Optimization of bioethanol production using stress-tolerant yeast strains isolated from household alcoholic beverages (Tella, Tej, and Areke) and molasses (as substrate)

Mulugeta Fentahun; Berhanu Andualem

doi:10.12688/f1000research.146910.2

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Research Article

Revised

Optimization of bioethanol production using stress-tolerant yeast strains isolated from household alcoholic beverages (Tella, Tej, and Areke) and molasses (as substrate)

[version 2; peer review: 1 approved, 1 not approved]

Mulugeta Fentahun ^1,2, Berhanu Andualem³

PUBLISHED 16 Dec 2024

Author details Author details

¹ Department of Biology, University of Gondar, Gondar, Amhara, Ethiopia
² Department of Biology, Debre Markos University, Debre Markos, Amhara, Ethiopia
³ Institute of Biotechnology, University of Gondar, Gondar, Amhara, Ethiopia

Mulugeta Fentahun
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing – Original Draft Preparation

Berhanu Andualem
Roles: Conceptualization, Project Administration, Supervision, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Energy gateway.

Abstract

Background

Yeast strains that are tolerant to several environmental stresses are extensively employed to produce bioethanol from sugar-rich feedstock. Bioethanol production efficiency in Metehara and Fincha Sugar Factories (the two major sugar factories in Ethiopia) is 50% lower than that of similar factories in other parts of the world.

Methods

In our previous research findings, five wild indigenous strains of multi-stress tolerance yeast were isolated from traditional fermented alcoholic beverages (Tella, Tej, and Areke) in Ethiopia to the purpose of evaluate their potential use for bioethanol using sugarcane molasses as a substrate.

Result

The optimal parameters for maximum ethanol production were pH of 4.5, 25°Brix, 30°C, 4.0 g/l ammonium sulfate, supplement with yeast extract, and 72 h of incubation period. Under these optimum conditions, 13.13 ± 0.08% (v/v) ethanol was obtained. The yield of hydrous and anhydrous bioethanol from fermented molasses samples were produced at 94.20–95.60% (v/v) and 99.05–99.56% (v/v), respectively.

Conclusion

According to this finding, Saccharomyces cerevisiae isolate MUT15, Saccharomyces cerevisiae isolate MUT18F, and Saccharomyces cerevisiae isolate R9MU strains have been good candidates for production of industrial bioethanol.

Keywords

Bioethanol, Fermentation, Molasses, Optimization, S. cerevisiae, Stress tolerance

Corresponding author: Mulugeta Fentahun

Competing interests: No competing interests were disclosed.

Grant information: This research was supported by University of Gonder and Debre markos University.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Fentahun M and Andualem B. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Fentahun M and Andualem B. Optimization of bioethanol production using stress-tolerant yeast strains isolated from household alcoholic beverages (Tella, Tej, and Areke) and molasses (as substrate) [version 2; peer review: 1 approved, 1 not approved]. F1000Research 2024, 13:286 (https://doi.org/10.12688/f1000research.146910.2) First published: 17 Apr 2024, 13:286 (https://doi.org/10.12688/f1000research.146910.1) Latest published: 16 Dec 2024, 13:286 (https://doi.org/10.12688/f1000research.146910.2)

Revised Amendments from Version 1

We have updated the references and grammatical corrections of the article. In addition to new information is added in response to the reviewer’s feedback and comments.

See the authors' detailed response to the review by Evika Sandi Savitri
See the authors' detailed response to the review by Boris U. Stambuk

1. Introduction

Due to resource depletion as the result of population growth and environmental pollution caused by fossil fuels, it is necessary to find out other eco-friendly renewable and sustainable sources of energy.¹ Liquid biofuels, which account for 40% of all energy sources worldwide, were prioritized among renewable energies.² The use of liquid biofuels helps in the reduction of greenhouse gas emissions, energy security, creation of job opportunities, regional development, rural development and reduction of poverty.³ Bioethanol is a promising alternative energy sources and a popular biofuel for transportation globally since it is a renewable, nontoxic, biodegradable resource and it is oxygenated.⁴ Production and use of bioethanol for transport fuel have recently attracted attention worldwide.⁵

Starch, cellulose, and sucrose can all be used for the production of bioethanol, which may be found in molasses, coconut, sugar palm, cassava, banana stems, sweet potatoes, sugarcane bagasse, sunflower, woodchips and most recently microalgae.⁶^,⁷ Among the different substrates available for production bioethanol sugarcane molasses is a good substrate because of cheap raw materials, readily available and compared to starchy or cellulosic materials for industrial bioethanol fermentation, ready for conversion with limited pretreatments.⁸ Molasses contains minerals, organic acids and high sugar content, which is 34-54%,⁹ especially the fructose content in molasses varies between 5-12%, sucrose 25-40%, and total reduced sugar 50-65%.¹⁰ Due to this, sugarcane molasses have gained major attention high prospects for making bioethanol.⁶^,¹¹

Several factors affect the process of fermentation, as well as the bioethanol yield such as the production microorganism strain, temperature, pH, oxygen, media composition, fermentation duration and initial concentration of sugar.¹² Therefore, it is important to use optimal values among these parameters, for each selected production microorganism, to be able to produce the highest amounts of the required product.¹³ The choice and development of the efficient bioethanol production of yeast with a resistance multi-stress tolerance are crucial importance.¹⁴ Yeasts strain tolerant to high temperature, low pH, osmotic pressure and ethanol are ideal for industrial bioethanol production.¹⁵

In Ethiopia, Metehara and Fincha Sugar Factories have been using sugarcane molasses as a substrate for the production of ethanol after diluting it to 12–15 °Brix. However, these factories are currently producing bioethanol with a yield of less than 7% (v/v), in contrast to in other countries that produce bioethanol with yields of 12–14% (v/v).¹⁶ This means is that the efficiency of Ethiopian Factories is 50% lower than that of factories in other parts of the world. This study intends to find out potential multi-stress-tolerant yeasts that could serve as a starter culture and substitute for commercial yeast strains. Therefore, the research objective to maximize production bioethanol from molasses using stress-tolerant yeast strains from household alcoholic beverage preparations (Tella, Tej, and Areke) under optimum conditions. Five yeast strains were used in this study, specifically Saccharomyces cerevisiae strains with accession numbers from the National Center for Biotechnology Information, such as strains R9MU (OR143320.1), strain R20MU (OR143322.1), strain MUT15F (OR209276.1), strain MUT18F (OR209286.1), and strain R19MU (OR143321.1). These strains of Saccharomyces cerevisiae were obtained in our stock cultures from the previous study because to their noteworthy tolerance to ethanol, 26% (v/v), glucose, 70% (w/v) and temperature, 45°C.¹⁷

2. Methods

2.1 Collection sugar cane molasses

The study was carried out at Department of Biology, University of Gondar, Northwestern Ethiopia. A sample of 70 liter of molasses was acquired from Ethiopia’s Methara Sugar Factory through September to January 2023. The molasses from sugarcane was collected in a clean, durable plastic container and kept at room temperature to the laboratory of microbiology at the University of Gondar for further use ( Figure 1).

Figure 1. Molasses sample.

2.2 Characterization of sugar cane molasses

The physicochemical parameters of sugarcane molasses, including pH, specific gravity, Brix, moisture content, sucrose, and sulfated ash were estimated according to Refs. 18, 19. The quantity of total sugar in molasses samples was determined through Fehling method.²⁰ The reducing sugar concentration in the molasses was determined by the 3,5-dinitrosalicylic acid (DNS) method.²¹ The moisture content molasses sample was determined thought oven (MB45, OHAUS, Switzerland) drying method. Sucrose content in the molasses sample was measured by measuring optical rotation in a polarimeter. A sample of molasses weighing around 10 g was placed in a beaker, and after adding water and 50 mL of Pb (NO₃)₂ solutions stirred to settle out the heavy portion of molasses. After filtering the slurry, the clear solution was added to the polarimeter and the sucrose levels were measured. Brix was measured with the help of a refractometer (Atagodensimeter model 2312; Atago Co. Ltd., Tokyo, Japan). Specific gravity of molasses samples were estimated by using baume universal hydrometer. The pH of the molasses samples were determined in the laboratory using pH meter (PHS-3C Digital) at ambient temperature. The ash content was established by muffle furnace at 650°C for 2 hours until constant weight.

2.3 Bioethanol production from molasses

2.3.1 Pretreatment of molasses

Distilled water was used to dilute raw molasses (1:1) w/w and pretreated with 99.8% sulfuric acid (H₂SO₄) until the pH reach 3.5 for the purpose of removing unwanted particles, dirt, and microbial contaminants.²² The mixture was heated to 90°C in a water bath (Gemmy industrial Corp, Taiwan) with continuous mixing for 30 minutes. Then it was allowed to stay for 24 h to cool and decant. The required Brix was achieved through the dilution of raw molasses with distilled water, and the medium was supplemented with 0.7 g/L ammonium sulfate (101217; MilliporeSigma) and then homogenized with a magnetic stirrer. Finally, the pH was adjusted at 4.5 using 0.5 M NaOH and autoclaved at 121°C for 15 min. The treated medium was used for the propagation of yeast strains and fermentation to produce ethanol.

2.4 Best selected yeast strain for production of bioethanol

Five native wild strains of Saccharomyces cerevisiae designated as R9MU (OR143320.1), R20MU (OR143322.1), MUT15F (OR209276.1), MUT18F (OR209286.1), and R19MU (OR143321.1) were selected in our stock cultures from the previous study. The capacity of these strains to ferment molasses was assessed, which were previously isolated from homemade alcoholic beverages (Tella, Tej, and Areke). Moreover, they were also selected for their remarkable tolerance because of their multi-stress tolerance yeast strains. The MT industrial yeast strain was used as a control for comparison with other strains.

2.5 Propagation of yeast strains

A loop full of 48 h old cultures has been taken on yeast extract peptone dextrose (YEPD) solid media and inoculated into each flask containing 100 ml of treated and sterilized molasses. The flasks were incubated for 24 hrs at 30°C. Then, to increase the cell number, propagated yeast cultures were transferred to the second stage of propagation in Erlenmeyer flasks of 500 mL containing 100 mL of 30 °Brix molasses medium supplemented with 0.7 g/L ammonium sulfate (101217; MilliporeSigma) and sterilized at 121°C for 15 min and incubated at 30°C for another 24 hrs. Then after, optical density of propagated cultures was estimated using a UV-Vis spectrometer at 600 nm (Abron ISO 9001:2008) ( Table 1). Finally, 48 h of propagated yeast culture which is ready as inoculum was transferred to 800 mL bottles containing 300 mL of sterilized and diluted molasses for optimization of feed-batch molasses fermentation to produce ethanol.²³

Table 1. Propagated yeast cells as indicated by OD (nm) for five strains (Initial OD reading was done immediately after inoculation, one OD =1x10⁸ CFU/ml).

Yeast strains	Initial (0 h) OD	Final (48 h) OD
Saccharomyces cerevisiae isolate R9MU	0.420 ± 0.03^b	1.817 ± 0.03^c
Saccharomyces cerevisiae isolate R20MU	0.645 ± 0.05^c	1.885 ± 0.23^ba
Saccharomyces cerevisiae isolate MUT15F	0.542 ± 0.03^a	1.846 ± 0.14^a
Saccharomyces cerevisiae isolate MUT18F	0.463 ± 0.13^ba	1.710 ± 0.33^b
Saccharomyces cerevisiae isolate R19MU	0.424 ± 0.20^b	1.892 ± 0.03^a
MT standard yeast	0.523 ± 0.10^a	1.744 ± 0.03^bc

2.6 Optimization of fermentation process parameters

The fed-batch fermentation system was used to optimize the process parameters, including molasses concentration (15, 20, 25, 30 and 35 °Brix), temperature (25, 30, 35, and 40°C), pH (3.5, 4.0, 4.5, 5.0, and 5.5), ammonium sulfate (101217; MilliporeSigma) supplement (1, 2, 3, 4 and 5 gL-1), different supplementation such as, yeast extract (VWR; 97063-370), urea (Koch Fertilizer, LLC), and ammonium nitrate (EM1.01187.5000; MilliporeSigma), and incubation periods (24, 48, 72, 96, and 120 h) by applying the one variable at a time method (VAT).²⁴ Eight hundred milliliter bottles containing 300 ml of sterilized, diluted molasses were inoculated with 48 h of propagated yeast cells. The inoculum of each yeast strain was prepared by growing it in 200 ml of molasses medium for 48 h at 30°C. The inoculated bottles were plugged with sterilized cotton and incubated for 72 h in an incubator (J.P. Selecta Incubator, Spain). Samples were withdrawn after 72 h and analyzed for ethanol content. All treatments were studied in triplicate, and the mean and standard error were calculated.

