Biofuels

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Ethanol production by syngas fermentation

in a continuous stirred tank bioreactor using
Clostridium ljungdahlii

Bimal Acharya, Animesh Dutta & Prabir Basu

To cite this article: Bimal Acharya, Animesh Dutta & Prabir Basu (2017): Ethanol production by
syngas fermentation in a continuous stirred tank bioreactor using Clostridium ljungdahlii, Biofuels,
DOI: 10.1080/17597269.2017.1316143

To link to this article: http://dx.doi.org/10.1080/17597269.2017.1316143

Published online: 16 May 2017.

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BIOFUELS, 2017
https://doi.org/10.1080/17597269.2017.1316143

Ethanol production by syngas fermentation in a continuous stirred tank

bioreactor using Clostridium ljungdahlii
Bimal Acharyaa, Animesh Dutta a
and Prabir Basub
a
School of Engineering, University of Guelph, Guelph, ON, Canada; bFaculty of Engineering, Dalhousie University, Halifax, NS, Canada

ABSTRACT ARTICLE HISTORY

Ontario biomass could be thermochemically processed by dry and wet torrefaction to produce Received 21 November 2016
high quality solid biofuel. These solid fuels or raw biomass could also be gasified to produce Accepted 17 February 2017
syngas. This study analyzes and demonstrates a successful and efficient way of producing KEYWORDS
bioethanol from syngas fermentation using Clostridium ljungdahlii in a laboratory scale Syngas fermentation;
continuous stirred tank bioreactor having an innovative gas supply and effluent extraction bioethanol; mass transfer;
structures. At the beginning of the experiment, a batch process was conducted to grow Clostridium ljungdahlii
microorganisms and allow the growth of the microorganisms to reach to maximum cell density
in a reactor without supplying a gas. Ethanol production was observed by supplying two
different gas compositions which included 100% CO and simulated syngas, mimicking the
composition of syngas extracted from lignocellulosic biomass having 60% CO, 35% H2, and 5%
CO2. CO and syngas were fermented with different gas flow (5–15 mL/min), effluent flow (0.25–
0.75 mL/min), and media flow rates and stirrer speed (300–500 rpm) at atmospheric pressure
and 378C. The gas flow rate, media and effluent flow rate, pH level, and stirrer speed were
controlled during the fermentation process. The exhaust gas was reused for the improvement
of residence time using a loop-back system for improving the gas–liquid mass transfer.
Excessive foam was observed during the fermentation process which was controlled using
diluted antifoam-204. Maximum cell concentration reached 2.4 g/L. The mass transfer
coefficient showed better performance during syngas fermentation than CO fermentation.
More bioethanol production was observed by syngas fermentation than CO fermentation. CO
fermentation produced 0.17–1.33 g/L-effluent ethanol and 8.92–23.67 g/L-effluent acetic acid
whereas syngas fermentation produced 0.85–3.75 g/L-effluent ethanol and 8.89–14.97 g/L-
effluent acetic acid.

Introduction thermochemical conversion process has many advan-

tages over the biochemical conversion process: the
Biofuels produced from lignocellulosic biomass are
possibility of using 100% lignocellulosic biomass
good options for the future replacement of fossil fuels.
including lignin; independence of feedstock composi-
Bioethanol and biodiesel are produced from lignocellu-
tions; elimination of complex pretreatment and high
losic biomass through biochemical and thermochemi-
enzyme costs; and independence of H2:CO:CO2 ratio in
cal transformation processes. Both processes have
syngas fermentation [5]. However, the main constraints
merits and demerits but the biochemical process is a
of syngas fermentation using biocatalysts are low gas–
more matured and dominating process than the ther-
liquid mass transfer, production of inhibitory products
mochemical process in bioethanol production [1]. Eth-
during fermentation, and low productivity associated
anol produced from syngas fermentation using
with cell density [6]. Another likely limiting factor for
industrial off-gas or natural gas or syngas extracted
ethanol production is its high concentration of undis-
from biomass could be a cheaper feedstock solution
sociated acetic acid in the bioreactor [7].
[2]. There are two paths to produce bioethanol from
Syngas, a mixture of carbon monoxide, hydrogen
syngas, using either a metallic catalyst or a biological
and carbon dioxide, is used in the biochemical conver-
catalyst. Metallic catalysts, either metal based or modi-
sion process in the presence of a biocatalyst or microor-
fied methanol catalysts, are expensive and require
ganism to produce biofuel. Syngas fermentation is
higher operating temperatures and pressures [1,3].
defined as the process to convert syngas into various
Bioethanol produced from syngas using a biological
chemical products like ethanol, alcohol, and organic
catalyst is economical and operable at ambient tem-
acid. Commonly used biological catalysts or microorgan-
perature and atmospheric pressure using low grade
isms in syngas fermentation are Clostridium ljungdahlii,
feedstocks including lignocellulosic biomass and
Clostridium ragsdalei, Clostridium carboxidivorans,
municipal wastes [4,5]. Syngas fermentation using the

