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Bioethanol production through syngas fermentation in a tar free bioreactor using

Clostridium butyricum

Minhaj Uddin Monir, Azrina Abd Aziz, Fatema Khatun, Abu Yousuf

PII: S0960-1481(20)30797-7
DOI: https://doi.org/10.1016/j.renene.2020.05.099
Reference: RENE 13588

To appear in: Renewable Energy

Received Date: 10 July 2019

Revised Date: 8 May 2020
Accepted Date: 19 May 2020

Please cite this article as: Monir MU, Aziz AA, Khatun F, Yousuf A, Bioethanol production through
syngas fermentation in a tar free bioreactor using Clostridium butyricum, Renewable Energy (2020), doi:
https://doi.org/10.1016/j.renene.2020.05.099.

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Credit Author Statement

Minhaj Uddin Monir: Conceptualization, Methodology, Software, Validation, Formal

Analysis, Investigation, Data Curation, Writing- Original Draft, Writing- Review & Editing,
Visualization
Azrina Abd Aziz: Conceptualization, Methodology, Validation, Resources, Writing-
Original Draft, Writing- Review & Editing, Visualization, Supervision, Project
Administration, Funding Acquisition
Fatema Khatun: Formal Analysis
Abu Yousuf: Writing-Original Draft
Bioethanol production through syngas fermentation in a tar free bioreactor using Clostridium

butyricum

Minhaj Uddin Monira, Azrina Abd Azizb,c,*, Fatema Khatunb and Abu Yousuf d

a
Department of Petroleum and Mining Engineering, Jashore University of Science and

Technology, Jashore-7408, Bangladesh

b
Faculty of Civil Engineering Technology, Universiti Malaysia Pahang, 26300 Gambang,

Malaysia
c
Earth Resources & Sustainability Center, Universiti Malaysia Pahang, 26300, Gambang,

Malaysia
d
Department of Chemical Engineering and Polymer Science, Shahjalal University of Science

and Technology, Sylhet-3114, Bangladesh

*Corresponding author: azrinaaziz@ump.edu.my (Azrina Abd Aziz)

1
 1 Abstract

2 Biomass-generated syngas conversion through fermentation is a promising technique for

3 bioethanol production due to increasing demand for clean and sustainable energy.

4 However, lignocellulosic biomass is difficult to degrade wholly, and traditional pretreatment

5 process has several drawbacks. The present study emphasizes on bioethanol production from

6 lignocellulosic biomass-based syngas including the main composition of N2 (45.58 %), CO

7 (22.92%), CO2 (7.9%), H2 (13.05%), and CH4 (1.13%). Field emission electronic microscopic

8 analysis was used to characterize freshly cultured Clostridium butyricum for syngas

9 fermentation and experiment was run in a bioreactor (TFB). The obtained yield of bioethanol

10 was analyzed by nuclear magnetic resonance and gas chromatography-mass spectrometry

11 analyses. For this syngas fermentation, treated syngas was preferred, as most of the

12 Clostridium butyricum grown on best fermentation conditions. The results show that except

13 0.03% of CO2, other gases were dissolved entirely. It is also found that extracted bioethanol

14 was identified by corresponding NMR (1H) spectra of methyle group (CH3-), methylene

15 group (-CH2-) and hydroxyl group (OH). The yield of bioethanol was 29.94 mmol from 1L of

16 syngas. Hence, this biomass-generated syngas is the appropriate renewable energy source for

17 the meetup of future energy needs.

18 Keywords: Bioethanol; Clostridium butyricum; Syngas; Syngas fermentation; TFB.

19

20

21

22

2
 1 1. Introduction

2 Biomass is one of the most promising alternative sources of bioenergy for the production of

3 bioethanol due to future fuel insecurity [1]. It is the versatile renewable energy resource that

4 is reliance on fossil fuels and mitigates global warming [2, 3]. Less sulfur containing biomass

5 is considered as a source of clean and sustainable bioenergy [3]. Furthermore, fossil-based

6 fuel resources contributed more than 80% of global energy demand, while renewable energy

7 contributed only 15% [4, 5]. Mostly available lignocellulosic biomass is usually used for

