Industrial Crops & Products 139 (2019) 111507

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Industrial Crops & Products

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

Bioethanol production potential in Bangladesh from wild date palm (Phoenix T

sylvestris Roxb.): An experimental proof
A.M. Swaraza, , Mohammed A. Satterb, Md. Mahfuzur Rahmana, Mohammad Asadullah Asadc,
⁎

Imran Khand,e, Md. Ziaul Amina

a
Department of Genetic Engineering and Biotechnology, Jashore University of Science and Technology, Jashore - 7408, Bangladesh
b
Institute of Food Science and Technology (IFST), Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka - 1205, Bangladesh
c
Institute of Fuel Research and Development (IFRD), Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka - 1205, Bangladesh
d
Department of Electrical and Electronic Engineering, Jashore University of Science and Technology, Jashore - 7408, Bangladesh
e
Centre for Sustainability, University of Otago, Dunedin - 9016, New Zealand

ARTICLE INFO ABSTRACT

Keywords: To sustain economic growth in Bangladesh, energy demand is expected to increase rapidly, resulting in more
Bioethanol greenhouse gas emissions, as the energy sector, including transport and electricity, is dominated by fossil fuels.
Wild date palm In order to reduce these emissions, the government of Bangladesh has already formed a long-term energy plan,
Food versus fuel debate part of which is introducing a 5% blend of bioethanol with gasoline in the transport sector. However, the source
Energy security
of this bioethanol has not yet been identified. An investigation has thus been conducted to evaluate the potential
Bioenergy
of bioethanol production using wild date palm fruit pulp and sap, sugary biomass of a perennially grown plant in
Bangladesh that has been grown for decades without regular agronomic practice. A male plant produced
140.42 kg sugary biomass per year with 29.85% total sugar whereas a female plant yielded 195.56 kg, con-
taining an average of 43.61% of total sugar. A batch fermentation study using Saccharomyces cerevisiae found
that fruit pulp extract and sap could be conveniently fermented without nutritional supplementation to po-
tentially yield ethanol. An average of 0.278 g/g ethanol was yielded from the fruit pulp extract and sap fer-
mentation. From these findings, it was estimated that minimum 8076.62 L of ethanol could be obtained from an
orchard comprising 500 plants per hectare with the planting ratio of male: female plants of 1:4. This indicates
that this biomass of the wild date palm is a highly promising sugary feedstock for the potential production of
bioethanol.

1. Introduction
A global low-carbon future would be challenging without a proper
combination of technologies and policy measures (IEA, 2017). Bioe-
nergy could play a vital role in achieving a sustainable low-carbon fu-
ture, but it must be produced in a sustainable way. Bioenergy, and
particularly bioethanol, is an important option in the transportation
(Månsson et al., 2014) and electricity (Moreira et al., 2016; Dias et al.,
2013) sectors, where decarbonization is essential not only at the na-
tional level but also globally to ensure United Nations’ (UN) sustainable
development goals (SDG)1 . However, the incremental trends of bioe-
thanol deployment in the transport and electricity generation sectors
are limited in scale, particularly in the developing world (Saravanan
et al., 2018). It was reported by the International Energy Agency (IEA)
that “Transport biofuel consumption needs to triple by 2030 to ensure that
biofuels’ share of transport fuel demand, which was 3% in 2017, reaches
10% by 2030 as required by the SDS (Sustainable Development Sce-
nario)” (IEA, 2018a). In an earlier report, the IEA estimated that
“….decarbonisation of the energy system will require a fivefold increase in
the supply of biomass feedstock for modern bioenergy uses compared to
today….. Wastes and residues can provide around two-thirds of this re-
quirement, but supply will also be needed from other sources” (IEA, 2017).
In line with this global requirement, Bangladesh needs to take in-
itiatives in developing greener and more varied fuels in the near future
(Baeyens et al., 2015), and the government is planning to allow a 5%
blend of bioethanol with octane and gasoline in the transport sector
(Biofuels International, 2018; Islam, 2017). However, the sources of
this bioethanol remain unclear and this raises the question: “whether or

⁎
Corresponding author.
E-mail address: a.m.swaraz@just.edu.bd (A.M. Swaraz).
1
https://sustainabledevelopment.un.org/sdgs