CONTACT Bimal Acharya bacharya@uoguelph.ca

© 2017 Informa UK Limited, trading as Taylor & Francis Group
2 B. ACHARYA ET AL.
Clostridium autoethanogenum, and Alkalibaculum bacchi
[7–10]. These biocatalysts play an important role to
metabolize syngas into bioethanol and acetic acid
through the wood–ljungdahl pathway [11]. There are
number of challenges to economically operate continu-
ous fermentation by using biomass extracted syngas in
a bioreactor because it needs complex distillation due
to low ethanol concentration, advanced cleaning of
toxic contaminants in biomass extracted syngas, and
cheaper growth medium or media ingredients [12–15].
The continuous fermentation process is better than
the batch process because continuous flow of nutrition
never allows the decrease of nutrition levels in the
reactor for the microorganism. This continuous flow of
nutrition process favors the continuous growth of the
biocatalyst at the highest concentration level. How-
ever, a batch culture requires more time to re-grow the
fermenting organism after each fermentation cycle.
For commercial ethanol production, batch culture may
be unrealistic. Similarly, a two-stage continuous culture
could be a better option than a single stage-continu-
ous fermentation process because separate treatments
can be made for acetogenesis and solventogenesis
stages like separate pH level, temperature, working vol-
ume level, dilution rate, nutrition level, growth rate, cell
density, and reactor productivity [7].
The mass transfer in substrate and microbes
depends on the amount of dissolution of syngas in the
fermentation media. This process brings in more gas–
liquid interfacial area and retention time in the fermen-
tation media. The mass transfer rates and microbe
accessibility to syngas can also be improved by creat-
ing finer bubbles or decreased rising velocity in the fer-
mentation media with an increase in agitation speed
[5]. To improve the mass transfer of gasses into the liq-
uid medium, different bioreactor designs such as bub-
ble columns, packed columns, trickle beds, hollow
fiber reactors, and continuously stirred tank reactors
with micro-spargers are utilized [6–8,11,16–18]. Richter
et al. concluded that continuous culture has more
advantages than batch culture in syngas fermentation
technology [7]. Even though there is improvement in
the mass transfer by increasing the agitation speed or
gas flow rate, the overall process may not be commer-
cially viable due to high energy consumption or high
power to volume ratio and stress to the biocatalysts
[19,20]. If the amount of cells in the bioreactor is not
sufficient to consume the gas provided, then the
increase in syngas flow rate means an eventual
increase in the loss of syngas at the exhaust system,
resulting in lower gas conversion efficiency. The pro-
ductivity of a biocatalyst decreases with an increase in
stress either by mechanical means or an increase in
cell density [18,21]. In orthodox syngas fermentation
technology, a single orifice or multiple orifices are used
to release the gas into the fermentation broth in both
the batch and continuous fermentation process
[18,22]. Shen et al. and Richter et al. used a hollow fiber
membrane for the gas supply system [7,16]. Trickle
bed reactors (TBR) are used in syngas fermentation to
produce hydrogen using photosynthetic bacterium
e.g. Rhodospirillum rubrum and Rubrivivax gelatinosus;
CH4 using tri-cultures of R. rubrium; acetate using Pep-
tostreptococcus productus; and alcohol using Clostrid-
ium ragsdalei [8]. There are no accessible literatures on
the study of fermentation using a continuous feed gas
loop-back supply system with continuous stirred tank
reactor (CSTR). Hence, this study tries to utilize a simpli-
fied continuous stirred tank bioreactor employing a
novel gas flow with feedback gas supply system and
effluent abstraction schemes to improve gas–liquid
mass transfer, ease inhibitory stress, and increase bioe-
thanol production and compare the productivity of
bioethanol by using carbon monoxide and syngas. Fur-
ther, the influence of gas and liquid flow rates on gas
conversion efficiency, bioethanol yield, acetic acid
yield, pH level, and mass transfer are analyzed.
Mass transfer and product yield
One of the major limitations of syngas fermentation
technology is the low mass transfer between substrate
and microbes. A number of studies were conducted to
enhance the mass transfer [5,7,8,11,16]. Researchers
observed an improvement in mass transfer rates by
improving the residence time of syngas with biocata-
lyst, gas supply system, gas flow rate, reactor design,
and gas supply system design. Increasing the agitation
speed of the stirrer up to the optimum limit improves
the accessibility of the syngas to the biocatalyst by
forming finer bubbles and reducing the rising velocity
in the fermentation media. The mass transfer coeffi-
cients, k,CO a/VLand kL,H2 a/VL are the major parameters
for determining the mass transfer rate which can be
calculated by using following Equations (1) (general)
and (2) (boundary layer theory) [23]:
h i
kL;i :a  V :
1 dni
dt
¼2
L
3 (1)
VL
4Cout;i 5
 Cin;i
Cout;i
ln Cin;i
 sffiffiffiffiffi

VL kL;i :aVL Di
 i ¼ (2)
VL kL;i :aVL j Dj
Similarly, the overall mass transfer coefficient value
can be determined for a hollow fiber membrane by
 
C
Equation (3) [24]. The slope of ln Cout;iout;i
Cin;i vs: time
can be used to determine the mass transfer coefficient:
   
Cout; i Q L
ln ¼ exp kL;i a t (3)
Cout; i  Cin; i VL VL
 BIOFUELS 3

where outlined by Liu et al. [10]:

dni total moles of ethanol produced

¼ instantaneous molar rate of gas componets i Ethanol Yield ¼ total moles of CO consumed
 100% (5)
dt transfered into the medium; mmol 1 mole of ethanol produced
h 6 moles of CO consumed
Maximum cell mass  Initial cell mass
VL ¼ dynamic liquid holdup volume in the Cell Mass Yield ¼
moles of CO consumed
bioreactor; L
(6)
kL; i orj :a VL ¼ volumetric mass transfer coefficient
Total moles of ½Gasi consumed
of supplied gas; h1 ½Gasi utilization % ¼  100%
Total moles of ½Gasi supplied

i ¼ components of composition of gas e:g: CO; H2 ; (7)

