Biotechnology Reports 40 (2023) e00815

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Biotechnology Reports
journal homepage: www.elsevier.com/locate/btre

Research Article

Isolation, screening and identification of ethanol producing yeasts from

Ethiopian fermented beverages
Dagnew Bitew a, b, *, Anteneh Tesfaye c, d, Berhanu Andualem d
a
Department of Biology, Mizan-Tepi University, P. BOX: 260, Ethiopia
b
Institute of Biotechnology, University of Gondar, P.BOX: 196, Ethiopia
c
Institute of Biotechnology, Addis Ababa University, P.BOX: 1176, Ethiopia
d
BioTEI, Winnipeg, Manitoba, Canada

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

Keywords: The growing demand for renewable energy sources such as bioethanol is facing a lack of efficient ethanologenic
Banana peel microbes. This study aimed to isolate and screen ethanologenic yeasts from Ethiopian fermented beverages. A
Bioethanol progressive screening and selection approach was employed. Selected isolates were evaluated for bioethanol
Biomass
production using banana peel waste as substrate. A total of 102 isolates were obtained. Sixteen isolates were
S. cerevisiae
Renewable energy
selected based on their tolerance to stress conditions and carbohydrate fermentation and assimilation capacity.
Most found moderately tolerant to 10 %, but slightly tolerant at 15 and 20 % (v/v) ethanol concentration. They
yield 15.3 to 20.1 g/L and 9.1 ± 0.6 to 12.9 ± 1.3 g/L ethanol from 2 % (w/v) glucose and 80 g/L banana peel,
respectively. Molecular characterization identified them as Saccharomyces cerevisiae strains. Results demonstrate
insight about their potential role in the ethanol industry. Optimization of the fermentation conditions is
recommended.

1. Introduction
Energy is a pacemaker for mankind’s activity and a nation’s devel­
opment [1]. Global energy demand has been entirely dependent on fossil
fuels for centuries [2]. The growing global population and industriali­
zation led to the rapid depletion of fossil fuels, which in turn created a
global energy crisis. The annual global crude oil production is projected
to decline to 5 billion barrels in 2050 [3]. On the other hand, fossil fuels,
unevenly distributed all over the world, are responsible for the emission
of greenhouse gases into the common environment that everybody
shares [4,5]. Nowadays, the problem of climate change is a phenomenon
that we are seeing and feeling in terms of devastating events [6]. As a
result of the energy crisis and climate change, which are currently
troubling the world, clean and safe alternative energy supplies have
become the concern of all countries [7,8].
Bioethanol can be produced from sugar and/or starch-based energy
crops and lignocellulosic biomass [9]. Bioethanol can replace fossil fuels
partially or fully [10]. It is recognized as an important transportation
fuel as a result of its high octane number, oxygen content (35 %),
emissions of less greenhouse gases [11], blendability with gasoline and
petrol [11,12], and can be produced from diversified lignocellulosic
materials [13]. Indeed, it is biodegradable, less toxic, and ideal for
meeting the energy demands of fossil-fuel poor countries [14]. Thus,
bioethanol production is growing from 13.2 billion liters in 2000 [15] to
109.8 billion liters in 2019 [16]. However, bioethanol production is
hampered by numerous factors such as sustainability of substrates,
biomass recalcitrance [17], lack of pretreatment methods to efficiently
release fermentable sugars [18,19], co-hydrolysis of inhibitor sub­
stances [20,21], and poor performance of fermentative microbes [19].
Global bio-energy production is principled on the exploitation of
viable and cheap substrates to produce high-quality energy products
[22]. Waste biomass is critically important since it is cheap, abundantly
found, and sustainably available [23]. Utilizing of such substrates avoids
the conflict between whether edible materials are for human con­
sumption or industrial purposes [24]. Fruit waste is considered an
attractive biomass for bioethanol production since it has a high amount
of fermentable sugars, cellulose and hemicellulose with low lignin
content [25,26]. In particular banana fruit waste contains significant
amount of fermentable sugar and investigated as an important substrate
for the production of high quality bioethanol [27–29]. These wastes are
abundantly found in fruit markets, juice vendors, and juice processing
industries and impose environmental problems. Hence, generating

* Corresponding author at: Institute of Biotechnology, University of Gondar, P.BOX: 196, Ethiopia.
E-mail address: btdagne@gmail.com (D. Bitew).