https://doi.org/10.1016/j.indcrop.2019.111507
Received 25 February 2019; Received in revised form 24 May 2019; Accepted 22 June 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
A.M. Swaraz, et al.
not feedstock will be sourced solely from waste?” (Biofuels International,
2018; Islam, 2017). It underscores the need to explore possible biomass
sources for bioethanol production. Investigating the potential of bioe-
thanol production from locally available and plentifully produced
feedstock, which will not impact food security in Bangladesh, is timely
research, and is the main focus of this study.
Bangladesh is a transitional lower middle-income country which is
experiencing rapid economic growth in advancing towards a middle-
income country by 2021, in spite of its enormous daunting local and
global challenges and threats. To achieve Vision 2021 (GED, 2012),
SDGs and Vision 2041 (Hamid, 2016) the country has to meet its in-
creasing energy demands. As a “strategic commodity”, a continuous
flow of clean and secure energy is a prerequisite for sustainable tranquil
rapid development. At present, the primary energy sources of Bangla-
desh are natural gas, oil, coal, hydro, biomass and solar which are in
inadequate quantities, and can provide only 308 kW h per capita energy
consumption, which is among the lowest levels in the world (BPDB,
2017). In 2016, electricity generation in Bangladesh was about
64327 GW h, of which, 1102, 9666, 52823, 566, and 170 GW h were
generated from coal, oil, natural gas, hydro, and solar and others, re-
spectively (IEA, 2018b). Currently, only 2.95% produced energy in
Bangladesh comes from renewable sources. This breakdown is illu-
strated in Fig. 1 (SREDA, 2018). On the other hand, 2344 and 1104
thousand tonnes of oil equivalent energy from oil and natural gas were
used in the transport sector in 2016 (IEA, 2018b). Importantly, no re-
newable energy sources, such as bioethanol, have yet been used in the
transport sector in Bangladesh. In order to ensure sustainable energy
security as well as to meet the increasing energy demand, primary
cleaner energy sources of the country need to be realistically expanded
to reduce negative environmental impacts, climate change and the
carbon footprint of the country. In this regard, Bangladesh aims to in-
crease its renewable energy capacity of about 10% by 2020 through
solar photovoltaic (solar PV), onshore wind, biomass, biogas and hy-
dropower technology, all of which are far behind the increasing global
trends of renewable energy. Renewable energy sources supply about
23.7% of the total world energy demand and accounted for 70% of net
additions to global power generating capacity in 2017 (REN21, 2018;
IEA, 2018c). Among various renewable energy resources, biomass-
generated energy accounts for the least amount (0.10% each) of the
total renewable energy in Bangladesh (cf., Fig. 1).
In the case of Bangladesh, the available biodiesel (i.e., biofuel)
feedstock sources were reviewed, and mustard, jatropha, rapeseed, se-
same, castor, cotton seed, neem, algae, coconut, and groundnut were
identified as potent (Habibullah et al., 2015). One study found that
biofuel can be a potential energy source for the country (Ferdous et al.,
2012), and some studies of biodiesel production options from different
sources, such as rubber seed (Hevea barasiliensis) (Morshed et al., 2011),
karanja (Pongamia pinnata) (Nabi et al., 2009), jatropha (Jatropha
curcas) and castor (Ricinus communis) (Haque et al., 2009) have been
Fig. 1. Electricity generation fuel mix in Bangladesh. HSD: High Speed Diesel, HFO: Heavy Fuel Oil. [Data source: (SREDA, 2018)].
2
Industrial Crops & Products 139 (2019) 111507
reported. However, no previous study has investigated the potential of
bioethanol production with supporting experimental investigation in
Bangladesh. As Bangladesh is unable to afford bioethanol production
from its primary food source, which certainly poses a great threat to its
vulnerable food security. Thus, for sustainable, fuel grade bioethanol
production there is an urgent need to use minor crops or nonfood su-
gary and starchy crops that are grown perennially with abundant yield
potential from marginal land, in order to establish sustainable bioe-
thanol production without hampering food security.
Among possible sugary feedstock plants, palms possess high utility,
as they have been used as sugar producing plants throughout the tro-
pical world from ancient times (Dalibard, 2018). Sugary feedstock of
palms is usually its sap, and suitable plants are coconut palm (Cocos
nucifera), palmyra palm (Borassus flabellifer), sugar palm (Arenga pin-
nata), nipa palm (Nypa fructicans), wild date palm (Phoenix sylvestris)
and commercial date palm (Phoenix dactylifera) (Sriroth and
Piyachomkwan, 2013). Most of the sugar-producing palms, except date
and coconut palm, are nonmajor crops of the growing region; they grow
plentifully in harsh habitats, and their sap contains approximately 10%
to 20% sugar (Dalibard, 2018). Palm sap is collected by tapping the
palm inflorescence spadix and foliage region of the trunk top without
damaging the plant. Plants can be tapped when they are five years old
and can continue producing sap for up to 30 to 100 years (Dalibard,
2018). Except for the labor-intensive process of tapping in exploiting
sap collection, palm sap could be the best alternative of major food
crop-based bioethanol feedstock of the growing region.
Wild date palm (Phoenix sylvestris Roxb.) is a monocotyledonous,
dioecious, perennial tall tree plant that belongs to the Arecaceae family
and is one of the dominant palm species among twelve Phoenix genera
widely grown in Bangladesh and other parts of South Asia (Krueger,
2011; Henderson, 2009). Its sweet sap is either consumed as fresh juice
or processed into a broad array of products, including spread, syrup,
vinegar, organic acids, sugar, and wine (Krueger, 2011). Its sweet fruit
is consumable but usually unutilized and left as waste under the tree.
Wild date palm has naturalized in fallow inarable lands, around
homestead and farmland boundaries, along roads and canals, in the
crop field along with other crops, in foreshore areas and orchards (Islam
et al., 2014). This palm has adapted to a wide range of environmental
stresses, including salinity, drought and climate change, alleviating the
threat of its continuous production due to climate change (Elshibli and
Korpelainen, 2011). Moreover, its production is not interrupted by re-
plantation or crop rotation, both of which are the main drawbacks of
classical sugar feedstock production. Even though wild date palm sap
has many domestic uses, its fruit- and sap-based industrial potential
remains unexplored. Establishing wild date palm fruit- and sap-based
bioethanol production could provide a sustainable solution to the for-
midable challenges of secure energy diversification in Bangladesh. This
study has two objectives: first, to investigate the potential of wild date
palm as a sugary feedstock of bioethanol through experimental
A.M. Swaraz, et al.
investigation; and second, to explore whether using this feedstock can
potentially yield ethanol with minimum expense and effort.
2. Materials and methods
2.1. Study site and habitat
The study site was in Kotchandpur Upazila of the Jhenaidah district,
one of the ancient wild date palms growing regions in Bangladesh. It is
situated between 23°22′ and 23°33′ north latitudes and between 88°55′
and 89°06′ east longitudes, and is an entirely plane area. The annual
average temperature varies from a maximum of 37 °C to a minimum of
11 °C, with an annual average rainfall of 60.51 in. Physiochemical
property analysis of the soil was conducted randomly throughout the
study site, and the soil was found to be characteristically sandy-loam in
texture with a slightly high pH, decent organic matter, and moderately
high amounts of minerals, HCO3 and SO42−. The average moisture
content in the soil was 84% and the average electrical conductivity was
113.33 μS cm −1. Most of the land of the selected site is used for the
cultivation of rice, wheat, jute and sugarcane, as well as various fruits
and vegetables.
2.2. Selection of wild date palm cultivars
The wild date palm trees over the entire study area are grown
naturally on crop fields, crop field boundaries and sloping terrain sides
of crop fields. The palm population was composed of progeny only from
seedlings, had probably never undergone selection based on individual
qualities, and thus has persisted with considerable genetic diversity. To
date, there are no scientific reports defining good quality sap and fruit
yield traits in wild date palms. The inflorescence is the only morpho-
logical feature that can distinguish male and female plants. Mature fruit
morphology could be a good candidate trait for defining female culti-
vars. There were dozens of morphologically different fruit-producing
female plants identified in the study site. Among these, six different
female cultivars were selected for this study (see Appendix, Fig. S1.
[A]). Plants aged about twenty years of those six cultivars were ran-
domly selected from three sites of the study area to study their fruit and
sap yield. Fruits and sap yields were calculated for four consecutive
years. There was the possibility of a wide range of genetic diversity of