CO2 ; CH4
Cin; i ¼ liquid phase concentration of gas reactant i Methodology
in equilibrium by Henry’ s law with the gas at
the inlet of bioreactor, mmol CSTBR fabrication
h
Cout; i ¼ liquid phase concentration of gas reactant i The continuous stirred tank bioreactor (CSTBR) was
0
in equilibrium by Henry s law With the gas designed and fabricated at Bio-Renewable Innovative
at the outlet of bioreactor, mmol
h Lab (BRIL), University of Guelph, ON. The fabricated 3L
Di, Dj = diffusivities for i and j gases e.g. at 37  C, CO laboratory scale low cost reactor (made with transpar-
(3.26 £ 10¡9) and H2 (6.48 £ 10¡9) m2/s, ent PVC pipe type PVC-9002-86-20 for tank and Plexi-
Q = liquid recirculation rate glass sheet type VH-100 Acrylic resin for base and lid)
L = length of gas supply pipe line, m with working volume of 2L broth media is handy, sim-
t = sampling time, s ple, robust, and easy for maintenance, data reading,
a = specific surface area of gas supply system, m2. and cleaning. A low cost innovative gas diffuser was
positioned at 1 cm and on the fringe, and an in situ cell
Equation (1) indicates that an increase in the inlet
retention filter at 15 cm height of the reactor. The reac-
syngas increases the volumetric mass transfer coeffi-
tor was connected to a temperature and pH meter
cient. However, Devarapalli et al. [8] observed that kL,
(PHE-1411, Omega Environmental Inc., Laval, QC, Can-
CO a/VL increased by 24% when the syngas flow rate
ada) and digital pressure gauge (PHH-222, Omega
was doubled while kL,H2 a/VL decreased by 46% which
Environmental Inc., Laval, QC, Canada). A gas flow
may be due to limitations of concentration of hydro-
meter (PMR1-0106018, Cole Parmer Inc., USA) and a
gen by large concentration of liquid phase carbon
micro-pump (GE-F155001, Gilson Inc, USA) of the
monoxide. Orgill et al. concluded that a decrease in
desired rate by using F117938 tubes were connected
the mass transfer coefficient kL,i a/VL for both carbon
to the reactor to monitor the gas flow amount and
monoxide and hydrogen is due to high liquid holdup
insertion/extraction of the media/product. Membrane
volume, VL, a result of the increased liquid recirculation support was positioned at two-thirds level of the biore-
rate [17]. The hydrogenase activity of CO limits the mix-
actor to install the membrane on the higher level of
ing ability of hydrogen gas into the media [25]. Simi-
the fermentation broth. For a continuous supply of
larly, the mass transfer coefficient declines with the
media and extraction of effluent, modified 2L anaero-
rise in media flow rate in a pure CO environment [17].
bic glass jars were used.
Klasson et al. used Equation (4) to determine the
mass transfer coefficient (kL) for a slightly soluble gas-
eous substrate [26]: Microorganism and media preparation
An anaerobic bacterium biocatalyst, Clostridium ljung-
1 dNsG kL a  G 
¼ Ps  PsL
VL dt H
where
NsG ¼ molar substrate transfered from gas phase
ðmolÞ
VL ¼ volume of the bioreactor (L)
PsG and PsL = partial pressure of the gaseous substrate
in gas and liquid phase (atm)
H ¼ Henry's laws constant (L.atm/mol).
During CO and syngas fermentation, the product
yield can also be expressed by using Equations (5–7) as
(4) dahlii, an American Type Culture Collection
(ATCC#55380) was collected from Cedarlane, Burling-
ton, Ontario. Cedarlane supplied the broth media
agents and preparation manual. About 1L of broth
media was prepared by adding 10 g of peptone, 10 g
of beef extract, 3 g of yeast extract, 5 g of salt, 1 g of
soluble starch, 3 g of sodium acetate, 4 ml of 0.0025%
resazurin as an oxygen indicator, and 1000 ml of deion-
ized water. After all ingredients dissolved, the medium
was boiled for 10 min to drive off oxygen. The medium
was cooled by bubbling with oxygen free gas, 0.5 g of
L-cysteine HCl as a reducing agent was added and the
pH adjusted to 6.8. This was dispensed under an
4 B. ACHARYA ET AL.
oxygen free environment and autoclaved at 121 C. The
freeze dried 0.5 ml of biocatalyst was transferred asep-
tically into the broth media in test tubes, cultured for
72 h and disseminated anaerobically in the broth
media jar at 37 C. A Bunson burner was used to create
an aseptic environment, and an incubator was used to
maintain a constant temperature environment. Finally,
the cultured microorganism was transferred anaerobi-
cally into the CSTBR for the syngas fermentation
process [6].
Experimental setup and procedure
A schematic diagram of the experimental setup for
syngas fermentation is presented in Figure 1. All appa-
ratus for the experiment including CSTBR and accesso-
ries were sterilized in an autoclave at 121 C for 20 min.
Nitrogen gas was used inside the reactor to create an
anaerobic environment. Initially, the prepared and ster-
ilized 1.9 L fresh broth media was transferred to the 3 L
CSTBR in a nitrogen environment. An inoculum of
100 mL was added in the medium in the same environ-
ment. The medium plus inoculum was kept airtight. To
drive off the oxygen, nitrogen was continuously
pumped into the reactor with medium for 2 h to create
a completely anaerobic environment. The reactor was
placed in a temperature controlled environment for 48
h to allow propagation of microorganisms in the biore-
actor. The batch was then ready to run in the reactor
with different fermenting gases, carbon monoxide,
and syngas in a continuous fermentation process. Car-
bon monoxide was run in the first batch at the begin-
ning then switched to syngas in the second batch
fermentation processes. After 48 h of each batch, the
reactor was operated continuously for 45 days.
Pure carbon monoxide was supplied to the reactor
in the first cycle and then switched to syngas with 60%
CO, 35% H2, and 5% CO2 at rates of 5–15 mL/min, and
bioethanol production was recorded. Throughout the
Figure 1. CSTBR semi-continuous fermentation setup.
experiment, an anaerobic environment was created at
atmospheric pressure. The pH level was set to 4.5 § 0.5
by using base-NaOH or acid-HCl whenever necessary
and the stirrer speed was adjusted from 300 to
500 rpm [7,18]. The medium and effluent extraction
flow rates were set at 0.5 § 0.25 mL/min. Pressure was
controlled by using a back-pressure regulator in the
reactor. To capture the unwanted exhaust products, a
bubbler was used.
The main aim of syngas fermentation here is to
extract bioethanol from the gasification of biomass
products. The composition of syngas released from the
gasification of all types of biomass consists of CO, H2,
CO2, CH4 and few other gases with tar and ashes [18].
A similar composition of syngas from a gas supplier
was used during the experiment. For the investigation
of products, gas and liquid samples were collected
intermittently. The nutrient level and activities of the
microorganism was maintained by continuously sup-
plying broth media using a micro-pump. The cell mass,
excluding the suspended cells in the bioreactor, could
be captured in a droplet, form a biofilm or be sus-
pended in the medium. Gas flow rates of 5, 10, and 15
mL/min; media flow rates of 0.25, 0.5, and 0.75 mL/
min; and stirrer speeds of 300 and 500 rpm were
tested. Different batches of testing were conducted for
different combinations: Batch 1 and 2 (B11 and B21)
for 5 mL/min gas, 0.25 mL/min media and effluent flow
rate, and 300 rpm of stirrer; Batch 3 and 4 (B12 and
B22) for 10 mL/min gas, all other parameters
unchanged. Batch numbers are numbered as B1x for
the fermentation of carbon monoxide and B2x for the
fermentation of syngas (x = 1–18 signifying each batch
having fresh broth medium with varying gas flow rate,
media/affluent flow rate, and stirrer speed). Cell density
increased to a maximum level with the addition of
fresh medium in each batch. Foam formation was con-
trolled by using diluted antifoam-204 (1/100 mL dilu-
tion with deionized water).