2.6.1 Effect of substrate concentration

The yeast strains were grown at varying molasses medium concentrations (15, 20, 25, 30, and 35 °Brix), which is an optimum operating condition at 30°C to investigate the effects of substrate concentration on ethanol production. The pH was adjusted to 4.5 before inoculation of all strains, and incubated for 72 h. The concentration of ethanol (v/v %) was determined at the end of the fermentation period for each set of concentrations. The amount of ethanol (v/v %) was measured at the end of the fermentation time.

2.6.2 Effect of temperature

Each yeast strain was grown on a molasses medium that was diluted by 30% and incubated at various temperatures (25, 30, 35, and 40°C). All yeast strains were incubated for 72 h at different temperatures, and the initial pH was adjusted to 4.5. The concentration of ethanol (v/v %) was determined at the end of the fermentation period at various temperatures.

2.6.3 Effect of pH

The effect of different pH (3.5, 4.0, 4.5, 5.0, and 5.5) on ethanol production from diluted molasses (30%) by the yeast strains was evaluated at 30°C for 72 hrs. The pH was adjusted using 1N HCl or 0.1N NaOH. At the end of the fermentation time the percentage of ethanol was measured.

2.6.4 Effect of ammonium sulfate supplement

The effects of supplement concentrations of ammonium sulfate (101217; MilliporeSigma) (1, 2, 3, 4, and 5 g/l) on ethanol production by each yeast strain were determined. All yeast strains were grown on a 30% molasses with an initial pH of 4.0 for 72 h at 30°C. At the end of the fermentation period, the percentage of ethanol concentration for each supplement concentration was measured.

2.6.5 Effect of different supplements

The effects of different supplements such as, 1 g/l of yeast extract (VWR; 97063-370), urea (Koch Fertilizer, LLC), and ammonium nitrate (EM1.01187.5000; MilliporeSigma) on ethanol production by each yeast strain were conducted. All yeast strains were grown on a 30% molasses dilution with an initial pH of 4.5 for 72 h at 30°C. The effect of these supplements on ethanol concentration was evaluated after measuring the ethanol content of the fermented broth.

2.6.6 Effect of fermentation period

The yeast strains were cultured on 30% molasses and incubated at 30°C for various fermentation times (24, 48, 72, 96, and 120 h). At the end of the fermentation period, the amount of ethanol in each fermentation period was measured as a percentage.

2.7 Analytical methods

The Brix of fermented samples was measured using a refractometer (Atagodensimeter model 2312; Atago Co. Ltd., Tokyo, Japan). The optical density of propagated cultures was determined using a UV-Vis spectrometer at 600 nm (Abron ISO 9001:2008). The ethanol level of fermented molasses was determined by the measurement of specific gravity using a Baume universal hydrometer.²⁵ The levels of ethanol produced from the distillate were measured using an alcoholmeter. The calculation of ethanol concentration (ABV%) and bioethanol yield were calculated according to the Refs. 25 and 26 equation, respectively.

(1)

ABV = (SGin - SG) \times 131.25

Where ABV is the alcohol by volume (%), SGin is the liquid’s initial specific gravity before to the addition of yeast, and SG is the liquid’s current specific gravity.

(2)

The distillation yield (%) = \frac{Total volume of distilled sample}{Initial sample volume} \times 100

2.8 Distillation of Fermented molasses

Bioethanol production was performed by scaling up the process in 3 L fermenter that each containing 2 L of pretreated molasses at a 30% molasses with an initial pH of 4.5 in anaerobic conditions at 30°C for 72 h. After preparing a loop full of 48-hour-old cultures on YEPD solid media, 500 ml of molasses medium was inoculated for 48 hrs at 30°C. Using a fractional distillation apparatus, bioethanol was extracted from 2.5 L of fermented molasses in 3 L Erlenmeyer flasks.

The distillation flask (Pyrex, 500 ml, England) was filled with 400 ml of the fermented sample and placed on the machine’s heating unit with the water flow connected. The temperature was maintained manually at 78°C (the evaporation temperature of ethanol). The vapor was passed over a fractional column and the bulb of the thermometer, at which point the vapor was determined. The vapor was condensed to a liquid in the horizontal condenser, which was cooled with a flow of cold water. The distillate was collected in a receiver. The distillate was measured for volume. Repeated distillations were carried out until 90% of the bioethanol recovered fulfilled American Standard and Testing Material (ASTM) requirements for fuel.²⁷ Furthermore, the resulting bioethanol was analyzed to determine the yield of the bioethanol. The levels of bioethanol produced from the molasses fermentation and distillation processes were measured using an alcoholmeter. A cylinder was filled with enough fresh molasses samples, and an alcohol meter was held by the tip, and placed into the molasses. The alcohol meter was left to float on its own until it reached equilibrium. Then the reading at the bottom of the meniscus was recorded.

2.9 Pre-concentration of the bioethanol in continuous distillation system

The initial concentrations of the bioethanol used in the first phase were ranged from 38.70 to 44.62% (v/v). Using a distillation column, the first bioethanol-diluted broth was pre-concentrated to a concentration that was nearly 95% (v/v) of the azeotropic point. During experimentation, samples were frequently taken out, and ethanol content was measured with an alcoholmeter. Near the azeotropic point, a distillate composition of 92.75% w/w was set,²⁸ with a 99.5% w/w recovery of the ethanol feed.

2.10 Dehydration of ethanol using 3A molecular sieve bead

The recovered bioethanol from the pre-concentration process, at a concentration close to 95% (v/v), was used in this study. Dehydrating 3A molecular sieve beads were used in this investigation. Molecular sieve beads were acquired from the Methara Sugar Factory in Ethiopia. The molecular sieves were dried in an oven (MB45, OHAUS, Switzerland) at 190–210°C for 24 h. The dried molecular sieves were kept in bottles, which were then stored in a glass chamber. Samples of distillate were dried using 3A molecular sieves overnight to capture water molecules. After being filtered, decanted, and redistilled to remove sieve dust and achieve anhydrous bio ethanol with less than 1% water.²⁹

2.11 Data analysis

All Data was analyzed using Statistical Package for Social Sciences software, version 23.0 (IBM SPSSInc., Chicago, IL, SPSS (RRID:SCR_002865), https://www.ibm.com/support/pages/downloading-ibm-spss-statistics-23). Analysis of variance (ANOVA) was conducted to compute the means and standard deviations of the triplicates analysis, and Tukey’s multiple range testing was used to assess if there were any significant differences between the means (p < 0. 05). The statistically significant difference was defined as p < 0. 05.

3. Results

The process parameters such as molasses concentration (15, 20, 25, 30, and 35 °Brix), temperature (25, 30, 35, and 40°C), pH (3.5, 4.0, 4.5, 5.0, and 5.5), other different ammonium sulfate supplement (1, 2, 3, 4 and 5 g L^-1), different supplementation (yeast extract, urea and ammonium nitrate), and period of incubation (24, 48, 72, 96, and 120 h) were all optimized using fed-batch fermentation ( Figure 2). In fed-batch fermentation using a 200 ml treated molasses sample as the inoculum, yeast cell propagation was accomplished with a cell count of 1.710 – 1.892 × 10⁸ cells per ml after 48 h ( Table 1).

Figure 2. Molasses fermentation.

The physicochemical parameters of sugarcane molasses from the Methara Sugar Factory in Ethiopia are presented in Table 2. Molasses was composed of 84.00 ± 1.53° Brix, 50.35 ± 0.12% total reducing sugars, 33.31 ± 1.94% sucrose, 14.32 ± 0.65% reduced sugars, 19.30 ± 0.26% water content, and 17.01 ± 0.69% sulfated ash (w/v). Moreover, the molasses pH was 5.72 ± 0.09.

Table 2. The physicochemical parameters of sugarcane molasses from Methara Sugar Factory, Ethiopia.

Physicochemical composition molasses
Parameter	Methara - Mean ± SE (w/v)
Brix	84.00 ± 1.53°
PH	5.72 ± 0.09
Specific Gravity	1.34 ± 0.02
Total reduced sugar	50.35 ± 0.12%
Sucrose	33.31 ±1.94%
Reducing sugar	14.32 ± 0.65%
Water content	19.30 ± 0.26%
Sulphated Ash	17.01 ± 0.69%

3.1 Effect of molasses concentration, temperature, and pH on ethanol concentration

As shown in Table 3, various molasses percentages (Brix of 15%, 20%, 25%, 30%, and 35%) were utilized in fed batch fermentation to examine the optimization of ethanol concentration at 30°C and a pH of 4.5. Based on the present study, the five yeast strains from molasses with a Brix of 15–35 (^oB) were produced ethanol in amounts ranging from 7.80 to 12.47% (v/v) of the concentration of ethanol. The standard industrial yeast strain was used as a control for comparison with other strains. Saccharomyces cerevisiae isolate MUT15F, 12.47 ± 0.07% (v/v) and Saccharomyces cerevisiae isolate R9MU, 11.81 ± 0.16% (v/v) have been shown statistically (p ≤ 0.05) greater ethanol concentrations than the remaining strains. Production of ethanol in all yeast strains was significantly (p ≤ 0.05) reduced in either low concentration (15–20%) or high concentration (30–35%). Higher or lower concentrations of molasses have a significant negative effect on ethanol production. These findings have been shown that the optimal molasses concentration for all yeast strains to produce ethanol was 25% ( Table 3).

Table 3. Effect of different molasses concentration, temperature and pH in ethanol production process at optimum temperature 30°C, 30 °Brix, and pH 4.5 after 72 h in fed batch culture.

	Molasses concentration (°Brix)
Yeast strain	15 °Brix	20 °Brix	25 °Brix	30 °Brix	35 °Brix
Saccharomyces cerevisiae isolate R9MU	8.66 ± 0.06^a	10.63 ± 0.18^ad	11.81 ± 0.16^b	11.15 ± 0.08^b	10.13 ± 0.08^b
Saccharomyces cerevisiae isolate R20MU	8.27 ± 0.02^ad	9.84 ± 0.04^b	10.76 ± 0.04^ad	10.24 ± 0.08^a	9.19 ± 0.01^a
Saccharomyces cerevisiae isolate MUT15F	9.19 ± 0.04^b	11.16 ± 0.28^d	12.47 ± 0.07^c	11.94 ± 0.06^c	10.64 ± 0.08^c
Saccharomyces cerevisiae isolate MUT18F	7.79 ± 0.09^c	9.19 ± 0.08^c	10.50 ± 0.17^a	9.84 ± 0.16^d	7.88 ± 0.08^d
Saccharomyces cerevisiae isolate R19MU	8.14 ± 0.08^d	10.50 ± 0.46^a	11.29 ± 0.11^bd	10.50 ± 0.09^a	9.19 ± 0.1^a
MT standard yeast	8.53 ± 0.11^a	10.37 ± 0.03^ab	11.16 ± 0.04^d	10.11 ± 0.12^a	8.27 ± 0.04^f