the male wild date palm in the study area, and similarly for female
plants. However, due to the absence of any similar phenotypic features
in female plants, male cultivars were selected, based on higher sap
yielding efficacy. In this investigation, a batch of six moderately higher
sap yielding males of about twenty years of age were selected to cal-
culate their sap yield over two consecutive years.
2.3. Wild date palm fruit pulp and sap collection
Mature fruits of the wild date palm of various cultivars were col-
lected from spikelets from late May to early July when fruits become
mature and amber in color (see Appendix, Fig. S1. [B]). Their fresh
weights were measured immediately after collection. Pulps were se-
parated from seeds and their weights were separately measured. Pulps
were dried in a drier box at 55 °C until they turned crispy and were
suitable for making into a fine, dry powder. The dry weight of pulp was
also measured and subsequently ground into a fine power by an electric
grinder. This pulp powder is used for sugar extraction and other further
uses.
Wild date palm sap from male and female cultivars was collected
using the zig-zag tapping method, beginning in mid-November and
continuing until mid-March. In this method, fronds were removed using
sharp, heavy machetes from the base of the petiole on one side of the
palm crown, leaving other side with flower productive capacity
(Appendix Fig. S2. [A]). Later, the petiole bases were cleaned until the
peripheral region of the palm heart was reached. A sharp, thin machete
3
Industrial Crops & Products 139 (2019) 111507
was used to make a clean tapping surface, and this was tanned by sun
drying for one week (Appendix Fig. S2. [B]). Before making the incision
and inserting the spout, the tapping surface was again cleaned for
several days until the sap completely dried from the cleaned tapping
surface (Appendix Fig. S2. [C]). Two semi-circular incisions were made
in the middle of the cleaned tapping surface, and a bamboo collection
spout was inserted into the middle of the incisions (Appendix Fig. S2.
[D]). The exuding sap ran through the spout into a clay pot (Appendix
Fig. S2. [E]). New incisions were made at three- to five-day intervals.
Until the earlier incision had dried up, the second incision was made
approximately one inch above the base of the first incision, and the
same cycle was repeated until the palm heart was reached, which de-
veloped a stepped appearance (Appendix Fig. S2. [F]). Three to five
cuts complete an incision cycle and approximately 10 incisions were
produced a year, so 30 to 50 cuts can be obtained per year.
The following year, the same treatment was completed on the other
side of the tree, producing a zig-zag tapping pattern (Appendix Fig. S2.
[G]). Incisions were made just before sunset and the sap was collected
at 12-h intervals, continuing until the incised surface had dried up. The
collected sap was measured using a measuring cylinder as the total sap
collected from each plant, each month, and the total sap yield of all
tapping months was considered to be the yearly sap yield from each
plant. Sap samples that were used for chemical analysis and fermen-
tation study were mixtures of male and female sap collected in the later
weeks of tapping period and preserved at −20 °C in a refrigerator im-
mediately after collection.
In addition, sugarcanes were also collected from the study site in the
same tapping month and used for comparative study with wild date
palm fruit pulp and sap. After collection, sugarcanes were washed in
running tap water and milled to collect juice, which was filtered and
preserved at −20 °C in a refrigerator for chemical analysis and fer-
mentation study.
2.4. Analysis of sugar, approximate and minerals contents of wild date palm
fruit pulp and sap
The sap samples were collected before sunrise, immediately trans-
ferred into an insulated icebox to protect fermentation, and taken to the
laboratory to be preserved in a refrigerator. The box-dried fruit pulp
samples were ground into a fine, dry powder by an electric grinder.
Sugar was extracted by the water extraction method from the pulp
powder. Sugarcane juice was collected through the milling process. All
prepared samples including wild date palm fruit pulp extract, sap and
sugarcane juice were immediately stored in a freezer at −20 °C before
being used for analysis and fermentation study. Samples were used
within a week to retain their composition.
The sugar content from various samples of wild date palm and su-
garcane juice were analyzed using the reverse phase liquid chromato-
graphy system (SHIMADZU Prominence-i, LC-2030C 3D), equipped
with a refractive index detector (RID-20A) and Shim-pack GIST NH2 -
5 μm column (250 × 4.6 mm). The High-Performance Liquid
Chromatography (HPLC) grade acetonitrile and water at 80:20 ratio
was used as the mobile phase, and the flow rate was set at 1.0 ml/min.
The injection volume was 10 μl and the column temperature was set at
40 °C. The retention time for the standard fructose (6.432 min), glucose
(7.026 min) and sucrose (8.929 min) was detected at 254 nm. The total
sugar content of individual samples was calculated by summing all the
sugar (glucose, sucrose, fructose) contained in the sample.
Total crude protein and ash content of wild date palm fruit pulp, sap
and sugarcane juice were analyzed by following the standard food
analysis methods as described in an earlier work (Swaraz et al., 2017).
Crude lipid contents of wild date palm fruit pulp sap and sugarcane
juice were analyzed according to the method described in AOAC In-
ternational (OMA, 2005).
Contents of minerals – (Na+, K+, Ca+, Mg2+, Fe2+, Zn2+) – in
various samples were determined by the Atomic Absorption
A.M. Swaraz, et al.
Spectrophotometer (AAS), (Spectr AA 220, Varian, USA). Briefly, ash
residues of each sample were digested with a perchloric acid and nitric
acid (1:4) solution. The samples were then left to cool, and the contents
filtered through Whatman® grade 42 filtration paper. Each sample so-
lution was made up to a final volume of 25 ml with deionized water.
The aliquot was used to determine the mineral contents separately.
2.5. Batch fermentation and ethanol yield estimation
The yeast (Saccharomyces cerevisiae) was obtained from local market
as activated dry yeast and used for culture initiation. A loop of a single
colony of yeast that was grown on a solidified medium [D - glucose
40 g/l + Peptone 10 g/l + pH 7.0 and solidified with 15 g/l Agar] was
transferred to a 10 ml nutrient broth containing falcon tube [D - glucose
40 g/l + Peptone 10 g/l and pH 7.0] and incubated overnight at 28 °C
and 150 rpm in a shaking incubator. The culture was then transferred to
100 ml of the same nutrient broth containing 250 ml in a conical flask
to incubate for a period of 12–18 h at 28 °C and 100 rpm in order to
harvest yeast cells. Harvested yeast cells were inoculated to the fer-
mentation samples ensuring the inoculated samples contain approxi-
mately 1 × 107 cells/ml that were determined using UV–vis spectro-
photometer at OD 600 nm. All yeast work procedures were carried out
aseptically.
Fermentations were conducted by batch in 250 ml conical flasks
with the same conditions used for pulp extract, sap, sugarcane juice and
sucrose, where sugarcane juice and sucrose were used as the control.
The conical flask was air tightened with a cotton plug. The initial sugar
contents of pulp extract, sap, sugarcane juice and sucrose were adjusted
Fig. 2. Bioethanol production process from wild date palm.
4
Industrial Crops & Products 139 (2019) 111507
to 140 g/l. Samples were further diluted by adding nutrients (yeast
extract 120 g/l + malt extract 120 g/l + peptone 200 g/l) prior to
sterilization through autoclaving at 121 °C for 15 min. Throughout the
procedure, the pH of all cultures were adjusted to 5.0 with 1 N NaOH or
1 N HCl solution before autoclaving, and the incubation temperature
was maintained at 28 °C for 60 h without any agitation. After the in-
cubation period (60 h) all samples were collected and immediately
exposed to a water bath at 100 °C for five minutes to heat kill active
yeast cells. Later, samples were stored at −20 °C in a freezer for further
analysis. The methodology of bioethanol production stages from wild
date palm fruit pulp extract and sap is illustrated in Fig. 2.
The ethanol concentrations of various samples were determined
using UltiMate 3000 HPLC system (Thermo Fisher Scientific Inc.)
equipped with a Shodex RI-101 Refractive Index detector and
HyperREZ XP Organic Acids, 8 μm, 100 × 7.7 mm column. In this
method, 0.0001 mM H2SO4 eluent was used as the mobile phase and the
flow rate was set at 0.7 ml/min. Column temperature was fixed at 30 °C.
The injection volume was 20 μl. The run time was selected as 10 min for
each sample injection. The ethanol peak was detected by the RI detector
at 5.90 min. The ethanol yields were calculated using Eq. (1) (Shahirah
et al., 2015) and fermentation efficiencies were determined as the ac-
tual ethanol yield divided by the theoretical ethanol yield (Appiah-
Nkansah et al., 2018; Laopaiboon et al., 2007).
Final ethanol concentration (g /l)
Ethanol yield (%) = × 100
Total initial sugar (g /l) × 0.511 (1)
A.M. Swaraz, et al. Industrial Crops & Products 139 (2019) 111507