The pH level of the fresh medium was set to 6.8.
After the addition of inoculum, the pH level decreased
rapidly and stabilized at 4.4 § 0.4. This may be due to
formation of number of acidic compounds like propa-
nol-C3H8O, propionic acids-C3H6O2, nonanoic acid-
C9H18O2, benzaldehyde including bioethanol-C2H6O, or
acetic acid-CH3COOH [4,6]. During the culturing of the
microorganism in the fresh medium in the first 48 h,
the pH level changed from 5.8 to 5.0. This may be due
to rapid formation of acetic acid at the beginning. The
pH level lowered from 5.0 to 4.4 during the continuous
process due to formation of bioethanol and acetic
acid. The addition of base-NaOH was made when the
pH level dropped below 4.0 as shown in Figure 1. The
variation of pH level could be due to formation of ace-
tic compounds, and flow of media, affluent, and fer-
menting gas.
Analytical methods
Cell growth
Cell growth of Clostridium ljungdahlii was observed by
quantifying the cell dry weight of it. Three samples
were taken from the bioreactor in each batch and the
average was plotted with standard deviation. Optical
cell density was analyzed using a spectrophotometer
at 600 nm and the cell concentration or cell dry weight
was determined using a standard calibration curve.
Solvent analysis
The amounts of bioethanol and acetic acid were ana-
lyzed using a GC-MS system, equipped with a Bruker
BR-SWAX column (30 m x 0.25 mm internal diameter
with a 0.25 mm phase thickness), gas chromatograph
(7890A) and mass spectrometer (59756MS) of Agilent
Technologies, USA. Oven temperature was set at 44 C
and held for 3.5 min. Intermittent temperature was set
at 200 C with ramp of 5 C/min. Helium carrier gas was
supplied at a rate of 1 mL/min and the sample was
inoculated in an un-fragmented mode at 280 C. The
0.5 mL solvent samples were transferred to 15 mL vials
and incubated at 75 C for 5 min which was then equili-
brated with a 75 mm carboxen-polydimethyle-siloxane
(CAR-PDMS) fiber dipped in a headspace for 20 min.
The volatile compounds were thermally desorbed in
the injector port by manually injecting and exposing
the fiber for 8 min. The mass spectrometer scanned
10–150 m/z at an interval of 1 s. Ionization was formed
by an electronic impact at 200 C, while the transfer
line was kept at 250 C. The +ve ion mode data were
collected and products were extracted physically by
using a SPME holder (Supelco, USA), a hotplate, and a
metal support with clamps.
Gas analysis
Gas samples were analyzed using 8610C GC (SRI Instru-
ments, Torrance, CA) having two columns molecular
BIOFUELS 5
sieve. There were two detectors: a flame ionization
detector and a thermal conductivity detector. A stan-
dard gas mixture was utilized for the standardization
of the analyzer readings. A Hamilton gas tight syringe
(Hamilton Co., Revo, NV, USA) was used to inject
100 mL in a helium gas environment. The gas samples
were collected from the gas inlet and outlet of the bio-
reactor. Helium gas at 2 mL/min was supplied automat-
ically during the experiment from the gas cylinder. Gas
analysis data were collected from the experiment.
Results and discussion
Operation of the bioreactor
The bioreactor was operated continuously at 37 C after
48 h of bacterial growth. Fresh broth medium was
pumped at different liquid flow rates into the bioreac-
tor after achieving an appropriate cell population dur-
ing the first 48 h. The stirrer rate was set at 300 rpm for
one set of continuous process and 500 rpm for another
set of continuous process. During the continuous pro-
cess, liquid flow rates were chosen from 0.25, 0.5, and
0.75 mL/min; and gas flow rates were chosen from 5,
10, and 15 mL/min. The pH of the bioreactor was kept
at 4.0–4.8. During the experiment, in the first week of
the first run, excessive foam was observed in the biore-
actor. Diluted antifoam liquid was used to control the
foam, but all biocatalyst were dead after the addition
of 10 drops of 1/100 diluted antifoam liquid. This may
be due to an excessive amount of antifoam liquid. The
antifoam liquid was 1/100 diluted with deionized water
and added 1–2 drops at a time into the bioreactor
whenever excessive foam was observed. The experi-
ment was repeated without considering any of the pre-
vious data. During the second continuous run, i.e. after
45 days, the experiment had to be repeated due to
loss of microorganism caused by air leakage in the
experimental setup. With an increase in the liquid flow
rate, the increased dilution reduced cell population
and productivity. Out of the three liquid flow rates, the
optimum was 0.5–0.75 mL/min. The optimum condi-
tion for bioethanol production was observed at a stirrer
rate of 300 rpm, gas flow rate of 10–15 mL/min, and
liquid flow rate of 0.25–0.5 mL/min.
Cell concentration and pH
Measurements of cell optical density or cell dry weight
(cell concentration) were taken in each batch when the
condition changed. Cell concentration in the first batch
(B11 and B21) of each run reached a maximum level.
After the first 48 h, the cell concentration never crossed
the cell concentration of first batch (2.4 g/L). This may
be due to the lowering of pH by the formation of acidic
components, stirring, bubbling of gas, and depleting of
nutrition levels of the medium. Nutrition levels of the
6 B. ACHARYA ET AL.
medium were maintained in the reactor by continu-
ously supplying fresh medium to the reactor at a speci-
fied rate for each cycle (or batch). Cell growth rate
decreased with a decrease in pH value and an increase
in temperature, so continuous monitoring of the pH
level and temperature was carried out and the pH level
was maintained by addition of base. The pH of fermen-
tation is a significant parameter in controlling the sub-
strate metabolism and changing the physiological
state. Cell growth, selectivity of product, and metabolic
by-products discharge are affected by pH level [18].
The pH level decreased during the first cycle of the fer-
mentation process due to formation of weak organic
acids which may permeate through the cell wall, accu-
mulate inside the cell, and reduce the inside pH by H+
ions. This phenomenon helps to convert the acetogen-
esis phase to the solventogenesis phase.
During CO and syngas fermentation, pH decreased
rapidly during the first 48 h from 5.80 to 5.0 during the
batch process at a gas flow rate of 5 mL/min, media/
affluent circulation rate of 0.25 mL/min, and stirrer
speed of 300 rpm. The pH level further decreased and
reached an almost stable condition at 4.4 § 0.4 for CO
and syngas fermentation during the continuous pro-
cess as shown in Figure 2. The rapid change in pH level
at the beginning indicates that acid formation is high
which may be due to an increase in formation of acetic
acid. A continuous variation in pH level was observed
throughout the experiment which may be due to a var-
iation in acid formation during the fermentation pro-
cess and disturbances caused to the biocatalyst by
variation of media and affluent in/out process, stirrer
speed, and gas flow rate. Lowering the pH (indicating
the formation of acetic acid) triggered a transformation
in cell metabolism from acetogenesis to solventogene-
sis. Cell concentration and ethanol yield are directly
proportional to pH variation. The pH level is always at
the higher level during syngas fermentation than CO
Figure 2. pH profiles and cell concentration during CO and syngas fermentation.
fermentation. A similar observation was found with cell
concentration. This may be due to higher formation of
acetic acid during CO fermentation than syngas
fermentation.
Product profiles
The acetyl-CoA biochemical or wood–ljungdahl path-
way is followed by Clostridium ljungdahlii for cell
growth, acetic acid formation, and adenosine triphos-
phate (ATP) during the growth stage of syngas fermen-
tation at pH 4–7 [27]. The conversion of acetyl-CoA to
acetate needs two major steps, where the first step fol-
lows conversion from acetyl-CoA to acetate then acet-
aldehyde with reduced ferredoxin and finally to
ethanol via alcohol dehydrogenase (ADH), and the sec-
ond step follows direct conversion of acetyl-CoA to
acetaldehyde then to ethanol by reducing acetalde-
hyde [28]. Adjusting the pH level by adding base to the
bioreactor causes ethanol production to be improved
but the growth of unwanted acetic acid was also
observed. Acetic acid production during CO fermenta-
tion is more than acetic acid formation during syngas
fermentation. More acetic acid formation during CO
fermentation may be due to the less conversion of ace-
tic acid in to the ethanol caused by thin layer formation
or by low reactivity with CO or high CO flow rate.
In each batch of continuous process, the products
are bioethanol and acetic acid. Production of bioetha-
nol and acetic acid increased with cell concentration,
CO and syngas utilization in a linear pattern, which
increases or decreases according to pH level, cell con-
centration level, gas flow, and liquid flow. This signifies
that continuous production of bioethanol and acetic
acid occurs in each cycle. Each batch of cycle was
refreshed after 120 h by introducing fresh broth media.
The production of bioethanol varied from 0.30 § 0.07
to 1.3 § 0.05 g/L and acetic acid varied from 8.60 §

