a high cellulose and hemicellulose content and is readily available, et al., 2018; Ramamoorthy et al., 2019). Among these fungi, oleaginous
making it a potential renewable and inexpensive resource for bio­ fungi with lignocellulolytic activity are suitable for pretreating ligno­
products and biorefineries (Intasit et al., 2020a,b). Before using EFB as cellulosic biomass due to their simultaneous production of fungal oils,
cellulose pulp, lignin and hemicellulose fractions have been separated which have high potential as biodiesel feedstocks (Cheirsilp and Kitcha,
using a variety of chemical and hydrothermal pretreatments in an effort 2015; Intasit et al., 2020a,b).
to increase their enzymatic digestibility (Yuan et al., 2019; Joy and Currently, despite much research having been done for bioenergy
Krishnan, 2022; Jin et al., 2022). production, one reason that biomass processing costs remain high is due

* Corresponding author.
E-mail address: [email protected] (B. Cheirsilp).

https://doi.org/10.1016/j.indcrop.2023.116503
Received 6 October 2022; Received in revised form 9 February 2023; Accepted 23 February 2023
Available online 4 March 2023
0926-6690/© 2023 Elsevier B.V. All rights reserved.
B. Cheirsilp et al. Industrial Crops & Products 196 (2023) 116503

to the high cost of enzymes and the ineffective pretreatment and 2. Materials and methods
fermentation processes. Furthermore, most studies process biomass into
just one product. This means that the biomass is not fully utilized. In 2.1. Microorganisms
fact, biomass by nature often consists of many components with
different potentials. Therefore, the effective fractionation and valoriza­ An oleaginous fungus, Aspergillus tubingensis TSIP9, was obtained
tion techniques based on the biorefinery concept should be developed to from the Bioprocess Engineering Laboratory, Faculty of Agro-Industry,
fully utilize components of the biomass. A combined refining technology Prince of Songkla University, Thailand. The fungus was grown on
would help convert cellulose, hemicellulose, and lignin in biomass into commercial Potato dextrose broth (HiMedia, India) at pH 5 and their
high-value products. This study aimed to develop a prototype of bio­ spores were used as an inoculum. The spore suspension with suitable
energy production from EFB considering the use of environmentally concentration was prepared by using haemocytometer (Cheirsilp and
friendly technologies. Firstly, EFB was delignified using oleaginous Kitcha, 2015). Ethanol-producing yeast Saccharomysis cerevisiae and
fungi and the hemicellulose in EFB was fractionated by dilute acid post Candida shehatae were obtained from Microbiology Laboratory, Faculty
treatment. Subsequently, the pretreated EFB was used to produce bio­ of Agro-Industry, Prince of Songkla University, Thailand.
ethanol via simultaneous saccharification and fermentation (SSF). It Ethanol-producing yeast Kluyveromyces marxiamas TISTR 5178 and
should be noted that increasing sugar concentration is a straight method lactic acid bacterium Lactobacillus pentosus TISTR 920, were obtained
to achieve a higher ethanol titer, which is economically beneficial for from the Thailand Institute of Scientific and Technological Research
subsequent downstream process. To increase sugar concentration and (TISTR, Thailand). Yeast inoculum was prepared using YPD medium, pH
ethanol productivity, the repeated-prehydrolysis prior to SSF was 5, which was composed of 1 % yeast extract, 2 % tryptone, and 10 %
attempted. In addition, the fungal oils were evaluated for their biodiesel glucose. The yeast culture was incubated at 30 ◦ C and at 150 rpm for
properties, and the hemicellulose hydrolysate was used to produce 18–24 h (Yuan et al., 2019). The lactic acid bacteria inoculum was
ethanol and lactic acid. The chemical composition, functional groups, prepared by transferring stock culture to MRS broth at pH 6, which was
and crystallinity of raw and pretreated EFB were compared. The concept composed of 1 % beef extract, 1 % proteose peptone, 0.5 % yeast extract,
of fungal pretreatment and acid post-treatment for fractionation and 2 % glucose, 0.5 % sodium acetate, 0.2 % ammonium citrate, 0.2 %
biovalorization of EFB into bioethanol and lactic acid is shown in Fig. 1. dipotassium phosphate, 0.1 % polysorbate 80, 0.01 % magnesium sul­
fate, and 0.005 % manganese sulfate. The bacterium was incubated at
37 ◦ C under anaerobic conditions for 18–24 h (Chen et al., 2019).

Fig. 1. Concept of fungal pretreatment and acid post-treatment for fractionation and biovalorization of EFB into bioethanol and lactic acid.

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B. Cheirsilp et al. Industrial Crops & Products 196 (2023) 116503