2.6. Data collection and statistical analysis
Fruit and sap yield data were collected over four consecutive years,
approximate analysis carried out in triplicate, mineral and batch fer-
mentation study carried out once with the same fermentation condi-
tions for all samples. Collected data were subjected to one-way ANOVA
(Analysis of Variance) wherever necessary after setting the statistical
significance level at P ≤ 0.05 followed by DMRT (Duncan’s Multiple
Range Test) using IBM SPSS Statistics v.20 software. Further, graphic
and flowchart presentations were prepared using Graph Pad Prisom
(version 7), Microsoft XL and Visio software.
3. Results and discussion
3.1. Fruit pulp and sap yield in wild date palm
In the present study site both sexes of wild date palm started
flowering at the beginning of January, fruit setting was completed in
the early weeks of February and fruits matured by late May. Variation
of the female wild date palm cultivars was categorized based on fruit
morphology, and dozens of morphologically distinct date fruit-produ-
cing female wild date palm cultivars were found in the study site. From
these six readily available different female cultivars (see Appendix Fig.
S1. [A]) were selected for fruit and sap yield study. At the same time,
male cultivars were selected based on higher sap yielding capability.
Both male and female cultivars were about 20 years of age.
Each of the female palm cultivars produced plenty of dates every
year, which remained unused and degraded under palm trees due to the
lack of scientific information available to the local population regarding
their potential as a valuable agro-industrial product. Fruit yields of the
tapped and nontapped plant of each type of the six female wild palm
cultivars are shown in Table 1.
There were significant variations in fruit yields, fresh pulp yield,
pulp percentage and dry pulp yields found among the cultivars.
However, insignificant variations of these attributes were found be-
tween the tapping and nontapping plants of individual cultivars, except
for the total fruit yield, fresh and dry pulp yield. In tapping plants, fruit
yield, fresh pulp and dry pulp yield were greatly reduced compared to a
nontapped plant of each individual cultivar. This reduction of fruit yield
was due to the inability of inflorescence development on the tapping
side, as inflorescence buds are removed while preparing the tapping
surface (Appendix Fig. S2. [A–C]). Notably, total fresh pulp yield does
not only depend on the volume of the fruit produced; rather, it depends
on pulp percentage and pulp moisture content, along with total fruit
yield of individual cultivar. The highest fruit yield was observed from
cultivar 1 (FCV1) with 56.15 ± 1.95 kg/plant, whereas cultivar 2
(FCV2) exhibited maximum pulp percentage and dry pulp yield with
54.95 ± 0.55% and 11.16 ± 0.68 kg/plant, respectively from
nontapped plant. However, these higher yields were greatly reduced in
all tapped plants, including the highest fresh pulp yielding cultivar
FCV1, which produced 5.45 ± 0.65 kg/plant dry pulp, and the highest
pulp percentage containing cultivar FCV2, which yielded
8.74 ± 0.60 kg/plant of dry pulp. An average 27.84 ± 2.78 kg/plant
fruit was found from a tapped plant, yielding 5.25 ± 0.59 kg/plant of
dry pulp. In contrast, an average 41.54 ± 1.18 kg/plant fruit was
yielded from a nontapped plant that produced 7.75 ± 0.84 kg/plant
dry pulp. These results indicate that an average of 32.98% yield loss
occurred due to tapping. Variations of pulp percentage in tapped and
nontapped plant of individual cultivars were insignificant. The average
pulp percentage of tapped plants of the six cultivars was
49.14 ± 1.22%; whereas for nontapped plant it was 48.93 ± 1.36%.
The reason of these variations of fruit pulp yield, pulp percentage and
total dry pulp yield among cultivars could be due factors including
genotype specificity of each cultivar. However, reduction of fruit yield
in tapped plants was solely linked to tapping, as inflorescence buds are
removed from half of the palm crown to prepare the tapping surface.
The volume of fruit biomass obtained from the wild date palm in the
present study was promising, and it could be priced to be used as
bioethanol feedstock. In addition, it seems that it will not exert any
pressure on food scarcity of the country, as this feedstock usually re-
mains unused and rotten under the tree.
Traditionally, wild date palm is prized for its sweet sap, collected by
tapping. In this study, the wild date palm was tapped for only 135–150
days in the winter season from November to March in the study area, so
the yearly sap yield means that the sap is yielded from each plant only
in the winter months. The sap yield of the six male and female cultivars
are presented in Fig. 3. It was found that the yearly average sap yields
were higher in female wild date palms than males. The highest sap yield
was recorded in female cultivar 4 (FCV4), with 216.10 ± 7.18 L/plant.
Male cultivar- MCV3 yielded the highest volume of sap
with154.25 ± 3.58 L/plant. The average sap yield of the male cultivars
was 140.42 ± 2.67 L/plant; whereas, in female cultivars it was
190.31 ± 3.71 L/plant. These variations of sap yield among the male
and female plant could be due to genotypic specificity of individual
cultivars. Additionally, the superior sap yields in female plants could be
due to genetics, along with the prolonged tapping period in female
plants, as these exhibited moderate sap exudation until end of the
season, while male plants exhibited gradual reduction of sap exudation
after completion of blooming (mid-February). As a seasonal sap pro-
ducing palm, the yearly sap yield in wild date palms is much more
competitive regarding total biomass produced compared with other
tapped palm species: the seasonal sap-producing palmyra palm with
150 L/plant (Davis and Johnson, 1987) the year-round sap-producing
palms, such as the nipa palm with 180 L/plant sap (Tamunaidu et al.,
2013; Das et al., 2000), and the African oil palm with 100 L/plant sap
(Eze and Ogan, 1988).