0.40 to 23.67 § 0.80 g/L during CO fermentation
whereas production of bioethanol varied from 0.80 §
0.02 to 3.75 § 0.08 g/L and acetic acid varied from 5.20
§ 0.80 to 14.20 § 0.50 g/L during syngas fermentation.
In the continuous extraction process, when the stirrer
speed is set at 300 rpm (B11–B19 and B21–B29), the
production of ethanol and acetic acid batch was from
0.41 to 1.33 g/L in B11–B19 and from 0.92 to 3.75 g/L
in B21–B29. In each gas flow with the 300 rpm stirrer,
ethanol production increased with the increase in liq-
uid flow (media in and affluent out) rate. When the gas
flow doubled from 5 to 10 mL/min, ethanol production
increased by 58% during CO fermentation, whereas
49% during syngas fermentation.
The acetic acid formation is more at the beginning
of the experiment. Such acetic acid formation contin-
ued during the continuous processes. This may be due
to biofilm formation and acetogenesis process. Liquid
flow rate varied from 0.25 to 0.75 mL/min; there was
11–29% variation in bioethanol and acetic acid forma-
tion with variation of the liquid flow. This may be due
to an increase in nutrition to the cells and an increase
in gas–liquid interaction. Similarly, an increase in the
gas flow rate increased production of bioethanol by
30–60% and increased production of acetic acid by 3%
to 40%.
From the plot of the above experimental results and
data, the following models were extracted to represent
bioethanol and acetic acid production:
YCO ¼ 8  1012 t4  2  108 t3 þ 2  105 t 2
 0:0044 t þ 0:73
R2CO ¼ 0:924
16 6 12 5 9 4
ZCO ¼ 5  10 t  2  10 t þ 3  10 t
6 3
 3  10 t þ 0:0011 t  0:187 t
2 converted into ethanol, acetate and carbon dioxide
þ 19:614
R2CO ¼ 0:9945
11 4 8 3 6
YSyngas ¼  1  10 t þ 2  10 t  9  10
þ 0:0036 t þ 0:615
R2Syngas ¼ 0:987
ZSyngas ¼  3  1013 t5 þ 1  109 t 4  1  106 t3
þ 5  103 t2  0:0814 t þ 13:18
R2Syngas ¼ 0:9862
where
Y = bioethanol yield in g/L either by CO or syngas
fermentation
Z = acetic acid yield in g/L either by CO or syngas
fermentation
BIOFUELS 7
t = time duration of continuous fermentation in bio-
reactor (48–1088 h)
R = R-squared value or coefficient of determination
Suffix = production from CO or syngas e.g. YCO
means bioethanol from carbon monoxide.
R-squared value indicates that the proposed model
is valid or near to the experimental results. Figures 3a
and 3b show that bioethanol production increased
with an increase in the gas flow rate up to an optimal
flow rate. The optimum gas flow and liquid flow/
extraction rate for the production of bioethanol was
found to be 10–15 mL/min and 0.25–0.5 mL/min. How-
ever, liquid flow rate and stirrer speed has less impact
on bioethanol production than gas flow rate. Acetic
acid formation was almost proportional to bioethanol
formation. However, the production of acetic acid was
higher with higher gas flow rate during CO fermenta-
tion and lower during syngas fermentation. This may
be due to more effective solventogenesis during syn-
gas fermentation than CO fermentation. The maximum
bioethanol during CO fermentation was 1.33 g/L at 10
mL/min gas flow rate, 0.25 mL/min liquid flow rate,
and 300 rpm. The maximum bioethanol during syngas
fermentation was 3.75 at 15 mL/min gas flow rate, 0.25
mL/min liquid flow rate, and 300 rpm. Acetic acid for-
mation reached 23.67 g/L during 15 mL/min gas flow
rate, 0.75 mL/min liquid flow rate, and 300 rpm during
CO fermentation whereas acetic acid formation was
14.97 mL/min during syngas fermentation at 10 mL/
min gas flow rate, 0.25 mL/min liquid flow rate, and
300 rpm.
(8a)
(8b) Product formation and gas conversions
During the fermentation process, carbon monoxide is
through the wood–ljungdahl pathway using biocatalyst
(9a) Clostridium ljungdahlii. Supplied gas carbon monoxide
and hydrogen are useful for the cell growth of the
(9b)
microorganism. The concentration of carbon monoxide
t 2
continuously decreases with the increase in concentra-
tion of carbon dioxide [16,18]. Carbon dioxide may
(10a) react with the hydrogen as an electron donor to yield
(10b) ethanol and acetate.
(11a) i. 6CO þ 3H2 O ! CH3 CH2 OH þ 4CO2
ii. 4CO þ 2H2 O ! CH3 COOH þ 2CO2
(11b) iii. 4CO þ 6H2 ! 2CH3 CH2 OH þ 2CO2
iv. 2CO2 þ6H2 ! CH3 CH2 OH þ 3H2 O
v. 2CO2 þ4H2 ! CH3 COOH þ 2H2 O
Hydrogen intake increased after a couple of days of
operation of the continuous fermentation process,
which confirmed the utilization of homoacetic fermen-
tation pathway to produce anaerobic oxidation of
hydrogen and carbon dioxide by Clostridium ljungdahlii
8 B. ACHARYA ET AL.
Figure 3. (a) Bioethanol yield during CO and syngas fermentation; (b) acetic acid yield during CO and syngas fermentation.
[18]. Cell activity was governed by the fermentation
pH, concentration of enzymes, substrates, cofactors,
effectors and inhibitors. Richter et al. stated that etha-
nol production can be improved by using the alde-
hyde:ferredoxin oxidoreductase and ADH route, which
consist of the requisite manufacturing of acetate and a
further reduction of un-dissociated acetic acid to etha-
nol [29]. The provision of more electrons into the cul-
ture in the presence of 5.07 mM of cysteine-HCl in the
medium having a pH of 5.9 increases ethanol produc-
tion by 48% [30].
Ethanol production increased with an increase in
the consumption of CO and H2. However, acetic acid
production decreased with an increase in ethanol pro-
duction as shown in Figure 4a. Maximum CO consump-
tion occurs when maximum ethanol production is
reached. The production of ethanol and acetic acid not
only depends on syngas conversion but also on pH, liq-
uid flow, and gas flow. The variability observed may be
due to the variation of other factors. Similarly, during
the CO fermentation, bioethanol production increased
with an increase in CO conversion and achieved