2.2. Pretreatment of EFB the amount of enzyme liberating 1 mol of pNP under the assay condi­
tions (Çelik et al., 2016). For commercial cellulase, the FPase activity
EFB after separation of fresh fruit, were obtained from Chumporn was measured using filter paper (Whatman No.1 with size of
Palm Oil Industry Public Co. Ltd. in Chumporn Province, Thailand. 1 cm × 6 cm) as substrate. One unit of FPase activity (FPU) is defined as
Dried EFB was shredded and cut into 2–5 cm size before use. The main the amount of enzyme liberating 1 µmol of glucose per min under the
compositions of EFB were as follows: 48.9 ± 4.2 % cellulose; 23.6 ± 1.9 specified conditions (Ezeilo et al., 2020). Lignin peroxidase was
% hemicellulose, 16.8 ± 0.8 % lignin, 4.6 ± 0.4 % ash, and 7.86 analyzed by measuring the oxidation of veratryl alcohol to vera­
± 0.67 mg-oil/g-EFB. Fungal pretreatment of EFB was conducted traldehyde. One unit of lignin peroxidase activity is defined as that
through solid state fermentation by adding modified mineral salt solu­ amount of enzyme which produces 1 μmol of veratraldehyde from
tion (MS solution) to control moisture adjustment at 65 %, and inocu­ veratryl alcohol in one minute (Mohamad Ikubar et al., 2018).
lating fungal spores at 106-107 spores/g-EFB (Intasit et al., 2020a). The To determine fungal biomass and fungal oils, the fungal biomass was
modified MS solution contained 1 g/L yeast extract, 1.7 g/L (NH4)2SO4, eluted from the fungal pretreated EFB using Tween 80 solution (0.1 %)
2.0 g/L KH2PO4, 0.5 g/L MgSO4⋅7 H2O, 0.2 g/L CaCl2⋅2 H2O, 0.01 g/L at a solid loading of 10 %, and the mixture was vortexed for 5 min. The
FeSO4⋅7 H2O, 0.01 g/L ZnSO4⋅7 H2O, 0.001 g/L MnSO4⋅4 H2O, elution was based on the property of Tween 80 that it can disperse fungal
0.0005 g/L CuSO4⋅5 H2O, 1 g/L Tween-80, pH 5.5 (Cheirsilp and aggregates from the biomass (Intasit et al., 2020a). The fungal biomass
Kitcha, 2015). The culture was statically incubated at 30 ± 2 ◦ C for 6 was dried and used to extract fungal oil by chloroform-methanol solvent
days. After fungal pretreatment, the fungal biomass and enzymes was extraction method. The extraction was repeated and the solvent phase
eluted from the fermented solids by using citrate buffer (50 mM, pH was combined. The solvent was then evaporated to obtain fungal oils.
5.0). The fermented solids, namely fungal pretreated EFB (FPEFB), were The yield of fungal oils was calculated as oil weight in milligrams per
dried at 60 ◦ C before use. gram-EFB.
Post-treatment of FPEFB by dilute acid was carried out in order to Microscopic pictures were taken to determine the surface
fractionate hemicellulose from cellulose pulp (Chen et al., 2018). In morphology of raw and pretreated EFB. The chemical functional groups
short, the biomass was added with 1% (w/w) sulfuric acid at 10% solid of raw and pretreated EFB were assessed by a Fourier-Transform
loading and sterilized at 121 ◦ C for 30 min. After the treatment, the Infrared Spectroscopy (FT-IR) analyzer (Agilent Technologies Cary
slurry was neutralized to pH 7.0 with NaOH. The solid fraction was 630 FT-IR, California, USA), at a frequency range of 500–4000 cm− 1 and
separated, dried at 60 ◦ C to a constant weight, and named acid-fungal a resolution of 4 cm− 1. The crystallinity of the raw and pretreated EFB
pretreated EFB (AFPEFB). The liquid fraction was named hemicellu­ (40–60 mesh) was measured using an X-ray diffractometer (XRD) in a
lose hydrolysate. Shimadzu XRD-700 MaximaX series, pro diffractometer set at 40 kV,
using an ultrafast detector at 2θ range of 5–90◦ with a 5◦ min− 1 scan
2.3. Ethanol production via simultaneous saccharification and rate. Formulation was used to determine the degree of crystallinity as
fermentation (SSF) and repeated-prehydrolysis prior to SSF (RPH-SSF) the ratio of the combined area of all crystalline peaks to the total area
(Soha et al., 2021).
First, SSF was performed by adding AFPEFB in buffer at 10 % solid Fatty acid compositions were analyzed by converting fungal oils into
loading and yeast extract at 0.3% as a nitrogen source. The cellulase was fatty acid methyl esters (FAME) prior to GC analysis. The fungal oils
added together with yeast inoculum at 5, 10 and 15 FPU/g. The yeast were transesterified using acid catalyst, sulfuric acid at 90 ◦ C for 90 min.
culture was incubated at 30 ◦ C and at 150 rpm. After fermentation for A DB-5-MS capillary column and a flame ionization detector (FID) were
24 h, the flask was tightly sealed to keep anaerobic condition during used. The temperature profiles for oven were: initial temperature held at
ethanol production for 72 h. To increase the initial sugar concentration, 80 ◦ C, 5 min, raised to 290 ◦ C at 4 ◦ C/min, and held at 290 ◦ C, 5 min.
the RPH-SSF was performed by replacing AFPEFB every 72 h. Additional The injector temperature and detector temperature were 250 ◦ C and
enzyme mixture was added to maintain constant activity during each 230 ◦ C, respectively. FAME compositions were then used to calculate
cycle of RPH. After RPH for 4 cycles, the yeast was inoculated with the important biodiesel properties of the fungal oils based on the
AFEFB in the fifth cycle. Subsequently, SSF was performed as mentioned empirical equations (Karpagam et al., 2015). The iodine value (IV) and
above. saponification value (SV) were calculated using percentage of each fatty
acid methyl ester (%FA), molecular weight (MW), and the number of
2.4. Analytical methods double bonds (D) in each FAME component.