8 biofuel production due to the exhaustion of fossil fuels and emissions of greenhouse gases

9 (GHGs) through petrochemical processes [6]. Moreover, the uses of fertilizer, seed, pesticide,

10 and burning of fossil fuels or biomass are the sources of GHGs emission [7]. The emissions

11 of these gases can be reduced by mixing a percentage of coal with biomass during co-

12 gasification [8]. However, incessant advancement of petroleum exploitation technology and

13 discovery of new natural resources increased fuel production could not appease the required

14 energy demand [9, 10]. Moreover, Serrano-Ruiz [11] reported that limited fossil-based

15 resources will be diminished in near future.

16 There are two main conversion routes for bioethanol production including biochemical and

17 thermo-biochemical [12]. Energy conversion through gasification involves some frequent

18 processes which are drying, pyrolysis, oxidation and reduction, and yield products are

19 obtained in three stages: low molecular weight liquids, gas fuels and solid residues [13, 14].

20 Syngas is produced through gasifiers; such as fixed bed gasifier (updraft, downdraft, and

21 cross-draft), fluidized bed gasifier (circulating and bubbling) and entrained flow gasifier [15,

22 16]. Moreover, gas cleaning is the most challenging part, and energy conversion is very

23 difficult because of tar and particles present in syngas [17, 18]. It has a great difference in

24 their composition of industrially cleaned and biomass-based raw syngas. The main

25 compositions of industrial syngas are hydrogen (H2), carbon monoxide (CO), carbon dioxide

3
 1 (CO2) and methane (CH4). On the other hand, raw syngas contained solid compounds,

2 condensable volatiles and other gases [19]. Besides, other compounds are acetylene (C2H2),

3 benzene (C6H6), methane (CH4), ethane (C2H6), hydrogen sulfide (H2S), ethylene (C2H4),

4 hydrogen cyanide (HCN), carbonyl sulfide (COS), oxygen (O2), ammonia (NH3), sulfur

5 dioxide (SO2), nitrogen (N2), water (H2O), mono-nitrogen oxides (NOx), chlorine

6 compounds, tars, and ash [19].

7 Biofuels are categorized as the first generation, second generation, third generation and fourth

8 generation based on biomass types and process employed [10, 20]. The first generation

9 biofuels are bio-methanol, bio-ethanol, bio-propanol, bio-butanol, fatty acid esters, etc., are

10 converted from simple sugars, starch, fats and vegetable oils [21]. Inderwildi and King

11 [22] stated that second-generation biofuels, such as ethanol are produced from lignocellulosic

12 biomass. In addition, the production of bioethanol is cost-effectiveness compared to fossil

13 fuels [23]. Moreover, Rastogi and Shrivastava [24] reported that utilization of lignocellulosic

14 biomass to produce various energy like biogas, biodiesel, bioethanol etc. has been increased

15 day by day. The yield of ethanol is increased considering efficient recovery methods reported

16 by Shen, Brown and Wen [25]. Sepehri and Sarrafzadeh [26] worked on the nitrifying-

17 enriched activated sludge (NAS) and stated that membrane fouling in membrane bioreactors

18 which is depended on the microbial cell density and its population structure. Bioethanol

19 (second-generation) is produced from lignin-rich biomass through syngas fermentation [27].

20 There are various types of microorganisms that used for syngas fermentation. These

21 microorganisms are capable of bioethanol production through gas fermentation [28, 29].

22 Sun, Atiyeh, Zhang, Tanner and Huhnke [30] reported that biochar supplied nutrients to the

23 microorganisms during syngas fermentation. Moreover, microorganisms grow well in CO

24 than H2, and syngas impurities reduce the fermentability [27, 31]. The advanced technique of

25 syngas fermentation, syngas is used as a raw material that is produced from biomass

4
 1 gasification [10, 29, 32]. Bioethanol producing model microorganisms that are

2 Saccharomyces cerevisiae, Clostridium sp., Bacillus sp., Escherichia coli, Fusarium

3 oxysporum and Trichoderma reesei [33-35]. Clostridium butyricum is an important

4 microorganism because of its hydrogen productivity [36]. Furthermore, microorganisms

5 generate various alcohols like ethanol, butanol, hexanol, propanol and some organic acids

6 [23, 30, 37]. Some stoichiometric equations that are usually responsible for ethanol and acetic

7 acids production are represented in the following equations (Eq. 1-Eq. 4) [23].