	Incubation temperature
Yeast strain	25°C	30°C	35°C	40°C
Saccharomyces cerevisiae isolate R9MU	9.40 ± 0.06^b	11.15 ± 0.08^b	10.70 ± 0.10^b	9.90 ± 0.10^a
Saccharomyces cerevisiae isolate R20MU	8.70 ± 0.13^a	10.24 ± 0.08^a	9.03 ± 0.29^a	8.60 ± 0.07^b
Saccharomyces cerevisiae isolate MUT15F	9.98 ± 0.11^c	11.94 ± 0.06^c	11.11 ± 0.05^b	10.20 ± 0.1^a
Saccharomyces cerevisiae isolate MUT18F	8.22 ± 0.05^a	9.84 ± 0.16^d	9.23 ± 0.05^a	8.78 ± 0.09^bc
Saccharomyces cerevisiae isolate R19MU	8.52 ± 0.04^a	10.50 ± 0.09^a	9.60 ± 0.05^a	9.12 ± 0.02^c
MT standard yeast	8.75 ± 0.08^a	10.11 ± 0.12^a	9.40 ± 0.03^a	8.50 ± 0.06^b

	pH
Yeast strain	pH 3.5	pH 4	pH 4.5	pH 5	pH 5.5
Saccharomyces cerevisiae isolate R9MU	6.54 ± 0.06^ac	8.67 ± 0.1^ac	11.15 ± 0.08^b	10.50 ± 0.29^b	9.90 ± 0.07^b
Saccharomyces cerevisiae isolate R20MU	6.21 ± 0.11^c	8.54 ± 0.05^b	10.24 ± 0.08^a	9.40 ± 0.03^a	8.09 ± 0.11^c
Saccharomyces cerevisiae isolate MUT15F	7.40 ± 0.06^b	9.08 ± 0.06^c	11.94 ± 0.06^c	11.19 ± 0.06^c	10.45 ± 0.09^b
Saccharomyces cerevisiae isolate MUT18F	6.08 ± 0.02^c	8.32 ± 0.13^ad	9.84 ± 0.16^d	9.03 ± 0.13^a	8.65 ± 0.12^ca
Saccharomyces cerevisiae isolate R19MU	6.50 ± 0.11^ca	8.34 ± 0.12^b	10.50 ± 0.09^a	9.09 ± 0.08^a	8.00 ± 0.14^c
MT standard yeast	6.90 ± 0.18^a	8.13 ± 0.06^d	10.11 ± 0.12^a	9.60 ± 0.12^a	8.88 ± 0.16^a

The effect of temperature at 25, 30, 35, and 40°C in the production process of ethanol was also investigated. The optimum ethanol production was 30°C as shown in Table 3. A subsequent increase in temperature reduced the production of ethanol. According to this finding, Saccharomyces cerevisiae isolate MUT15F, 11.94 ± 0.06% (v/v) and Saccharomyces cerevisiae isolate R9MU, 11.15 ± 0.08% (v/v) were statistically (p ≤ 0.05) greater in ethanol concentration than the other strains, which was included strain MT (the commercial strains) at 30°C after 72 h of the fermentation period. Saccharomyces cerevisiae isolate MUT18F has been shown a statistically lower (p ≤ 0.05) amount of ethanol, 9.84 ± 0.16% (v/v) at 30°C than the other yeast strains. There was no statistically significant (p ≥ 0.05) difference between strains of Saccharomyces cerevisiae R19MU, Saccharomyces cerevisiae R20MU, and MT (standard) at 30°C. Below 25°C or above 30°C, the production of ethanol by all yeast strains was significantly (p ≤ 0.05) lower. According to this finding, the optimum incubation temperature for all yeast strains was 30°C.

The effect of different pH values on ethanol productions is shown in Table 3. The ethanol production gradually increases along with the increase in pH and reaches a maximum at a pH 4.5. The production was decreased slightly for pH values higher than 5. The yeast strains had shown a different pattern of ethanol production of 6.08–11.94% (v/v) from pH 3.5 to pH 5.5. Based on the present study, ethanol produced by yeast strain Saccharomyces cerevisiae isolate MUT15F, 11.94 ± 0.06% (v/v) and Saccharomyces cerevisiae isolate R9MU, 11.15 ± 0.08% (v/v) were shown a statistically significant (p ≤ 0.05) higher than the production by all the remaining strains at pH 4.5. Saccharomyces cerevisiae isolate R20MU has been shown statistically (p ≤ 0.05) lower ethanol concentration than all other strains. There was no statistically significant (p ≥ 0.05) difference between strains Saccharomyces cerevisiae isolate R19MU, Saccharomyces cerevisiae isolate R20MU, and the MT (standard) at pH 4.5. All the strains were more effective to produce high amount of ethanol at pH 4.5 and lower ethanol produced at pH 3.5 ( Table 3).

3.2 The effect of supplement concentration, different supplement, and incubation periods on ethanol concentration

In Table 4, the effect of ammonium sulfate as a nitrogen source is shown. Under optimal conditions, 4.0 g/l of ammonium sulfate produced the highest amount of ethanol. As the ammonium sulfate concentration increased from 1.0 to 4.0 g/l, the production ethanol also significantly (p ≤ 0.05) increased and then reduced in amount as concentration of ammonium sulfate increased. In this study, the amount of ethanol production by Saccharomyces cerevisiae isolate MUT15F was increased from 11.95 ± 0.1 to 13.13 ± 0.08% (v/v) between 1.0 and 4.0 g/l ammonium sulfate. According to this finding, Saccharomyces cerevisiae isolate MUT15F (13.13 ± 0.08% (v/v) has been shown statistically (p ≤ 0.05) greater ethanol production than that of the other strains including strain MT (the commercial strains). Saccharomyces cerevisiae isolate MUT18F was shown significantly (p ≥ 0.05) less ethanol concentration, (10.76 ± 0.03% (v/v)) at 4.0 g/l ammonium sulfate than all other strains.

Table 4. The effect of supplement concentration, different supplement, and incubation periods in ethanol production process at optimum temperature 30°C, 30 ^oBrix, and pH 4.5 after 72 h in fed batch culture.

	Ammonium sulphate (NH₄SO₄) concentration
Yeast strain	1g NH₄SO₄	2g NH₄SO₄	3g NH₄SO₄	4g NH₄SO₄	5g NH₄SO₄
Saccharomyces cerevisiae isolate R9MU	11.17 ± 0.08^b	11.45 ± 0.16^b	11.93 ± 0.07^b	12.39 ± 0.22^a	12.10 ± 0.12^b
Saccharomyces cerevisiae isolate R20MU	10.24 ± 0.08^a	10.47 ± 0.04^a	10.79 ± 0.05^ad	11.36 ± 0.21^b	10.85 ± 0.03^ad
Saccharomyces cerevisiae isolate MUT15F	11.95 ± 0.1^c	12.43 ± 0.19^c	12.70 ± 0.12^c	13.13 ± 0.08^c	12.84 ± 0.10^c
Saccharomyces cerevisiae isolate MUT18F	9.84 ± 0.02^a	10.06 ± 0.21^a	10.50 ± 0.12^d	10.76 ± 0.03^b	10.60 ± 0.06^d
Saccharomyces cerevisiae isolate R19MU	10.56 ± 0.12^da	10.90 ± 0.06^ab	11.22 ± 0.13^a	12.34± 0.17^a	11.26 ± 0.15^a
MT standard yeast	10.17 ± 0.12^a	10.38 ± 0.22^a	10.86 ± 0.03^ad	12.21± 0.12^a	11.34 ± 0.06^fa

	Different nitrogen sources
Yeast strain	Yeast extract	Urea	NH₄NO₃	Control
Saccharomyces cerevisiae isolate R9MU	11.60 ± 0.06^b	11.05 ± 0.06^a	10.40 ± 0.23^a	9.85 ± 0.1b^a
Saccharomyces cerevisiae isolate R20MU	10.43 ± 0.12f	10.13 ± 0.06^ac	10.02 ± 0.02^ad	9.51 ± 0.07^bad
Saccharomyces cerevisiae isolate MUT15F	12.24 ± 0.08^c	11.91 ± 0.12^b	11.34 ± 0.03^b	10.43 ± 0.48^a
Saccharomyces cerevisiae isolate MUT18F	9.95 ± 0.09^d	9.67 ± 0.02^c	9.48 ± 0.09^c	8.22 ± 0.13^c
Saccharomyces cerevisiae isolate R19MU	10.83 ± 0.02^af	10.56 ± 0.32^ad	9.77 ± 0.07^cd	9.25 ± 0.16^bc
MT standard yeast	10.89 ± 0.12^a	10.21 ± 0.08^cd	9.88 ± 0.04^cd	8.53 ± 0.12^cd

	Fermentation time
Yeast strain	24 h	48 h	72 h	96 h	120 h
Saccharomyces cerevisiae isolate R9MU	7.90 ± 0.03^b	8.90 ± 0.05^ac	11.15 ± 0.08^b	10.90 ± 0.03^b	10.05 ± 0.16^a
Saccharomyces cerevisiae isolate R20MU	8.90 ± 0.03^c	9.43 ± 0.12^ab	10.24 ± 0.08^a	9.03 ± 0.30a	8.60 ± 0.03^b
Saccharomyces cerevisiae isolate MUT15F	8.52 ± 0.12^a	9.89 ± 0.05^b	11.94 ± 0.06^c	12.02 ± 0.13^c	11.34 ± 0.09^c
Saccharomyces cerevisiae isolate MUT18F	6.56 ± 0.07^d	8.67 ± 0.07^cd	9.84 ± 0.16^d	9.63 ± 0.02^ad	8.55 ± 0.12^b
Saccharomyces cerevisiae isolate R19MU	7.21 ± 0.05^f	9.23 ± 0.09^ad	10.50 ± 0.09^a	10.77 ± 0.04^b	10.62 ± 0.13^d
MT standard yeast	7.63 ± 0.09^b	8.76 ± 0.17^cd	10.11 ± 0.12^a	9.85 ± 0.16^d	8.59 ± 0.1^b

Ammonium nitrate, urea, and yeast extract are different nitrogen sources and their effects were studied at a concentration of 1 g supplementation per liter, as presented in Table 4. Yeast extract supplement was produced high levels of ethanol than other nitrogen sources and the control. Compared to the other supplemented media, an ammonium nitrate-supplemented medium was shown the lowest level of ethanol. Saccharomyces cerevisiae isolate MUT15F (12.24 ± 0.08% (v/v)) and Saccharomyces cerevisiae isolate R9MU (11.60 ± 0.06% (v/v)) were statistically (p ≤ 0.05) greater in ethanol production from yeast extract supplementation than stains MT (the commercial strains) (10.89 ± 0.12% (v/v)) and Saccharomyces cerevisiae isolate R19MU (10.83 ± 0.02% (v)). The lowest ethanol concentration was observed (10.43 ± 0.12% (v/v)) by strains Saccharomyces cerevisiae isolate R20MU and Saccharomyces cerevisiae isolate MUT18F (9.95 ± 0.09% v/v) from supplementation of yeast extract compared to the rest of the yeast strains. The maximum ethanol concentration was observed (11.95 ± 0.1% (v/v)) by Saccharomyces cerevisiae isolate MUT15F and Saccharomyces cerevisiae isolate R9MU (11.17 ± 0.08% (v/v)) from the supplementation of urea than the rest of the yeast strains. All yeast strains grown without nitrogen sources (control) had the lowest ethanol concentration in comparison to other supplemented media in this study.

Incubation periods effect on ethanol production after fermentation processes were carried out for 24, 48, 72, 96, and 120 h is presented in Table 4. Ethanol production increased during incubation periods up to 72 h and then decreased again to reach the minimum levels at 120 h of incubation. After 72 h of fermentation, the maximum amount of ethanol was produced. Therefore, the optimum period of incubation to produce ethanol was 72 h except for yeast strains Saccharomyces cerevisiae isolate MUT15F (12.02 ± 0.13% (v/v)) and Saccharomyces cerevisiae isolate R19MU (10.77 ± 0.04% (v/v)), which achieved their maximum production after 96 h. Saccharomyces cerevisiae isolate R9MU (11.15 ± 0.08% (v/v)) showed statistically (P ≤ 0.05) higher ethanol production.

3.3 Pre-concentration and dehydration of bioethanol

The yield of anhydrous and hydrous bioethanol generated from molasses sample is shown in Table 5. A continuous distillation was utilized to accomplish fractional distillation on each sample of fermented molasses, yielding various distillate quantities and hydrous bioethanol ( Figure 3). After pre-concentration, the distillation column concentrates hydrous bioethanol up to a final concentration of 94.20–95.60% (v/v) was determined using an alcoholmeter. The volume of bioethanol produced using different yeast strains in molasses samples varied significantly (p ≤ 0.05). Anhydrous bioethanol of 99.05–99.56% (v/v) was achieved by further distillation employing a 3A molecular sieve bead ( Figure 4). Saccharomyces cerevisiae isolate R9MU has been shown the highest anhydrous bioethanol concentration (99.56% (v/v)) after dehydration, while Saccharomyces cerevisiae isolate MUT18F produced the least amount of anhydrous bioethanol (99.05% (v/v)) as shown in Table 5. The maximum yield of bioethanol (8.50% v/v) was produced by S. cerevisiae isolate R9MU. All the bioethanol produced was clear and colorless.

Table 5. The pre-concentration, dehydration and percentage bioethanol yield.

