Bioresource Technology 320 (2021) 124298

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Two-step pretreatment of oil palm trunk for ethanol production by

thermotolerent Saccharomyces cerevisiae SC90
Afrasiab Khan Tareen a, Imrana Niaz Sultan a, Kiettipong Songprom a, Nikhom Laemsak b,
Sarote Sirisansaneeyakul a, Wirat Vanichsriratana a, Pramuk Parakulsuksatid a, *
a
Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, 50 Ngam Wong Wan Rd, Ladyaow, Chatuchak, Bangkok 10900, Thailand
b
Department of Forest Product, Faculty of Forestry, Kasetsart University, 50 Ngam Wong Wan Rd, Ladyaow, Chatuchak, Bangkok 10900, Thailand

H I G H L I G H T S

• Steam explosion breaks down the biomass structure and removes hemicellulose.
• Alkaline extraction improved delignification process by removing 71.67% of lignin.
• Thermotolerent SC 90 performed well at 40 and 45 ◦ C with no significant difference.
• SSF reduces end-product inhibition of the enzymatic hydrolysis and overall cost.
• SSF produced higher ethanol concentration of 44.25 g/L than PSSF 31.22 g/L.

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

Keywords: Oil palm (Elaeis guineensis) trunk chips were processed by steam explosion under different steam conditions,
Oil palm trunk followed by alkaline extraction and fermentation to produce efficient lignocellulosic ethanol as sustainable
Steam explosion alternative energy resource. The optimum condition of steam explosion was attained at 210 ◦ C for 4 min
Alkaline extraction
(α-cellulose: 58.83% and lignin: 27.12%). Taguchi 3 factor design [(sodium hydroxide concentration (NaOH),
Simultaneous saccharification and
temperature and time)] was performed to optimize alkaline extraction. The optimum condition at 15% NaOH,
fermentation (SSF)
Pre-hydrolysis simultaneous saccharification 90 ◦ C for 60 min gave highest percentage α-cellulose: 87.14% and lowest percentage of lignin: 6.13%. Simul­
and fermentation (PSSF) taneous saccharification and fermentation (SSF) involved 10% dry weight pretreated fibers, Celluclast 1.5L (15
FPU /gram substrate), Novozyme 188 (15 IU/gram substrate) and Saccharomyces cerevisiae SC90. The highest
ethanol concentration (CP) produced during SSF was 44.25 g/L. Nonetheless, pre-hydrolysis simultaneous
saccharification and fermentation gave 31.22 g/L (CP). All results suggested that optimized two step pretreat­
ment produced efficient ethanol.

1. Introduction biomass is industrially preferred than the first generation bioethanol

which competes with food and lead to serious concerns regarding the
Depleting fossil fuels have aggravated the fuel transition towards socio-economic and environmental consequences (Robak and Balcerek,
bioethanol utilization. Even though the ethanol production industry is 2018). For this reason, oil palm trunk (OPT) has grasped the attention of
currently based on sugar and starch feedstocks, yet lignocellulosic researchers because of massive biomass production. The average pro­
biomass may play an important role as a renewable, rich source of ductive life-span of OPT is about 25 to 30 years (Maluin et al., 2020) and
carbohydrate. With the aim of commercializing the bioethanol produced new seedlings are replanted after cutting the sterile oil palm trees
from lignocellulose, the global science community has achieved signi­ (Woodham et al., 2019). An estimated per annum OPT biomass of 1.44
fcant goals in developing effectual conversion technologies (Moreno million tons is obtained which is equivalent to 258.12 ktoe/year crude
et al., 2017). oil potentials and 604 million kW-h/year electrical energy generation.
The second generation bioethanol, produced from the lignocellulosic Cellulose along with hemicellulose and lignin comprised of the major

* Corresponding author.
E-mail address: fagipmp@ku.ac.th (P. Parakulsuksatid).

https://doi.org/10.1016/j.biortech.2020.124298
Received 30 August 2020; Received in revised form 15 October 2020; Accepted 17 October 2020
Available online 22 October 2020
0960-8524/© 2020 Published by Elsevier Ltd.
A.K. Tareen et al. Bioresource Technology 320 (2021) 124298