4‫ ܱܥ‬+ 2‫ܪ‬ଶ ܱ → ‫ܪܥ‬ଷ ‫ ܪܱܱܥ‬+ 2‫ܱܥ‬ଶ Eq. 1

2‫ܱܥ‬ଶ + 4‫ܪ‬ଶ → ‫ܪܥ‬ଷ ‫ ܪܱܱܥ‬+ 2‫ܪ‬ଶ ܱ Eq. 2

6‫ ܱܥ‬+ 3‫ܪ‬ଶ ܱ → ‫ܥ‬ଶ ‫ܪ‬ହ ܱ‫ ܪ‬+ 4‫ܱܥ‬ଶ Eq. 3

2‫ܱܥ‬ଶ + 6‫ܪ‬ଶ → ‫ܥ‬ଶ ‫ܪ‬ହ ܱ‫ ܪ‬+ 2‫ܪ‬ଶ ܱ Eq. 4

8
9 Liao, Mi, Pontrelli and Luo [38] studied the exploration technique for next-generation biofuel

10 production using microorganisms (such as bacteria and fungi). In this regards, Kennes,

11 Abubackar, Diaz, Veiga and Kennes [23] informed that biomass feedstock is gasified by

12 thermochemical alternative and yield of syngas comprised CO, CO2 and H2 which are

13 fermented anaerobically, usually by Clostridia Sp. to ethanol or other yields. Shen, Brown

14 and Wen [36] reported that C. carboxidivorans also plays an important role for the

15 production of bioethanol. Moreover, Kanchanasuta, Prommeenate, Boonapatcharone and

16 Pisutpaisal [39] stated that biohydrogen was produced by adding of Clostridium

17 butyricum into non-sterile food waste. These are cultured to produce desired products using a

18 fermenter. It is designed to give the suitable environment for optimal microbial cell growth

19 and their metabolic activity. Continuous stirred tank reactor, bubble column reactor, trickle

20 bed reactor, monolithic biofilm reactor, membrane-based system reactor and microbubble

21 dispersion stirred-tank reactor are usually used as fermenter [40, 41].

5
 1 According to the literature, insufficient studies have been focused on syngas fermentation

2 with Clostridium butyricum using biomass-based syngas. Although, few number of studies

3 have been performed on the yield of bioethanol through syngas fermentation [32, 36, 42].

4 Therefore, the aim of this study is to produce clean, sustainable and environmental-friendly

5 bioethanol through syngas fermentation (using Clostridium butyricum). However, from

6 previous study it was found that produced syngas contained an unwanted co-product tar

7 compounds, some impurities and ash content during co-gasification of lignocellulosic

8 biomass and charcoal [3, 15, 43, 44]. As a result, syngas causes serious effects on further

9 processing to other fuels due to the presence of tar and particles. Consequently, impure

10 syngas is not capable of biofuel production efficiently. Moreover, produced syngas can be

11 used in gasoline or diesel engines with modifications to the existing engine which is costly.

12 Whole biomass including lignin is not converted into bioethanol in their biochemical

13 pathway. In addition, conventional fermentation process is not feasible due to the complex

14 pretreatment and high enzyme cost. Therefore, tar-free syngas is needed to perform syngas

15 fermentation by adding a purification system on the fed-batch mode bioreactor to produce

16 bioethanol. In this study, syngas fermentation using Clostridium butyricum was performed in

17 a tar free bioreactor (TFB) under varying operational conditions for best syngas fermentation,

18 with the variables including effect of temperature, effect of pH, syngas impurity (treated and

19 untreated syngas), effect of total organic carbon and growth condition, effect of syngas

20 composition (before and after syngas fermentation) were investigated. The objective of this

21 study is to evaluate the production of bioethanol using TFB bioreactor through syngas

22 fermentation.