Strains	S. cerevisiae isolate R9MU	S. cerevisiae isolate R20MU	S. cerevisiae isolate MUT15F	S. cerevisiae isolate MUT18F	S. cerevisiae isolate R19MU	MT standard yeast
Distillated molasses vol.	400 ml	400 ml	400 ml	400 ml	400 ml	400 ml
Volume of hydrous bioethanol	44.62 ± 0.1	43.60 ± 0.04	43.70 ± 0.04	38.70 ± 0.09	41.60 ± 0.25	40.62 ± 0.04
Hydrous bioethanol con. (v/v %)	95.60 ± 0.28	94.70 ± 0.04	95.10 ± 0.07	94.20 ± 0.14	94.90 ± 0.09	95.15 ± 0.39
Volume of anhydrous bioethanol	34 ± 0.09	29 ± 0.07	31 ± 0.05	28.20 ± 0.05	29.30 ± 0.01	30 ± 0.1
Anhydrous bioethanol con.	99.56 ± 0.01	99.15 ± 0.1	99.40 ± 0.05	99.05 ± 0.1	99.20 ± 0.06	99.30 ± 0.03
Bioethanol yield (%)	8.50 ± 0.06	7.25 ± 0.04	7.75 ± 0.10	7.05 ± 0.6	7.33 ± 0.21	7.50 ± 0.03

Figure 3. Distillation of molasses fermentation.

Figure 4. Dehydration of ethanol.

4. Discussion

Different molasses concentrations were used to determine their effect on the ethanol concentration using the optimized pH of 4.5. The finding indicated that 25 °Brix was the most suitable sugar concentration for isolate of Saccharomyces cerevisiae MUT15F and Saccharomyces cerevisiae R9MU to produce ethanol concentrations of 12.47 ± 0.07% (v/v) and 11.81 ± 0.16% (v/v) at 72 h, respectively. The ethanol concentration in this study was higher than the findings of other authors.³⁰^,³¹ However, Gu et al.³² used yunnan molasses and recorded the highest production of ethanol was 16% (v/v) by strain 1912 and 13.7% (v/v) by strain 1190 at 30% molasses sugar for 72 h. Muruaga et al.³³ also reported that for the A10 strain isolated from molasses at 250 g/L of initial sugar, the highest level of ethanol production was 13.20% (v/v). The production of ethanol by all yeasts significantly decreased at high molasses concentrations (35%). This may be due to high substrate concentrations being inhibitory to the fermentation process of yeasts due to osmotic stress.

Temperature is the key factor that affects the growth, metabolism, and ethanol production capability of the fermenting organisms. During this investigation, ethanol production by all yeast strains increased with the increase in temperature up to 30°C. This finding is in line with the findings of.³⁴^,³⁵ Below 25°C or above 30°C, the production of ethanol by all yeast strains was significantly reduced. This might be due to the fact that denaturation happens at high temperatures and reduced enzyme activity at low temperatures.

Optimal pH values are essential for the activity of plasma membrane-based proteins, including enzymes and transport proteins.³⁶ Based on the present finding, the production of ethanol gradually increased with the increase in pH to 4.5, and then started to decline. An optimum pH of S. cerevisiae was thus obtained at pH 4.5. This is in line with the report of other investigators.³⁵^,³⁷^,³⁸ In contrast to this investigation, Ercan et al.³⁹ found that optimum ethanol production using S. cerevisiae was obtained at pH 5.5. The most favorable pH of S. cerevisiae for ethanol production ranges from 4.0 to 5.0.²⁹

Nitrogen limitation has been shown to affect cell growth and biomass formation as well as directly affect the fermentation rate.⁴⁰ Although most of the nutrients needed for yeast development are present in molasses, sufficient nitrogen is frequently added to promote yeast growth and the production of more ethanol.⁴¹ Numerous investigations were conducted in previous years to optimize supplies of nitrogen and other supplements.⁴² The obtained data revealed that the addition of 1.0 to 4.0 g/L ammonium sulfate to the fermentation medium also increased ethanol production. In the present finding, the amount of ethanol production by Saccharomyces cerevisiae isolate MUT15F increased from 11.95 ± 0.1 to 13.13 ± 0.08% (v/v) between 1.0 and 4.0 g/L ammonium sulfate. Our study agrees with previous studies by Ref. 43 the optimal medium for S. cerevisiae to produce ethanol was 4 g/L of ammonium sulfate with 10% (v/v) ethanol content. Anupama et al.⁴⁴ was reported that the usage of ammonium sulfate at a concentration of 3 g/l resulted in an optimal 5.6% yield of ethanol.

Nutrients are quite effective in the production of ethanol from sugar cane molasses.²⁴ Different nitrogen sources (yeast extract, urea, and ammonium nitrate) were investigated as possible nitrogen supplements for sugar cane molasses. This study revealed that all nitrogen sources investigated had a positive effect on the production of ethanol. The maximum ethanol production was recoreded with yeast extract supplement and by Saccharomyces cerevisiae isolate MUT15F (12.24 ± 0.08% (v/v)) and Saccharomyces cerevisiae isolate R9MU (11.60 ± 0.06% (v/v)). These results were in agreement with Rasmey et al.,³⁷ who found that yeast extract addition significantly improved the ethanol concentration. In this finding, yeast extract provides convenient growth factors for yeast growth and it is in line with Ortiz-Muñiz et al.⁴⁵ report. On the other hand, it was also detected that ammonium sulfate was a good nitrogen source that stimulated ethanol production from sugar cane molasses. An effective and common source of nitrogen for yeast development is ammonium sulfate; it is cheap and has been chosen for future experiments.⁴¹

Controlling a number of variables can result in increased ethanol fermentation activity. At 30°Brix of molasses, 30°C, 4 g NH4SO4, 4.5 pH, and 72 hours of fermentation, Saccharomyces cerevisiae isolate MUT15F produced a maximum ethanol production of 13.13 (v/v) as compared to 12.21 (v/v) MT industrial yeast strain. Temperature, pH, substrate concentration, and fermentation time also greatly influences ethanol production by yeasts.¹²^,⁴⁶ According to this finding, ethanol concentration was reaching its maximum amount after 72 h except for Saccharomyces cerevisiae isolate MUT15F (12.02 ± 0.13% (v/v)) and Saccharomyces cerevisiae isolate R19MU (10.77 ± 0.04% (v/v)), which achieved their maximum production after 96 hrs. After three days of growth, it was determined that yeasts produce the highest amounts of ethanol by fermenting sugary substrates with actively growing yeast cells.⁴⁷ Ethanol production increased gradually, but a slight decrease in ethanol concentration with an increasing incubation period could be due to the loss of yeast cell viability and consumption of it by the yeast cells as time passed.⁴⁸ The current findings showed that optimization of temperature, pH, incubation duration, and substrate concentration is quite important for the maximization of ethanol production, as previously reported by other studies.⁴⁹^,⁵⁰

The result of our study indicated that hydrous bioethanol was concentrated in the distillation column to a final concentration of 94.20–95.60% (v/v). Further distillation using a 3A molecular sieve bead resulted in anhydrous bioethanol of about 99.05-99.56% (v/v). The highest anhydrous bioethanol concentration was obtained from Saccharomyces cerevisiae isolate R9MU (99.56% (v/v)) after distillation and dehydration. These results were in agreement with Yang et al.,⁵¹ who produced bioethanol in the form of hydrous ethanol (96% v/v).

5. Conclusion

Saccharomyces cerevisiae isolate MUT15F was produced maximum ethanol (13.13 ± 0.08% (v/v)) at 30 °Brix of molasses, 30°C, 4 g/L NH₄SO₄, 4.5 pH, and 72 hrs. In contrast to unoptimized conditions, the concentration of ethanol increased approximately by 77.4% after optimization. The candidate yeast strains designated as Saccharomyces cerevisiae isolate MUT15F, Saccharomyces cerevisiae isolate R9MU, and Saccharomyces cerevisiae isolate MUT18F produced maximum alcohol under the optimized fermentation conditions and have the potential to be used for industrial bioethanol production. Saccharomyces cerevisiae isolate R9MU produced the highest bioethanol yield (8.50% v/v). Traditional alcoholic beverages such as Tella, Tej, and Areke can could thus serve as a potential yeasts source that are able to produce high concentration of bioethanol.

Declarations

Author contribution statement

Conceptualization, M.F. and B.A.; Methodology, M.F.; Data analysis, M.F.; Investigation, M.F.; Resources, M.F.; Writing – original draft, M.F.; Writing – review & editing, M.F. and B.A.; Visualization, M.F.; Supervision, B.A.; Funding acquisition, M.F. All authors have read and agreed to the published version of the manuscript.

Ethical approval and consent

Ethical approval and consent were not required.

Data availability

Underlying data

Figshare: Molasses concentration (15, 20, 25, 30 and 35 °Brix), and pH (3.5, 4.0, 4.5, 5.0, and 5.5) of data analysis. https://doi.org/10.6084/m9.figshare.24941358.⁵²

This project contains the following underlying data:

- Molasses concentration (15, 20, 25, 30 and 35 °Brix), and pH (3.5, 4.0, 4.5, 5.0, and 5.5) of data analysis.
- SPSS data analysis of supplements, and period of incubation (24, 48, 72, 96, and 120 h).
- SPSS data analysis of temperature (25, 30, 35, and 40°C), and ammonium sulfate supplement (1, 2, 3, 4 and 5 g L-1).

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgments

We want to express our gratitude to Methara Sugar Factory for support molecular sieves and molasses.

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Berhanu Andualem
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This research was supported by University of Gonder and Debre markos University.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Fentahun M and Andualem B. Optimization of bioethanol production using stress-tolerant yeast strains isolated from household alcoholic beverages (Tella, Tej, and Areke) and molasses (as substrate) [version 2; peer review: 1 approved, 1 not approved]. F1000Research 2024, 13:286 (https://doi.org/10.12688/f1000research.146910.2)

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The strength of this article is that it uses a strain of yeast that was discovered, namely specifically Saccharomyces cerevisiae strains with accession numbers from the National Center for Biotechnology Information, such as strains R9MU (OR143320.1), strain R20MU (OR143322.1), strain MUT15F (OR209276.1), strain MUT18F (OR209286.1), and strain R19MU (OR143321.1)

INTRODUCTION

• Authors should also take care in the use of references:

The choice and development of the efficient bioethanol production of yeast with a resistance multi-stress tolerance are crucial importance14.

14. Chohan NA, Aruwajoye GS, Kana EBG: Valorisation of potato peel wastes for bioethanol production using simultaneous saccharification and fermentation: Process optimization and kinetic assessment. Renew. Energy 2020; 146: 1031–1040. Publisher Full Text

should use more specific literature for of yeast with a resistance multi-stress tolerance

These strains of Saccharomyces cerevisiae were obtained in our stock cultures from the previous study because to their noteworthy tolerance to ethanol, 26% (v/v), glucose, 70% (w/v) and temperature, 45°C.
• Authors should also references for this statement
• should provide citations from previous research for the characteristics of the yeast

METHODS

• Figures 1, 2, 3 and 4 are totally useless, they should be removed.

The levels of bioethanol produced from the molasses fermentation and distillation processes were measured using an alcoholmeter.

• Alcoholmeter need more detail

CONCLUSION
Saccharomyces cerevisiae isolate MUT15F yielded a maximum ethanol production of 13.13 0.08% (v/v) at 30 °Brix of molasses, 30°C, 4g NH4SO4, 4.5 pH, and 72 h fermentation

• In the discussion sub-chapter, it should be discussed in more depth how the characteristics of the recommended yeast compare with the standard yeast as a commercial yeast strain was used as a control for comparison.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: biotechnology, plant biotechnology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

CITE

Report a concern

Author Response 16 Dec 2024

Mulugeta Fentahun, Department of Biology, Debre Markos University, Debre Markos, Ethiopia

16 Dec 2024

Author Response
Mulugeta Fentahun Nega
Dr. Evika Sandi Savitri thank you very much for your insightful comments and the time you dedicated to review.
I greatly appreciate your feedback and suggestions. Please ... Continue reading
Mulugeta Fentahun Nega
Dr. Evika Sandi Savitri thank you very much for your insightful comments and the time you dedicated to review.
I greatly appreciate your feedback and suggestions. Please find my responses to your comments below:
Introduction Revision:

Comment: Authors should also take care in the use of references: Example the choice and development of the efficient bioethanol production of yeast with a resistance multi-stress tolerance are crucial importance. References or citation 14.

Response: Thank you for this suggestion We corrected it as Alfenore S, Molina C, Guillouet SE, Uribelarrea JL, et al.: Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process. Appl Microbiol Biotechnol.2002; 60: 67–72.