Table 1
Fruit pulp yield of wild date palm. Data taken four consecutive years (two year for tapped and two year for nontapped plant) each with ≈ 20 year age five plants of
same cultivar. Here, FCV: Female Cultivar and number indicates cultivar number.
Plant genotype no. Total fresh fruit yield (kg/ plant) Fresh pulp yield (kg/ plant) Pulp percentage (weight %) Total pulp dry weight (kg/ plant)

Tapped Non - Tapped Tapped Non - Tapped Tapped Non - Tapped Tapped Non - Tapped

FCV1 31.45 ± 3.25bc 56.15 ± 1.95c 13.67 ± 1.55bc 24.37 ± 0.59c 43.42 ± 0.44a 42.26 ± 0.06a 5.45 ± 0.65bc 9.68 ± 0.36b
FCV2 42.45 ± 2.15d 54.00 ± 3.00c 23.17 ± 1.50d 29.48 ± 2.06d 54.55 ± 0.79c 54.95 ± 0.55d 8.74 ± 0.60d 11.16 ± 0.68b
FCV3 33.35 ± 1.65c 53.95 ± 3.95c 16.75 ± 1.42c 27.08 ± 1.83cd 50.22 ± 0.27b 50.85 ± 0.60bc 6.44 ± 0.50c 10.36 ± 0.71b
FCV4 23.35 ± 2.85ab 32.30 ± 1.90b 11.43 ± 1.40ab 15.80 ± 0.91b 48.95 ± 0.05b 49.20 ± 0.30b 4.10 ± 0.51ab 5.66 ± 0.35a
FCV5 15.90 ± 1.90a 19.35 ± 2.55a 8.40 ± 0.87a 10.22 ± 1.18a 52.96 ± 0.84c 52.43 ± 0.03c 3.31 ± 0.35a 4.02 ± 0.48a
FCV6 20.55 ± 1.95ab 33.50 ± 3.50b 9.22 ± 1.07ab 14.96 ± 1.24ab 44.77 ± 0.97a 43.88 ± 0.78a 3.47 ± 0.38a 5.62 ± 0.48a

Note: Each value represents the mean ± standard error (SE). Mean followed by the same letter in the same column are not significantly different from each other at
P ≤ 0.05 level according to Duncan’s multiple range test.