23

24 2. Materials and Methods

25 2.1. Microbial Cultivation and Growth Media

6
 1 Clostridium butyricum was grown anaerobically in a Reinforced Clostridial Medium (RCM)

2 at Cell Culture Laboratory (Universiti Malaysia Pahang, Malaysia). The medium contained

3 chemical components (Table 1) for the preparation of microbial growth media and these

4 mixed with deionized water at a ratio of 38 g/L. Subsequently, the medium was sterilized by

5 autoclaving at 121°C for 20 minutes. Prior to cultivation, Clostridium butyricum was

6 reactivated by transferring 2 mL of the stock culture into 20 mL of RCM. The cultural serum

7 bottle was flushed with nitrogen gas (N2) for 2 minutes to create anaerobic condition and

8 incubated at 37 °C for 24 h and stored at 4 °C. Freshly cultivated Clostridium butyricum cell

9 was analyzed by field emission scanning electron microscope (FESEM) analysis.

10 2.2 Experimental Setup and Bioethanol Extraction

11 The syngas fermentation was conducted in a tar free bioreactor (TFB) followed by fed-batch

12 system (Fig. 1). In this syngas fermentation study, forest residue (70%) and charcoal (30%)

13 producing syngas were used [45] that collected from previous co-gasification [15]. The used

14 syngas compositions were hydrogen (H2)-13.05% Mole, carbon monoxide (CO)-22.92%

15 Mole, carbon dioxide (CO2)-7.90% Mole, methane (CH4)-1.13% Mole, nitrogen (N2)-45.58%

16 Mole and other gases 9.42 % Mole. In this study, charcoal was also used as a nutrient for

17 Clostridium butyricum that was also collected from previous co-gasification based study [15].

18 The 20 mL, 100 mL, 250 mL, 500 mL, and 1000 mL stored syngas were used as the main

19 carbon sources for Clostridium butyricum. A peristaltic pump (Brand: OEM, power supply:

20 AC220V 50/60HZ) was used to flow (1mL - 250mL/min) syngas into the TFB and mixed

21 with fermentation broth properly every 24h interval during the fermentation process. After

22 mixing these (nutrients and charcoal), medium was autoclaved at 121 °C for 20 min. Then,

23 syngas was passed through methanol and acetone for the purification of tar compounds. The

24 fine particles from syngas were cleaned through cotton filter. The effect of temperature, pH,

7
 1 colony forming unit (CFU) using untreated and treated syngas, total organic carbon (TOC)

2 etc. were investigated throughout the whole process.

3 Prior to the experimental run, medium of fermentation broth for Clostridium butyricum was

4 prepared. An impinger bottle (500 mL) was used for the experiments where 80% filled with

5 fermentation broth and remaining (20%) was used as a working volume. The syngas

6 fermentation medium was made by mixing of nutrient broth (10gm in 400 mL of DI water)

7 and charcoal. Subsequently, pure syngas was passed through TFB and Clostridium butyricum

8 was inoculated. The pH of 4.0-6.0 was controlled in the initial stage of syngas fermentation.

9 Subsequently, shaking incubator was used to perform the experiment. The retained

10 temperature was 37 °C and rotational speed of shaking incubator was 200 rpm. Syngas was

11 recycled throughout the process to increase their overall efficiency. The whole process was

12 run for 16 days. The yield product was extracted at the end of the process. Liquid-liquid

13 extraction (LLE) was applied for the separation of organic compounds from aqueous

14 solution. In this study, deuterochloroform, CDCl3 (Sigma-Aldrich) and n-hexane

15 (chromatogram pure grade, Sigma Chemicals, USA) were used to extract bioethanol from

16 fermentation broth. During extraction process, CDCl3 or n-hexane (5 ml) was mixed with

17 fermentation broth (40 ml), and then vigorous uniform vortex was performed through vortex

18 mixer (Vortex-Genie 2, Scientific Industries Inc., USA) for 15 minutes. Subsequently,

19 solvent phase was separated and transferred to the sample vial for GC-MS and NMR (1H)

20 analysis.

21 2.3 Analytical Procedure

22 The morphology of newly cultured Clostridium butyricum was observed by FESEM (Brand:

23 JEOL, Model: JSM-7800F). The samples were rinsed with deionized water and dried in the

24 air, then coated with Pt to make the samples conductive before loading into the instrument.

