Bioresource Technology 172 (2014) 41–49

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Enrichment and optimization of anaerobic bacterial mixed culture for

conversion of syngas to ethanol
Ashish Singla a,b, Dipti Verma b, Banwari Lal a,b, Priyangshu M. Sarma a,b,⇑
a
TERI University, 10 Institutional Area, Vasant Kunj, New Delhi 110 070, India
b
TERI, Darbari Seth Block, India Habitat Centre, New Delhi 110 003, India

h i g h l i g h t s

 Anaerobic bacterial mixed culture enriched for conversion of syngas to ethanol.

 Operational parameters optimized for enhancing ethanol production from syngas.
 Semi-continuous fermentation study done for getting increased ethanol production.
 Up-scaling studies done for further enhancing ethanol production from syngas.

a r t i c l e i n f o a b s t r a c t

Article history: The main aim of the present study was to enrich anaerobic mixed bacterial culture capable of producing
Received 13 June 2014 ethanol from synthesis gas fermentation. Screening of thirteen anaerobic strains together with enrich-
Received in revised form 18 August 2014 ment protocol helped to develop an efficient mixed culture capable of utilizing syngas for ethanol pro-
Accepted 19 August 2014
duction. Physiological and operational parameters were optimized for enhanced ethanol production.
Available online 27 August 2014
The optimized value of operational parameters i.e. initial media pH, incubation temperature, initial syn-
gas pressure, and agitation speed were 6.0 ± 0.1, 37 °C, 2 kg cm2 and 100 rpm respectively. Under these
Keywords:
conditions ethanol and acetic acid production by the selected mixed culture were 1.54 g L1 and 0.8 g L1
Syngas
Mixed culture
respectively. Furthermore, up-scaling studies in semi-continuous fermentation mode further enhanced
Optimization ethanol and acetic acid production up to 2.2 g L1 and 0.9 g L1 respectively. Mixed culture TERI SA1
Ethanol was efficient for ethanol production by syngas fermentation.
Up-scaling Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction
Synthesis (syngas) gas, primarily a mixture of CO, CO2 and H2 is
a major feedstock in production of many fuels and chemicals. It can
be produced from gasification of several materials such as coal,
wood, municipal solid waste and lignocellulosic biomass (Phillips
et al., 1993). The essential syngas components CO, CO2 and H2
can be biologically converted into ethanol and other value added
compounds such as acetic acid, 2-butanol, n-propanol and polyhy-
droxyalkanoate (PHA) (Kundiyana et al., 2010). Catalytic processes
Abbreviations: PHA, polyhydroxyalkanoate; PBM, Pfennig’s basal media; TCD,
thermal conductivity detector; FID, flame ionization detector; OD, optical density;
CO, carbon monoxide; H2, hydrogen; rs, resparged; AA, acetic acid; EtOH, ethanol;
SD, Sludge; CD, cow dung; CF, chicken faeces; VFA, volatile fatty acids.
⇑ Corresponding author at: TERI University, 10 Institutional Area, Vasant Kunj,
New Delhi 110 070, India. Tel.: +91 11 24682100; fax: +91 11 24682144.
E-mail address: priyanms@teri.res.in (P.M. Sarma).
are also used to convert syngas components into a variety of fuels
and chemicals such as hydrogen, methane, methanol, ethanol, and
acetic acid (Klasson et al., 1992). Biological processes, although rel-
atively slower than chemical reaction have several advantages over
catalytic process such as elimination of expensive metal catalyst,
higher specificity of biocatalyst, lower energy costs, greater resis-
tance to catalyst poisoning and independence of H2/CO ratio
(Wolfrum and Watt, 2002). The biological reaction occurs under
ambient condition of pH, temperature and pressure with the for-
mation of specific products. However, direct production of fuels
and chemicals from gasification technology is economically unfa-
vorable and requires very large infrastructure as about 60% of the
total investment cost in modern methanol plants is accounted for
syngas generation (Vannby and Winter Madsen, 1992). Therefore,
the economical advantage of biological processes through develop-
ment of suitable biocatalyst to ferment gaseous substrate to valu-
able products can be considered. Previous study indicates that FT
process has a relative overall energy efficiency of 45%, while gas