5
A.M. Swaraz, et al.
Fig. 3. Sap yield of wild date palm. Data taken for two tapping years each with
≈ middle-aged plants. Each value represents the mean ± standard error (SE).
Above the mean bar with the same letter are not significantly different from
each other at P ≤ 0.05 level according to Duncan’s multiple range test. Here
MCV and FCV correspond to male cultivar and female cultivar where number
indicate cultivar number.
3.2. Sugar profile, approximate and mineral content of wild date palm fruit
pulp and sap
Freshly collected date pulp powder, cooled sap and juice were used
for sugar and approximate analysis. Table 2 shows the sugars content
and approximate amounts of fruit pulp, sap and these were compared
with sugarcane juice, which is grown in the study site. Supplementary
Fig. S3. in the Appendix depicts sugar profiles (HPLC chromatograms)
of the samples. It was found that pulp contains glucose and fructose of
30.60 ± 0.60% and 30.47 ± 0.12%, respectively, yielded
61.09 ± 0.73% of total sugar. No sucrose was detected in the pulp
samples. Sucrose was found to be the dominant sugar
(24.08 ± 0.10%), followed by fructose (2.05 ± 0.02%) and glucose
(1.84 ± 0.00%) that resulted in a total of 27.98 ± 0.13% sugar in
wild date palm sap (c.f., Table 2).
The sugarcane juice is mainly composed of sucrose
(16.47 ± 0.06%) with a small amount of glucose (2.67 ± 0.07%) and
fructose (2.34 ± 0.01%) as detected in HPLC that yielded
21.49 ± 0.10% of total sugar.
Findings from sugar profiling show the presence of glucose and
fructose and the absence of sucrose in wild date fruit pulp (see
Supplementary Fig. S3. in the Appendix), which is in line with the re-
sults of date fruits that showed absence of sucrose in mature dry fruits
in (Haider et al., 2014; Ghnimi et al., 2017). Even though wild dates
contained a lesser amount of total sugar compared to dates, which
contain more than 81% of total sugar (Assirey, 2015), wild dates pos-
sess great potential as a sugary biomass producing plant since it is an
inarable land growing, non-major perennial crop. Thus, wild date palm
fruit may have an important agro-industrial future as a potential source
of sugary feedstock for bioethanol production.
Table 2
Approximate contents (%) of wild date palm fruit pulp, sap and sugarcane juice.
Samples Sugar
Sucrose Glucose
Sugarcane juice 16.47 ± 0.06 2.67 ± 0.07
Wild date palm dry fruit pulp 0.0 ± 0.0 30.60 ± 0.75
Wild date palm sap 24.08 ± 0.10 1.84 ± 0.00
Note: Data taken for three consecutive years from same maturation stage of ≈ 20-year age and same cultivar and each value represents the mean ± SE. Sugarcane
juice proximate was analyzed to compare with wild date palm samples.
Industrial Crops & Products 139 (2019) 111507
Sugar profiling in wild date palm sap shows the dominance of su-
crose and the results are similar to those of other sap producing palms,
including palmyra palm, date palm, and nipa palm which were reported
elsewhere (Tamunaidu et al., 2013; Rao et al., 2009). The total sugar in
sap of wild date palms was highest among the various sugary saps such
as nipa palm with 18.6% (Tamunaidu et al., 2013), palmyra palm and
date palm with 10.93% and 11.50% (Rao et al., 2009) and oil palm with
17.2% (Shahirah et al., 2015). This total is even more sugar than
classically used feedstock such as sweet sorghum juice, which contains
13.0% (Appiah-Nkansah et al., 2018), sugarcane 19.10% (Chauhan
et al., 2002) and sugar beet 20% (Marzo et al., 2019) of total sugar.
Thus, wild date palm sap could be considered as the best alternative of
major food crop-based sugary feedstock for bioethanol production, as
its production is not interrupted by replantation or crop rotation, which
are the main drawbacks of such production.
The approximate analysis found that fruit pulp contains
3.79 ± 0.078% total crude protein, 2.73 ± 0.07% crude lipid and
2.37% ash; whereas sap contains 0.948 ± 0.039% total crude protein
and 0.18 ± 0.004% ash. Among the three sugary biomasses, sugarcane
contains the least amount of total crude protein (0.42 ± 0.014%). It
was noticed that lipid was absent in both sap and juice samples (cf.,
Table 2).
The elemental analysis demonstrated that saps and fruit pulp of wild
date palm contain a valuable amount of minerals (see Table 3). The
calcium concentration was the highest (191.218 mg/kg), followed by
magnesium (176.280 mg/kg), potassium (84.553 mg /kg), iron
(16.214 mg/kg), sodium (3.017 mg /kg) and zinc (2.773 mg/ kg) in
wild date fruit pulp powder. Magnesium was the most abundant ele-
ment, followed by potassium, sodium, calcium, iron and zinc in the sap
sample, which were 15.549 mg/kg, 10.078 mg /kg, 3.416 mg /kg,
2.007 mg/kg, 0.556 mg /kg and 0. 1345 mg/kg, respectively. This data
implies that wild date fruit pulp and sap contain most of the elements at
higher concentrations than sugarcane juice and other sugary sap pro-
ducing palm such as nipa palm (Tamunaidu et al., 2013), coconut palm
(Ranasinghe and Waidyanatha, 2003; Chongpraditnun et al., 2001) and
palmyra palm (Rao et al., 2009), as well as fruits of the date palm
(Assirey, 2015; Souli et al., 2014; Salvi and Katewa, 2012; Thabet et al.,
2009). The higher approximate and mineral contents of wild date palm
fruit pulp extract and sap indicate that those feedstocks could be ad-
vantageous for cost efficient production of bioethanol, as no external
nutritional supplement may be necessary for fermentation.
3.3. Fermentation profile of wild date fruit pulp and sap
The optimum fermenting capability in yeast (Saccharomyces cerevi-
siae) was achieved at pH 5.0 and temperature between 28 °C – 37 °C in a
number of studies (Lin et al., 2012; Liu and Shen, 2008; Zabed et al.,
2014). Conventional batch fermentation processes were thus conducted
to ferment fruit pulp extract and sap in supplemented and non-sup-
plemented nutritional conditions adjusted to pH 5.0 and temperature
28 °C in conical flasks without agitation for 60 h. The ethanol yields and
ethanol conversion efficiency of the pulp extract and sap in supple-
mented and non-supplemented nutrient conditions are depicted in
Protein Lipid Ash
Fructose Total sugar
2.34 ± 0.01 21.49 ± 0.10 0.42 ± 0.014 0.0 0.469 ± 0.016
30.47 ± 0.12 61.09 ± 0.73 3.79 ± 0.078 2.73 ± .07 2.37 ± 0.14
2.05 ± 0.02 27.98 ± 0.13 0.948 ± 0.039 0.0 0.18 ± 0.004
6
A.M. Swaraz, et al. Industrial Crops & Products 139 (2019) 111507