25 FESEM images were attained with an acceleration voltage of 10 kV.

8
 1 The end product (bioethanol) was analyzed by NMR (1H) spectra and its volume was

2 estimated through GC-MS analysis. This product analysis was conducted using GC-MS

3 analyzer (Brand: Agilent, Model: 7890A). The initial instrumental temperature was set as 70

4 °C and final temperature was at 325 °C. The GC oven temperature was set as 325 °C. The

5 program was set as 100 °C (2 minutes), then 5 °C/minute to 120 °C for 0 min, and finally 5

6 minutes for post run the program. Analytical run time was set for 6 minutes. Standard ethanol

7 (99.99%) was used for quantitative analysis. MS fraction of samples was matched with

8 standard ethanol (99.99%) and finally bioethanol was estimated. In this study, NMR (1H)

9 analysis was used for final bioethanol quantification and 1H NMR (500 MHz) spectra were

10 analyzed through BRUKER-500 spectrometer (Model: Bruker Ultra Shield Plus 500 MHz).

11 The chemical shifts were specified comparative to CDCl3 (TMS, 0.00 ppm).

12 The syngas composition was analyzed using gas chromatography-thermal conductivity

13 detector (GC-TCD) (Brand: Shimadzu, Model: GC-2014) considering before and after

14 fermentation. The attached TCD sensor changes thermal conductivity of the column that

15 compares with reference flow of carrier gas (helium). The analyzed gases were mainly

16 hydrogen (H2), carbon dioxide (CO2), carbon monoxide (CO), methane (CH4), etc. During

17 GC-TCD analysis, injected syngas volume was 1 µL. The temperature and pH were studied

18 using EutechTM pH 700 meter. In this study, colony forming unit (CFU) and total organic

19 carbon (TOC) were analyzed through StuartTM colony counter and MERCK Spectroquant®

20 Pharo 300 analyzer, respectively.

21

22 3. Results and Discussion

23 3.1 Microorganisms Preparation and Characterization

24 The freshly cultured Clostridium butyricum, its cell growth on petri-plate and FESEM

25 characterization results are shown in Fig. 2. After the inoculation of Clostridium butyricum

9
 1 (Fig. 2a), the medium colour was changed to creamy yellow (Fig. 2b). The physical

2 observation (CFU) on petri plate was investigated and colony counted throughout the whole

3 process to determine the number of Clostridium butyricum colony (Fig. 2c). From this

4 investigation it was found that the number of Clostridium butyricum colony was increased

5 and then decreased gradually with time. For the confirmation of Clostridium butyricum,

6 FESEM analysis was performed which is represented in Fig. 2d and 2e. FESEM images

7 revealed the Clostridium butyricum that was cultured effectively and suited for syngas

8 fermentation. The average wide and length of the Clostridium butyricum cell ranges are from

9 474.96nm to 695.03nm and 1511.51nm to 2097.99nm, respectively (Fig. 2d and 2e). The

10 morphological characteristics of Clostridium butyricum were agreed with the literature [46].

11 3.2 Effects of Syngas Fermentation Performance

12 The effect of treated and untreated syngas on the growth of Clostridium butyricum was

13 investigated throughout the process (Fig. 3). The experiment was performed for 16 days, and

14 syngas impurity effect was investigated through bacterial colony counting. The bacterial cell

15 growth was rapidly increased until the 2nd day and then cells trends to the stationary phase.

16 The maximum cell growth was observed in this stage (Fig. 3). Subsequently, bacterial cell

17 growth reduced rapidly due to utilizing untreated syngas. After that, it was slowly decreased,

18 and it remained steady until the 11th day and bacteria was not visible. On the other hand, lag

19 phase was found when treated syngas was used (Fig. 3) and this phase is responsible for

20 bioethanol production [47]. From this analysis it is found that bacterial cell growth was 500

21 times higher when treated syngas was used instead of untreated syngas. As a result, untreated

22 syngas was strongly affected on the yield of bioethanol during syngas fermentation. In the

23 literature, Xu, Tree and Lewis [19] reported that bacterial cell growth is intensely affected by

24 tar and nitric oxide (NO) containing syngas. The bacterial cell growth can be tolerated if NO

25 in syngas is less than 40 ppm during syngas fermentation [48]. Therefore, impurities were

10
 1 cleaned from syngas before running the fermentation process. A similar effect was also found

2 in the literature as reported by Xu, Tree and Lewis [19].