https://doi.org/10.1016/j.btre.2023.e00815
Received 3 June 2023; Received in revised form 25 August 2023; Accepted 2 October 2023
Available online 4 October 2023
2215-017X/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
D. Bitew et al.
energy from it while cleaning the environment is like hitting two birds
with one stone [30].
One of the challenges in large-scale bioethanol production is
obtaining efficient microorganisms that are able to ferment a variety of
sugars released during hydrolysis while also tolerating stress conditions
[31,32]. Yeasts are known and preferred over bacteria for ethanol pro­
duction as a result of their good fermentation capacity and tolerance to
ethanol, low pH, and other fermentation byproducts. Saccharomyces
cerevisiae, in particular, is a workhorse microbe that has been used by
industries to produce bioethanol from lignocellulosic biomasses [33,34].
It has gained support due to its tolerance to a wide range of stress
conditions, ethanologenic characteristics, and good fermentation per­
formance [35,36]. However, commercialized ethanologenic yeasts have
faced criticism due to their susceptibility to thermophilic conditions,
co-hydrolyzed inhibitory chemicals, and inability to ferment pentose
sugars, which make up a significant portion of fermentable sugars
generated from lignocellulosic biomass [37,38]. Hence, there is a need
for new ethanologenic microbes as bioethanol production advances [36,
38].
Fermented food and beverages are considered an important source of
ethanologenic yeasts that are able to tolerate a wide range of stress
conditions, such as nutrient starvation and complexity, low pH, tem­
perature fluctuations, and high osmotic stress [39–41]. Therefore, this
study was designed to isolate ethanologenic yeast from Ethiopian
traditional fermented beverages, evaluate their ethanol productivity
using banana peel as a substrate, and identify the best-performing iso­
lates for bioethanol industrial applications.
2. Materials and methods
2.1. Sample collection and isolation of yeasts
Samples of traditional fermented beverages, i.e. tella, tej, korefe, areki
tinsese, terahi, bubegne, shamita, borde, and cheka were collected from
different sites in Amhara Regional State and Arba Minch town, Gamo
Gofa Zone, South Nations Nationalities and People’s Regional State, and
transported to the Cellular and Microbial Laboratory, Institute of
Biotechnology, University of Gondar with an ice box to avoid the dy­
namics of microbes in the sample. All samples were kept at 4 ◦ C until
processed [42].
All collected fermented beverages were serially diluted (10− 1 to
− 5
10 ) using peptone water as diluents. Twenty microliters (20 µL) of
sample from 10− 4 and 10− 5 dilution factors in each beverage were
spread on yeast extract peptone dextrose agar (YPDA) (containing yeast
extract, 10 g/L; peptone, 10 g/L; D-glucose, 20 g/L, and 1 L sterile
distilled water, 50 µg chloramphenicol/mL and adjusted to pH 5 before
autoclaving). Inoculated agar plates were incubated at 30 ◦ C for 72 hrs.
Representative colonies that showed comparatively different cultural
characteristics were sub-cultured onto different YDPA plates and incu­
bated at 30 ◦ C for 72 hrs [43].
2.2. Morphological characterization, designation and preservation of
yeast isolates
A separately grown colonies of each isolate were examined for cul­
tural characteristics (colony shape, margin, elevation, size, color and
surface texture). Once characterized, yeast isolates were designated
based on the site and type of fermented beverage they were isolated
from. Then each isolate was preserved in malt extract agar slants (40 g/
L) using test tubes and in vials with trypetone soya broth containing 20
% glycerol (v/v) for further use [44].
2.3. Physiological and biochemical characterization of isolates
2.3.1. Pre-selection of yeast isolates
Purified yeast isolates were screened for glucose fermentation
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Biotechnology Reports 40 (2023) e00815
performance according to [45]. A 1 mL of 24 hrs old culture of each
yeast isolate (adjusted to 108 CFU/mL) was inoculated to 40 mL YEPD
broth (yeast extract 10 g/L, peptone 10 g/L, glucose 20 g/L and 1 L
distilled water, 4 µg/L phenol red adjusted to pH 5)) in test tubes con­
taining a 5 mL size inverted Durham tube. Inoculated tubes were incu­
bated at 30 ◦ C for 48 hrs. Gas formation was checked every 12 hrs and
isolates were selected based on the volume of gas in the Durham tube
after 48 hrs of incubation [46].
2.3.2. pH requirement evaluation
A protocol developed by [47] was used to examine the pH tolerance
and requirement of isolates in terms of their glucose fermentation and
growth at YPD broth adjusted to different pH values (3, 3.5, 4, 4.5, 5,
5.5, 6 and 6.5). A 1 mL of each yeast isolate (108 CFU/mL, adjusted by
0.5 McFarland standard) was inoculated separately into 40 mL YEPD
broth medium in bottles and test tubes and incubated at 30 ◦ C.
Fermentation performance response was estimated by measuring the
formation of gas (in terms of the amount of liquid replaced by gas) in a 5
mL Durham tube after 48 hrs of incubation. The formation of gas was
checked every 12 hrs. The growth response of isolates was evaluated via
variation in their dry biomass at different pH values after 72 hrs.
2.3.3. Temperature requirement evaluation
The yeast isolates’ fermentation performance and growth response at
different incubation temperatures were examined. A 1 mL of each yeast
isolate (108 CFU/mL) was inoculated separately into 40 mL YEPD broth
(adjusted to pH 4.5) in bottles and test tubes and incubated at different
temperatures. Growth response was evaluated via variation in their dry
biomass, and fermentation response was estimated by measuring the
formation of gas (in terms of the amount of liquid replaced by gas) in the
Durham tube. The formation of gas was evaluated every 12 hrs [47].
2.3.4. Carbohydrate fermentation
Carbohydrate molecules such as glucose, sucrose, maltose, galactose,
fructose, lactose, mannose, arabinose and xylose were used for the
carbohydrate fermentation test. This test was conducted according to
[48] with slight modification. A 1 mL of 24 hrs old culture of each yeast
isolate (adjusted to 108 CFU/mL) was inoculated to 40 mL YPD broth
medium (yeast extract 10 g/L, peptone 10 g/L, carbohydrate molecules
20 g/L and 1 L distilled water; 4 µg/L phenol red adjusted to pH 4.5) in
test tube and incubated at 30 ◦ C for 72 hrs. The formation of gas was
checked every 12 hrs. The fermentation of each carbohydrate molecule
by each yeast isolate was estimated via observing the formation of gas in
the Durham tube with or without medium color change after 72 hrs of
incubation [49].
2.3.5. Carbohydrate assimilation test
A modified auxanographic method developed by [50] was used to
evaluate the carbohydrate assimilation test. A modified yeast nitrogen
base (YNB) medium (containing ammonium sulfate 5 g/L, potassium
phosphate monobasic 0.85 g/L, potassium phosphate dibasic 0.15 g/L,
magnesium sulfate 0.5 g/L, sodium chloride 0.1 g/L, calcium chloride
0.1 g/L, yeast extract (fermentable carbohydrate free) 1 g/L and Agar
15 g/L) was used to check the isolate’s carbohydrates assimilation
spectrum. A modified YNB medium containing 2 % (w/w/) carbohy­
drate molecules (glucose, sucrose, maltose, lactose, fructose, galactose,
mannose, arabinose, mannitol, starch and xylose) was sterilized and
poured at 4 mm thickness aseptically onto 90 mm Petri plates and
allowed to solidify. Then the dried medium was inoculated with 10 µL of
a 24 hrs old culture of each yeast isolate (adjusted to 12 × 108 CFU/mL)
and incubated at 30 ◦ C and examined for growth every 2 days for up to 5