composition of OPT biomass that can be renewed through fermentation further cut down into small chips of 20 × 20 × 5 mm3 by the help of a
to bioethanol. The distortion of OPT structure through pretreatment is wood chipper and stored at room temperature.
necessary for better accessibility of cellulolytic enzymes to the OPT fi­
bers. The pretreatment stage is hence crucial to increase the release of
2.2. Biomass pretreatment
fermentable sugars from biomass (Noparata et al., 2017).
Steam explosion pretreatment through pressure change mechanism
2.2.1. Effect of temperature and time on steam explosion of OPT
causes vast breakdown of internal cell structures of biomass. It is
The steam explosion (SE) of OPT fibers was performed in a 10 L
generally carried out at 160–260 ◦ C with 0.69–4.83 MPa pressure,
pressurized vessel. 150 g dry weight fibers were used in each batch. The
ranging from few seconds to several minutes (Kuboon et al., 2018).
vessel was heated with steam at 200 ◦ C and 210 ◦ C for 2, 4 and 6 min, to
Afterwards, high pressure is released to the atmosphere, causing an
allow the breakdown of OPT chips and exposing lignin, cellulose and
explosion, resulting in disruption of cell wall structure. Hemicellulose is
hemicelluloses (the major components of cell wall) in elevated yields.
mostly removed by steam explosion, however lignin is hardly removed
The severity of steam explosion was designated by a single factor called
and delignification is required to enhance cellulose hydrolysis (Matsakas
(log R0) which uses the relationship of time and temperature. Each
et al., 2018). Higher the temperature of the steam explosion, greater the
condition was replicated thrice and the severity factor (log R0) was
pre-treatment intensity. Therefore, it is easy to dissolve lignin in
calculated by using Equation (1):
delignification process due to steam explosion (Cara et al., 2007).
An appropriate pretreatment for lignocellulose is dependent on the log R0 = log(t.exp(T − 100/14.75) ) (1)
nature of biomass, desired product and it must be economically feasible
(Choi et al., 2019). In comparison with other pretreatment methods, where t is time (min), T is the temperature (◦ C), 100 is the base tem­
alkaline pretreatment is a prominent method with several advantages perature (◦ C), and 14.75 is an empirical parameter associated with
including low pressure, time and temperature that considerably improve activation energy of first-order kinetics (Overend et al., 1987).
the enzyme hydrolysis (Kim et al., 2016). Prior studies on lignin have The hemicellulose removal from OPT fibers was carried out by hot
suggested alkaline pretreatment, or its combination with any other re­ water washing technique. The technique was performed by placing
agent such as H2O2 (alkaline oxidation/alkaline peroxide pretreatment), dried steam exploded fibers in a 2000 mL beaker, containing deionized
for effective lignin removal (Robak and Balcerek, 2020). In addition, it is water with 1:8 solid to liquid ratio. The beaker was heated up for 60 min
testified that alkaline pretreatment exerts effective impact on wood in a water bath at 80 ◦ C. The slurry was poured into a muslin bag and
materials and remove the acetyl group from hemicellulose (Wirawan squeezed to remove hemicellulose. The extracted fibers were stored in
et al., 2020). Mild alkaline pretreatment in contrast with acid pre­ sealed bags at − 20 ◦ C.
treatment produces less inhibitors at low temperatures and pressure
(Fan et al., 2020). For successive enzymatic hydrolysis and fermentation 2.2.2. Optimization of alkaline extraction of steam exploded OPT by
(two step method) technique, alkaline pretreatment is a good choice for Taguchi method
breakdown of lignocellulosic biomass (Li and Kim, 2011). The steam exploded fibers were pretreated with alkaline extraction
Simultaneous saccharification and fermentation (SSF) is an effica­ at varied temperature of 70 ◦ C, 80 ◦ C and 90 ◦ C in a water bath for 30, 60
cious method for lignocellulosic ethanol production. The chief advan­ and 90 min with sodium hydroxide (NaOH) concentration of 15%, 20%,
tages of conducting enzymatic hydrolysis and fermentation together, as and 25%. The solid to liquid ratio was 1:8 and each condition was car­
opposed to a separate step after hydrolysis, are the abridged end-product ried out with three replications. The orthogonal array of L9 design of
inhibition of the enzymatic hydrolysis, and the reduction in overall in­ three factors by Taguchi method was used. The slurry was stirred every
vestment expenditures. Conversely, the foremost shortcomings are the 3–5 min, filtered and washed after cooling down at room temperature to
technical hitches in finding the favorable temperature and pH for attain neutral pH. The alkaline extracted fibers were weighed after
enzymatic hydrolysis and fermentation. The hunt for thermotolerant drying and stored at 4 ◦ C until further use.
yeasts to elevate ethanol yield is a huge challenge, involving numerous The impact of sodium hydroxide (NaOH) concentration, temperature
factors such as growth temperature, initial pH, cell concentration, and and extraction duration was studied on the optimum steam exploded
sugar concentration (Techaparin et al., 2017). Generally, Saccharomyces fibers. The performance attribute of “the larger – the better” and “the
cerevisiae has an optimum temperature near 30 ◦ C (Azhar et al., 2017). smaller- the better” for cellulose and lignin, respectively were used to
S. cerevisiae TISTR5606, an example of a thermotolerant yeast, can acquire the optimum conditions. The S/N for “the larger- the better” and
effectively produce ethanol at elevated temperature i.e. 40 ◦ C to 43 ◦ C, “the smaller- the better” performances attribute were estimated using
during fermentation (Techaparin et al., 2017). Additionally, difficulties the Equation (2) and (3), respectively.
in recycling the fermenting microbe and enzymes are the obstacles in ∑
S/N = log 10( (1/Yi )2 /n) (2)
attaining cost-effective bioethanol production.
To the best of author’s knowledge, optimization of alkaline extrac­ ∑
tion using Taguchi method, particularly on oil palm trunk, is very S/N = log 10( (Yi )2 /n) (3)
limited in the literature. The present study emphasizes optimization of
two step pretreatment (steam explosion and alkaline extraction) of oil where Yi is a variable for either combination or comparison in experi­
palm trunk with different parameters (reaction time, temperature and ment, i is a specific combination of controlled factor levels and n is the
concentration) by using statistically aligned experiments and the number of experiments conducted.
Taguchi method for optimum release of celluloses. The optimized
alkaline pretreated OPT fibers were subjected to hydrolysis and 2.3. Strain, media and enzymes
fermentation for high ethanol yield by a thermotolerant yeast Saccha­
romyces cerevisiae SC90. Saccharomyces cerevisiae SC90 (industrial yeast) was used for ethanol
fermentation in simultaneous scarification and fermentation (SSF) and
2. Materials and methods pre-saccharification followed by simultaneous saccharification and
fermentation (PSSF). Pre-cultured active SC90 cells, grown (30 ◦ C, 150
2.1. Raw material rpm, 18 h) on yeast extract, peptone and dextrose (YPD) growth medium
containing (g/L) yeast extract: 10; peptone: 20; and glucose: 20, were
An oil palm (Elaeis guineensis Jacq.) trunk (~25 years old) was ob­ used for inoculation. The commercial enzymes Celluclast 1.5 L and
tained from Plai Phraya District Krabi province, Thailand. The OPT was Novozyme 188 were obtained from Novozymes (Denmark).