http://dx.doi.org/10.1016/j.biortech.2014.08.083
0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
42 A. Singla et al. / Bioresource Technology 172 (2014) 41–49
fermentation has an overall energy efficiency of 57%, in terms of
energy in feedstock converted to final product (Griffin and
Schultz, 2012).
Microorganisms act as biocatalysts to convert syngas into
chemicals and fuels. Anaerobic bacteria such as Clostridium
ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans,
Clostridium aceticum, Acetobacterium woodii and Clostridium ther-
moaceticum convert biomass generated syngas composed of CO,
CO2 and H2 to ethanol and acetic acid (Kundiyana et al., 2010).
Bioethanol production through acetogenic fermentation of the
gaseous substrates (CO, CO2 and H2) follows the Wood-ljungdahl
and acetyl-CoA pathways. Syngas fermentation for ethanol pro-
duction based on the acetyl-CoA metabolic pathway is an emerg-
ing area and the process require significant research
interventions. Several research groups have explored the use of
anaerobic bacteria to convert syngas to ethanol (Gaddy and
Clausen, 1992; Younesi et al., 2005). However, these studies have
yet to define a methodology for generating high ethanol produc-
tion levels with a stable culture. The selection of appropriate
microbes for efficient syngas fermentation is often a challenging
task. Therefore, the isolation and engineering of new microbial
species, which are more productive and robust, need to be
employed. Results from published literature show that tempera-
ture, pH, reducing agents, redox potential, agitation speed, gas
partial pressure, gas compositions and media components have
significant effects on cell growth and ethanol production (Hurst
and Lewis, 2010). Optimization of these parameters is critical
for assessing the potential sustainability of ethanol from syngas.
In the recent years, a general understanding regarding cell
growth and metabolism leading to higher conversion of syngas
to liquid fuel has been made. Syngas fermentation using Clostrid-
ium P7 showed that increase in CO partial pressure from 0.35 to
2.0 atm. resulted in 440% improvement in cell growth and etha-
nol production shifted from non-growth associated to growth
associated phase (Hurst and Lewis, 2010). Studies on agitation
speed showed 50% decrease in ethanol production when agitation
speed was increased to 250 rpm from 150 rpm (Ramachandriya,
2006). Similar studies on bioreactor design and up-scaling
parameters showed six fold increase in ethanol production when
syngas fermenting strain Clostridium P11 was scaled up from 7.5 L
to 100 L pilot scale fermentor (Kundiyana et al., 2010). Further-
more, studies on the aspects of process optimization have also
enhanced the conversion rates and ethanol production
(Kundiyana et al., 2011).
Based on the points highlighted above, the present study was
aimed at developing anaerobic mixed bacterial culture that have
the capability to utilize syngas for ethanol production. However
there is a considerable number of studies on syngas fermentation
using mesophilic pure cultures (Tanner, 2005; Maddipati et al.,
2011). Limited studies using mixed-cultures have been reported,
although mixed culture fermentation is more suited for industrial
applications, when compared to pure culture fermentation. Some
of the advantages are: (i) no need for highly sterile cultivation,
(ii) presence of high microbial diversity, which offers increased
adaptation capacity, (iii) possibility of mixed substrates co-fermen-
tation, and (iv) higher capacity for continuous processing. The
mixed culture presents an opportunity for higher alcohols produc-
tion from syngas. Semi-continuous fermentations in a 3 L fermen-
tor with the mixed culture and CSL medium resulted in a twofold
more total alcohol production than in the YE medium. The synergy
between strain CP15 and Clostridium propionicum in the mixed cul-
ture in bottle fermentations resulted in 50% higher efficiency in
converting propionic acid, butyric acid and hexanoic acid to their
respective alcohol (Liu et al., 2013). Further, different operational
parameters such as pH, temperature, syngas pressure and agitation
speed were optimized for enhanced ethanol production from syn-
gas. In addition, semi-continuous and scale-up fermentation stud-
ies from 130 mL to 1 L volumes were also carried out to enhance
the ethanol production.
2. Methods
2.1. Source of inoculum and fermentation medium
Micro-organisms used for the present study were taken from