days. A carbohydrate free medium was used as a control. Assimilation
was considered positive if there was considerable growth in the plate
containing the carbohydrate as compared with the control and negative
if there was no growth.
D. Bitew et al.
2.3.6. Osmotolerance evaluation
A 5 mL YPD broth medium containing (60 %, 70 % and 80 % (w/w)
of glucose in a separate flask and adjusted to pH 4.5), was dispensed into
screw-cap test tubes and sterilized. Each test tube was inoculated with 1
mL of each yeast isolate (standardized at 108 CFU/mL) and incubated at
30 ◦ C for 72 hrs. The growth of each yeast isolate in each glucose con­
centration was determined qualitatively by observing its turbidity and
quantitatively by measuring the dry mass [51,52].
2.3.7. Ethanol tolerance test
Ethanol tolerance of isolates was examined in terms of growth,
fermentation performance response, and survival percentage in YPD
broth containing different concentrations (10 %, 15 % and 20 % (v/v))
of absolute ethanol. A 40 mL YPD broth medium containing those
concentrations of absolute ethanol separately and adjusted to pH 4.5,
was dispensed into screw-cap test tube and a 300 mL glass bottle for
fermentation and growth response respectively. Each test tube was
inoculated with 1 mL of each yeast isolate (standardized at 108 CFU/mL)
separately and incubated at 30 ◦ C for 72 hrs. The amount of gas formed
was considered to evaluate isolates’ fermentation performance and
indirectly their tolerance to each ethanol concentration tested. On the
other hand, growth was determined by measuring the dry mass after 72
hrs of incubation [41].
Similarly, 1 mL (108 CFU/mL) of each isolate was inoculated into 40
mL of YPD broth containing different concentrations of ethanol as
mentioned above and incubated at 30 ◦ C. Then, after 72 hrs of incuba­
tion, the culture broth of each isolate was diluted (10− 1 to 10− 3) with
phosphate buffer saline, and a 20 µL sample from the 10− 3 dilution was
spread on YPDA plate and incubated at 30 ◦ C for 72 hrs. The survival
percentage of each isolate was estimated by comparing it with its
counterpart grown in pure YPD broth and incubated at 30 ◦ C for 72 hrs.
The survival percentage was calculated as Eq. (1):
Total cell count after stressed
Survival Percentage = X100 (1)
Total cell count of unstressed/control
Ethanol tolerance was determined based on the percentage of sur­
vival: highly tolerant (>50 % survival), moderately tolerant (25–50 %
survival), and slightly tolerant (<25 % survival) [53].
2.3.8. Evaluation of ethanol productivity
Yeast isolates that withstand the above mentioned stressful envi­
ronment and aggressively ferment and assimilate tested carbohydrate
molecules were selected. These isolates were further screened for
ethanol production using yeast extract broth containing 2 % glucose. A
1 mL of 24 hrs old cultures of each isolate (adjusted to 12 × 108 CFU/
mL) was inoculated into a 300 mL of broth (pH 4.5). The inoculated
bottles were sealed with parafilm to create anaerobic condition and
incubated at 30 ◦ C for 72 hrs under shaking condition at150 rpm. Then,
the produced ethanol was separated from the aqueous solution at
78.4 ◦ C using fractional distillation. The amount of ethanol produced
was measured colorimetrically using the potassium dichromate method
[54]. Unassimilated glucose was estimated using the 3, 5- dini­
trosalicylic acid (DNS) method, according to [55]. Ethanol and glucose
standard curves were constructed using absolute ethanol and glucose,
analytic grade quality.
2.4. Flocculation property of isolates
The flocculence of isolates were checked at 24, 48 and 72 hrs ac­
cording to [56] with little modification. A 1 mL of 24 hrs culture of each
isolate (108 CFU/mL) was inoculated separately into 40 mL of YPD broth
and incubated at 30 ◦ C. To determine cells in suspension, inoculated
broth were serially diluted (10− 1 to 10− 3) after 24, 48 and 72 hrs of
incubation. Then 20 µL from the 10− 3 dilution factor was spread onto
YPDA plate and incubated at 30 ◦ C for 72 hrs.
Total flocculated cells were determined after overnight cold induced
3
Biotechnology Reports 40 (2023) e00815
lagering at the end of fermentation. Yeast cell mass was harvested by
centrifugation 16,000 rpm for 10 min, washed three times with physi­
ological saline and 1 g of washed cell mass of each strain was suspended
in 40 mL of saline and serially diluted (10− 1 to 10− 3). A 20 µL from the
10− 3 dilution factor was spread plated and incubated at 30 ◦ C for 72 hrs.
Total colonies were counted in both cases and flocculation percentage of
each isolate was determined by the following formula [57].
Total cell count BL − Total cell count AL
Floculation Percentage = X 100
Total cell count BL
(2)
Where, BL-Before laagering and AL-After laagering
2.5. Evaluation bioethanol production potential of selected isolates
2.5.1. Banana peel collection and processing
Banana peel waste was collected from fruit selling areas in Azezo
district, Gondar, Ethiopia. Collected peel waste was rinsed with tap
water to clean soil and associated impurities and sun dried. Following,
the dried peel waste was oven-dried at 60 ◦ C for 10 min and ground with
a grinder. The obtained powder waste was sieved, and the fine powder
was subjected to steam pretreatment [29].
2.5.2. Pretreatment and hydrolysis
Banana peel powder was steam pretreated and acid hydrolyzed ac­
cording to [1]. Eighty gram of peel powder was suspended in 1 L of
distilled water in screw caped bottles and autoclaved at 121 ◦ C and 15
psi pressure for 30 min. Then, pretreated peel powder was acid hydro­
lyzed with 1.5 % (v/v) sulfuric acid and heated at 91 ◦ C for 21 min. The
obtained hydrolysate was filtered with Whatman Grade 1 filter paper
(11 μm) and adjusted to pH 4.5 (optimum pH) with 1 M NaOH.
2.5.3. Banana peel chemical characterization
The pretreated banana peel sample was dried in hot air oven at 72 ◦ C.
The dried sample was then pulverized to a particle size smaller than 1
mm in a mechanical grinder [58]. Banana peel cellulose, hemicellulose,
soluble lignin and pectin content was analyzed following the protocols
of [59]. Total sugar and reduced sugar amounts were quantified by the
sulfuric phenol method and the 3–5 dinitrosalicylic acid (DNS) method
according to [18,55], respectively. The ash content was determined by
the Association of Official Analytical Chemists (AOAC) method 2000
[60]. This assay was conducted in triplicate.
2.5.4. Fermentation and determination of ethanol content
A batch fermentation system was employed, and the obtained hy­
drolysate was directly used for fermentation. Fermentation was done in
300 mL capped glass bottles containing 250 mL hydrolysate. Each bottle
was inoculated with a 2 % (v/v) 24 hrs old culture of each isolate (108
CFU/mL initial cell density). Inoculated fermentation bottles were
sealed with parafilm (Bemis, USA) to ensure anaerobic condition, and
incubated in CO2 incubator at 30 ◦ C for 72 hrs under shacking condition
at 150 rpm [61]. The supernatant was separated from yeast cell mass
and insoluble solid mater via centrifugation at 4000 rpm for 10 min.
Then the produced ethanol was separated from the aqueous solution at
78.4 ◦ C using fractional distillation, and its amount was estimated
colorimetrically using the potassium dichromate method [54]. Ethanol
standard curve was constructed using standard solutions of absolute
ethanol [62]. Unfermented residual sugar was estimated using the 3, 5-
dinitrosalicylic acid (DNS) method, according to [55]. Fermentation
parameters such as sugar utilization percentage, fermentation effi­
ciency, ethanol yield, and ethanol productivity were calculated ac­
cording to [63,64].
Sugar utilization% =
S1 − S2
X100 (3)
S1
D. Bitew et al. Biotechnology Reports 40 (2023) e00815