3 The temperature effect on syngas fermentation was investigated and maximum bacterial

4 growth was found at a temperature of 37 °C (Fig. 4). Hence, syngas fermentation process was

5 done at the temperature of 37 °C for getting highest bioethanol production. This result agreed

6 with the literature value reported by Acharya, Dutta and Basu [42]. The pH effect on the

7 whole fermentation process was observed in this study (Fig. 5). The initial pH was set for

8 Clostridium butyricum was 5.6. The pH level was decreased to 4.4 at the end of syngas

9 fermentation. The maximum pH level was observed when the inoculation of microbes to the

10 fermentation broth after 24 hrs (1st day). After that the pH level was rapidly decreased at

11 5.08. Then, pH level was fluctuating within the ranges of 5.08 to 4.85 from 3rd day to 11th

12 day, and at the 12th day it was reduced to 4.5. Subsequently, the pH level remains unchanged

13 until 16th day (Fig. 5). The ability of acetic acid production is decreased at the pH level of

14 4.5, and bioethanol yield is not increased when pH decreased to 4.5 reported by

15 Asimakopoulos, Gavala and Skiadas [32]. Therefore, bacterial cell tends to use liquid carbon

16 from charcoal that mixed with fermentation broth and gaseous carbon from syngas (CO, CH4,

17 and CO2). The initial medium of pH had significant effects on the metabolism process of

18 Clostridium butyricum. More acidic pH level lead to slower growth rates and lower

19 bioethanol yields also reported by Asimakopoulos, Gavala and Skiadas [32]. Therefore, yield

20 of bioethanol was confirmed by the change of pH throughout the process.

21 The total organic carbon (TOC) analytical results are shown in Fig. 6. The TOC level reduced

22 from initial to the final stage of the experiment. In this study, two main carbon sources were

23 syngas (CO, CO2, CH4) and charcoal (mainly carbon) that used during syngas fermentation.

24 These sources provided carbon nutrient to the Clostridium butyricum. The organic carbon

25 content was decreased from 713 gm/L to 47 gm/L from the 1st day to 16th day (Fig. 6), and

11
 1 Clostridium butyricum received gaseous carbon gradually throughout the whole process. The

2 TOC level was slowly reduced from 713 gm/L to 704 gm/L and 704 gm/L to 546 gm/L, until

3 5th and 11th day, respectively. Subsequently, TOC level was rapidly decreased (from 546

4 gm/L to 145 gm/L) within the 11th to 14th day (Fig. 6). Finally, TOC was 47 gm/L at the end

5 of the experiment. Therefore, Clostridium butyricum was taken carbon nutrient from syngas

6 and charcoal gradually and active upto 16th day and bacteria died due to the decreasing trend

7 of carbon sources. Thus, throughout their lifetime bioethanol was produced, and it was

8 confirmed by GC-MS and NMR (1H) analyses.

9 The effect of syngas composition was observed on before and after fermentation (Fig. 7). In

10 this study, it is evidently found that composition of CO, CO2, and CH4 were gradually diluted

11 with fermentation broth, and bacteria received dissolved carbon substantially. At the end of

12 fermentation, remaining syngas was analysed. It is noticeably revealed that syngas

13 composition was decreased from the initial value, and it was obtained that except CO2, the

14 other two carbon-containing gases (CO and CH4) were dissolved with the broth (Fig. 7).

15 Hence, it is evident that carbon-containing gases (CO, CO2, and CH4) were mixed perfectly,

16 and bacteria was taken gaseous carbon significantly.