TERI’s culture collection. TERI is a referral centre and culture repos-
itory for bacterial strains in India (Sarma et al., 2004; Agrawal et al.,
2010; Kaur et al., 2009; Singh et al., 2014). An additional approach
of enrichment was also employed using sludge (SD), cow dung
(CD) and chicken faeces (CF) samples collected from Gwal pahari
(Gurgaon, Haryana, 28°280 N 77°1.90 E) India. Pfennig’s basal media
(PBM) medium containing (per liter) 50 mL mineral stock solution,
10 mL trace metal stock solution, 10 mL vitamin stock solution, 1 g
yeast extract, 1 mL resazurin (0.1%) and 20 mL L-cysteine HCl
(2.5%) was used for the enrichment of microbial strains. The com-
position of the mineral, vitamin and trace metal stock solutions has
been previously reported (Gaddy and Clausen, 1992). Unless spec-
ified, all the experiments were performed in 130 mL Wheaton
serum bottles containing 40 mL of the liquid medium. Anaerobic
condition during media preparation was maintained as described
by (Singh et al., 2014). The bottles were sealed with butyl rubber
stopper and aluminum cap and then sterilized at 121 °C and
15 psi for 15 min. Vitamin solution was added after sterilization.
The initial pH of the medium was adjusted at 6.0 ± 0.1 with 2 N
KOH and 2 N HCl solutions.
2.2. Screening and enrichment of syngas utilizing microbial mixed
culture for ethanol production
In the present study initially 13 microbial cultures were col-
lected from TERI’s culture collection (Sarma et al., 2004; Agrawal
et al., 2010; Kaur et al., 2009; Singh et al., 2014) and screened for
syngas fermentation to ethanol. An additional approach of enrich-
ment was also designed. Sludge, cow dung and chicken faeces sam-
ples were used for the enrichment of syngas utilizing microbial
mixed culture. All samples were subjected to multiple enrichment
cycles by adding (10% w/v) sample inoculum into 125 mL of Whea-
ton serum bottle with 40 mL of freshly prepared medium. The cul-
ture bottles were sparged and headspace pressurized up to
1 kg cm2 with commercial syngas (CO 20%, CO2 15%, H2 20%,
CH4 3% balance N2). Incubation was done at 37 °C in a rotary shaker
at 150 rpm. After 5 days (10% v/v) enriched culture was subse-
quently transferred into fresh medium and this process was
repeated for 6 cycles. During each sub-culturing cycle the culture
bottles were analyzed for syngas utilization, Volatile fatty acids
(VFA) and solvent production (as described in Section 2.6). The
enrichment cycles were done in duplicates.
2.3. Optimization of operational parameters for enhanced ethanol
production by the selected mixed culture
Based on enrichment results, selected syngas fermenting mixed
culture was taken up for further study. Different operational
parameters were investigated for enhanced ethanol production.
To investigate the effect of temperature on growth and ethanol
production by the selected mixed culture, the experimental set
was incubated at temperatures 30 °C, 37 °C and 50 °C at pH 6.0,
agitation speed 150 rpm and syngas pressure in the reactor bottle
headspace of 1 kg cm2. Effect of initial medium pH on ethanol
production by selected mixed culture TERI SA1 was studied with
the pH 5.0, 6.0, 7.0 and 8.0. The pH was adjusted with 2 N HCl
 A. Singla et al. / Bioresource Technology 172 (2014) 41–49
and 2 N KOH solutions. Similarly the effect of different initial syn-
gas pressure in the reactor bottle headspace of 0.5, 1.0, 1.5, 2.0, 2.5
and 3.0 kg cm2 and different rate of agitation (100, 150 and
200 rpm) were studied for analyzing the effect on ethanol produc-
tion. All the experiments were performed in duplicate and the data
points are average of the duplicate ± standard deviation (less than
5% of average).
2.4. Semi-continuous fermentation studies for enhanced ethanol
production by mixed culture TERI SA1
The experiment was conducted in semi-continuous mode
(sparged with fresh syngas after every 24 h and fermentation in
batch mode) for enhanced ethanol production by the selected
mixed culture. The change in pH, cell biomass, acetic acid and eth-
anol production was analyzed at regular time interval of 24 h for a
period of 216 h. The culture bottles were resparged after every 24 h
with fresh syngas and maintained with the syngas pressure of
2 kg cm2 in the headspace. The experiment was performed in
duplicates.
2.5. Scale up studies using mixed culture TERI SA1 for increased
ethanol production
Scale up studies for the selected mixed culture was performed
in 1 L wheaton serum bottles with 300 mL of medium in semi-