Glucose fermentation (mL)

4.5
4
3.5
3
2.5
2
1.5
1

NMKD2
BTD1
BTD4

NMTD2
NMTD5
NMTjD3
NMTjD4
BTjD1
AKD2
ATD1
ATD2

BTjD2

MABD1
MABD3

AMBD2
RTj1D5
RTj2D2
RTj2D4
GK1D5

BTrD1

DBTj1D1
BTrD3
GT1D1
GT1D2
GT1D3
GT1D4
GT3D2

GB2D5
GTj1D2
GTj1D3
GTj2D2
GTj2D3
GTj3D1
GTj3D2
GTj3D3
GTj3D5
GB2D3

DGTD2
DGTjD1
Fig. 1. Glucose fermentation performance of pre-selected yeasts isolates.

Where S1 is the initial sugar concentration in the hydrolysate and S2 is Data Sheet). PCR products were analyzed by loading 5 μL onto 2 %
the unconsumed residual sugar concentration in the fermented broth. agarose gels containing 3 μL ethidium bromide and visualized under UV
light.
Practical Yield
Fermentation efficiency = X100 (4)
Theortical Yield
2.6.3. Sequencing and phylogenetic tree construction
Where practical yield is the ethanol produced and theoretical yield is PCR products were purified and sequenced using standard
s 0.511 per gram of sugar consumed. sequencing. Sequenced data were edited using BioEdit software package
Ethanol Concentration (g/L) in fermented broth and checked for similarity via BLAST search program (https://10blast.
Practical ethanol yield = ncbi.nlm.nih.gov/Blast.cgi) to those previously deposited sequences
Sugar Consumed (g/l)
from GenBank databases. Species determination was done by consid­
(5)
ering identity percentage ≥ 99 %, E- value = 0 and query coverage ≥ 95
Ethanol Concentration (g/L) in fermented broth %. Finally, sequence of each isolate was submitted at GenBank of NCBI.
Ethanol Productivity =
Fermentation time (hrs) Then ITS sequences of isolates were aligned with the multiple alignment
program CLUSTAL W and neighbor-joining method was applied to
(6)

Ethanol Conc. (g/L) in fermented broth

Yield of ethanol (g/kg) of dry biomass = x1000 (7)
Dry weight of substrate biomass

The commercial strain, S. cerevisiae ©DB (Sc ©DB) obtained from construct a phylogenetic tree using MEGA software version 11.0.
Dashen Brewery Factory, Gondar, Ethiopia was used as a control. Ba­
nana peel hydrolysate fermentation and ethanol yield parameters assay
2.7. Statistical analysis
were conducted in triplicate.

Experimental data were generated in triplicate and analyzed using

2.6. Molecular identification of ethanologenic yeasts
2.6.1. DNA extraction
The genomic DNA of selected isolates was extracted using the Gen­
EluteTM Fungal/Plant Genomic DNA Miniprep Kit (Sigma Aldrich). The
concentration and quality of extracted DNA were determined using
NanoDrop and gel electrophoresis and kept at -20 ◦ C until it was needed
for PCR amplification.
2.6.2. Amplification of ITS region
The internal transcribed spacer regions (ITS 1 and ITS 2) of the rRNA
gene were used as a barcode and amplified using universal primers
ITS1F (5′-CGGGATCCGTAGGTGAACCTGCGG-3′) and ITS4R (5′-
CGGGATCCTCCGCTTATTGATATGC-3′) (Sigma Company). A 20 µL
containing (5 x FIREPol® Master Mix 4 μL, forward primer 0.3 μL (10
pmol/μL), reverse primer 0.3 μL (10 pmol/μL), 3 µL (30 ng), template
DNA, and 13.4 µL nuclease-free water. The PCR was run for about 30
cycles with initial denaturation at 95 ◦ C for 5 min, denaturation at 95 ◦ C
for 30 second, annealing temperature at 54 ◦ C for 30 second, extension
at 72 ◦ C for 2 min, and final extension 72 ◦ C for 7 min (Solis BioDyne
one-way ANOVA analysis using SPSS version 23. Tukey post hoc mul­
tiple comparison test was used for mean comparison. The significant
difference among variables was considered at p ≤ 0.05. The results are
expressed as mean ± standard deviation
3. Results and discussions
3.1. Isolation, screening and progressive selection of yeast isolates for
ethanol production
The shocking impacts of climate change and the lack of security in
fossil fuel supply have forced countries across the world to look for
environmentally friendly energy sources such as bioethanol [65].
However, its large scale high-quality production with reasonable costs is
hindered by a lack of suitable ethanologenic microbes [66,67]. This
urged searching for robust ethanologenic yeasts that are able to meet the
demands of the bioethanol industry [19]. Thus, in the present study, a
total of 102 yeast isolates were isolated from Ethiopian traditional fer­
mented beverages: tella, tej, korefe, areki tinsese, terahi, bubegne, shamita,
borde, and cheka. Colony morphological characterization revealed that

4
D. Bitew et al. Biotechnology Reports 40 (2023) e00815

Table 1
Ethanol tolerance and survival percentage of selected yeast isolates.
Isolate Tolerance to different ethanol concentrations Survival percentage of isolates
Glucose fermentation (mL) Growth (in terms of dry weight in mg)
10 15 20 10 15 20 10 15 20