The concentration of yeast cells was measured using a haemocy­ 3. Results and discussion
tometer. The ethanol concentration was determined using a gas chro­
matography (GC, Shimadzu GC-2014) equipped with a Stabilwax 3.1. Fungal pretreatment of EFB
column (Noomtim and Cheirsilp, 2011; Deesuth et al., 2016; Malik et al.,
2021). The concentrations of sugars and products in the supernatant In general, the cellulose content in lignocellulosic biomass is covered
were determined using a high pressure liquid chromatography (HPLC) with lignin. Therefore, pretreatment is required to remove lignin and
equipped with an Aminex HPX-87H column (Bio-Rad, USA). Total acid reduce cellulose crystallization. The removal of lignin can be done in a
was determined by the titration method. Reducing sugar and total sugar number of ways. Compared with other methods, the biological method
concentrations were analyzed using the dinitrosalicylic (DNS) method, is more environmentally friendly. Therefore, EFB was biologically pre­
and the phenol sulfuric method, respectively. treated through solid state fermentation by the oleaginous fungus
The fungal enzymes, including cellulase, xylanase, β-glucosidase, A. tubingensis TSIP9 as previously described (Intasit et al., 2020a,b). It
and lignin peroxidase, in the fermented liquid were determined. The should be noted that this biological method also simultaneously pro­
activities of cellulase and xylanase were determined using carboxy­ duced fungal oils and lignocellulosic enzymes (Fig. 1).
methylcellulose (CMC) and xylan, respectively. The mixture of enzyme
and substrate was incubated at 50 ◦ C for 10 min. One unit of cellulase 3.1.1. Composition changes of raw and pretreated EFB
(IU) was defined as the amount of enzyme liberating 1 μmol of glucose The composition of EFB after fungal pretreatment is shown in
per min, and one unit of xylanase was defined as the amount of enzyme Table 1. The content of lignin decreased, and this resulted in a higher
liberating 1 μmol of xylose per min (Ezeilo et al., 2020). Fungal content of cellulose and hemicellulose. As shown in Fig. 2, after
β-glucosidase was analyzed using p-nitrophenyl-β-D-glucopyranoside delignifying using fungi, the EFB bundles broke down and released
(pNPG) solution as substrate. One unit of β-glucosidase was defined as holocellulose fiber. Depolymerization and aromatic ring cleavage are

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Table 1
Comparison of raw empty fruit bunches (EFB), fungal pretreated EFB (FPEFB), acid-fungal pretreated EFB (AFPEFB) before and after ethanol production.
Materials Residual yield Cellulose (%) Hemicellulose (%) Lignin (%) Crystallinity (%)

Raw EFB 1.00 48.9 ± 4.2 23.6 ± 1.9 16.8 ± 0.8 24.51
FPEFB 0.79 54.6 ± 2.6 32.8 ± 0.5 7.01 ± 0.15 17.98
AFPEFB 0.40 68.3 ± 0.7 2.9 ± 0.7 7.11 ± 0.09 14.54
AFPEFB after ethanol production 0.21 57.2 ± 0.1 3.9 ± 0.1 14.9 ± 0.03 16.98

Fig. 2. Appearance of empty fruit bunches (EFB), fungal pretreated EFB (FPEFB) and acid-fungal-pretreated EFB (AFPEFB) before and after ethanol fermentation.

phases in the delignification process carried out by fungus using a va­ hemicellulose hydrolysate obtained after the acid post-treatment con­
riety of extracellular enzymes. First, lignin’s -O-4 bonds are oxidized to tained total sugars of 25.4 ± 0.8 g/L.
produce arylglycerol molecules. Second, aromatic rings are cut along the
"-ketoadipate pathway," and third, the "-O-4 oxidation" of the "cleaved 3.1.2. Evaluation of fungal oils as biodiesel feedstock
aromatic rings" results in the "cyclic carbonate structures" (Javaid et al., In addition to biological pretreatment, the fungi also accumulated
2019). Additionally, in non-enzymatic oxidation reactions, the fungi oils as high as 68 mg/g-EFB. The production of biodiesel, namely FAME,
have been claimed to be able to partially oxidize lignin through aromatic from fungal oils involves two consecutive steps of hydrolysis and
ring demethylation that breaks down lignin. It should be emphasized esterification with methanol. The FAME compositions of fungal oils are
that the fungi can delignify materials either selectively or indifferently. shown in Table 2. The main FAMEs found in fungal oils were long chain
Selective delignification removes lignin without significantly reducing fatty acids with carbon atom of 16–18 including palmitic acid (42.54
cellulose, whereas a non-selective delignifying process degrades all of ± 0.06%), oleic acid (41.04 ± 0.08 %), stearic acid (5.78 ± 0.04 %),
the primary cell wall constituents (Priyanga and Kannahi, 2018). and linoleic acid (8.51 ± 0.02 %). Biodiesel fuel properties include
In this study, to provide an alternative use of xylose, the hemicel­ iodine value (IV), saponification value (SV), cetane number (CN), high
lulose fraction was separated from the cellulose pulp and used as carbon heating value (HHV), long chain saturation factor (LCSF), cold filter
source for either xylose-assimilating yeasts or lactic acid bacteria. plugging point (CFPP), and oxidation stability (OS). These fuel proper­
Furthermore, prior to enzymatic hydrolysis, the cellulose pulp in EFB ties can be used to assess the suitability of the fungal oils as biodiesel
should be decrystallized to increase enzymatic accessibility. In this feedstocks (Table 2). The SV value represents the number of milligrams
study, the fungal pretreated EFB (FPEFB) was then further fractionated of alkali such as potassium hydroxide that are used to react with 1 g of
into hemicellulose hydrolysate and cellulose pulp by using acid post- triglycerides into soap. The SV value of fungal oils was 206.69 ± 0.03
treatment. The compositions of EFB, FPEFB, and acid-fungal pre­ mgKOH/g oil. IV indicates the level of unsaturated fatty acids and the
treated EFB (AFPEFB) are compared in Table 1. After acid post- possible oxidation that forms partially oxidized compounds causing
treatment of FPEFB, the cellulose content of FPEFB increased from deposits in the engine injection system. A high IV indicates a high
54.6 ± 2.6 % to 68.3 ± 0.7 %, while the amount of hemicellulose content of unsaturated fatty acids and rancidity (lipid oxidation). Ac­
decreased from 32.8 ± 0.5 % to only 2.9 ± 0.7 %. The cellulose content cording to the European biodesel standard, the IV value should not
of AFPEB in this study was significantly higher than that of EFB pre­ exceed 120 g-I2/100 g-oil. As shown in Table 2, the IV value of fatty
treated by other methods such as alkaline and hydrogen peroxide (Zhai acids derived from the fungus A. tubingensis TSIP9 is 53.04 ± 0.08 g-I2/
et al., 2020). Obviously, after acid post-treatment, the cellulose pulp 100 g-oil, which is considered suitable and does not oxidize easily, and
with smooth surface was obtained (Fig. 2). Typically, when lignocellu­ this value also meets the international standard requirements.
losic materials are used for ethanol production by the yeast, the ligno­ The CN value presents the biodiesel combustion characteristic in
cellulosic materials need a saccharification step to produce fermentable engine, which relates to the FAME compositions and depends on the
sugars (Joy and Krishnan, 2022; Jin et al., 2022; Yuan et al., 2019). The degree of unsaturated fatty acids and the chain length. A high value of