Comment: Authors should provide citations from previous research for the characteristics of the yeast strains of Saccharomyces cerevisiae were obtained in our stock cultures from the previous study because to their noteworthy tolerance to ethanol, 26% (v/v), glucose, 70% (w/v) and temperature, 45°C?

Response: Thank you for this valuable suggestion. We corrected it as Fentahun M, Andualem B: Characterization of wild indigenous yeasts that can withstand different stress from traditional fermented beverages in the Amhara Region, Ethiopia. Food Sci. Appl. Biotechnol. 2024; 7(2): 262- 277. 10.30721/fsab2024.v7.i2.4052024

Methodology Revision:

Comment: Figures 1, 2, 3 and 4 are totally useless, they should be removed.

Response: I appreciate your suggestion. The article's figures were compiled based on comments of the reviews of F1000 research.

Comment: Alcoholmeter need more detail.

Response: Thank you for your comments: We have updated the method according to your comments (refer the revised article).

Discussion Revision:

Comment: In the discussion sub-chapter, it should be discussed in more depth how the characteristics of the recommended yeast compare with the standard yeast as a commercial yeast strain was used as a control for comparison.

Response: Thank you for the suggestion. We have made revisions (refer the revised article).

Thank you once again for your valuable feedback. Your comments have significantly contributed to improving the quality of this paper.
Best regards,
Authors
Mulugeta Fentahun Nega
Dr. Evika Sandi Savitri thank you very much for your insightful comments and the time you dedicated to review.
I greatly appreciate your feedback and suggestions. Please find my responses to your comments below:
Introduction Revision:

Comment: Authors should also take care in the use of references: Example the choice and development of the efficient bioethanol production of yeast with a resistance multi-stress tolerance are crucial importance. References or citation 14.

Response: Thank you for this suggestion We corrected it as Alfenore S, Molina C, Guillouet SE, Uribelarrea JL, et al.: Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process. Appl Microbiol Biotechnol.2002; 60: 67–72.

Comment: Authors should provide citations from previous research for the characteristics of the yeast strains of Saccharomyces cerevisiae were obtained in our stock cultures from the previous study because to their noteworthy tolerance to ethanol, 26% (v/v), glucose, 70% (w/v) and temperature, 45°C?

Response: Thank you for this valuable suggestion. We corrected it as Fentahun M, Andualem B: Characterization of wild indigenous yeasts that can withstand different stress from traditional fermented beverages in the Amhara Region, Ethiopia. Food Sci. Appl. Biotechnol. 2024; 7(2): 262- 277. 10.30721/fsab2024.v7.i2.4052024

Methodology Revision:

Comment: Figures 1, 2, 3 and 4 are totally useless, they should be removed.

Response: I appreciate your suggestion. The article's figures were compiled based on comments of the reviews of F1000 research.

Comment: Alcoholmeter need more detail.

Response: Thank you for your comments: We have updated the method according to your comments (refer the revised article).

Discussion Revision:

Comment: In the discussion sub-chapter, it should be discussed in more depth how the characteristics of the recommended yeast compare with the standard yeast as a commercial yeast strain was used as a control for comparison.

Response: Thank you for the suggestion. We have made revisions (refer the revised article).

Thank you once again for your valuable feedback. Your comments have significantly contributed to improving the quality of this paper.
Best regards,
Authors
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 16 Dec 2024

Mulugeta Fentahun, Department of Biology, Debre Markos University, Debre Markos, Ethiopia

16 Dec 2024

Author Response
Mulugeta Fentahun Nega
Dr. Evika Sandi Savitri thank you very much for your insightful comments and the time you dedicated to review.
I greatly appreciate your feedback and suggestions. Please ... Continue reading
Mulugeta Fentahun Nega
Dr. Evika Sandi Savitri thank you very much for your insightful comments and the time you dedicated to review.
I greatly appreciate your feedback and suggestions. Please find my responses to your comments below:
Introduction Revision:

Comment: Authors should also take care in the use of references: Example the choice and development of the efficient bioethanol production of yeast with a resistance multi-stress tolerance are crucial importance. References or citation 14.

Response: Thank you for this suggestion We corrected it as Alfenore S, Molina C, Guillouet SE, Uribelarrea JL, et al.: Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process. Appl Microbiol Biotechnol.2002; 60: 67–72.

Comment: Authors should provide citations from previous research for the characteristics of the yeast strains of Saccharomyces cerevisiae were obtained in our stock cultures from the previous study because to their noteworthy tolerance to ethanol, 26% (v/v), glucose, 70% (w/v) and temperature, 45°C?

Response: Thank you for this valuable suggestion. We corrected it as Fentahun M, Andualem B: Characterization of wild indigenous yeasts that can withstand different stress from traditional fermented beverages in the Amhara Region, Ethiopia. Food Sci. Appl. Biotechnol. 2024; 7(2): 262- 277. 10.30721/fsab2024.v7.i2.4052024

Methodology Revision:

Comment: Figures 1, 2, 3 and 4 are totally useless, they should be removed.

Response: I appreciate your suggestion. The article's figures were compiled based on comments of the reviews of F1000 research.

Comment: Alcoholmeter need more detail.

Response: Thank you for your comments: We have updated the method according to your comments (refer the revised article).

Discussion Revision:

Comment: In the discussion sub-chapter, it should be discussed in more depth how the characteristics of the recommended yeast compare with the standard yeast as a commercial yeast strain was used as a control for comparison.

Response: Thank you for the suggestion. We have made revisions (refer the revised article).

Thank you once again for your valuable feedback. Your comments have significantly contributed to improving the quality of this paper.
Best regards,
Authors
Mulugeta Fentahun Nega
Dr. Evika Sandi Savitri thank you very much for your insightful comments and the time you dedicated to review.
I greatly appreciate your feedback and suggestions. Please find my responses to your comments below:
Introduction Revision:

Comment: Authors should also take care in the use of references: Example the choice and development of the efficient bioethanol production of yeast with a resistance multi-stress tolerance are crucial importance. References or citation 14.

Response: Thank you for this suggestion We corrected it as Alfenore S, Molina C, Guillouet SE, Uribelarrea JL, et al.: Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process. Appl Microbiol Biotechnol.2002; 60: 67–72.

Comment: Authors should provide citations from previous research for the characteristics of the yeast strains of Saccharomyces cerevisiae were obtained in our stock cultures from the previous study because to their noteworthy tolerance to ethanol, 26% (v/v), glucose, 70% (w/v) and temperature, 45°C?

Response: Thank you for this valuable suggestion. We corrected it as Fentahun M, Andualem B: Characterization of wild indigenous yeasts that can withstand different stress from traditional fermented beverages in the Amhara Region, Ethiopia. Food Sci. Appl. Biotechnol. 2024; 7(2): 262- 277. 10.30721/fsab2024.v7.i2.4052024

Methodology Revision:

Comment: Figures 1, 2, 3 and 4 are totally useless, they should be removed.

Response: I appreciate your suggestion. The article's figures were compiled based on comments of the reviews of F1000 research.

Comment: Alcoholmeter need more detail.

Response: Thank you for your comments: We have updated the method according to your comments (refer the revised article).

Discussion Revision:

Comment: In the discussion sub-chapter, it should be discussed in more depth how the characteristics of the recommended yeast compare with the standard yeast as a commercial yeast strain was used as a control for comparison.

Response: Thank you for the suggestion. We have made revisions (refer the revised article).

Thank you once again for your valuable feedback. Your comments have significantly contributed to improving the quality of this paper.
Best regards,
Authors
Competing Interests: No competing interests were disclosed. Close
Report a concern

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Reviewer Report 12 Jul 2024

Boris U. Stambuk, Federal University of Santa Catarina, Florianopolis, Brazil

Not Approved

https://doi.org/10.5256/f1000research.161041.r292438

The manuscript by Fentahun and Andualem “Optimization of bioethanol production using stress-tolerant yeast strains isolated from household alcoholic beverages (Tella, Tej, and Areke) and molasses (as substrate)” describes experiments performed to produce ethanol from molasses by yeasts isolated in Ethiopia. In this reviewer point of view, the manuscript needs extensive English revision. Since the manuscript does not have line numbers, is hard to point out which sentences have problems, I have mark some of them in the attached document (https://f1000research.s3.amazonaws.com/linked/663424.146910_-_Optimization_of_bioethanol_production_using_stress-tolerant_yeast_strains_isolated_from_household_alcoholic_beverages_%28Tella%2C_Tej%2C_and_Areke%29_and_molasses_%28as_substrate%29_-_Boris_Stambuk.pdf)

Authors should also take care in the use of references:
-For example, in the first paragraph of the Introduction they claim “Bioethanol is a promising alternative energy sources and a popular biofuel for transportation globally since it is a renewable, nontoxic, biodegradable resource and it is oxygenated” and they cite ref. 4, but this reference deals with Lactobacillus paracasei, metabolic syndrome and rats! (?)
-In the second paragraph they are describing the composition of molasses, and ref. 10 is cited, but this reference deals with ultrasonic and ozonation pretreatments of molasses for methane production…… In the context of bioethanol production, I believe that these references (Amorim HV, Basso LC, Lopes ML. Sugar cane juice and molasses, beet molasses and sweet sorghum: composition and usage. In: The Alcohol Textbook. Nottingham: Nottingham University Press; 2009; https://doi.org/10.1186/s13068-018-1221-x; and https://doi.org/10.1038/s41598-023-37618-8) would be more appropriated.

-In the same paragraph they claim “In recent years, sugarcane molasses’ high prospects for making bioethanol have gained major attention from researchers.” and cite ref. 11, but this reference deals with vinegar production! (?)

-In the same paragraph they claim “12–35% reducing sugar and the level of total reduced sugar reaches 50-65%”….. Confusing!

-In the Discussion section, second paragraph, they claim “This finding is in line with the findings of.” and cite ref. 32 and 33, but this last reference is from an Anesthesiology journal (?), with no title of the manuscript.

-At the beginning of the third paragraph of the Discussion they claim “Optimal pH values are essential for the activity of plasma membrane-based proteins, including enzymes and transport proteins.” and cite ref. 34, but this reference is not related to yeasts, but lactic acid bacteria (?)

-Several paragraphs later they claim “Ethanol production increased gradually, but a slight decrease in ethanol concentration with an increasing incubation period could be due to the loss of yeast cell viability and consumption of it by the yeast cells as time passed” and cite ref. 46, which seams also inappropriate!

-Indeed, SEVERAL of the references (# 3, 4, 8, 9, 16, 17, 30, 33, 35, 42, 44) are with problems, lacking article title and/or journal name and/or volume and/or page numbers……

-In the fourth paragraph of the Introduction they claim “other countries that produce bioethanol with yields of 12–14% (v/v)”, this should have a reference!

-At the end of the Introduction they claim “from the previous study because to their noteworthy tolerance to ethanol, 26% (v/v), glucose, 70% (w/v) and temperature, 45°C.”
and in item 2.4 they also claim “from the previous study”…..

-Item 2.3.1 “and pretreated with 99.8% sulfuric acid” pretreated how?

-The manuscript is highly repetitive, “ammonium sulfate (101217; MilliporeSigma)” for example is repeated at least 4 times, section 2.6 (and 2.6.1 to 2.6.6) is also repetitive!

-Item 2.4 mentions a “commercial yeast strain” without any indication of which one is (also “MT standard yeast”)

-Item 2.5 a “YEPD solid media” is mentioned, without any description! At the end of this item (and in several other sections) is claimed “feed-batch molasses fermentation”, but they did not do that, they performed batch fermentations, there was no feeding…..!

-Item 2.6 they claim “The anaerobic condition was maintained for 3 days”, but certainly no “anaerobic” condition was indeed used!

-“polarimeter’ and “alcoholmeter” needs more details....

-Figures 1, 2, 3 and 4 are totally useless, they should be removed.

-The paragraph after Table 2 reproduces all the data presented in Table 2, should be removed!
Etc., etc., etc., etc………

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Fuel ethanol production

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

CITE

Report a concern

Author Response 16 Dec 2024

Mulugeta Fentahun, Department of Biology, Debre Markos University, Debre Markos, Ethiopia

16 Dec 2024

Author Response
Mulugeta Fentahun Nega
Thanks to Prof. Boris U. Stambuk for your constructive comments. We conducted a number of changes.
I greatly appreciate your feedback and suggestions. Please find my responses ... Continue reading
Mulugeta Fentahun Nega
Thanks to Prof. Boris U. Stambuk for your constructive comments. We conducted a number of changes.
I greatly appreciate your feedback and suggestions. Please find my responses to your comments below:

Introduction Revision:

Comment: The manuscript needs extensive English

Response: Thank you for your comments: We have corrected grammatical errors throughout the manuscript and made the necessary corrections to enhance clarity and readability accordingly (refer the revised article).