GT1D3 2 ± 0.00b - - 81.0 ± 3.00b - – 41.6 ± 8.5c 6.1 ± 3.9b 1.6 ± 1.4b
GT1D4 2 ± 0.43b - - 75.3 ± 4.04b - – 50.3 ± 2.8d 5.9 ± 3.4b –
GT3D2 2.5 ± 0.50b - - 77.0 ± 2.65b - – 57.1 ± 4.3f 9 ± 0.8b 3.2 ± 1.1c
GTj1D3 1.8 ± 0.25b - - 79.3 ± 3.51b - – 50.5 ± 7.4d 7.4 ± 2.1b –
GTj2D2 1.9 ± 0.96b - - 75.3 ± 3.21b - – 65.6 ± 6.9e 11.5 ± 6.9c 2.8 ± 1.0c
AKD2 3.5 ± 0.50c - - 81.3 ± 2.51b - – 56.5 ± 11.0d 5 ± 2.1b 0.3 ± 0.6a
ATD2 3.4 ± 0.36c - - 78.0 ± 1.73b - – 40.7 ± 8.3c 4.2 ± 0.9a –
GK1D5 2.3 ± 1.15b - - 76.3 ± 1.53b - – 57.7 ± 1.8d 8.7 ± 4.0b 1.8 ± 0.4b
BTD1 4.0 ± 0.50c - - 76.7 ± 3.78b - – 56.6 ± 11.0d - –
BTrD1 2 ± 0.50b - - 78.0 ± 3.00b - – 31.2 ± 11.3b 9.5 ± 0.3b –
NMTD5 2.6 ± 0.51b - - 78.0 ± 2.64b - – 21.7 ± 5.0a - –
NMTjD4 2.3 ± 0.26b - - 76.3 ± 2.5b - – 27.3 ± 6.1b - –
MABD1 2.1 ± 0.17b - - 79.0 ± 1.73b - – 37.8 ± 8.3c 4.7 ± 2.8a –
RTj2D2 2.8 ± 1.10c - - 75.7 ± 5.13b - – 29.1 ± 5.7b - –
DGTjD1 3.5 ± 0.25c - - 76.7 ± 6.11b - – 31.9 ± 10.8b 8 ± 6.4b –
AMBD2 3.3 ± 0.26c - - 76.0 ± 4.00b - – 39.2 ± 5.5c 4.5 ± 2.7a –
Sc©DB 1.6 ± 0.40b – – 68.7 ± 4.72b – – 33.3 ± 5.5b –

Key: Mean values superscripted with different letters across the column are significantly different at P-value ≤ 0.05. (-) denotes no growth, fermentation and survival.

all are circular shaped and most are white colored with an entire margin
and a smooth texture. Morphologically distinct isolates from a given
fermented beverage were considered different yeast species and/or
strains and were designated differently (data not shown). This is in
agreement with several reports that states traditional fermented bever­
ages are an important niche for yeasts in general and ethanol producing
yeasts in particular [47,68,69].
3.1.1. Pre-selection of isolates
Glucose fermentation has been regarded as a confirmatory test for
ethanol production [41]. For that reason, glucose fermentation was used
as a pre-selection hurdle to exclude non-fermenters and weak fermen­
ters. Among 102 yeast isolates, 39 (38 %) formed gas that ranged be­
tween 3.7 ± 0.29 and 5 ± 0.00 mL after 48 hrs of incubation at 30 ◦ C
(Supplementary data (Sd) Table 5, Fig. 1). These were selected and
subsequently screened for pH and temperature requirement and
tolerance.
3.1.2. pH and temperature requirement and tolerance
The bioethanol production imposes stressful conditions on ethano­
logenic yeasts [70]. Hence, apart from fermentation efficacy and ethanol
productivity, tolerance to stressing factors such as pH and temperature
are recognized as desirable features of ethanologenic yeasts [33,71,72].
Thus, extensive screening of yeast isolates for these physiological
characteristics is considered as a pre-requisite so as to get an ideal
ethanologenic strain to be used in industrial scale bioethanol production
[73,74].
Yeast are acidophilus and they love the initial medium pH (4.5 − 5.5)
during ethanol production. However, the ultimate lowered pH as a result
of organic acid production imposes a deleterious effect on their viability
and fermentation performance [75,76]. Therefore, tolerance to low pH
is an essential feature of ethanol producing yeast [77]. Pre-selected
isolates were screened for their pH requirements and tolerance. They
were able to grow and ferment glucose in all tested pH values, with dry
weight and gas formed ranging from 52.3 ± 6.43 (at pH 6.5) to 95.7 ±
5.13 mg (at pH 4.5) and 0 (at pH 6.5) to 5.0 ± 0.0 mL (at pH 4.5),
respectively. Both fermentation and the growth of isolate decreased as
the acidity of the medium decreased. The maximum growth and
fermentation performance were recorded at pH 4.5, suggesting that this
is an optimum pH environment for these isolates (Sd Table 1 and 2). The
results of this study are in line with the fact that nearly all yeasts prefer
to grow and ferment in acidic conditions [78,79]. The considerable
growth and fermentation potential of pre-selected isolate at low pH (3
and 3.5) revealed that they are acidophilus; and this is critically
important in avoiding bacterial contamination during the ethanol
fermentation process.
Temperature is the key factor for fermentation [80]. Keeping the
optimum temperature during the fermentation process is a difficult task
in all bioethanol plants [81]. Deviating from the ideal temperature af­
fects the specific growth rate of yeast strains, which in turn affects
ethanol production [82,83]. Hence, screening yeast isolates for their
temperature requirement and tolerance is a basic component in the
search and development of ethanologenic yeasts. Results indicated that
the growth and fermentation responses of pre-selected yeast isolates
increased as the temperature increased to 35 ◦ C. However, their growth
was sharply declined at 40 ◦ C, while fermentation completely ceased.
This is consistent with [80] and [83,84], reported that yeast growth,
viability and the fermentation potential are repressed as the temperature
increases above 35 ◦ C. This decrease in viability and fermentation is
attributed to the synergetic inhibitory effect of high temperature and the
production of ethanol [85]. Maximum growth and fermentation per­
formance were recorded at 30 ◦ C. (Sd Table 3 and 4). This agrees with
[79,86,87], stated that most yeasts showed better growth and fermen­
tation performance at 30 ◦ C.
3.1.3. Carbohydrate fermentation and assimilation
Yeasts vary in their fermentation and assimilation of carbohydrate
molecules. A broader carbohydrate fermentation and assimilation
spectrum of ethanologenic yeast is crucial for the effective conversion of
various sugars in the biomass to ethanol [88]. Results of this study
showed that pre-selected isolates ferment glucose, sucrose, maltose,
fructose, mannose and galactose to different extents after 48 hrs of in­
cubation at 30 ◦ C. Regarding their carbohydrate assimilation spectrum,
they were able to grow on glucose, sucrose, maltose, galactose, fructose,
and mannose. However, none of them ferment and assimilate arabinose,
mannitol, lactose, xylose and starch (Sd Table 5 and 6). This is in line
with the reports of [41,42,47], investigated that most yeast isolates
ferment and assimilate glucose, sucrose, maltose, fructose and galactose.
In addition, [89] investigated that among 90 Saccharomyces species
tested, the majority of strains showed a very poor metabolic index for
arabinose and xylose. Carbohydrate fermentation and assimilation
profiles are a species-dependent properties; any variation in carbohy­
drate utilization may emanate from their taxonomic variation. Accord­
ing to their carbohydrate fermentation results, 30 better-performing
isolates were selected among 39 pre-selected isolates and further
screened. The carbohydrate fermentation and assimilation pattern of the
investigated isolates and the reference strain, Sc ©DB, were the same.
This might be due to they are belongs to the same taxonomic group.