Figure 4. (a) Bioethanol, acetic acid, CO and H2 conversion during syngas fermentation; (b) Bioethanol, acetic acid, and CO conver-
sion during CO fermentation.
highest levels at 75% CO conversion. However, acetic
acid production increased with CO conversion which
may be due to thin film formation at the top of the bio-
reactor or less activities of the microorganism during
the CO fermentation process (see Figure 4b). During
CO fermenation, the production of ethanol is lesser
than the acetic acid. Pressure buildup during the fer-
mentation process is due to an increase in the gas sup-
ply rate which also retards cell activities. Experimental
results show that the utilization of hydrogen is less
than the amount of CO during syngas fermentation.
This matches thermodynamically because electron
BIOFUELS 9
production from CO is more suitable than H2 without
dependency on gas partial pressure and pH value [31].
Younesi et al. found that a higher amount of hydrogen
is used after depletion of carbon monoxide during syn-
gas fermentation using Clostridium ljungdahlii [32].
However, such a situation was never experienced here
due to continuous supply of syngas from the same
tank.
Due to the higher concentration of cells at the
beginning, hydrogen consumption was more at the
beginning than in later stages/cycles. However, con-
sumption declined slightly because of the decrease in
10 B. ACHARYA ET AL.
cell concentration. This change in cell concentration
was due to the change in the environment including
pH level. However, CO conversion increased slightly
with ethanol production during syngas fermentation.
Cells consume CO as it starts to flow in the bioreactor
and reduces the inhibition effect of CO on hydroge-
nase. CO conversion decreased with an increase in ace-
tic acid production during CO fermentation. This may
be due to an increase in CO flow. The experiment was
conducted with a loop-back system which increased
the consumption of CO and H2 due to double interac-
tion of the gas with the cell-liquid of the bioreactor.
The liquid flow rate was changed every 120 h, but
keeping same stirrer and gas flow. The ethanol
Figure 5. (a) Comparative study of ethanol yield-CO vs. syngas fermentation (Batch 1–9); (b) Comparative study of ethanol yield-CO
vs. syngas fermentation (Batch 10–18).
production varies upto 22% with the liquid flow and
43% with the gas supply.
Product yields and comparisons
The cell density reaches at maximum during the cell
growth during first 48 h of experiment. After 48 h, the
CO and syngas supplied then the ethanol and acetic
acid formation starts in the bioreactor. The measure-
ments are carried out at different stirrer speed of
300 rpm and 500 rpm to observe the effect of liquid
flow and gas flow. Gas flow rate has more impact on
ethanol and acetic acid production than liquid flow
rate (Figures 5a–6b). Ethanol production increased by

Figure 6. (a) Comparative study of acetic acid yield-CO vs. syngas fermentation (Batch 1–9); (b) Comparative study of acetic acid
yield-CO vs. syngas fermentation (Batch 10–18).
29–41% when liquid flow rate was doubled or tripled.
Ethanol production increased by 85–151% when gas
flow rate doubled. However, ethanol production
declined after an increase in gas rate more than 100%.
Acetic acid production increased 18–22% by increasing
liquid flow rate, and increased by 82% when gas flow
rate increased by 100–200%. This may be due to the
disturbance of excessive flow of gas or formation of
acetic acid due to the continuous supply of fresh
media or formation of biofilm on the bioreactor. Acetic
acid formation declined during syngas fermentation
whereas it continued to grow during CO fermentation.
During CO fermentation, maximum ethanol production
was 1.33 g/L during batch 1–9 at 10 mL/min gas flow,
BIOFUELS 11
0.5 mL/min liquid flow at 300 rpm; and 1.19 g/L during
batch 10–18 at 10 mL/min gas flow, 0.25 mL/min liquid
flow at 500 rpm. During syngas fermentation, maxi-
mum ethanol production was 3.75 g/L during batch 1–
9 CO fermentation at 15 mL/min gas flow, 0.25 mL/min
liquid flow at 300 rpm; and 3.05 g/L during batch 10–
18 at 10 mL/min gas flow, 0.75 mL/min liquid flow at
500 rpm. The optimum flow rate for syngas fermenta-
tion was 10–15 mL/min and liquid flow at 0.5–0.75mL/
min and stirrer speed above 300 rpm but below
500 rpm. CO fermentation produced less ethanol and
more acetic acid so is not recommended to produce
ethanol. However, it could be a good option for acetic
acid production.
12 B. ACHARYA ET AL.

Table 1. Ethanol, acetic acid concentration andgas conversion efficiency in syngas fermentation.
CO fermentation with feedback loop (B1X) Syngas fermentation with feedback loop (B2X)
Stirrer CO flow Liquid flow CO CO Syngas Syngas CO H2
Batch speed rate mL/ rate mL/ bioethanol CO acetic conversion Syngas flow bioethanol g/ acetic acid conversion conversion
X Hours rpm min min g/L acid g/L % rate mL/min L g/L % %
1 0–120 300 5 0.25 0.41 8.92 60 5 0.92 9.14 57 59
2 121–240 300 5 0.5 0.53 10.11 67 5 1.34 11.23 63 61
3 241–360 300 5 0.75 0.75 12.15 71 5 1.55 13.44 66 45
4 36W80 300 10 0.25 0.76 14.44 60 10 1.89 14.97 70 43
5 481–600 300 10 0.5 1.33 13.88 75 10 2.84 13.88 68 42
6 601–720 300 10 0.75 1.28 14.57 68 10 3.11 12.09 71 47
7 721–840 300 15 0.25 1.32 16.27 55 15 3.75 11.64 79 38
8 841–960 300 15 0.5 1.13 18.22 57 15 3.61 10.99 75 41
9 961–1080 300 15 0.75 1.07 21.53 56 15 3.05 10.41 74 42
10 0–120 500 5 0.25 0.49 9.12 65 5 1.09 8.89 68 55
11 121–240 500 5 0.5 0.57 11.99 71 5 1.12 10.43 66 39
12 241–360 500 5 0.75 0.64 12.61 65 5 1.47 12.91 68 43
13 361–480 500 10 0.25 1.19 15.87 64 10 2.34 14.08 71 42
14 481–600 500 10 0.5 1.02 19.34 66 10 2.47 13.72 72 42
15 601–720 500 10 0.75 0.97 20.09 68 10 3.05 11.75 75 41
16 721–840 500 15 0.25 0.95 18.45 62 15 2.87 11.01 69 37
17 841–960 500 15 0.5 0.86 21.21 55 15 2.54 11.23 67 39
18 961–1080 500 15 0.75 0.81 23.67 58 15 2.12 11.34 65 45