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Table 2 CN means that the biodiesel requires a shorter time for ignition. The
Fatty acid composition and estimated biodiesel fuel properties of fungal oil. calculated CN value in this study was 60.77 ± 0.01 which meets with
Fatty acids Relative content (%) the international standard of ASTM D6751 that require a minimum of 47
(Karpagam et al., 2015). The DU value of the fungal oils was 58.56
Caprylic acid (C8:0) 0.01 ± 0.00
Nonanoic acid (C9:0) 0.01 ± 0.00 ± 0.11, which ensures the stability against oxidizing agents during
Capric acid (C10:0) 0.02 ± 0.00 long-term storage (Talebi et al., 2013). The LCFP and CFPP values show
Lauric acid (C12:0) 0.08 ± 0.01 the biodiesel filterability under low temperature condition. These values
Myristic acid (C14:0) 0.23 ± 0.01 depend on the degree of saturated fatty acids such as palmitic and steric
Pentadecoic acid (C15:0) 0.05 ± 0.00
Palmitic acid (C16:0) 42.54 ± 0.06
acids. Higher CFPP values indicate poorer flow performance at low
Pamitoleic acid (C16:1) 0.11 ± 0.00 temperatures as the oil precipitates into the filter (Karpagam et al.,
Heptadecanoic acid (C17:0) 0.14 ± 0.00 2015). The results showed that the CFPP and LCSF values of fatty acids
Steric acid (C18:0) 5.78 ± 0.04 from A. tubingensis TSIP9 were 8.73 ± 0.04 and 10.95 ± 0.12, respec­
Oleic acid (C18:1) 41.04 ± 0.08
tively. These parameters have proven that the fungal oils obtained
Linoleic acid (C18:2) 8.29 ± 0.03
Linolenic acid (C18:3) 0.22 ± 0.01 during biological pretreatment in this study, have high potential to be
Arachidic acid (C20:0) 0.49 ± 0.01 used as biodiesel feedstocks. The biodiesel properties of fungal oil in this
Eicosenoic acid (C20:1) 0.14 ± 0.01 study were consistent with those previously reported in terms of CN
Behenic acid (C22:0) 0.19 ± 0.00 value of 50–69, which complies with the ASTM D6751 international
Erucic acid (C22:1) 0.26 ± 0.01
Lignoceric acid (C24:0) 0.41 ± 0.02
standard (a minimum of 47) (Kamat et al., 2013; Intasit et al., 2020b;
C16-C18 98.12 ± 0.07 Srinivasan et al., 2021).
Saturated fatty acid (SFA) 49.95 ± 0.10
Unsaturated fatty acid (UFA) 50.05 ± 0.10 3.1.3. Fungal enzyme production during pretreatment
Monounsaturated fatty acid (MUFA) 41.55 ± 0.08
In addition to pretreatment of EFB and producing fungal oils, the
Polyunsaturated fatty acid (PUFA) 8.51 ± 0.02
SFA/UFA 1.00 ± 0.00 fungi could also produce fungal cellulase and fungal xylanase as shown
Estimated biodiesel fuel properties Value in Fig. 3. Fig. 3 shows that the fungi produced the xylanase enzyme at
Saponification value (SV) 206.69 ± 0.03 the maximum level on day 4 of fermentation, 61.5 ± 1.7 units/g-EFB.
Iodine value (IV) 53.04 ± 0.08 While the maximum cellulase level was achieved on day 5 of fermen­
Cetane number (CN) 60.77 ± 0.01
tation at 23.0 ± 1.0 units/g-EFB, no beta-glucosidase activity was
Degree of unsaturation (DU) 58.56 ± 0.11
Long chain saturation factor (LCSF) 8.73 ± 0.04 found. The majority of the enzymes secreted by the fungi depend not
Cold filter plugging point (CFPP) 10.95 ± 0.12 only on the chemical composition of the biomass but also on its struc­
tural complexity. The biomass should contain accessible inducers and a
low level of hydrolytic products (Shah et al., 2017). The higher xylanase
activity than cellulase activity might be due to the substrate structure,

Fig. 3. Enzyme production during fungal pretreatment of EFB. EFB was inoculated with oleaginous fungus A. tubingensis TSIP9 and fermented under solid state
condition for 6 days.