17 3.3 Bioethanol Production and Analysis

18 Bioethanol was produced from Clostridium butyricum-based syngas fermentation considering

19 best fermentation conditions. Final product of bioethanol was separated at the end of the

20 syngas fermentation. The bioethanol formation was proved by NMR (1H) and yield was

21 computed by GC-MS analysis (Fig. 8). It was detected by 1H NMR spectrum when syngas

22 was fermented by Clostridium butyricum. From this study, it is distinctly found that

23 bioethanol donates a triplet signal (1.27 ppm), which indicated to methyl group (-CH3) along

24 with adjacent methylene group (-CH2-) (Fig. 8a; Table 2). The presence of methylene group

25 (-CH2-) referred to a quartet signal and peak position of this signal was at 3.75 ppm. It was

12
 1 further checked by methylene group which was connected with an oxygen atom (Fig. 8a).

2 Furthermore, a singlet peak corresponding to 2.20 ppm with one proton integral which

3 indicated the presence of hydroxyl group in the ethanol molecule. These results are agreed

4 with the literature value reported by Zuriarrain, Zuriarrain, Villar and Berregi [49] and Monir,

5 Abd Aziz, Yousuf and Alam [35].

6 GC-MS analysis has been done for final confirmation of bioethanol molecule (Fig. 8b). In

7 this study, it was observed that MS fraction of Clostridium butyricum-based bioethanol was

8 15.02:29.01:31.00:45.01 (Fig. 8b) which were comparable as the standard MS fraction of

9 15:29:31:45. MS for ethanol is 45 and achieved MS value from GC-MS was 45.0 from this

10 study. Moreover, from the fragmentation data, it is also observed that MS 31 is corresponding

11 to the [CH2-OH]+. In addition, fragmentation of 31 is referred to [CH2OH]+ that was changed

12 to more stable cation of [CH3=O]. Furthermore, MS of 15 is corresponding to [CH3]+ and MS

13 of 29 is consistent with [CH3CH2]+.

14 Therefore, it is concluded that Clostridium butyricum generated bioethanol from biomass-

15 based syngas potential for yield product. Charcoal contributed carbon nutrient to bacteria

16 during syngas fermentation. The yield of bioethanol using Clostridium butyricum were 1.50

17 mmol/L, 2.99 mmol/L, 7.48 mmol/L, 14.97 mmol/L and 29.94 mmol/L, from 50mL, 100mL,

18 250mL, 500mL and 1L of syngas, respectively as shown in Fig. 9. Following the approach

19 describe in the literature reported by Martin, Richter, Saha and Angenent [50] and Monir,

20 Abd Aziz, Yousuf and Alam [35]. Furthermore, Acharya, Dutta and Basu [42] reported that

21 the CO fermentation produced 0.17-1.33 g/L of ethanol whereas syngas fermentation

22 produced 0.85-3.75 g/L of ethanol. Therefore, it is confirmed that Clostridium butyricum is

23 suitable for bioethanol production through syngas fermentation in a TFB bioreactor.

24

25 4. Conclusions

13
 1 Syngas fermentation yielded bioethanol, which can be used as a biofuel. The yield of

2 bioethanol produced from Clostridium butyricum based syngas fermentation. The (1H) NMR

3 spectra revealed the corresponding protons from methyle group, CH3- produced by the

4 methylene group, -CH2-. The yield of bioethanol production using Clostridium butyricum

5 were 1.50 mmol/L, 2.99 mmol/L, 7.48 mmol/L, 14.97 mmol/L and 29.94 mmol/L from

6 various amount of syngas (50mL to 1L). Consequently, lignocellulosic biomass of FR and

7 charcoal producing syngas is the potential source of renewable bioenergy in the form of

8 bioethanol for the fulfillment of future energy need. In this study, syngas fermentation

9 process is limited by syngas-to-liquid mass transfer which results low bioethanol

10 concentration. Further kinetic study and techno-economic analysis (TEA) is needed for the

11 evaluation of commercial scale processing plant through this process. This research work can

12 be further extended to optimize the whole process and compared with various potential

13 biomass-based syngas to bioethanol or other chemicals, and its consistency can be increased

14 with the advancement of new models for syngas fermentation. Therefore, the obtained result

15 in this study has a great advantage in the diverse areas of syngas fermentation, and ultimately

16 more efficient in bioethanol production for future energy demand.