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D. Bitew et al. Biotechnology Reports 40 (2023) e00815

Glucose utilization (%)

Ethanol concentration (g/L)
100 24

20
80

Ethanol concentration (g/L)

Glucose utilization (%)

16
60
12
40
8

20
4

0 0

Fig. 2. Ethanol production and glucose consumption performance of selected isolates.

3.1.4. Osmotic and ethanol tolerance evaluation of isolate
Ethanologenic yeasts always face osmotic stress as a result of high
sugar and solute concentrations, particularly during high-gravity
fermentation. Later, the accumulation of ethanol further imposes
stress and affects yeast viability and vitality [89]. In high-gravity
fermentation, a high ethanol yield is expected, however, the hyper­
osmotic condition imposes a deleterious effect on yeast proliferation and
viability. This eventually decreases sugar to ethanol conversion effi­
ciency and the final ethanol titer [72]. Hence, prevailing under high
osmotic pressure and ethanol concentration are other important features
of ethanologenic yeasts, particularly in the case of high-gravity bio­
ethanol fermentation [90–92]. Thus, screening yeast isolates for toler­
ance to the aforementioned stress factors is very critical to get efficient
ethanologenic strains. Pre-selected isolates were able to grow at each
glucose concentration with dry weight (mg) ranging from 3.7 ± 0.58 to
8.2 ± 0.68, 1.8 ± 0.29 to 4.7 ± 0.58 and 0.9 ± 0.10 to 2.7 ± 0.28
respectively (Sd Table 7). This agrees with [51], reported yeast strains
that are able to grow in sugar concentrations up to 80 % (w/w) sugar.
Since osmotic tolerance is a species-dependent feature, screening of
isolates at elevated concentrations makes the selection of tolerant iso­
lates easier. The tolerance of these isolates to high sugar concentration
showed that they are a potential candidate for high-gravity industrial
production of bioethanol.
Ethanol tolerance assessment revealed that among 16 selected iso­
lates 5 were highly tolerate, and 10 were moderately tolerant and 1 was
slightly tolerant to 10 % (v/v) ethanol concentration with survival

24 hrs 48 hrs 72 hrs

100

80
Floculation (%)

60

40

20

Fig. 3. Flocculation percentage of isolate after 24, 48 and 72 hrs of incubation.

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D. Bitew et al. Biotechnology Reports 40 (2023) e00815

Table 2 3.1.6. Flocculation property of selected isolates

The chemical composition banana peel powder.
Composition Amount ( % dry matter)
Cellulose 10.5 ± 1.2
Hemicellulose 8.2 ± 2.1
Lignin 4.6 ± 1.4
Total sugar 57.2 ± 5.7
Reduced sugar 46.2 ± 1.5
Pectin 5.2 ± 2.6
Ash 6.8 ± 2.3
Other solids 7.5 ± 1.2
percentages ranging from 21.7 ± 5.0 to 65.6 ± 6.9. But they were
slightly tolerant at 15 % and 20 % (v/v) ethanol concentration with an
insignificant survival rate (Table 1). Ethanol is toxic to the cell, and a
reduction in growth, fermentation performance, and survival percent­
age is expected when ethanol concentration increases. The findings of
this study are consistent with [41,83], who found that S. cerevisiae iso­
lates survived better at 10 % ethanol concentrations while their growth
declined when the ethanol concentration increased beyond 12 %. This is
also in line with [93], reported that about 86 %, 35 %, and 17 % of
isolates grow well in yeast malt extract medium (YM) containing 10 %,
12 %, and 14 % (v/v) ethanol respectively. Differently, the study of [41]
stated that seventeen isolates, isolated from bio-wastes and co-products
of sugar factories were highly tolerant to16 % ethanol concentration and
one was exceptionally tolerant to 20 % ethanol. The discrepancy in
ethanol tolerance might be due to variation in species and/or strains,
genetic makeup and sources they are isolated from.
3.1.5. Ethanol productivity screening
Screening for ethanol production demonstrated that all selected
isolates produce a considerable amount of ethanol with significant
glucose consumption. The obtained ethanol yield ranged from 15.3 to
20.1 g/L, with the degree of glucose consumption varying between 73
and 100 % after 72 hrs of incubation at 30 ◦ C (Fig. 2). The results are in
line with the findings of [94], reported ethanol production ranging from
1.09 % (10.9 g/L) to 2.49 % (24.9 g/L) using YPD medium containing 2
% (w/w) glucose. [95] also reported that ethanol production ranged
from 12.99 ± 0.37 to 23.10 ± 0.25 g/L and 13.83 ± 0.34 to 25.94 ±
0.52 g/L by osmotolerant S. cerevisiae strains using YPD broth containing
5.5 % glucose and prepared by reverse osmosis water and sea water,
respectively.
Flocculence is another desired trait of ethanologenic yeasts [96]. It is
a spontaneous, reversible aggregation of single yeast cells into clumps
and subsequent sedimentation to the conical bottom of the fermenter.
This makes it easier to clarify yeasts from the medium at the end of bulk
fermentation and prepare for repitching. The timing of flocculation is
critically important, and it should be neither too early nor too late [97].
The early flocculating yeasts tend to settle and clump together very
quickly and fail to complete the fermentation of fermentable sugar,
which then results in low-quality bioethanol. Results of the present
study showed that all selected yeast isolates are bottom fermenters, with
flocculation percentages ranging from 53.6 ± 9.4 to 89.7 ± 1.5 %, 77.7
± 1.5 to 94.2 ± 2.8 % and 89.7 ± 4.0 to 98.6 ± 0.9 % after 24 hrs, 48 hrs
and 72 hrs incubation, respectively. Their flocculence increased as time
went on until the end of fermentation (Fig. 3). The results of the present
study were higher than the flocculation percentage of transgenic wine
yeast strains BM45-F5A and VIN13-F5A, which demonstrated 72.1 ±
3.9 % and 59.4 ± 2.7 % flocculation after 48 h of incubation at 30 ◦ C
[98]. The variance in flocculation percentage might be attributed to
differences in genetic makeup, particularly variation in
flocculin-encoding FLO genes, cell wall composition [99], and the
physicochemical environment [100].
3.2. Bioethanol production from banana peel
3.2.1. Banana peel characterization
As shown in Table 2 the chemical composition revealed that banana
peel waste is an important source of cellulose, hemicellulose, lignin and
total and reduced sugars that would be converted in to bioethanol. The
cellulose, hemicellulose, lignin, pectin and ash content detected in this
study agree with previous reports [1,27]. However, lower with the re­
ports of [58]. The amount of total sugar obtained in this study is also
lowered as compared with [25]. This might be attributed to variation in
banana variety and sccharification approach employed. The lowered
amount of cellulose, hemicellulose and lignin can be due to their
transformation into monomers, which in turn related to the effectiveness
of pretreatment and hydrolysis approach. The ash content is related to
mineral compositions. Studies reported that the ash content of banana
peel is ranged between 6.4 to 12 % for different banana variety [60,
101]. The investigated ash content is within the reported range.
3.2.2. Sugar utilization and ethanol production efficiency
Banana peel waste is rich in fermentable sugar and recognized as an
important feedstock for the production of bioethanol [102]. However,