Comment: Authors should also take care in the use of references. The first paragraph of the introduction they claim “Bioethanol is a promising alternative energy sources and a popular biofuel for transportation globally since it is a renewable, nontoxic, biodegradable resource and it is oxygenated” and they cite ref. 4, but this reference deals with Lactobacillus paracasei, metabolic syndrome and rats! (?)

Response: Thank you for this valuable suggestion. We corrected it as Scott A, Bryner M: Alternative Fuels: Rolling out Next-Generation Technologies. Chem. Week. 2006; 27: 17-21.

Comment: In the second paragraph they are describing the composition of molasses, and ref. 10 is cited, but this reference deals with ultrasonic and ozonation pretreatments of molasses for methane.

Response: Thank you for your comments. We corrected it as Lino FS, Basso TO, Sommer, MO: A synthetic medium to simulate sugarcane molasses. Biotechnology for Biofuels. 2018; 11(1). 221. 10.1186/s13068-018-1221-x

Comment: In the same paragraph they claim “In recent years, sugarcane molasses’ high prospects for making bioethanol have gained major attention from researchers.” and cite ref. 11, but this reference deals with vinegar production! (?)

Response: Thank you for your comments. We corrected it as Akbas MY, Stark BC: Recent trends in bioethanol production from food processing byproducts. J. Ind. Microbiol. Biotechnol. 2016. 10.1007/s10295-016-1821-z

Comment: In the same paragraph they claim “12–35% reducing sugar and the level of total reduced sugar reaches 50-65%”….. Confusing!

Response: Thank you for your critical insight. we corrected it based on your suggestions (refer the revised article).

Comment: In the fourth paragraph of the Introduction, they claim “other countries that produce bioethanol with yields of 12–14% (v/v)”, this should have a reference!

Response: Thank you for your critical insight. we cited as Kotarska K, Czupryński B, Kłosowski G: Effect of various activators on the course of alcoholic fermentation. J. Food Eng. 2006; 77: 965-971.

Comment: At the end of the Introduction, they claim “from the previous study because to their noteworthy tolerance to ethanol, 26% (v/v), glucose, 70% (w/v) and temperature, 45°C.” and in item 2.4 they also claim “from the previous study”.

Response: Thank you for your critical insight. We cited it as Fentahun M, Andualem B: Characterization of wild indigenous yeasts that can withstand different stress from traditional fermented beverages in the Amhara Region, Ethiopia. Food Sci. Appl. Biotechnol. 2024; 7(2): 262- 277. 10.30721/fsab2024.v7.i2.4052024

Methodology Revision:

Comment: Figures 1, 2, 3 and 4 are totally useless, they should be removed.

Response: I appreciate your suggestion. The article's figures were compiled based on comments of the reviews of F1000 research.

Comment: Alcoholmeter and polarimeter need more detail.

Response: Thank you for your comments: We have updated the method according to your comments (refer the revised article).

Comment: Item 2.6 they claim “The anaerobic condition was maintained for 3 days”, but certainly no “anaerobic” condition was indeed used!.

Response: Thank you for your comments: Yes. No “anaerobic” condition was used. We made correction based on your comments.

Comment: Item 2.5 a “YEPD solid media” is mentioned, without any description! At the end of this item (and in several other sections) is claimed “feed-batch molasses fermentation”, but they did not do that, they performed batch fermentations, there was no feeding…..!

Response: Thank you for your critical insight. We corrected YEPD solid media (refer the revised article) and Fed-batch fermentations were carried out, and feeding was done to boost the number of cells. A propagated yeast culture in 200 milliliters was used by adding 100 ml of sterile new batch molasses in to 100 ml inoculated flask for 24 hrs at 30°C. (refer item 2.5 propagation of yeast strains page 4 the revised article).

Comment: Item 2.4 mentions a “commercial yeast strain” without any indication of which one is (also “MT standard yeast”).

Response: Thank you for your comments: We have updated the method according to your comments (refer the revised article).

Comment: -Item 2.3.1 and pretreated with 99.8% sulfuric acid” pretreated how?

Response: Thank you for your comments: Because the pH raw molasses was adjusted at 3.5 using 99.8% H₂SO_4, and then the treated medium was standing overnight for decantation. The clear upper suspension of the broth was then carefully poured into a newly sterile fermentation flask.

Discussion Revision:

Comment: In the discussion section, second paragraph, they claim “This finding is in line with the findings of.” and cite ref. 32 and 33, but this last reference is from an Anesthesiology journal (?).

Response: Thank you for the suggestion. We have made revisions based on your comments as Abdel-Banat BMA, Hoshida H, Ano A, et al.: High-temperature fermentation: How can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Appl Microbiol Biotechnol. 2010; 85:861–7.

Comment: At the beginning of the third paragraph of the discussion they claim “Optimal pH values are essential for the activity of plasma membrane-based proteins, including enzymes and transport proteins.” and cite ref. 34, but this reference is not related to yeasts, but lactic acid bacteria (?).

Response: Thank you for your comments. We corrected it as Lopandic K, Zelger S, Bánszky L K, et al.: Identification of yeasts associated with milk products using traditional and molecular techniques. Food Microbiol. 2006; 23:341–50. 10.1016/J.FM.2005.05.001.

Comment: Several paragraphs later they claim “Ethanol production increased gradually, but a slight decrease in ethanol concentration with an increasing incubation period could be due to the loss of yeast cell viability and consumption of it by the yeast cells as time passed” and cite ref. 46, which seams also inappropriate!

Response: Thank you for your comments. We corrected it as Zabed H, Faruq G, Sahu JN, et al.: Bioethanol production from fermentable sugar juice. Sci. World J. 2014; 10.1155/2014/957102

Thank you once again for your valuable feedback. Your comments have significantly contributed to improving the quality of this paper.
Best regards,
Authors
Mulugeta Fentahun Nega
Thanks to Prof. Boris U. Stambuk for your constructive comments. We conducted a number of changes.
I greatly appreciate your feedback and suggestions. Please find my responses to your comments below:

Introduction Revision:

Comment: The manuscript needs extensive English

Response: Thank you for your comments: We have corrected grammatical errors throughout the manuscript and made the necessary corrections to enhance clarity and readability accordingly (refer the revised article).

Comment: Authors should also take care in the use of references. The first paragraph of the introduction they claim “Bioethanol is a promising alternative energy sources and a popular biofuel for transportation globally since it is a renewable, nontoxic, biodegradable resource and it is oxygenated” and they cite ref. 4, but this reference deals with Lactobacillus paracasei, metabolic syndrome and rats! (?)

Response: Thank you for this valuable suggestion. We corrected it as Scott A, Bryner M: Alternative Fuels: Rolling out Next-Generation Technologies. Chem. Week. 2006; 27: 17-21.

Comment: In the second paragraph they are describing the composition of molasses, and ref. 10 is cited, but this reference deals with ultrasonic and ozonation pretreatments of molasses for methane.

Response: Thank you for your comments. We corrected it as Lino FS, Basso TO, Sommer, MO: A synthetic medium to simulate sugarcane molasses. Biotechnology for Biofuels. 2018; 11(1). 221. 10.1186/s13068-018-1221-x

Comment: In the same paragraph they claim “In recent years, sugarcane molasses’ high prospects for making bioethanol have gained major attention from researchers.” and cite ref. 11, but this reference deals with vinegar production! (?)

Response: Thank you for your comments. We corrected it as Akbas MY, Stark BC: Recent trends in bioethanol production from food processing byproducts. J. Ind. Microbiol. Biotechnol. 2016. 10.1007/s10295-016-1821-z

Comment: In the same paragraph they claim “12–35% reducing sugar and the level of total reduced sugar reaches 50-65%”….. Confusing!

Response: Thank you for your critical insight. we corrected it based on your suggestions (refer the revised article).

Comment: In the fourth paragraph of the Introduction, they claim “other countries that produce bioethanol with yields of 12–14% (v/v)”, this should have a reference!

Response: Thank you for your critical insight. we cited as Kotarska K, Czupryński B, Kłosowski G: Effect of various activators on the course of alcoholic fermentation. J. Food Eng. 2006; 77: 965-971.

Comment: At the end of the Introduction, they claim “from the previous study because to their noteworthy tolerance to ethanol, 26% (v/v), glucose, 70% (w/v) and temperature, 45°C.” and in item 2.4 they also claim “from the previous study”.

Response: Thank you for your critical insight. We cited it as Fentahun M, Andualem B: Characterization of wild indigenous yeasts that can withstand different stress from traditional fermented beverages in the Amhara Region, Ethiopia. Food Sci. Appl. Biotechnol. 2024; 7(2): 262- 277. 10.30721/fsab2024.v7.i2.4052024

Methodology Revision:

Comment: Figures 1, 2, 3 and 4 are totally useless, they should be removed.

Response: I appreciate your suggestion. The article's figures were compiled based on comments of the reviews of F1000 research.

Comment: Alcoholmeter and polarimeter need more detail.

Response: Thank you for your comments: We have updated the method according to your comments (refer the revised article).

Comment: Item 2.6 they claim “The anaerobic condition was maintained for 3 days”, but certainly no “anaerobic” condition was indeed used!.

Response: Thank you for your comments: Yes. No “anaerobic” condition was used. We made correction based on your comments.

Comment: Item 2.5 a “YEPD solid media” is mentioned, without any description! At the end of this item (and in several other sections) is claimed “feed-batch molasses fermentation”, but they did not do that, they performed batch fermentations, there was no feeding…..!

Response: Thank you for your critical insight. We corrected YEPD solid media (refer the revised article) and Fed-batch fermentations were carried out, and feeding was done to boost the number of cells. A propagated yeast culture in 200 milliliters was used by adding 100 ml of sterile new batch molasses in to 100 ml inoculated flask for 24 hrs at 30°C. (refer item 2.5 propagation of yeast strains page 4 the revised article).

Comment: Item 2.4 mentions a “commercial yeast strain” without any indication of which one is (also “MT standard yeast”).

Response: Thank you for your comments: We have updated the method according to your comments (refer the revised article).

Comment: -Item 2.3.1 and pretreated with 99.8% sulfuric acid” pretreated how?

Response: Thank you for your comments: Because the pH raw molasses was adjusted at 3.5 using 99.8% H₂SO_4, and then the treated medium was standing overnight for decantation. The clear upper suspension of the broth was then carefully poured into a newly sterile fermentation flask.

Discussion Revision:

Comment: In the discussion section, second paragraph, they claim “This finding is in line with the findings of.” and cite ref. 32 and 33, but this last reference is from an Anesthesiology journal (?).

Response: Thank you for the suggestion. We have made revisions based on your comments as Abdel-Banat BMA, Hoshida H, Ano A, et al.: High-temperature fermentation: How can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Appl Microbiol Biotechnol. 2010; 85:861–7.

Comment: At the beginning of the third paragraph of the discussion they claim “Optimal pH values are essential for the activity of plasma membrane-based proteins, including enzymes and transport proteins.” and cite ref. 34, but this reference is not related to yeasts, but lactic acid bacteria (?).

Response: Thank you for your comments. We corrected it as Lopandic K, Zelger S, Bánszky L K, et al.: Identification of yeasts associated with milk products using traditional and molecular techniques. Food Microbiol. 2006; 23:341–50. 10.1016/J.FM.2005.05.001.

Comment: Several paragraphs later they claim “Ethanol production increased gradually, but a slight decrease in ethanol concentration with an increasing incubation period could be due to the loss of yeast cell viability and consumption of it by the yeast cells as time passed” and cite ref. 46, which seams also inappropriate!