Table 3
Mineral contents in fresh and fermented wild date palm fruit pulp and sap. Here sugarcane juice was use as control.
Minerals (mg/kg) Sugarcane juice Wild date palm fruit pulp Wild date palm sap

Fresh Fermented Fresh Fermented Fresh Fermented

Na 2.339 2.202 3.017 2.657 3.416 1.632

K 9.373 7.318 84.553 14.633 10.078 3.386
Ca 12.800 7.836 191.218 5.575 2.007 0.569
Mg 61.755 5.945 176.280 22.473 15.549 4.540
Fe 1.353 0.561 16.214 0.186 0.556 0.162
Zn 0.408 0.281 2.773 0.036 0.1345 0.073

Fig. 4, and relevant HPLC chromatograms presented in Supplementary
Fig. S4 in the Appendix. It was found that ethanol yield and ethanol
conversion efficiency from nutrient supplemented samples were lower
from their non-supplemented counterparts. In general, no inclusion of
nutrient supplementation achieved increased fermentation in all sample
types except pure sucrose. The addition of nutrition supplement most
likely played an important negative pressure in increasing the overall
metabolism in yeast, leading to poorer ethanol yield and efficiency
(Theerarattananoon et al., 2008).
Clearly, ethanol production increases with fermentation time, as
shown in Fig. 4. Among all the samples, wild date palm sap achieved
the highest ethanol yield (0.323 g/g) with maximum ethanol conver-
sion efficiency (63.40%); whereas wild date fruit pulp yielded (0.232 g/
g) ethanol with 45.47% conversion efficiency after 60 h of fermentation
in nutrition non-supplemented condition (see Fig. 4). The sugarcane
juice samples resulted in a lower ethanol yield and conversion rate
compared to sap samples with 0.272 g/g ethanol yield and 53.34%
conversion efficiency in the same fermentation condition. The pure
sucrose samples without addition of nutrients yielded only 0.024 g/g
ethanol with 4.8% conversation efficiency.
Notably, all nutrient supplemented samples except pure sucrose
samples exhibited poor fermentation trends and ethanol yield com-
pared with non-supplemented samples. Among the nutrition supple-
mented samples, the highest fermentation suppression observed in wild
date palm sap yielded 0.08 g/g ethanol, followed in ascending order by
fruit pulp extract with 0.11 g/g ethanol yield and sugarcane juice with
0.18 g/g ethanol yield. On the other hand, as expected, ethanol yield as
well as fermentation efficiency increased in sucrose samples in nutrition
supplemented conditions.
Overall, the average fermentability of wild date palm sugary bio-
masses exhibited superior potential for bioethanol production com-
pared to the conventional bioethanol fermentation feedstock: sugarcane
juice. Moreover, wild date palm showed greater fermentation potential
without an external supply of nutrition, which might be due to the
presence of more naturally occurring fermentation promoting nutrients
and their consumption in the fermentation process in wild date palm

Fig. 4. Ethanol yield and fermentation efficiency. WNS: with nutrition supplement; NNS: no nutrition supplement. (Readers are suggested to see online version for better
understanding).