The present results as shown in Table 1 match with
other publications. Shen et al. reported that ethanol
production increases with an increase in gas flow rate
up to a point, then decreases with a further increase in
gas flow rate [16]. Shen et al. also observed a reduction
of bioethanol concentration with an increase in liquid
flow rate due to an increase in cell dilution. They sug-
gested 10–15 mL/min could be the optimum limit of
gas flow rate for CO and syngas fermentation for a bio-
reactor. Obviously, gas flow rate varies with the design
parameters of the bioreactor. Bredwell et al. and Klas-
son et al. observed that gas conversion is adversely
affected by high gas flow during the fermentation pro-
cess [20,33]. Alsaker et al. and Ungerman and Heindel
observed lower ethanol concentrations by increasing
the gas flow rate and stirrer speed due to an increase
in stress on the biocatalyst [19,34]. The ethanol produc-
tivity of the fermentation was improved by lowering
the cell dilution, i.e. by lowering the liquid flow rate
[7,35]. However, acetic acid production increased even
with an increase in media flow rate and gas flow rate
during the CO fermentation process after the optimum
limit of flow rates [16].
The results of the present investigation are com-
pared with results of others in Table 2. Acetic acid for-
mation in the present research is higher than other
findings during the CO fermentation process. In almost
all observation, syngas fermentation shifts from aceto-
genic phase to solventogenic phase with the decreas-
ing pH level of the culture [9].
From this study (Table 2), the maximum concentra-
tions of ethanol and acetic acid were 1.33 and 23.67 g/
L during the CO fermentation and 3.75 and 14.97 g/L
during syngas fermentation. Maximum ethanol and
acetate were 7.52 and 3.43 g/L [32] during CO fermen-
tation and 9.6 and 6.1 g/L [38] during syngas fermenta-
tion in a CSTR using Clostridium ljungdahlii as
biocatalyst. Richter et al. reported the ethanol produc-
tion as 2.2 g/L and Younessi et al. reported 0.55 g/L
using C. ljungdahlii in a stirred tank reactor [32].
Devarapalli et al. reported 5.7 g/L of ethanol using a
TBR with the same biocatalyst [8]. The highest produc-
tion of ethanol reported was 23.93 g/L by Shen et al.
during syngas fermentation using a hollow fiber mem-
brane reactor and the same biocatalyst [16]. Aghbashlo
et al. observed the maximum exergy efficiency of a
continuous bioreactor at 8 exergetic productive index
at media flow rate of 0.55 mL/min [36].
Mass transfer efficiency, which depends on the reac-
tor configuration, agitation speed, gas flow, and media
flow rate, is another important technical parameter to
improve ethanol productivity [5,7,16]. Hollow fiber
membrane is an effective option to improve gas–liquid
mass transfer [7,16]. The bubbling size and retention
time of syngas in the fermentation media can also
improve gas–liquid mass transfer. These factors are
dependent on the bioreactor design. Hence, bioreactor
design is a primary factor to improve gas–liquid mass
transfer. The position of the gas supply system in the
bioreactor may also have an impact on gas retention
time or gas–liquid mass transfer. Another limiting factor
of ethanol production during syngas fermentation is
the characteristics of the biofilm [16]. Double gas supply
throughout the fringe of the reactor enhances the gas
retention time compared to the gas supply system
installed at the center of the reactor. This may be due
to the greater velocity of the gas bubbles at the fringe
than at the center. Prior to the rupturing of the bubble
on the surface layer of the media on the bioreactor, the
peripheral gas bubble travels horizontally and voyages
longer time and distances. A gas loop-back system
improves gas–liquid mass transfer by improving reten-
tion time (due to improving the travel time of the bub-
bles) better than a single gas supply system. Sudiyo and
Andersson observed the lateral movement of bubbles
towards the center due to stirrer vortex [37].
Cell retention and growth of the cell depends on the
nutrition of the media. A continuous flow of fresh
media also helps with cell density. Maintaining cell
density improves bioethanol production. The higher
Table 2 . Product yield with different reactors for CO fermentation and syngas fermentation.
CO fermentation Syngas fermentation
Reactor Biocatalyst Operation Yield (g/L) Ref Reactor Biocatalyst Operation Yield (g/L) Ref
Continuous stirred tank Clostridium ljungdahlii Yeast medium Eth:1.33 This study Continuous stirred tank Clostridium ljungdahlii Yeast medium Eth:3.77 This
pH: 4.6 AA:23.67 pH: 4.6 AA:14.97 study
Continuous stirred tank Clostridium ljungdahlii pH: 4 Eth: 7.52g/L [32] Two stage continuous still Clostridium ljungdahlii pH: 4.75 Eth:2.2 [7]
Temp: 37 Ac: 3.43g/L tank Temp: 37 AA:1.9
Cell density: 10g/L
Continuous stirred tank Butyribacterium pH: 6–6.8 Acetate: 7.96 [32] Continuous stirred tank Clostridium ljungdahlii pH: 4.5 Eth+ AA:11 Cell density: [43]
methylotrophicum Temp: 37 mmol/L Temp:37 2g/L
Butyrate: 5.95 g/L
Gas lift reactor Eubacterium limosum pH: 6.8 Acetate: 0.1mmol/ [38] Continuous stirred tank Clostridium ljungdahlii - Eth:0.55 AA:1.3 [32]
Temp: 37 L Cell density: 10g/L
Eth: 38mmol/L
Continuous gas feed Clostridium pH:4.75 Eth: 0.91 [9] Stirred tank Clostridium ljungdahlii Yeast medium Eth:0.2 [22]
autoethanogenum Tungstem AA: 0.91 pH: 5.5 AA:2.2
Continuous CO feed Clostridium pH: 5.75 Eth: 5.55 [39] Trickle bed co-current Clostridium ragsdalei Yeast Extract, pH:46 Eth:5.7 [8]
carboxidivorans Butanol: 2.66 AA:12.3
Optimized medium and one single Clostridium pH: 4.75–5.75 Eth: 4.26 [40] Trickle bed counter-current Clostridium ragsdalei Yeast Extract, pH:46 Eth:3.0
gas-fed autoethanogenum AA: N/A AA:5.3
Gas lift reactor Eubacterium limosum pH: 6.8 Eth:0.2 [41] Stirred tank Clostridium ragsdalei Corn Steep Liquor, pH: no Eth:9.6 [38]
Temp: 37 AA:2.2 control AA:6.1
Cell density:
0.75g/L
Continuous stirred tank Carboxydothermus 6.8-7.0 Eth: 0.09mM [42] Stirred tank Clostridium Yeast Medium, pH: No Eth:2.2 [44]
hydrogenoformans Ac:0.51mM carboxidivorans control AA:1.9
Hollow fiber membrane Clostridium Yeast Medium, pH: 4.5–5.5 Eth: 23.93 [16]
stirred tank carboxidivorans AA: 4.79
Stirred tank Clostridium Modified Mineral Medium Eth:2.2 [9]
autoethanogenum pH: 4.75 AA:1.9
BIOFUELS
13
14 B. ACHARYA ET AL.
Figure 7. (a) Batch comparison of ethanol yield-CO vs. syngas fermentation (Batch 1–18); (b) Batch comparison of acetic acid yield-
CO vs. syngas fermentation (Batch 1–18).
ethanol concentration observed may be due to an
increase in the gas–liquid mass transfer by improving
the retention time due to the feedback gas flow system
and continuous extraction system.
The concentration profile of ethanol and acetic acid
throughout the experiment from batch B101–118 and
B201–218 are presented in Figures 7a and b. The con-
centration of acetic acid increased almost linearly for
stirrer speeds 300 and 500 rpm during the CO fermen-
tation; concentration of acetic acid is maximum for
batch B205 and B214. Ethanol production was slightly
better at 300 rpm than 500 rpm (Figures 7a,b). This
may be due to disturbance of the Clostridium
ljungdahlii resulting in less cell productivity. Ethanol
production at either stirrer speed increased with an
increase in liquid flow rate and gas flow rate. Liquid
flow rate has less impact on ethanol and acetic acid
production than gas flow rate in both fermentation
processes. Ethanol production started declining after
Batch #107 and #115 during syngas fermentation and
after Batch #105 and #112 during CO fermentation.
This may be due to declining acetic acid in the syngas
fermentation and increasing acetic acid formation in
the CO fermentation process. Acetogenesis is more
pronounced in CO fermentation, whereas solventogen-
esis is more pronounced in syngas fermentation.