Table 3
Sugar utilization and ethanol yield parameters of selected isolates.
Isolate RS (g/L) CS (g/L) SU% EC (g/L) YE (g/L) FE (%) EP (g/L/h) YoE (g/Kg)

GT1D3 14.0 ± 0.5a 32±0.5b 69.6 ± 1c 10.2 ± 0.5d 0.3 ± 0.0e 62±3.0h 0.1 ± 0.0f 127.8 ± 5.8b
GT1D4 13.6 ± 0.3a 32.4 ± 0.4b 70.5 ± 0.8c 11.4 ± 2.1d 0.4 ± 0.1e 69.2 ± 13.0f 0.2 ± 0.0 g 142.8 ± 26.1c
GT3 D2 13.9 ± 0.1a 32.1 ± 0.1b 69.8 ± 0.3c 9.1 ± 0.6d 0.3 ± 0.0e 54.8 ± 3.9e 0.1 ± 0.0f 113.8 ± 6.9a
GTj1D3 13.9 ± 0.4a 32.1 ± 0.4b 69.8 ± 0.9c 10.3 ± 1.8d 0.3 ± 0.1e 62.7 ± 10.4h 0.1 ± 0.0 g 128.3 ± 22.4b
GTj2D2 13.9 ± 0.3a 32±0.3b 69.7 ± 0.6c 11.4 ± 1.4d 0.4 ± 0.0e 69.3 ± 9.0f 0.2 ± 0.0 g 141.9 ± 17.3c
ATD2 14±0.4a 32±0.4b 70.4 ± 0.8c 9.7 ± 1.1d 0.3 ± 0.0e 59.5 ± 6.9h 0.1 ± 0.0f 121.5 ± 13.2b
AKD2 14±0.2a 32±0.2b 69.6 ± 0.4c 11±1.2d 0.3 ± 0.0e 64.9 ± 10.7h 0.2 ± 0.0 g 137.1 ± 15.5c
GK1D5 13.7 ± 0.2a 32±0.2b 70.1 ± 0.5c 11.6 ± 1.0d 0.4 ± 0.0e 70.6 ± 6.8f 0.2 ± 0.0 g 144.8 ± 12.4c
NMTD5 14±0.4a 32±0.4b 69.7 ± 1.0c 10.6 ± 2.3d 0.3 ± 0.1e 64.6 ± 14.1h 0.1 ± 0.0f 132.5 ± 29.3c
NMTjD4 13.8 ± 0.2a 32.2 ± 0.2b 70±0.5c 11.5 ± 0.9d 0.3 ± 0.0e 69.2 ± 6.2f 0.2 ± 0.0 g 143.2 ± 10.9c
BTD1 13.8 ± 0.4a 32.2 ± 0.4b 70±0.9c 11.8 ± 1.6d 0.4 ± 0.1e 71.7 ± 11.1f 0.2 ± 0.0 g 147±20.3c
BTrD1 13.7 ± 0.2a 32.2 ± 0.3b 70.1 ± 0.5c 11.1 ± 1.4d 0.3 ± 0.0e 67.2 ± 8.2f 0.2 ± 0.0 g 138.7 ± 17.9c
MABD1 13.8 ± 0.3a 32.2 ± 0.2b 70±0.6c 11.6 ± 2.5d 0.4 ± 0.1e 70.4 ± 15.3f 0.2 ± 0.0 g 145±30.9c
RTj2D2 13.8 ± 0.3a 32.2 ± 0.3b 70.1 ± 0.7c 12.7 ± 0.5d 0.4 ± 0.0e 75.7 ± 6.1 g 0.2 ± 0.0 g 159±6.8d
DGTjD1 13.9 ± 0.4a 32.1 ± 0.4b 69.8 ± 1.0c 11±2.0d 0.3 ± 0.0e 67.2 ± 12.7f 0.2 ± 0.0 g 138.4 ± 25.0c
AMBD1 13.6 ± 0.4a 32.4 ± 0.4b 70.5 ± 0.9c 12.9 ± 1.3d 0.4 ± 0.0e 77.6 ± 6.0 g 0.2 ± 0.0 g 161.4 ± 15.8d
Sc ©DB 8.8 ± 1.8b 37.2 ± 1.8a 80.8 ± 4.0d 17.2 ± 1.7f 0.5 ± 0.0e 90.0 ± 5.2k 0.2 ± 0.0 g 214.9 ± 21.3f

Key: Mean values superscripted with different letters across the column are significantly different at P-value 0.05. RS (Residual Sugar), CS (Consumed Sugar), SU%
(Sugar Utilization Percentage), EC (Ethanol Concentration), YE (Ethanol Yield), FE% (Fermentation Efficiency), EP (Ethanol productivity) and YoE (Yield of Ethanol
recovered from Kg of dry substrate).