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A.K. Tareen et al. Bioresource Technology 320 (2021) 124298

2.4. Simultaneous saccharification and fermentation (SSF) for ethanol factor of cellulose to glucose.
production
2.5. Pre-hydrolysis simultaneous saccharification and fermentation
2.4.1. Effect of temperature on thermotolerant Saccharomyces cerevisiae (PSSF) for ethanol production
SC90 in SSF
In the current study, SSF fermentation assays were performed. The To determine the effect Pre-hydrolysis prior to SSF, 10% of alkaline
experimental procedure for SSF was performed in a 500 mL Erlenmeyer extracted fibers were hydrolyzed using the enzyme blend of celluclast
flask containing a total volume of 300 mL YP medium (20 g/L peptone 1.5L (15 FPU/g substrate) and Novozyme188 (15 IU/g substrate) at
and 10 g/L yeast extract) of pH 4.8 (50 mM sodium citrate buffer for 50 ◦ C for 12 hrs. Afterwards, the temperature of the suspension was
maintaining pH). The liquid medium was loaded with 10% alkaline reduced to 40 ◦ C and Saccharomyces cerevisiae SC90 were added. An
extracted dry fibers and sterilized at 121 ◦ C for 15 mins in autoclave. A absolute volume of 300 mL YP medium (20 g/L peptone and 10 g/L
10% starter culture, celluclast 1.5L (15 FPU/g substrate), and Novo­ yeast extract at pH of 4.8, buffered with 50 mM sodium citrate buffer) in
zyme188 (15 IU/g substrate) were all together added into a 500 mL a 500 mL Erlenmeyer flask was used for conducting PSSF. A set of
Erlenmeyer flask. The SSF was carried out at 40, 45 and 50 ◦ C for 96 hrs triplicates were performed to avoid experimental discrepancies and
in the presence of Saccharomyces cerevisiae SC90, at 150 rpm in flask. attaining valid results for calculating the mean values.
The fermentation samples were collected with three replicates at 0, 2, 4,
6, 8, 10, 12, 15, 18, 24, 36, 48, 60, 72, 84, and 96 hrs. The kinetics of 2.6. Analytical methods
ethanol production was calculated according to the protocols of (NREL,
2008) and equations are given below: The chemical composition analysis of OPT fibers was performed in
The production rate of ethanol (QP) was calculated as given in accordance with the Technical Association of Pulp and Paper Industry
Equation (4): (TAPPI) standard methods. TAPPI T264 om-97, (1997) for moisture
content; T204 om-97 for extractives; T222 om-98 for Acid insoluble
Pt − P0
Qp (g/L/h ) = (4) lignin; T223 om-84 for pentosan (TAPPI, 1983d); TAPPI T 211 om-85
t − t0
(TAPPI, 1983b) for ash. The holocellulose was determined by acid –
Whereas, “P0” represents ethanol concentration at 0 hrs (g/L), “Pt” is chloride method of Browning. The alpha- cellulose was determined by
maximim ethanol concentration (g/L), “t0” indicates initial time (hrs) TAPPI T203 om-93 (TAPPI, 1983c).
and “t” is the duration of maximum ethanol concentration (hrs).
The yield coefficient (YP/S) was obtained from the initial and final 2.7. Analysis of cellobiose, glucose and ethanol by HPLC
corrected values of the relevant concentrations within SSF, depicted in
Equation (5). The cellobiose, glucose, xylose and ethanol were analyzed by High
Performance Liquid Chromatography (HPLC) (SHIMADZU, Japan) with
[EtOH t − EtOH0 ]
Yp/s = (5) Aminex HPX-87H column. The column temperature was maintained at
f [Biomass]1.11
50 ◦ C with mobile phase of 50 mM H2SO4 at a flow rate of 0.6 mL min− 1
whereas, “EtOH0” represents ethanol concentration at the begning (g/ (Xue et al., 2015).
L), “EtOHt” indicates maximim ethanol concentration (g/L), “f” Cellu­
lose fraction of dry biomass; Biomass: Fiber (g/L) at the beginning; 1.11: 2.8. Statistical analysis
is the theoretical conversion of cellobiose to glucose.
Theoretical ethanol yield [Equation (6)] was obtained from the The experimental setup was randomized, with analysis of variance
initial and final corrected values of the relevant concentrations within (ANOVA) and Fisher’s least significant difference test (SAS version 8.01)
SSF; thus: used to compare geometric means (GM), ethanol concentrations (g/L),
ethanol productivity (g/L/h), ethanol yield (g/g) and theoretical
Theoretical ethanol yield =
[EtOHt − EtOH0 ]
× 100 (6) ethanol yield (%). Level of statistical significance was set at 5% (p <
0.51f [Biomass]1.11 0.05).