Mass transfer analysis
The mass transfer coefficient of CO, syngas-CO, and
syngas-H2 was plotted and compared as in Figure 8.
Mass transfer is always higher in syngas-CO than CO
alone. The mass transfer coefficient increased with an
increase in run time and reached a maximum at B27
with 34.02 (1/h) during syngas fermentation, and B15
with 24.74 (1/h) during CO fermentation. Klasson et al.
and Bredwell et al. observed mass transfer coefficients
up to 35.5 (1/h). The mass transfer coefficient declined
during later batches. This may be due to formation of
more acetic acid in the CO fermentation than syngas
fermentation. The mass transfer coefficient increased
up to 38% with syngas-CO and 30% with CO with an
increase in the gas flow rates; and decreased up to
20% with syngas-CO and up to 11% with 100% CO in
tripling the liquid flow rate. Cell activity and gas uptake
also affected the coefficient. This may be due to an
increase in volume VL. The depleted nutrition level
decreased the cell density which consequently
decreased the mass transfer coefficient.
The major limiting factor in the fermentation pro-
cess is the potential bottleneck of the mass transfer of
gas into liquid. This is more severe for carbon monox-
ide, hydrogen, and its mixture in comparison with the
aerobic process because the solubility of CO and H2 on
mass basis with respect to oxygen is only 60% and 4%
respectively [20]. Mass transfer limitation may arise
due to gas–liquid interface, transport of gas to media,
diffusion of gas into the liquid layer, and diffusion of
the gaseous substrate to the intracellular reaction site
[45]. Improvements in impeller designs, liquid flow pat-
terns, mixing residence time, baffle design, aerated
power efficiency, and application of bubble dispersers
are but a few parameters to enhance the mass transfer
capability [5].
Figure 8. Mass transfer coefficient of CO, syngas (CO) and syn-
gas (H2).
BIOFUELS 15
To enhance the mass transfer, the presence of gas
has been increased by the gas loop-back system and
the cell density remains almost at the steady state due
to a continuous supply of nutrition with replacement
of fresh media. Gas–liquid mass transfer can be
improved by using a proper size of gas bubble diame-
ter because it enhances the specific surface area avail-
able for mass transfer. The present gas orifice system
generates small bubbles travelling slowly in a circular
fashion which increases the gas residence time in the
bioreactor and improves gas–liquid mass transfer
which then improves the mass transfer coefficient.
Orgill et al. noted a decrease in gas–liquid mass trans-
fer coefficient of CO and H2 with an increase in liquid
recirculation rate or liquid holdup volume VL [17]. Simi-
lar observations were found at higher liquid flow rates
during this experiment.
Conclusions
Continuous production of bioethanol in addition to
acetic acid from carbon monoxide fermentation and
syngas fermentation using a biocatalyst Clostridium
ljungdahlii in a designed and developed economical
continuous stirred tank bioreactor has been proved
successfully. We compared bioethanol production
using two different gases (CO and syngas) using an
innovative gas loop-back system. In both fermentation
processes, bioethanol production was closely moni-
tored by varying pH level, broth media flow rates, gas
flow rates, and stirrer speed. The pH level impacts bioe-
thanol production, gas consumption, and cell growth
environment. Maximum bioethanol production was
1.33 g/L during CO fermentation at 10 mL/min gas
flow, 300 rpm, 0.5 mL/min liquid flow and 3.75 g/L dur-
ing syngas fermentation at 15 mL/min gas flow,
300 rpm, 0.25 mL/min liquid flow. Similarly, the maxi-
mum acetic acid production was 23.67 g/L during CO
fermentation at 15 mL/min gas flow, 500 rpm, 0.75 mL/
min liquid flow and 14.97 g/L during syngas fermenta-
tion at 10 mL/min gas flow, 300 rpm, 0.25 mL/min liq-
uid flow. The higher amount of acetic acid is due to the
continuous process where the fresh broth media (addi-
tion of nutrients) is supplied continuously in the biore-
actor. The addition of nutrients favors the growth of
the microorganism. The maximum gas consumption
observed was 75% during CO fermentation and 79%
during syngas (65% CO, 30% H2, and 5% CO2) fermen-
tation. The syngas fermentation process is better than
the CO fermentation process for bioethanol production
because this process produced more ethanol. Further
research is recommended for the conversion of acetic
acid into bioethanol either by replacing the gas supply
system or by improving the engineering design param-
eters of the bioreactor to improve the mass transfer
coefficient.
16 B. ACHARYA ET AL.
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
Animesh Dutta http://orcid.org/0000-0002-9995-807X
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