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complexity, and availability. It seems that first the fungi have to secrete developed. It should also be noted that higher enzyme loading did not
xylanase to degrade the outer layer (hemicellulose) of the lignocellulosic significantly improve sugar conversion. This could be because of the
biomass before secreting cellulase to further degrade the inner layer saturation of enzyme adsorption on the cellulose surface (Rohrbach and
(cellulose). The lower cellulase activity might be due to the lower Luterbacher, 2021).
accessibility to cellulose. As β-glucosidase needs cellobiose to trigger To assess the feasibility of applying fungal enzymes for ethanol
their production, it was possible that there was no enough cellobiose in production from AFPEFB, the enzymes harvested after fungal pretreat­
the system (Santa-Rosa et al., 2018). Another possible reason would be ment were concentrated with cold acetone and used in the SSF of
the mass transfer limitation during solid-state fermentation. Other AFPEFB at 15 FPU/g. However, the ethanol production was only 2.82 g/
studies have reported the production of xylanase and cellulase by L (data not shown), which is much lower than the use of commercial
Aspergillus spp. using agricultural wastes. The xylanase production by cellulase. This may be due to the instability of the cellulase in the crude
A. tubingensis TSIP9 in this study, was comparable to those previously enzymes. At least, this study has shown that fungal enzymes can be used
reported N (Intasit et al., 2021a,b) and even higher than other studies to digest EFB into fermentable sugars for yeast to produce ethanol.
such as the xylanase production (54.32 U/g) by Aspergillus sp. LPB-5 However, more studies are needed to increase the stability of the fungal
cultivated on EFB (Colonia et al., 2019) and the cellulase production enzymes.
(0.063 U/mL) by A. tubingensis KY615746 cultivated on rice straw
(El-Nahrawy et al., 2017). The maximum lignin peroxidase activity of
5.96 U/g-EFB was obtained on day 5. It has been reported that lignin 3.3. Ethanol production via RPH-SSF
peroxidase is the primary catalyst in the de-polymerization of
non-phenolic lignin. The decrease in lignin content of EFB after fungal RPH-SSF of AFPEFB was conducted in order to increase the con­
pretreatment was likely due to the activity of lignin peroxidase. In centration of initial sugars without concentration of sugar solution by
addition, as lignin degradation is an oxidative process, solid-state evaporation. During RPH-SSF, the solid residues can be easily separated
fermentation that generally operates under minimal moisture but by sieving. Therefore, less energy is required when compared to the
maximum air exposure conditions, promotes better lignin degradation concentration of sugar liquid by evaporation. In RPH-SSF, each cycle of
(Mohamad Ikubar et al., 2018). However, it should be noted that the prehydrolysis was performed using only cellulase or a combination of
various enzyme activities detected also depend on the definition of the cellulase and β-glucosidase. After prehydrolysis in each cycle, the re­
enzyme activities. sidual fiber was replaced with the fresh AFPEFB for four cycles.
Incomplete hydrolysis of the fiber can be explained by several reasons,
such as unproductive enzyme adsorption, deactivation of enzymes,
3.2. Ethanol production via SSF using commercial cellulase and fungal decrease in availability of chain ends, and unaccessible inner crystalline
cellulase cellulose (Olofsson et al., 2008). Therefore, the residual fiber should be
further post-treated before reuse. From Fig. 5, it can be seen that the
Ethanol production was performed via SSF using different loadings enzymes were able to effectively hydrolyze AFPEFB and release sugars
of cellulase. The AFPEFB was added at 10 % in buffer and yeast extract during four cycles of RPH. The RPH successfully increased total sugar
was added at 0.3 % as a nitrogen source. The cellulase was added concentration up to the maximum level of 60.9 ± 1.9 g/L. This could be
together with yeast inoculum at 5, 10 and 15 FPU/g and the yeast cul­ because there was sufficient supply of substrate and, as the residual
ture was incubated at 30 ◦ C and at150 rpm for 24 h. The flask was enzymes could be reused, this also reduced the production cost. As a
covered with rubber stoppers to create anaerobic conditions and the result, the RPH used in this study is effective at increasing sugar con­
culture was shaken for 72 h. The results of yeast cell count and ethanol centration prior to SSF.
production are shown in Fig. 4. With increasing cellulase loading from 5 Compared with the use of cellulase alone, the combination of
FPU/g to 10 FPU/g, the yeast grew slightly better and produced more cellulase and β-glucosidase gave much higher sugar production. After
ethanol. A further increase in cellulase loading to 15 FPU/g did not the four cycles of RPH, yeast extract was added at 0.3 % and S. serevisiae
further increase yeast cell growth but increased ethanol production up to was inoculated to initiate SSF for ethanol production. From Fig. 6, it was
the maximum level of 8.5 g/L. However, this level was still very low, also found that the use of combined enzymes gave higher ethanol pro­
probably due to the relatively low sugar concentration in the system. It duction than the use of cellulase alone. The maximum ethanol produc­
was found that the maximum sugar concentration was only 13 g/L. tion obtained was 35.2 ± 2.7 g/L and 24.1 ± 0.4 g/L, respectively. It
Therefore, the process for increasing the sugar concentration should be can be seen that when only cellulase was added, in the third and the

Fig. 4. Yeast cell growth and ethanol production from acid-fungal pretreated empty fruit bunches (AFPEFB) via simultaneous saccharification and fermentation
(SSF) using various cellulase loadings.