7
A.M. Swaraz, et al. Industrial Crops & Products 139 (2019) 111507

sugary biomasses (Tables 2 and 3). This is in agreement with many
other studies that showed that Na+, K+, Ca+, Mg2+, Fe2+, Zn2+ are
required for cellular growth, and they fulfill specific metabolic and
structural roles in Saccaharomyces cerevisiae, which in turn stimulate the
fermentation rate and ethanol production (Somda et al., 2011; Breisha,
2010; Stehlik-Tomas et al., 2005, 2004).
From these findings, it could be hypothesized that fermentation
efficiency and ethanol yield potential depend on the efficient use of
bioavailable nutrient contents in fermentable sugar feedstock, at least
in the case of wild date palm fruit pulp extract and sap. However,
testing this hypothesis is beyond the scope of the current study. In
addition, these available nutrients in the wild date plants would po-
tentially reduce the cost of bioethanol production, and indicate that
these feedstocks can be used for sustainable cost-effective bioethanol
production.
ethanol-yielding fermentation protocol was established, and the plants
grown by following standard agricultural practices. Furthermore, ad-
ditional regulating factors such as concomitant maximum production of
sap and fruit in wild date palm should also be determined.
The estimated ethanol yield from wild date palm is much higher
compared to nipa palm sap (4550–9100 liter/hectare) (Tamunaidu
et al., 2013), sugarcane juice (5300–6500 liter/hectare), corn
(3100–3900 litter/hectare) (Marris, 2006), and sugar beet (5010–6680
liter/hectare) (Mussatto et al., 2010). A comparison between the results
obtained and findings in the literature is shown in Table 4.
In the light of the findings of this study, it can be summarized that
wild date palm has the greatest potential for use as a sugar-based
bioethanol feedstock, with minimal impact on biodiversity, carbon
footprint, arable land and food security, all of which have already been
terribly affected in recent years by climate change and global warming
in the region.

3.4. Bioethanol production potentials of wild date palm 4. Conclusion

Total bioethanol yield of wild date palm was estimated, based on Through experimental procedures, present study has established the
the results obtained in terms of yearly sap and fruit pulp yield, their wild date palm as a potential sugary feed stock for bioethanol pro-
corresponding total sugar contents and bioethanol yield. The average duction which was the primary objectives of the work. Our results show
sap yield of male wild date palm was 140.42 L/plant with 29.85% total that male wild date palm plants produced a lower amount of sap
sugar. The average dry pulp yield of tapped female wild date palms was compared to female plants through conventional tapping practice.
5.25 kg /plant, with 61.09% total sugar, and the average sap yield was There was an insignificant impact of tapping on fruit pulp content;
190.31 L/plant with 26.13% total sugar. The optimum bioethanol however, tapping reduced the total fruit yield and 32.98% of fruit pulp
yields achieved from the present study were of 0.323 g/g from sap and yield loss occurred due to tapping of female wild date palm. Sugar
0.232 g/g from fruit pulp extract through conventional batch fermen- profiling of wild date palm and sap found that mature fruits contained
tation process without an external supply of nutrition, and these values only reducing sugars whereas sap was composed of mainly sucrose with
were used as the maximum bioethanol yield to calculate the bioethanol little amount of reducing sugars. Fermentation study using yeast found
production potential of wild date palm. that, sap and fruit pulp extract of wild date palm can ferment without
It is possible that 500 wild date palm plants per hectare of land nutrient supplementation to potentially yield ethanol. In addition,
could be grown. Since biomass (sap and fruit) production potential is average fermentation efficiency and ethanol yield of wild date palm
higher in female plants, then plantation should be carried out by samples were outperformed compared to sugarcane juice and sucrose
planting male and female plants at a ratio of 1:4. The number of males from the present fermentation conditions.
in this ratio would be sufficient for successfully pollinating female Finally, based on our experimental findings, it was estimated that a
flowers to produce fruits in the given area. The average annual bioe- hectare of fully-grown wild date palm plants comprising 100 male and
thanol yield would be 13.53 L/plant from males and 16.80 L/plant from 400 female plants could yield 92269.04 kg sugary biomass, with
female plants. For female plants, out of 16.80 L, 16.06 L is from sap and 39.02% total sugar that could yield a minimum 8076.62 L of bioe-
0.74 L is from fruit pulp. One hectare of land would comprise 100 male thanol. We further hypothesized that ethanol yield could be attained in
and 400 female plants. Hence, the minimum bioethanol potential is bulk from wild date palm fruit pulp extract and sap if an optimum
estimated to be 8076.62 L/hectare, where 1353.93 L would be from the fermentation protocol for these feedstocks was established, since fer-
male plants and 6722.69 liter from the female plants. The estimated mentation efficiency and ethanol yield depends on many factors, in-
ethanol product potential could be even much higher if an optimum cluding fermentation process, temperature, total sugar concentration,
pH, fermentation duration, agitation status, amount of yeast and yeast
Table 4 strain type (Zabed et al., 2014). Further experimental investigation is
Ethanol yield for wild date palm compared with other sources in the literature
required to test this hypothesis. In addition, byproduct and coproduct
(Zabed et al., 2017; Mussatto et al., 2010; Tamunaidu et al., 2013).
from wild date palm fruit pulp and sap fermentation would need to be
Source Ethanol Yield (litter/hectare) explored and valorized.
Taken together, it can be concluded that the wild date palm has
Wild Date Palm (this study) 8076 (minimum)
Nipa Sap 4550–9100 great potential as a sugar-based feedstock for bioethanol production
Sugarcane 6190–7500 without threatening food security in Bangladesh. Such production
Sugar Beet 5010–6680 would generate thousands of jobs for the expanding population, and
Sweet Potatoes 1989–4800 would not raise the food versus fuel dilemma in the country.
Sweet Sorghum 3050–4070
Sorghum Bagasse 1796–6591
Jerusalem Artichoke 1821–5930 Acknowledgements
Yam 4800
Corn 3460–4180 Authors are thankful to wild date palm tappers who helped col-
Cassava 3310–4901
lecting sap and dates systematically. This research did not receive any
Sorghum Stalk 2062–2595
Arrowroot 2496 specific grant from funding agencies in the public, commercial, or not-
Wheat 1001–2590 for-profit sectors.
Potato 1600
Sorghum Grain 1099 Appendix A. Supplementary data
Corn Stover 1050–4400
Switch Grass 10760
Microalgae 46760–140290 Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.indcrop.2019.111507.

8
A.M. Swaraz, et al.
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