Response: Thank you for your comments. We corrected it as Zabed H, Faruq G, Sahu JN, et al.: Bioethanol production from fermentable sugar juice. Sci. World J. 2014; 10.1155/2014/957102

Thank you once again for your valuable feedback. Your comments have significantly contributed to improving the quality of this paper.
Best regards,
Authors
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 16 Dec 2024

Mulugeta Fentahun, Department of Biology, Debre Markos University, Debre Markos, Ethiopia

16 Dec 2024

Author Response
Mulugeta Fentahun Nega
Thanks to Prof. Boris U. Stambuk for your constructive comments. We conducted a number of changes.
I greatly appreciate your feedback and suggestions. Please find my responses ... Continue reading
Mulugeta Fentahun Nega
Thanks to Prof. Boris U. Stambuk for your constructive comments. We conducted a number of changes.
I greatly appreciate your feedback and suggestions. Please find my responses to your comments below:

Introduction Revision:

Comment: The manuscript needs extensive English

Response: Thank you for your comments: We have corrected grammatical errors throughout the manuscript and made the necessary corrections to enhance clarity and readability accordingly (refer the revised article).

Comment: Authors should also take care in the use of references. The first paragraph of the introduction they claim “Bioethanol is a promising alternative energy sources and a popular biofuel for transportation globally since it is a renewable, nontoxic, biodegradable resource and it is oxygenated” and they cite ref. 4, but this reference deals with Lactobacillus paracasei, metabolic syndrome and rats! (?)

Response: Thank you for this valuable suggestion. We corrected it as Scott A, Bryner M: Alternative Fuels: Rolling out Next-Generation Technologies. Chem. Week. 2006; 27: 17-21.

Comment: In the second paragraph they are describing the composition of molasses, and ref. 10 is cited, but this reference deals with ultrasonic and ozonation pretreatments of molasses for methane.

Response: Thank you for your comments. We corrected it as Lino FS, Basso TO, Sommer, MO: A synthetic medium to simulate sugarcane molasses. Biotechnology for Biofuels. 2018; 11(1). 221. 10.1186/s13068-018-1221-x

Comment: In the same paragraph they claim “In recent years, sugarcane molasses’ high prospects for making bioethanol have gained major attention from researchers.” and cite ref. 11, but this reference deals with vinegar production! (?)

Response: Thank you for your comments. We corrected it as Akbas MY, Stark BC: Recent trends in bioethanol production from food processing byproducts. J. Ind. Microbiol. Biotechnol. 2016. 10.1007/s10295-016-1821-z

Comment: In the same paragraph they claim “12–35% reducing sugar and the level of total reduced sugar reaches 50-65%”….. Confusing!

Response: Thank you for your critical insight. we corrected it based on your suggestions (refer the revised article).

Comment: In the fourth paragraph of the Introduction, they claim “other countries that produce bioethanol with yields of 12–14% (v/v)”, this should have a reference!

Response: Thank you for your critical insight. we cited as Kotarska K, Czupryński B, Kłosowski G: Effect of various activators on the course of alcoholic fermentation. J. Food Eng. 2006; 77: 965-971.

Comment: At the end of the Introduction, they claim “from the previous study because to their noteworthy tolerance to ethanol, 26% (v/v), glucose, 70% (w/v) and temperature, 45°C.” and in item 2.4 they also claim “from the previous study”.

Response: Thank you for your critical insight. We cited it as Fentahun M, Andualem B: Characterization of wild indigenous yeasts that can withstand different stress from traditional fermented beverages in the Amhara Region, Ethiopia. Food Sci. Appl. Biotechnol. 2024; 7(2): 262- 277. 10.30721/fsab2024.v7.i2.4052024

Methodology Revision:

Comment: Figures 1, 2, 3 and 4 are totally useless, they should be removed.

Response: I appreciate your suggestion. The article's figures were compiled based on comments of the reviews of F1000 research.

Comment: Alcoholmeter and polarimeter need more detail.

Response: Thank you for your comments: We have updated the method according to your comments (refer the revised article).

Comment: Item 2.6 they claim “The anaerobic condition was maintained for 3 days”, but certainly no “anaerobic” condition was indeed used!.

Response: Thank you for your comments: Yes. No “anaerobic” condition was used. We made correction based on your comments.

Comment: Item 2.5 a “YEPD solid media” is mentioned, without any description! At the end of this item (and in several other sections) is claimed “feed-batch molasses fermentation”, but they did not do that, they performed batch fermentations, there was no feeding…..!

Response: Thank you for your critical insight. We corrected YEPD solid media (refer the revised article) and Fed-batch fermentations were carried out, and feeding was done to boost the number of cells. A propagated yeast culture in 200 milliliters was used by adding 100 ml of sterile new batch molasses in to 100 ml inoculated flask for 24 hrs at 30°C. (refer item 2.5 propagation of yeast strains page 4 the revised article).

Comment: Item 2.4 mentions a “commercial yeast strain” without any indication of which one is (also “MT standard yeast”).

Response: Thank you for your comments: We have updated the method according to your comments (refer the revised article).

Comment: -Item 2.3.1 and pretreated with 99.8% sulfuric acid” pretreated how?

Response: Thank you for your comments: Because the pH raw molasses was adjusted at 3.5 using 99.8% H₂SO_4, and then the treated medium was standing overnight for decantation. The clear upper suspension of the broth was then carefully poured into a newly sterile fermentation flask.

Discussion Revision:

Comment: In the discussion section, second paragraph, they claim “This finding is in line with the findings of.” and cite ref. 32 and 33, but this last reference is from an Anesthesiology journal (?).

Response: Thank you for the suggestion. We have made revisions based on your comments as Abdel-Banat BMA, Hoshida H, Ano A, et al.: High-temperature fermentation: How can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Appl Microbiol Biotechnol. 2010; 85:861–7.

Comment: At the beginning of the third paragraph of the discussion they claim “Optimal pH values are essential for the activity of plasma membrane-based proteins, including enzymes and transport proteins.” and cite ref. 34, but this reference is not related to yeasts, but lactic acid bacteria (?).

Response: Thank you for your comments. We corrected it as Lopandic K, Zelger S, Bánszky L K, et al.: Identification of yeasts associated with milk products using traditional and molecular techniques. Food Microbiol. 2006; 23:341–50. 10.1016/J.FM.2005.05.001.

Comment: Several paragraphs later they claim “Ethanol production increased gradually, but a slight decrease in ethanol concentration with an increasing incubation period could be due to the loss of yeast cell viability and consumption of it by the yeast cells as time passed” and cite ref. 46, which seams also inappropriate!

Response: Thank you for your comments. We corrected it as Zabed H, Faruq G, Sahu JN, et al.: Bioethanol production from fermentable sugar juice. Sci. World J. 2014; 10.1155/2014/957102

Thank you once again for your valuable feedback. Your comments have significantly contributed to improving the quality of this paper.
Best regards,
Authors
Mulugeta Fentahun Nega
Thanks to Prof. Boris U. Stambuk for your constructive comments. We conducted a number of changes.
I greatly appreciate your feedback and suggestions. Please find my responses to your comments below:

Introduction Revision:

Comment: The manuscript needs extensive English

Response: Thank you for your comments: We have corrected grammatical errors throughout the manuscript and made the necessary corrections to enhance clarity and readability accordingly (refer the revised article).

Comment: Authors should also take care in the use of references. The first paragraph of the introduction they claim “Bioethanol is a promising alternative energy sources and a popular biofuel for transportation globally since it is a renewable, nontoxic, biodegradable resource and it is oxygenated” and they cite ref. 4, but this reference deals with Lactobacillus paracasei, metabolic syndrome and rats! (?)

Response: Thank you for this valuable suggestion. We corrected it as Scott A, Bryner M: Alternative Fuels: Rolling out Next-Generation Technologies. Chem. Week. 2006; 27: 17-21.

Comment: In the second paragraph they are describing the composition of molasses, and ref. 10 is cited, but this reference deals with ultrasonic and ozonation pretreatments of molasses for methane.

Response: Thank you for your comments. We corrected it as Lino FS, Basso TO, Sommer, MO: A synthetic medium to simulate sugarcane molasses. Biotechnology for Biofuels. 2018; 11(1). 221. 10.1186/s13068-018-1221-x

Comment: In the same paragraph they claim “In recent years, sugarcane molasses’ high prospects for making bioethanol have gained major attention from researchers.” and cite ref. 11, but this reference deals with vinegar production! (?)

Response: Thank you for your comments. We corrected it as Akbas MY, Stark BC: Recent trends in bioethanol production from food processing byproducts. J. Ind. Microbiol. Biotechnol. 2016. 10.1007/s10295-016-1821-z

Comment: In the same paragraph they claim “12–35% reducing sugar and the level of total reduced sugar reaches 50-65%”….. Confusing!

Response: Thank you for your critical insight. we corrected it based on your suggestions (refer the revised article).

Comment: In the fourth paragraph of the Introduction, they claim “other countries that produce bioethanol with yields of 12–14% (v/v)”, this should have a reference!

Response: Thank you for your critical insight. we cited as Kotarska K, Czupryński B, Kłosowski G: Effect of various activators on the course of alcoholic fermentation. J. Food Eng. 2006; 77: 965-971.

Comment: At the end of the Introduction, they claim “from the previous study because to their noteworthy tolerance to ethanol, 26% (v/v), glucose, 70% (w/v) and temperature, 45°C.” and in item 2.4 they also claim “from the previous study”.

Response: Thank you for your critical insight. We cited it as Fentahun M, Andualem B: Characterization of wild indigenous yeasts that can withstand different stress from traditional fermented beverages in the Amhara Region, Ethiopia. Food Sci. Appl. Biotechnol. 2024; 7(2): 262- 277. 10.30721/fsab2024.v7.i2.4052024

Methodology Revision:

Comment: Figures 1, 2, 3 and 4 are totally useless, they should be removed.

Response: I appreciate your suggestion. The article's figures were compiled based on comments of the reviews of F1000 research.

Comment: Alcoholmeter and polarimeter need more detail.

Response: Thank you for your comments: We have updated the method according to your comments (refer the revised article).

Comment: Item 2.6 they claim “The anaerobic condition was maintained for 3 days”, but certainly no “anaerobic” condition was indeed used!.

Response: Thank you for your comments: Yes. No “anaerobic” condition was used. We made correction based on your comments.

Comment: Item 2.5 a “YEPD solid media” is mentioned, without any description! At the end of this item (and in several other sections) is claimed “feed-batch molasses fermentation”, but they did not do that, they performed batch fermentations, there was no feeding…..!

Response: Thank you for your critical insight. We corrected YEPD solid media (refer the revised article) and Fed-batch fermentations were carried out, and feeding was done to boost the number of cells. A propagated yeast culture in 200 milliliters was used by adding 100 ml of sterile new batch molasses in to 100 ml inoculated flask for 24 hrs at 30°C. (refer item 2.5 propagation of yeast strains page 4 the revised article).

Comment: Item 2.4 mentions a “commercial yeast strain” without any indication of which one is (also “MT standard yeast”).

Response: Thank you for your comments: We have updated the method according to your comments (refer the revised article).

Comment: -Item 2.3.1 and pretreated with 99.8% sulfuric acid” pretreated how?

Response: Thank you for your comments: Because the pH raw molasses was adjusted at 3.5 using 99.8% H₂SO_4, and then the treated medium was standing overnight for decantation. The clear upper suspension of the broth was then carefully poured into a newly sterile fermentation flask.

Discussion Revision:

Comment: In the discussion section, second paragraph, they claim “This finding is in line with the findings of.” and cite ref. 32 and 33, but this last reference is from an Anesthesiology journal (?).

Response: Thank you for the suggestion. We have made revisions based on your comments as Abdel-Banat BMA, Hoshida H, Ano A, et al.: High-temperature fermentation: How can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Appl Microbiol Biotechnol. 2010; 85:861–7.

Comment: At the beginning of the third paragraph of the discussion they claim “Optimal pH values are essential for the activity of plasma membrane-based proteins, including enzymes and transport proteins.” and cite ref. 34, but this reference is not related to yeasts, but lactic acid bacteria (?).

Response: Thank you for your comments. We corrected it as Lopandic K, Zelger S, Bánszky L K, et al.: Identification of yeasts associated with milk products using traditional and molecular techniques. Food Microbiol. 2006; 23:341–50. 10.1016/J.FM.2005.05.001.

Comment: Several paragraphs later they claim “Ethanol production increased gradually, but a slight decrease in ethanol concentration with an increasing incubation period could be due to the loss of yeast cell viability and consumption of it by the yeast cells as time passed” and cite ref. 46, which seams also inappropriate!