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B. Cheirsilp et al. Industrial Crops & Products 196 (2023) 116503

advantages over traditional prehydrolysis-SSF, including smaller or

fewer reactors and lower utility consumption. When comparing with the
same substrate used for ethanol production, the ethanol production from
EFB in this study was much higher than those previously reported by
other studies (Kamoldeen et al., 2017; Pangsang et al., 2019; Zhai et al.,
2020; Polprasert et al., 2021), which were in the range of 4–16 g/L. The
use of fungal pretreated EFB and RPH-SSF in this study gave slightly
higher ethanol production than the use of sulfite-pretreated EFB
(32 g/L) (Cheng et al., 2014). Only one research study reported very
high ethanol production of 83.6 g/L from EFB pretreated by the For­
miline process (Cui et al., 2014). It should be noted that the fungal
pretreatment used in this study is more environmentally friendly and
can also produce fungal oils as biodiesel feedstocks.

3.4. Functional groups and crystallinity analysis of raw and pretreated

EFB

The functional groups present in raw and pretreated EFB were

analyzed by FT-IR. Raw EFB was composed of hemicellulose, lignin, and
celluloses, indicated by alkanes, ketones, and alcohols with different
oxygen-containing functional groups. Fig. 7 shows the spectra of raw
EFB (a), FPEFB (b), AFPEFB (c), and AFPEFB after ethanol production
(d). The broad peak (Peak 1) at wavenumber range between 3000 and
3500 cm− 1 can be associated with the O-H bond stretching vibration of
hydroxyl and carboxyl groups. In raw EFB, Peak 1 has quite a strong
intensity, which becomes reduced upon increasing steps of pretreat­
ment. Such a reduction in Peak 1 intensity indicates the removal of
hydroxyl and carboxyl groups through dehydration and decarboxylation
reactions, respectively. In general, removing hydroxyl and carboxyl
groups from biomass increases its hydrophobicity (Soha et al., 2021).
The peak that appears at wavenumber of around 2919 cm− 1 (Peak 2)
Fig. 5. Reducing sugar and total sugar production during repeated-
can be assigned to the C-H bond stretching vibration in aliphatic and
prehydrolysis prior to simultaneous saccharification and fermentation (RPH-
aromatic structures. Peak 3 (1725 cm− 1) on the other hand, can be
SSF) of acid-fungal pretreated empty fruit bunches (AFPEFB). The prehydrolysis
was repeated for four cycles prior to SSF in the fifth cycle. attributed to C–– O bonds in carbonyl or acetyl groups stretching in
hemicellulose. The intensity of Peak 3, which is relatively low in raw
EFB, becomes significantly stronger. The peaks located at wavenumber
fourth cycles the sugar concentration was likely constant, possibly due
of 1645 cm− 1 (Peak 4) and 898 cm− 1 (Peak 7) can be associated with the
to the product (cellobiose) inhibition to cellulase. When β-glucosidase
C–– C stretching vibrations and C-H out-of-plane bending, respectively,
was added together with cellulase, the cellobiose was further hydrolyzed
of the aromatic groups in lignin (Reza et al., 2014). In addition, the
to glucose, and this increased the reducing sugar concentration and also
peaks at wavenumbers of 1248 cm− 1 (Peak 5) and 1052 cm− 1 (Peak 6),
reduced the product inhibition to cellulase. Obviously, this higher sugar
on the other hand, correspond to C-O-C and C-O stretching vibrations in
concentration positively affected the yeast cell growth and hence
cellulose (Nakason et al., 2018). These two peaks become slightly more
ethanol production (Fig. 6). The residual pulp after prehydrolysis could
defined, which can be attributed to the removal of amorphous compo­
be reused and the residual pulp after SSF, together with yeast biomass,
nents in cellulose after ethanol production.
could be used as animal feed due to their high nutritional value (Shinya
Fig. 8 shows the powder X-ray diffraction patterns of raw EFB (a),
et al., 2022) (Fig. 1). The developed RPH-SSF also has several
FPEFB (b), AFPEFB (c), and AFPEFB after ethanol production (d). In this

Fig. 6. Yeast cell growth and ethanol production during repeated-prehydrolysis prior to simultaneous saccharification and fermentation (RPH-SSF) of acid-fungal
pretreated empty fruit bunches (AFPEFB). The prehydrolysis was repeated for four cycles prior to SSF in the fifth cycle.

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B. Cheirsilp et al. Industrial Crops & Products 196 (2023) 116503

Fig. 7. FT-IR spectra of raw empty fruit bunches (EFB) (a), EFB after fungal pretreatment (FPEFB) (b), EFB after fungal-acid pretreatment (AFPEFB) (c), and AFPEFB
after ethanol production (d).