7
D. Bitew et al.
Fig. 4. Phylogenetic tree of investigated ethanologenic S. cerevisiae strains (accession numbers from OQ085077-OQ085092) and selected closely related strains
scored ≥ 99 % identity similarity retrieved from NCBI database.
the amount of bioethanol produced depends on the amount of
fermentable sugar extracted, which in turn depends on the type of
sccharification method employed [27]. Acid hydrolysis is much faster
than enzymatic hydrolysis, and greater than 90 % fermentable sugar
yield can be achieved with concentrated acid hydrolysis with fewer toxic
degradation products than with dilute acid hydrolysis [103].
In this study, 0.57 g/g of dry banana peel powder equivalent to 46.2
± 1.5 g/L of reduced sugar, was liberated from 80 g banana peel powder
after steam pretreatment and acid treatment with 1.5 % (v/v) sulfuric
acid at 90 ◦ C for 20 min. This is low as compared with [104] obtained
45.088 g/L reduced sugar from 20 g of banana peel powder pretreated
and hydrolyzed under the same conditions. It is also lowered from [105],
who achieved 77.03 g/L of reduced sugar after acid hydrolyzing 50 g of
matooke variety banana peel powder at 1.5 (v/v) sulfuric acid, 70 ◦ C,
and 40 min. The variation in the amount of reduced sugar extracted
might be due to variations in banana peel sugar content and scchar­
ification conditions. [106] stated that the sugar content of banana fruit
varies with banana variety, growth, and environmental conditions.
The hydrolysate was fermented in batch condition. The obtained
8
Biotechnology Reports 40 (2023) e00815
ethanol concentration ranged from 9.1 ± 0.6 to 17.2 ± 1.7 g/L, the
lowest and the highest were recorded from GT3D2 and Sc ©DB,
respectively. The sugar to ethanol conversion efficiency varied between
54.8 ± 3.9 to 90.0 ± 5.2 % with ethanol yield (YE) 0.3 ± 0.0 and 0.5 ±
0.0 g/L and productivity ranging from 0.1 ± 0.0 to 0.2 ± 0.0 g/L/h. The
ethanol recovery from a kilogram of dry banana peel powder was ranged
from 113.8 ± 6.9 to 214.9 ± 21.3 g/kg (Table 3). This was in agreement
with the study of [27] where the reported ethanol concentration ranged
between 6.7 and 26.0 g/l obtained from enzyme-hydrolyzed banana
peel. It is also consistent with [107] reported 1.05 % (10.5 g/L), 1.52 %
(15.2 g/L), and 1.70 % (17.0 g/L), respectively, obtained from Raja
banana peel, Agung banana peel, and Nangka banana peel. But it is low
as compared with [108], reported 13 g/L and 11 g/L were achieved
within 10–12 hrs in simultaneous sccharification and fermentation using
S. cerevisiae and Kluyveromyces marxianus at 35 and 41 ◦ C, respectively,
from 10 % (w/w) banana peel powder. This might be due to the isolates
fermentation and assimilation capacity of a variety of carbohydrate
molecules released during hydrolysis. Moreover, there can also be var­
iations in the sugar content of banana variety peels and cell sensitivity to
D. Bitew et al.
inhibitory compounds produced during acid hydrolysis. The recorded
ethanol yield parameters of the reference strain were significantly
different from the investigated isolates. This might be due to variation in
tolerance of inhibitory substance co-produced during hydrolysis.
3.3. Molecular identification
Selected stress-tolerant and bioethanol producing isolates were
identified using the ITS regions of the rRNA gene as a barcode. Species
level was assigned based on BLASTn identity score match ≥ 99 % against
sequences in the GenBank. Ethanologenic yeast were identified as
S. cerevisiae isolates. Then, the sequence of each isolate was deposited in
GenBank in the NCBI database, and accession numbers were obtained
(Fig. 4). So as to determine their phylogenetic position and infer their
evolutionary history, sequences of the current isolates and related
strains were retrieved from GenBank, aligned with ClustalW, and a
phylogenetic tree was constructed using MEGA software version 11.0
[109] (Fig. 4). The phylogenetic tree was constructed from the evolu­
tionary distance data calculated from the Jukes-Cantor model [110]
using the neighbor-joining method [111].
4. Conclusion
The results of the present study demonstrate that traditional fer­
mented beverages are an important source of ethanologenic yeasts.
Molecular identification revealed that all selected isolates belong to the
species S. cerevisiae. The screening of these S. cerevisiae strains for ideal
properties of ethanologenic yeast, such as the ability to ferment and
assimilate carbohydrate molecules, grow and survive under different
stress conditions, and produce ethanol from glucose evidenced that
these strains are applicable in bioethanol industries. Ethanol production
evaluation using banana peel as a substrate showed that all tested iso­
lates produced a considerable amount of ethanol, ranging from 9.1 ± 0.6
to 12.9 ± 1.3 g/L and sugar to ethanol conversion efficiency varied
between 54.8 ± 3.9 to 77.6 ± 6.0 %. This ensures that they are a good
candidate for ethanol production using lignocellulosic biomass as a
substrate. Study on optimization of the fermentation process is
recommended.
Authors contribution
Conception of the research idea, design of the study, acquisition,
analysis and interpretation of data and manuscript drafting were done
by DB. B.A. and A.T. have supervised the laboratory work, edit and
approve the manuscript to be submitted.
Funding source
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
This work was part of PhD dissertation sponsored by Ministry of
Education, Ethiopia. Authors acknowledged Institute of Biotechnology
9
Biotechnology Reports 40 (2023) e00815
and Department of Chemistry, University of Gondar, Ethiopia for sup­
porting all necessary materials during conducting this study.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.btre.2023.e00815.
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