whereas 0.51 represents the theoretical change of glucose to ethanol. 3. Results and discussion

2.4.2. Enzymatic hydrolysis 3.1. Analysis of chemical composition

The enzymatic hydrolysis was conducted with celluclast 1.5L (15
FPU/g substrate), and Novozyme 188 (15 IU/g substrate) using a sub­ Moisture content of OPT fibers was observed as 5.8 wt%. Chemical
strate loading of 10%. The enzyme activity was assayed by following the composition on dry weight basis, measured as; 2.40 ± 0.01% ash, 8.53
National Renewable Energy Laboratory (NREL)’s protocol. A 10% dry ± 0.65% ethanol–benzene extractives, 73.0 ± 1.0% holocellulose. The
weight of fibers with simultaneous pouring of 270 mL 0.05 N citrate percentage of lignin i.e. 21.64% was found almost similar to the findings
buffer (pH 4.8) in a 500-mL Erlenmeyer flask was subjected to enzyme of Kim and Day (2011) on sweet sorghum, whereas that of cellulose i.e.
hydrolysis at 50 ◦ C and 150 rpm. For screening, the collection of test 40.83 was closer to the findings of Malherbe and Cloete (2002) on hard
samples was carried out at different intervals, from 0 hrs till 96 hrs wood.
(Kumneadklang et al., 2015). The enzyme hydrolysis was performed in
triplicates and the average results were listed. The enzyme digestibility 3.2. Effect of temperature and time on steam explosion of OPT
was calculated according to Equation (7):

Enzyme digestibility % = (Glucose) + 1.053(Cellobiose) The steam explosion of OPT fibers at 200 and 210 ◦ C for 2, 4 and 6
min was performed. Table 1 exhibits the analysis of the chemical
× 1001.111f (Biomass) (7)
composition of oil palm fibers after steam explosion. It was found that
the percentage of pulp yields ranged between 61 and 69% and the ratio
whereas (Glucose) indicates glucose concentration in (g/L); (Cellobiose)
of pentosan decreased when compared to the raw materials of OPT.
refers to cellobiose concentration in (g/L) released during enzyme hy­
Pentosan is a representative of hemicellulose, which serves the role of
drolysis; f is cellulose fraction in dry biomass (g/g); 1.053 is correction
linkage between cellulose and lignin. Once the structure of pentosan gets
factor of cellobiose to equals of glucose; and 1.111 is the conversion
weakened then it is easy to break it down which was observed after

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Table 1 OPT as substrate, it is highly recommended to use mild pretreatment