17

18 Acknowledgment

19 The authors would like to thanks the Faculty of Engineering Technology, Universiti Malaysia

20 Pahang, Malaysia for providing lab facilities (Cell Culture Laboratory; Chemistry

21 Laboratory). The authors would also acknowledge for the financial support of RDU (Grant

22 No. RDU170120) and GRS (Grant No. PGRS170370) received from Universiti Malaysia

23 Pahang, Malaysia. The authors are very grateful to Dr. Md. Shaheen Sarkar, Dr. Md. Aminul

24 Islam and Mostofa Tarek for their kind cooperation. We are also grateful to editor-in-chief

25 and anonymous reviewers for constructive comments on an earlier version of this manuscript.

14
 1

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16

17

21
1

2 Fig. 1. Experimental setup for bioethanol production through syngas fermentation in a TFB

3 using Clostridium butyricum.

22
1

3 Fig. 2. Cell culture of bacteria: (a) Before inoculation of Clostridium butyricum (b) After

4 inoculation of Clostridium butyricum (c) Cell culture on petri-plate (d) group of colony (e)

5 single colony.

23
1

2 Fig. 3. Effect of treated and untreated syngas on CFU during Clostridium butyricum -based

3 syngas fermentation.

24
1

2 Fig. 4. Temperature effect on syngas fermentation.

25
1

3 Fig. 5. Effect of pH on syngas fermentation.

26
1

3 Fig. 6. Effect of TOC on syngas fermentation.

27
1

2 Fig. 7. Effect of syngas composition before and after syngas fermentation.

28
 (a)

[3]
[2] [1]

(b)

2 Fig. 8. Syngas fermentation using Clostridium butyricum: (a) 1H NMR, 500 MHz (CDCl3): δ

3 = 3.75 (q, J = 7.00 Hz, 2H [CH2]), 2.20 (s, 1H [OH]), 1.27 (t, J = 7.00 Hz, 3H [CH3]) (b)

4 Clostridium butyricum-based bioethanol MS fraction (15.02:29.01:31.00:45.01).

29
1

2 Fig. 9. Bioethanol (mmol/L) production through syngas fermentation using Clostridium

3 butyricum.

30
1 Table 1. Composition of stock solutions for bacterial cell growth.

Medium components Amount (g/L)

Yeast extract 3.0

Lab-lemco powder 10.0

Peptone 10.0

Soluble starch 1.0

Glucose 5.0

Crysteine hydrochochloride 0.5

Sodium chloride 5.0

Sodium acetate 3.0

Agar 0.5

31
1 Table 2. 1H NMR data of bioethanol produced from Clostridium butyricum-based syngas

2 fermentation.

3
1
Peaks H NMR
(δH ppm)
1 1.27 (t, CH3)
2 3.75 (q, OCH2)
3 2.20 (s, OH)
4
5

32
Highlights

• Syngas fermentation was performed in a tar free bioreactor (TFB)

• Syngas and charcoal were used as a substrate of Clostridium butyricum

• Clostridium butyricum is one of the suited microorganisms for bioethanol production

• 500 times higher CFU was found when treated syngas was used instead of untreated

syngas

• Yield of bioethanol was 29.94 mmol from 1L of syngas

Dated: 12 April 2020

To,
The Editor-in-Chief
Renewable Energy

Dear Sir,

We wish to confirm that there are no known conflicts of interest associated with this publication

and there has been no significant financial support for this work that could have influenced its

outcome. We confirm that the manuscript has been read and approved by all named authors and

that there are no other persons who satisfied the criteria for authorship but are not listed. We

further confirm that the order of authors listed in the manuscript has been approved by all of us.

We understand that the Corresponding Author is the sole contact for the Editorial process

(including Editorial Manager and direct communications with the office). She is responsible for

communicating with the other authors about progress, submissions of revisions and final

approval of proofs. We confirm that we have provided a current, correct email address which is

accessible by the Corresponding Author.

Thanking you,

Sincerely Yours,

Assoc. Prof. Dr. Azrina Abd Aziz

Faculty of Civil Engineering Technology
Universiti Malaysia Pahang
26300 Gambang, Kuantan, Pahang, Malaysia
E-mail: azrinaaziz@ump.edu.my