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B. Cheirsilp et al. Industrial Crops & Products 196 (2023) 116503

study, the peaks of the X-ray signals of raw and pretreated EFB present However, both yeast strains produced very low ethanol (0.9–1.75 g/L)
the cellulose I pattern. Naturally, cellulose chains obtain both crystalline from hemicellulose hydrolysate with very low sugar consumption (<20
(ordered) regions, which are difficult for enzymes to digest, and amor­ %). It should be noted that after evaporation, the concentration of acetic
phous (less ordered) regions, which are easy to digest. The crystalline acid was as high as 10 g/L, which might inhibit the yeast cell growth and
cellulose structure is responsible for the three peaks labeled with (*) that ethanol production. As the hemicellulose hydrolysate was not suitable
appear at 2θ of around 15.5, 22.7, and 34.6◦ F. These three peaks look for ethanol production, its use for lactic acid fermentation was
sharper after pretreatment due to the removal of amorphous compo­ attempted. The hydrolysate was used directly without concentration. It
nents, i.e., hemicellulose, extractives, lignin, and amorphous cellulose was found that L. pentosus TISTR 920 could grow well and produce lactic
(Soha et al., 2021). The pretreatment mainly involves dehydration and acid of 18.9 g/L from 25.4 g/L sugar concentration corresponding to a
decarboxylation reactions, which contributed to hemicellulose and yield of 0.74, which was comparable to those in the literature (Abdel-­
amorphous cellulose decomposition. Nonetheless, it was found that the Rahman et al., 2016).
AFPEFB after ethanol production holds crystalline cellulose since most
of the amorphous cellulose was removed. The maximum crystallinity 3.6. Possible economic analysis
level (25.51 %) was found in raw EFB (Table 1). After fungal pretreat­
ment, the crystallinity degree decreased to 17.98 %, and after The chemicals and utility cost analysis for the production of 1 L-
acid-fungal pretreatment, the crystallinity degree further decreased to ethanol from EFB is presented in Table 3. The chemicals cost for fungal
14.54 %. However, the crystallinity degree of AFPEFB after ethanol pretreatment was 0.0329 $/L-EtOH, while that for acid pretreatment
production was slightly increased, reaching 16.98 %. Solubilization of was about threefold higher (0.108 $/L-EtOH). The chemicals cost for
the amorphous component of fibers under pretreatment may contribute yeast fermentation was as high as 0.505 $/L-EtOH, which was 63 % of
to the increasing crystallinity degree of AFPEFB (Fatriasari et al., 2017). the total chemicals and utility cost (0.8065 $/L-EtOH) for ethanol pro­
It should be noted that the cellulose content after ethanol fermentation duction from EFB. Enzymes and yeast extract were the main contribu­
decreased due to enzymatic hydrolysis by cellulase while the content of tors to the yeast fermentation cost. The high cost of ethanol production
hemicellulose and lignin increased. in this study was due to the relatively high cost of enzymes and yeast
extract. In Eggeman and Elander’s (2005) cost analysis model, the cost
3.5. Potential use of hemicellulose hydrolysate for ethanol and lactic acid of enzymes was assumed to be 0.04 $/L-EtOH as an estimate of a
production reasonable cost in future refineries. Therefore, if the price of enzymes
and yeast extract can be lowered, the production cost of ethanol would
The hemicellulose hydrolysate from the acid post-treatment was become much lower and be comparable to those previously reported,
used as a carbon source for ethanol and lactic acid fermentation. The which were in the range of 0.60–0.88 $/L-EtOH (Kumar and Murthy,
sugar composition in this hydrolysate was xylose of 13.93 ± 0.67 g/L 2011; Do and Lim, 2016; Syed abdullah et al., 2016). It should be noted
and arabinose of 1.05 ± 0.15 g/L. In addition, acetic acid was found to that the revenue from the co-product sales, i.e., fungal oil and lactic acid,
be released along with the sugar at a concentration of 3.14 ± 1.19 g/L. might significantly help offset the production cost of ethanol. The
Because ethanol production requires a high enough initial sugar to crystalline cellulose residues after ethanol fermentation might also be
stimulate yeast to produce ethanol, the hydrolysate was pH adjusted to one of the valuable co-products that could be applied in cellulose pulp
5.0 using NaOH and evaporated to adjust the total sugar concentration industry (Wang et al., 2021).
to 50 g/L and then used as a carbon source for ethanol production by
two xylose-assimilating yeasts, Candida shehatae and Kluyveromyces 4. Conclusions
marxiamas TISTR 5178. The yeast extract was added at 0.3 % as a ni­
trogen source. When using pure xylose as a carbon source, the ethanol This study has shown strategies to biovalorize palm biomass wastes
production by both strains were similar at 26.4 and 26.1 g/L, respec­ by using them as low-cost substrate for biofuel production. EFB was
tively which were consistent with the sugar utilization rate (70–71 %).
Table 3
Cost analysis for production of 1 L-ethanol (EtOH) from empty fruit bunches
(EFB).
Raw materials/ Amount Unit cost Cost Total cost
Chemicals (kg/L- ($/kg) ($/L-EtOH) ($/L-EtOH)
EtOH)

- EFB 10 - - -
Chemicals for fungal pretreatment 0.0329
- Yeast extract 0.0185 1.25 0.023125
- Tween 80 0.0185 0.002 0.000037
- Magnesium 0.00927 0.005 0.00004635
sulfate
- Calcium chloride 0.0037 0.01 0.000037
- Ammonium 0.03145 0.03 0.0009435
sulfate
- Dipotassium 0.037 0.085 0.003145
phosphate
- Water 18.5 0.0003 0.00555
Chemicals for acid pretreatment 0.108
- Sulfuric acid 1 0.088 0.088
- Sodium 2 0.01 0.02
hydroxide
Chemicals for yeast fermentation 0.505
- Enzymes 0.1 1 0.1
- Yeast extract 0.3 1.25 0.375
Fig. 8. Powder X-ray diffraction patterns of raw empty fruit bunches (EFB) (a), - Water 100 0.0003 0.03
Utility cost (kWh) 2.2 0.073 0.1606
EFB after fungal pretreatment (FPEFB) (b), EFB after fungal-acid pretreatment
Total cost for EtOH production 0.8065
(AFPEFB) (c), and AFPEFB after ethanol production (d).