Chemical composition of OPT fibers after steam explosion. conditions to avoid the degradation of starch (Eom et al., 2015). Steam
Log Ro Condition Pulp Composition (% Dry matter) explosion and alkaline extraction bring about changes in structure of
(Severity yield OPT fibers by the removal of hemicelluloses and significant amount of
Alpha- Pentosan Lignin Ash
factor)
Cellulose (AIL) lignin resulted in enlarged surface area and pore size of the fibers
(Fakirov and Bhattacharyya, 2007). Since alkaline pretreatment
3.24 200 ◦ C, 2 69.43 50.69c 15.73a 24.41c 0.68
min
removes most of lignin, thereby this technique is favored than acid
3.54 210 ◦ C, 2 61.19 55.51b 9.81b 26.28b 0.4 pretreatment methods which cause carbohydrate dissolution (Rizal
min et al., 2018). Lignin is a heterogeneous, phenolic and poly disperse
3.55 200 ◦ C, 4 64.39 56.03b 8.17b 26.74b 0.52 biopolymer which resists degradation on account of its complex struc­
min
a,b c a ture comprising of aromatic and highly branched configuration
3.72 200 ◦ C, 6 63.26 57.04 4.48 28.80 0.39
min (Schoenherr et al., 2018). In accordance with Wang et al. (2018), lignin
3.84 210 ◦ C, 4 65.68 58.83a 4.03c 27.12b 0.34 serves a vital role in determining the plant cell wall texture. Lai and Idris
min (2013) also reported that higher lignin content in OPT biomass is un­
4.02 210 ◦ C,6 62.96 56.53b 2.84c 26.47b 0.27 desirable and has several disadvantages i.e. lignin hinders the biode­
min
gradability of lignocellulosic biomass in the subsequent enzymatic
a b
, , and c superscripts of lowercase show the values are significantly different (p hydrolysis process. The existence of high quantity of lignin inhibits the
< 0.05). hydrolysis process due to the toxicity of lignin derivatives and its non-
specific adsorption of hydrolytic enzymes within the structure of lig­
steam explosion that had reduced the proportion of pentosans. nocelluloses (Kucharska et al., 2018).
Lignin is a multifarious molecule of three-dimensionally linked The relation of S/N ratio plots in Fig. 1 displayed the response of
phenylpropane units (Palmqvist and Hahn-Hagerdal, 2000). Thereby, it alkaline extraction to change in factor levels. Out of three levels (1, 2, 3)
needs to be removed to expose cellulose for degradation and subject to for three factors (A, B, C), only one could maximize the mean S/N ratio.
bioethanol production through pretreatment. After steam explosion and The condition that gave the highest cellulose content (Set A) and the
hot water washing, the substances that were still in the pulp were lowest lignin content (Set B) presented in Table 4.
leached out. Chemical composition after steam explosion revealed that The optimum conditions for alkaline extraction from two sets of
the alpha-cellulose content increased after steam explosion (Table 1). experiments were studied. The % dry weight of cellulose in Set A
The highest proportion of alpha cellulose was 58.83%. The pentosan (87.89%) was 0.75% higher than Set B (87.14%), % dry weight of lignin
percentage decreased as compared to the pulp after the steam explosion (Set A: 7.53%, Set B: 6.13%) in Set B was 1.40% lesser than Set A,
because hot water has solubility properties. The lowest pentosan content exhibited in Table 5. The results showed that the percentage of cellulose
was 2.84%. in both treatments was not much different. In addition, if the proportion
of lignin in a large amount, the enzyme activity is lower. The reduction
3.3. Optimization of alkaline extraction of lignin content will increase the yield of sugar in fermentation (Dien
et al., 2009).
The concentration of sodium hydroxide (NaOH), temperature and
time were extensively studied by the help of Taguchi method to deter­ 3.4. Chemical composition of treated and untreated oil palm samples
mine the optimum condition of alkaline extraction for lignin removal
(Table 2). Alkaline extraction expands the membrane, which is good for Chemical composition of OPT fibers changed from raw materials
the enzyme hydrolysis (Kim et al., 2003). After steam explosion followed when pretreated with steam explosion and alkaline extraction. The
by hot water washing, the OPT fibers were extracted with alkali at content of alpha cellulose increased from 40.83 to 87.14%. The pentosan
various conditions under all 9 experiments. The experimental results decreased from 29.53 to 1.40%. However, from multi-stage pretreat­
were analyzed for analysis of variance to investigate the variation ment, pulp yield was reduced to 23.01%. The content of ash also
caused by each factor. Each factor’s contribution to the effect of chem­ decreased from 2.40% in raw material to 1.40% after alkaline extrac­
ical composition (cellulose and lignin content) are displayed in Table 3. tion, moreover lignin content decreased from 21.64 to 6.13% which
Statistical analysis of cellulose and lignin content indicated that the demonstrates that OPT fibers are potential raw material to produce
strength of NaOH and temperature had a significant effect on lignin ethanol. Leschinsky et al. (2008) reported that the exclusion of hemi­
content (Table 6). Kumar et al. (2009) also reported a significant cellulose causes biomass structural characterization, developed
decrease in lignin content in hardwood by using NaOH extraction. dissemination of pulping chemicals and more rapid lignin removal on
Studies on pretreatment reveal that severity in pretreatment conditions mild conditions. The components pierce into the cell wall by means of
degrade the substrate to produce inhibitory products that may antago­ lumen, breaking down the approachable lignin in the secondary cell wall
nize the microbes during fermentation and also deter the activity of and sparing the leftover recalcitrant lignin in middle lemilla (Siqueira
cellulolytic enzymes. In order to obtain enhanced bioethanol yield from et al., 2011). The reduction of the pulp yield is related to the chemical