Response: Thank you for your comments. We corrected it as Zabed H, Faruq G, Sahu JN, et al.: Bioethanol production from fermentable sugar juice. Sci. World J. 2014; 10.1155/2014/957102

Thank you once again for your valuable feedback. Your comments have significantly contributed to improving the quality of this paper.
Best regards,
Authors
Competing Interests: No competing interests were disclosed. Close
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VERSION 2 PUBLISHED 17 Apr 2024

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Version 2 (revision) 16 Dec 24		read
Version 1 17 Apr 24	read	read

Boris U. Stambuk, Federal University of Santa Catarina, Florianopolis, Brazil
Evika Sandi Savitri, Universitas Islam Negeri Maulana Malik Ibrahim Malang, Malang, Indonesia

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Reviewer Report

4 Views

10 Feb 2025 | for Version 2

Evika Sandi Savitri, Universitas Islam Negeri Maulana Malik Ibrahim Malang, Malang, East Java, Indonesia

4 Views Cite this report Responses(0)

Approved

INTRODUCTION
Clear, provide citations from previous research for the characteristics of the yeast

METHODS
Cear, detail Alcoholmeter

RESULT AND DISCUSSION
Not yet, In the discussion sub-chapter, it should be discussed in more depth
Figure 1,2,3, should be removed

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

biotechnology, plant biotechnology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Reviewer Report

13 Views

27 Nov 2024 | for Version 1

Evika Sandi Savitri, Universitas Islam Negeri Maulana Malik Ibrahim Malang, Malang, East Java, Indonesia

13 Views Cite this report Responses(1)

Approved With Reservations

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

biotechnology, plant biotechnology

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Author Response

16 Dec 2024

Mulugeta Fentahun, Department of Biology, Debre Markos University, Debre Markos, Ethiopia

Mulugeta Fentahun Nega
Dr. Evika Sandi Savitri thank you very much for your insightful comments and the time you dedicated to review.
I greatly appreciate your feedback and suggestions. Please find my responses to your comments below:
Introduction Revision:

Comment: Authors should also take care in the use of references: Example the choice and development of the efficient bioethanol production of yeast with a resistance multi-stress tolerance are crucial importance. References or citation 14.
Response: Thank you for this suggestion We corrected it as Alfenore S, Molina C, Guillouet SE, Uribelarrea JL, et al.: Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process. Appl Microbiol Biotechnol.2002; 60: 67–72.
Comment: Authors should provide citations from previous research for the characteristics of the yeast strains of Saccharomyces cerevisiae were obtained in our stock cultures from the previous study because to their noteworthy tolerance to ethanol, 26% (v/v), glucose, 70% (w/v) and temperature, 45°C?
Response: Thank you for this valuable suggestion. We corrected it as Fentahun M, Andualem B: Characterization of wild indigenous yeasts that can withstand different stress from traditional fermented beverages in the Amhara Region, Ethiopia. Food Sci. Appl. Biotechnol. 2024; 7(2): 262- 277. 10.30721/fsab2024.v7.i2.4052024

Methodology Revision:

Comment: Figures 1, 2, 3 and 4 are totally useless, they should be removed.
Response: I appreciate your suggestion. The article's figures were compiled based on comments of the reviews of F1000 research.
Comment: Alcoholmeter need more detail.
Response: Thank you for your comments: We have updated the method according to your comments (refer the revised article).

Discussion Revision:

Comment: In the discussion sub-chapter, it should be discussed in more depth how the characteristics of the recommended yeast compare with the standard yeast as a commercial yeast strain was used as a control for comparison.
Response: Thank you for the suggestion. We have made revisions (refer the revised article).

Thank you once again for your valuable feedback. Your comments have significantly contributed to improving the quality of this paper.
Best regards,
Authors

View more View less

Competing Interests

No competing interests were disclosed.

Back to all reports

Reviewer Report

13 Views

12 Jul 2024 | for Version 1

Boris U. Stambuk, Federal University of Santa Catarina, Florianopolis, Brazil

13 Views Cite this report Responses(1)

Not Approved

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Fuel ethanol production

Respond to this report

Responses (1)

Author Response

16 Dec 2024

Mulugeta Fentahun, Department of Biology, Debre Markos University, Debre Markos, Ethiopia

Mulugeta Fentahun Nega
Thanks to Prof. Boris U. Stambuk for your constructive comments. We conducted a number of changes.
I greatly appreciate your feedback and suggestions. Please find my responses to your comments below:

Introduction Revision:

Comment: The manuscript needs extensive English
Response: Thank you for your comments: We have corrected grammatical errors throughout the manuscript and made the necessary corrections to enhance clarity and readability accordingly (refer the revised article).
Comment: Authors should also take care in the use of references. The first paragraph of the introduction they claim “Bioethanol is a promising alternative energy sources and a popular biofuel for transportation globally since it is a renewable, nontoxic, biodegradable resource and it is oxygenated” and they cite ref. 4, but this reference deals with Lactobacillus paracasei, metabolic syndrome and rats! (?)
Response: Thank you for this valuable suggestion. We corrected it as Scott A, Bryner M: Alternative Fuels: Rolling out Next-Generation Technologies. Chem. Week. 2006; 27: 17-21.
Comment: In the second paragraph they are describing the composition of molasses, and ref. 10 is cited, but this reference deals with ultrasonic and ozonation pretreatments of molasses for methane.
Response: Thank you for your comments. We corrected it as Lino FS, Basso TO, Sommer, MO: A synthetic medium to simulate sugarcane molasses. Biotechnology for Biofuels. 2018; 11(1). 221. 10.1186/s13068-018-1221-x
Comment: In the same paragraph they claim “In recent years, sugarcane molasses’ high prospects for making bioethanol have gained major attention from researchers.” and cite ref. 11, but this reference deals with vinegar production! (?)
Response: Thank you for your comments. We corrected it as Akbas MY, Stark BC: Recent trends in bioethanol production from food processing byproducts. J. Ind. Microbiol. Biotechnol. 2016. 10.1007/s10295-016-1821-z
Comment: In the same paragraph they claim “12–35% reducing sugar and the level of total reduced sugar reaches 50-65%”….. Confusing!
Response: Thank you for your critical insight. we corrected it based on your suggestions (refer the revised article).
Comment: In the fourth paragraph of the Introduction, they claim “other countries that produce bioethanol with yields of 12–14% (v/v)”, this should have a reference!
Response: Thank you for your critical insight. we cited as Kotarska K, Czupryński B, Kłosowski G: Effect of various activators on the course of alcoholic fermentation. J. Food Eng. 2006; 77: 965-971.
Comment: At the end of the Introduction, they claim “from the previous study because to their noteworthy tolerance to ethanol, 26% (v/v), glucose, 70% (w/v) and temperature, 45°C.” and in item 2.4 they also claim “from the previous study”.
Response: Thank you for your critical insight. We cited it as Fentahun M, Andualem B: Characterization of wild indigenous yeasts that can withstand different stress from traditional fermented beverages in the Amhara Region, Ethiopia. Food Sci. Appl. Biotechnol. 2024; 7(2): 262- 277. 10.30721/fsab2024.v7.i2.4052024

Methodology Revision:

Comment: Figures 1, 2, 3 and 4 are totally useless, they should be removed.
Response: I appreciate your suggestion. The article's figures were compiled based on comments of the reviews of F1000 research.
Comment: Alcoholmeter and polarimeter need more detail.
Response: Thank you for your comments: We have updated the method according to your comments (refer the revised article).
Comment: Item 2.6 they claim “The anaerobic condition was maintained for 3 days”, but certainly no “anaerobic” condition was indeed used!.
Response: Thank you for your comments: Yes. No “anaerobic” condition was used. We made correction based on your comments.
Comment: Item 2.5 a “YEPD solid media” is mentioned, without any description! At the end of this item (and in several other sections) is claimed “feed-batch molasses fermentation”, but they did not do that, they performed batch fermentations, there was no feeding…..!
Response: Thank you for your critical insight. We corrected YEPD solid media (refer the revised article) and Fed-batch fermentations were carried out, and feeding was done to boost the number of cells. A propagated yeast culture in 200 milliliters was used by adding 100 ml of sterile new batch molasses in to 100 ml inoculated flask for 24 hrs at 30°C. (refer item 2.5 propagation of yeast strains page 4 the revised article).
Comment: Item 2.4 mentions a “commercial yeast strain” without any indication of which one is (also “MT standard yeast”).
Response: Thank you for your comments: We have updated the method according to your comments (refer the revised article).
Comment: -Item 2.3.1 and pretreated with 99.8% sulfuric acid” pretreated how?
Response: Thank you for your comments: Because the pH raw molasses was adjusted at 3.5 using 99.8% H₂SO_4, and then the treated medium was standing overnight for decantation. The clear upper suspension of the broth was then carefully poured into a newly sterile fermentation flask.

Discussion Revision:

Comment: In the discussion section, second paragraph, they claim “This finding is in line with the findings of.” and cite ref. 32 and 33, but this last reference is from an Anesthesiology journal (?).
Response: Thank you for the suggestion. We have made revisions based on your comments as Abdel-Banat BMA, Hoshida H, Ano A, et al.: High-temperature fermentation: How can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Appl Microbiol Biotechnol. 2010; 85:861–7.
Comment: At the beginning of the third paragraph of the discussion they claim “Optimal pH values are essential for the activity of plasma membrane-based proteins, including enzymes and transport proteins.” and cite ref. 34, but this reference is not related to yeasts, but lactic acid bacteria (?).
Response: Thank you for your comments. We corrected it as Lopandic K, Zelger S, Bánszky L K, et al.: Identification of yeasts associated with milk products using traditional and molecular techniques. Food Microbiol. 2006; 23:341–50. 10.1016/J.FM.2005.05.001.
Comment: Several paragraphs later they claim “Ethanol production increased gradually, but a slight decrease in ethanol concentration with an increasing incubation period could be due to the loss of yeast cell viability and consumption of it by the yeast cells as time passed” and cite ref. 46, which seams also inappropriate!
Response: Thank you for your comments. We corrected it as Zabed H, Faruq G, Sahu JN, et al.: Bioethanol production from fermentable sugar juice. Sci. World J. 2014; 10.1155/2014/957102

Thank you once again for your valuable feedback. Your comments have significantly contributed to improving the quality of this paper.
Best regards,
Authors

View more View less

Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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Optimization of bioethanol production using stress-tolerant yeast strains isolated from household alcoholic beverages (Tella, Tej, and Areke) and molasses (as substrate)

Abstract

Background

Methods

Result

Conclusion

Keywords

Revised Amendments from Version 1

1. Introduction

2. Methods

2.1 Collection sugar cane molasses

Figure 1. Molasses sample.

2.2 Characterization of sugar cane molasses

2.3 Bioethanol production from molasses

2.4 Best selected yeast strain for production of bioethanol

2.5 Propagation of yeast strains

Table 1. Propagated yeast cells as indicated by OD (nm) for five strains (Initial OD reading was done immediately after inoculation, one OD =1x108 CFU/ml).

2.6 Optimization of fermentation process parameters

2.7 Analytical methods

(1)

(2)

2.8 Distillation of Fermented molasses

2.9 Pre-concentration of the bioethanol in continuous distillation system

2.10 Dehydration of ethanol using 3A molecular sieve bead

2.11 Data analysis

3. Results

Figure 2. Molasses fermentation.

Table 2. The physicochemical parameters of sugarcane molasses from Methara Sugar Factory, Ethiopia.

3.1 Effect of molasses concentration, temperature, and pH on ethanol concentration

Table 3. Effect of different molasses concentration, temperature and pH in ethanol production process at optimum temperature 30°C, 30 °Brix, and pH 4.5 after 72 h in fed batch culture.

3.2 The effect of supplement concentration, different supplement, and incubation periods on ethanol concentration

Table 4. The effect of supplement concentration, different supplement, and incubation periods in ethanol production process at optimum temperature 30°C, 30 oBrix, and pH 4.5 after 72 h in fed batch culture.

3.3 Pre-concentration and dehydration of bioethanol

Table 5. The pre-concentration, dehydration and percentage bioethanol yield.

Figure 3. Distillation of molasses fermentation.

Figure 4. Dehydration of ethanol.

4. Discussion

5. Conclusion

Declarations

Author contribution statement

Ethical approval and consent

Data availability

Underlying data

Acknowledgments

References

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Table 1. Propagated yeast cells as indicated by OD (nm) for five strains (Initial OD reading was done immediately after inoculation, one OD =1x10⁸ CFU/ml).

Table 4. The effect of supplement concentration, different supplement, and incubation periods in ethanol production process at optimum temperature 30°C, 30 ^oBrix, and pH 4.5 after 72 h in fed batch culture.