9
B. Cheirsilp et al. Industrial Crops & Products 196 (2023) 116503

biologically pretreated by oleaginous fungus, coupled with the pro­ El-Nahrawy, S., Metwally, M., El-Kodoos, R.Y.A., El-Sayed, B.B., Shabana, S.A., El-
Refai, I.M., 2017. Optimization of culture conditions for production of cellulase by
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substrate under solid state fermentation. Renew. Energy 156, 1301–1312.
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SSF. The RPH increased total sugar concentration up to 60.9 ± 1.9 g/L palm empty fruit bunches (EFB) to enhance its fermentable sugar production. Waste
and this higher sugar concentration led to higher ethanol production of Biomass Valoriz. 8, 379–391.
Hemansi, Gupta, R., Kuhad, R.C., Saini, J.K., 2018. Cost effective production of complete
35.2 ± 2.7 g/L in the subsequent SSF. These strategies may greatly cellulase system by newly isolated Aspergillus niger RCKH-3 for efficient enzymatic
contribute to the biovalorization of biomass wastes into biofuels and saccharification: medium engineering by overall evaluation criteria approach (OEC).
biochemicals. Biochem. Eng. J. 132, 182–190.
Intasit, R., Cheirsilp, B., Louhasakul, Y., Boonsawang, P., 2020a. Consolidated
bioprocesses for efficient bioconversion of palm biomass wastes into biodiesel
CRediT authorship contribution statement feedstocks by oleaginous fungi and yeasts. Bioresour. Technol. 135, 123893.
Intasit, R., Cheirsilp, B., Louhasakul, Y., Boonsawang, P., Chaiprapat, S., Yeesang, J.,
2020b. Valorization of palm biomass wastes for biodiesel feedstock and clean solid
Benjamas Cheirsilp: Conceptualization, Methodology, Funding biofuel through non-sterile repeated solid-state fermentation. Bioresour. Technol.
acquisition, Writing - review & editing. Asma Billateh: Investigation, 298, 122551.
Data curation, Writing- Original draft preparation. Rawitsara Intasit: Intasit, R., Cheirsilp, B., Suyotha, W., Boonsawang, P., 2021a. Synergistic production of
highly active enzymatic cocktails from lignocellulosic palm wastes by sequential
Review & editing. Apichat Upaichit: Review & editing. Piyarat solid state-submerged fermentation and co-cultivation of different filamentous fungi.
Boonsawang: Review & editing. Yasmi Louhasakul: Review & editing. Biochem. Eng. J. 173, 108086.
Intasit, R., Cheirsilp, B., Suyotha, W., Boonsawang, P., 2021b. Purification and
characterization of a highly stable fungal xylanase from Aspergillus tubingensis
cultivated on palm wastes through combined solid-state and submerged
Declaration of Competing Interest
fermentation. Prep. Biochem. Biotechnol., 1941105
Javaid, R., Sabir, A., Sheikh, N., Ferhan, M., 2019. Recent advances in applications of
The authors declare that they have no known competing financial acidophilic fungi to produce chemicals. Molecules 24, 786.
Jin, Y., Liu, J., Yang, H., Shi, Z., Zhao, P., Yang, J., 2022. Improving enzymatic
interests or personal relationships that could have appeared to influence
saccharification and ethanol production of bamboo residues with sulfomethylation-
the work reported in this paper. aided phosphoric acid pretreatment. Ind. Crops Prod. 177, 113733.
Joy, S.P., Krishnan, C., 2022. Modified organosolv pretreatment for improved cellulosic
Data availability ethanol production from sorghum biomass. Ind. Crops Prod. 177, 114409.
Kamat, S., Khot, M., Zinjarde, S., RaviKumar, A., Gade, W.N., 2013. Coupled production
of single cell oil as biodiesel feedstock, xylitol and xylanase from sugarcane bagasse
Data will be made available on request. in a biorefinery concept using fungi from the tropical mangrove wetlands. Bioresour.
Technol.. 135, 246–253.
Kamoldeen, A.A., Lee, C.K., Abdullah, W.N.W., Leh, C.P., 2017. Enhanced ethanol
Acknowledgements production from mild alkali-treated oil-palm empty fruit bunches via co-
fermentation of glucose and xylose. Renew. Energy 107, 113–123.
Karpagam, R., Preeti, R., Raj, K.J., Ashokkumar, B., Varalakshmi, P., 2015. Fatty acid
This research work was financially supported by the Agricultural
biosynthesis from a new isolate Meyerella sp. N4: Molecular characterization,
Research Development Agency (Public Organization) under Grant No. nutrient starvation, and fatty acid profiling for lipid enhancement. Energy Fuels 29
PRP6205012400 and the authors were supported by Thailand Research (1), 143–149.
Fund under Grant No. RTA6280014. Kumar, D., Murthy, G.S., 2011. Impact of pretreatment and downstream processing
technologies on economics and energy in cellulosic ethanol production. Biotechnol.
Biofuels 4, 27.
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