Table 2
The effect of alkaline extraction on oil palm trunk (OPT) fibers at different NaOH concentration, temperature and time.
NaOH (%) Temp (◦ c) Time (min) Pulp yield Composition (% Dry matter)

Alpha-cellulose Pentosans Lignin (AIL) Ash

15 70 30 25.07 79.92 3.04 7.03 0.74

15 80 60 24.37 86.67 3 7.34 0.62
15 90 90 22.74 87.61 3 6.63 0.64
20 70 60 25.52 81.21 2.19 8.82 0.89
20 80 90 23.53 84.4 2.27 9.36 0.69
20 90 30 23.66 82.31 2.28 8.89 0.78
25 70 90 22.51 83.28 1.91 9.68 1.06
25 80 30 24.49 84.24 2.08 11.03 0.84
25 90 60 22.49 83.08 1.72 9.25 0.83

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A.K. Tareen et al. Bioresource Technology 320 (2021) 124298

Table 3
Analysis of variance of cellulose and lignin content.
Factor df SS V F S’ Cont (%) Conf (%) Sig

Cellulose
NaOH (%) 2 0.048 0.024 1.076 0.003 0.633 51.83 –
Temperature (◦ C) 2 0.272 0.136 6.08 0.227 42.122 85.88 –
Time (min) 2 0.175 0.087 3.904 0.13 33.164 79.61 –
Other Error 2 0.044 0.022 33.164
Total 8 0.54 100
Lignin
NaOH (%) 2 15.03 7.51 359.18 14.98 89.03 99.72 **
Temperature (◦ C) 2 1.43 0.72 34.21 1.39 8.26 97.16 *
Time (min) 2 0.33 0.16 7.89 0.29 1.71 88.75 –
Other Error 2 0.04 0.02 1
Total 8 16.83 100

df = Degree of freedom; MS = Mean sum of squares; F = F-test statistic; S’ = Pure sum of squares * (P < 0.05).

Fig. 1. S/N ratio for various factors (A-C, Table 2) and levels (1–3, Table 2): (A) cellulose content; (B) lignin content. Optimal conditions are indicated by the peak
values of S/N ratio.

byproduct owing to the high temperature steam which further hydro­

Table 4
lyses xylan polymer into xylose and xylose oligomers dissolved in
Optimization of alkaline extraction by Taguchi Method.
hemicellulose solution (Vaithanomsat et al., 2009). The lignin quantity
Experiment Experimental levels Experimental factors reduced with each treatment particularly with alkaline extraction due to
Experimental Set A breakage of hemicellulose structure. Cellulose was slightly reduced as
NaOH concentration (%) 1 15 cellulose was evaporated at more than 100 ◦ C. The hot water washing
Temperature (◦ C) 2 80
did not affect lignin because lignin does not break down when heated.
Time (mins) 3 90
Experimental Set B There is a slight effect on pentosan, as the hemicellulose that is
NaOH concentration (%) 1 15 destroyed in the explosion process is also accumulated in the pulp.
Temperature (◦ C) 3 90 When extracted with hot water, the substance accumulated in the
Time (minutes) 2 60 pulp was leached in the form of a solution and the amount of cellulose
increased due to the breaking down of the hemicellulose, whereas the
proportions of lignin and pentosan decreased. Since both lignin and
Table 5 hemicellulose are soluble in alkaline solutions, as a result, the cellulose
Estimated and confirmed values of cellulose and lignin after optimized condi­ content increased. Cell wall is disrupted by alkaline treatment owing to
tions of alkaline extraction. (i) the removal of hemicellulose, lignin and silica; (ii) hydrolyzing
Experimental conditions Estimated Confirm test uronic acid and acetic acid esters; and (iii) swelling up of cellulose. The
value results alkaline treatment is responsible for α-ether bonds breakage between
The maximum cellulose content 88.12% 87.89% lignin and hemicellulose and it also ruptures the ester bonds between
(Experiment A) lignin and hydroxycinnamic acid such as p-coumaric acid and ferulic
The minimum lignin content (Experiment 6.57% 6.13% acid. Particularly, alkaline treatment is an environmental friendly
B)
approach which breaks down lignocellulose into valuable components
such as lignin, hemicellulose, and cellulose (Vu et al., 2017).
composition of the OPT fibers. Pentosan content indicated the condition
used for conformational changes in consequence of steam explosion. The
acetyl groups in hemicellulose molecules are converted into acetic acid

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3.5. Effect of temperature on thermotolerant Saccharomyces cerevisiae prolonging the exponential growth phase for 8 hrs. After 10 hrs, glucose
SC90 in SSF decreased rapidly and yeast cell also grew quickly. The ethanol was
produced continuously for 96 hrs, although the glucose concentration
To study the influence of temperature, SSF of pretreated oil palm reached to almost 0 g/L. The cellulose enzyme was still functional,
pulp 10% dry w/v was carried out at 40, 45 and 50 ◦ C to produce demonstrating from the ethanol profile. After 36 hrs, the yeast cells
ethanol. The results exhibited that the hydrolysis and fermentation were decreased gradually due to high temperature of 45 ◦ C, which is not
simultaneous. In the experiment, the pulp content was 10% and suitable for growth. Glucose was formed during the 96 hrs of fermen­
Saccharomyces cerevisiae SC90 was used for fermentation which gives tation and was used by yeast cells to produce ethanol till the end. At the
best growth rate at 30–36 ◦ C. end of fermentation, the concentrations of glucose, ethanol and live cells
In Fig. 2A, it was found that in the first 8 hrs, yeast was adapted to the were 0.02, 44.00 g/L and 1.98 × 109 CFU/L respectively. The fermented
new environment. The glucose concentration increased slowly due to pulp was 4.03 g dry weight as compared to the initial pulp 30 g dry
the fluffy structure of OPT causing poor mixing between cellulase en­ weight.
zymes and OPT fibers, which is also supported by Cara et al. (2007) who The least ethanol production was obtained at 50 ◦ C as compared to
reported that the enzyme initially does not work well enough to produce 40 and 45 ◦ C using OPT fibers. As shown in Fig. 2C, the yeast cells
glucose to meet the needs. After 8 hrs, the cells grew better because of decreased from the beginning and were found entirely dead at 48 hrs
releasing glucose and also started producing ethanol continuously, on (Rose and Harrison, 1971) because of high temperature, though at 40
the other hand after 48 hrs the glucose was not detected though the and 45 ◦ C, the viability of cells lasted till 96 hrs. The ethanol increased in
enzyme was still working to produce glucose. The missing glucose was responding to yeast cells and the glucose, no ethanol production was
consumed by the yeast to maintain its function through the entire 96 hrs observed after 48 hrs. At the end of fermentation, the concentrations of
of fermentation. In the end of fermentation, the concentrations of glucose, ethanol and live cells were 29.74, 12.72 g/L and 0.00 CFU/L,
glucose, ethanol and live cells were 0.02, 44.25 g/L and 1.50 × 1011 respectively. The fermented pulp was 6.80 g dry weight as compared to
CFU/L, respectively. The fermented pulp, left from the initial pulp (30 g the initial pulp 30 g dry weight.
dry weight) was calculated as 5.84 g dry weight.
Fig. 2B, shows the production of ethanol at 45 ◦ C. The results showed
that in the first 10 hrs, the cells were in adaptive range because of less 3.6. Enzymatic hydrolysis
glucose and the temperature was not appropriate. Reed and Peppler
(1973) reported that temperature starts to affect yeast cells at 43 ◦ C, by During enzyme hydrolysis the production rate of glucose increased
initially but the production rate of glucose gradually decreased due to

Fig. 2. Ethanol production during SSF fermentation at (A) 40 ◦ C, (B) 45 ◦ C, (C) 50 ◦ C, Ethanol (●), glucose (▴) and live cell (■).

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A.K. Tareen et al. Bioresource Technology 320 (2021) 124298

enzymatic inhibition of different sugars. After enzymatic hydrolysis of growth during PSSF rather than ethanol production. These results were
96 hrs, 71.57 g/L glucose, 11.17 g/L cellobiose and 0.14 g/L of xylose similar to that of Manzanares et al. (2011) who compared the SSF and
was produced (Fig. 3A), moreover 7.78 g/L of hydrolyzed pulp was left the PSSF fermentation of olive tree fibers by using hot water pretreat­
after enzyme hydrolysis from the initial pulp of 30 g weight. The high ment; the SSF fermentation gave 30% higher ethanol concentration than
residual pulp and cellobiose content showed reduced enzyme efficacy PSSF. However, when the fibers percentage was 23% loading, PSSF
(Garcia-Aparicio et al., 2006). The results were found better than the fermentation gave higher ethanol concentration of 77.17% in contrast
findings of Garcia-Aparicio et al. (2006) that managed to produce 15 g/L with the SSF. It was found that the SSF fermentation was suitable for less
of glucose after enzyme hydrolysis of pretreated rice straw barley. In fiber content whereas, the PSSF fermentation for high pulp content.
Fig. 3B, the maximum digestibility value was obtained as 88.51%. The The production of ethanol with oil palm pulp in SSF and PSSF
results were found higher than Bukhari et al. (2020) and Tareen et al. fermentation were summarized as kinetic parameters, shown in Table 6.
(2020) who studied sulfuric acid pretreatment and pretreatment with It was found that the use of 10% pulp by dry (w/v) SSF fermentation was
alkaline hydrogen peroxide on OPT exhibited 67.9% and 59.82% the best growth of S. cerevisiae SC90 at 40 ◦ C with a growth rate (μ) of
enzyme digestibility, respectively. On the basis of enzyme activity and 0.202 per hr, as it was close to that of the yeast. The higher the
digestibility, it was decided to keep the pre-hydrolysis step for 12 hrs fermentation temperature, the more effective it is for the enzyme to
prior to SSF. It was also found that the steam exploded and alkaline produce glucose. 10% pulp was used in this experiment. It was found
extracted oil palm pulps were suitable substrates for fermentation that fermentation at 40 ◦ C gave the highest ethanol (CP) of 44.25 g/L,
because of the enzymatic hydrolysis and high glucose content. representing the ethanol production (QP) of 0.45 g/L, ethanol yield (YP /
S) was 0.46 g ethanol and the theoretical ethanol yield of 90.34%. It was
found that at 40 and 45 ◦ C the results were not significantly different (p
3.7. Pre-hydrolysis simultaneous saccharification and fermentation ≤ 0.05). S. cerevisiae SC90 was able to resist temperature up to 45 ◦ C.
(PSSF) The SSF fermentation at 50 ◦ C did not last for more than 4 hrs and the
S. cerevisiae SC90 growth rate (μ) was lower (0.021). The results showed
The PSSF system is similar to the SSF system. The experiment was that ethanol production from SSF fermentation at 40 ◦ C was found to be
conducted with 10% (w/v) substrate, prehydrolysed at 50 ◦ C for 12 hrs, effective in producing ethanol from oil palm pulp, because of the
later the temperature was lowered to 40 ◦ C for the addition of SC90. The optimal temperature, yeast and enzymes work together effectively.
12 hrs pre-hydrolysis step was chosen for the study due to high enzyme
activity from 0 to 12 hrs during enzyme digestibility study. The results 4. Conclusion
showed that the yeast cells were started growing within the first 2 hrs,
after the addition of yeast which utilized glucose and produced This study exhibited the vital role of steam explosion pretreatment
continuous ethanol for 36 hrs. Subsequently, the yeast cells were con­ for hemicellulose isolation and reduction in biomass recalcitrance. The
stant and the growth profile was similar to SSF at 40 ◦ C. It was found that alkaline extraction treatment for achieving optimized delignification
the glucose content remained throughout 96 hrs period because the (71.67%) with improved cellulose content (46.85%) was attained at
yeast cells were constantly using glucose for the ethanol production. At 15% (w/v) NaOH at 90 ◦ C. The results were supported by enzyme hy­
the end of fermentation, the concentrations of glucose, ethanol and live drolysis which increased glucose concentration to 71.57 g/L. SSF gave
cells were 0.01, 31.22 g/L and 2.12 × 1011 CFU/L, respectively (Fig. 4). higher ethanol concentration i.e. 44.25 g/L than PSSF i.e. 31.22 g/L. In
Moreover, only 5.65 g dry weight of fermented pulp was left from the PSSF, the concentration of ethanol was low due to insufficient amount of
initial pulp 30 g dry weight. pulp, which may be improved with an increase in pulp content and
In PSSF, the adaptation time of yeast cells (2 hrs) was less than that of hydrolysis time before fermentation.
SSF at the same temperature because of the available glucose in the
PSSF. However, the ethanol concentration was lesser than the SSF,
which may be related to the greater concentration of glucose for ell

Fig. 3. The alkaline extracted oil palm trunk (OPT) pulp (A) enzyme hydrolysis, and (B) enzyme digestibility. (▴) glucose (g/L), (■) cellobiose (g/L) and (●) xylose
(g/L).

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A.K. Tareen et al. Bioresource Technology 320 (2021) 124298

Fig. 4. Ethanol production during PSSF fermentation. Ethanol (●), glucose (▴) and live cell (■) concentrations in PSSF.

for total utilization of high recalcitrant biomass by organosolv pretreatment. Renew.

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interests or personal relationships that could have appeared to influence Resources and Biorefineries. InTech.
the work reported in this paper. Kucharska, K., Rybarczyk, P., Hołowacz, I., Łukajtis, R., Glinka, M., Kamiński, M., 2018.
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