continuous mode at pH 6.0, temperature 37 °C and agitation of
100 rpm. The syngas pressure in the reactor bottle headspace
was maintained at 1 kg cm2. Sample was taken for analyzing
change in pH, cell growth, acetic acid and ethanol production
at regular time interval of 24 h for a period of 216 h. The reactor
bottle headspace gas was replaced with fresh syngas and main-
tained with same syngas pressure after every 24 h. The experi-
ment was performed in duplicate and the data points are
average of the duplicate ± standard deviation (less than 5% of
average).
2.6. Analytical methods
Gas phase concentrations in the reactor bottle headspace was
analyzed by using Agilent Technologies 7890A series gas chro-
matograph (GC) equipped with thermal conductivity detector
(TCD). GC was fitted with 4 m long Carbosieve-II (80/100 mesh
size) 11.80  2 mm SS column (Nucon, India). Argon was used
as carrier gas with the flow rate of 11.4 mL/min. The oven, injec-
tor and detector temperature were 100 °C, 120 °C and 150 °C,
respectively. For VFAs and solvent analysis, liquid samples were
centrifuged at 10,000 rpm for 10 min and supernatant was fil-
tered with 0.22 lm millipore membrane filters. Ethanol and
acetic acid concentration was analyzed using flame ionization
detector (FID) equipped with DB-WAXETR 30 m  0.53 mm 
1.0 micron packed column (Agilent 7890, USA). Argon was used
as the carrier gas at the flow rate of 0.947 mL/min. The injector
and detector temperature were 220 °C and 250 °C, respectively.
The oven temperature was set at 60 °C for 1 min and increased
to 170 °C and 200 °C at ramping rate of 20 °C/min and
10 °C/min respectively. The calibration curve obtained for all
the standards showed R2 value more than 0.998. The bacterial
growth was monitored by measuring the optical density (OD)
at 600 nm using UV visible spectrophotometer (UV-2450,
Shimadzu, Japan). Initial and final pH was checked by pH meter
(Mettler Toledo, India).
43
3. Results and discussion
3.1. Screening and enrichment of syngas utilizing microbial mixed
culture for ethanol production
The aim of the present study was to develop anaerobic mixed
bacterial culture capable of utilizing syngas as carbon source to
produce ethanol. In this study, natural anaerobes obtained from
sludge, cow dung and chicken faeces and thirteen cultures
obtained from TERI’s culture collection were examined for maxi-
mum ethanol production by utilizing syngas as carbon source. In
most natural environments, anaerobes are predominant and have
significant ability to transform organics to alcohols (Fan et al.,
2003). In addition, thirteen bacterial strains from TERI’s culture
collection were selected because this centre is a culture repository
of anaerobic microbes such as Clostridium sps., isolated from vari-
ous sources (Agrawal et al., 2010; Kaur et al., 2009; Singh et al.,
2014). Moreover, Chicken faeces are a good source for enrichment
of anaerobic mixed bacterial culture for conversion of syngas to
ethanol. Gaddy and Clausen (1992) have been previously reported
a mesophilic syngas fermenting bacterial strain (C. ljungdahlii)
belonging to a novel species from the similar source. Out of the
thirteen strains of TERI culture collection and three sample inocu-
lums, the mixed culture enriched from chicken faeces utilized syn-
gas as carbon source to produce maximum ethanol and acetic acid,
and was selected for subsequent studies (Fig. 1). Other enriched
strains did not utilize syngas efficiently to produce ethanol as they
might not have adapted for utilizing syngas as carbon source. After
4th enrichment cycle, the selected mixed culture TERI SA1enriched
from chicken faeces was capable of utilizing 100% of the initial sup-
plied CO. Each enrichment cycle was of 120 h. The maximum
amount of ethanol and acetic acid produced by mixed culture TERI
SA1 after 5th enrichment was 0.15 g L1 and 1.21 g L1 respec-
tively (Fig. 1). Thus, TERI SA1 being most efficient was taken up
for further optimization studies. Although similar study has been
done previously but there are crucial differences between the
strain reported in the previous study (Gaddy and Clausen, 1992)
and the microbial mixed culture enriched in the current study, (i)
The sample was collected from similar source but different geo-
graphical location, (ii) The bacterial diversity present in the sample
varies according to the climate conditions as well as eating habit of
the animal living in the particular region, (iii) The current study
was related to the enrichment and optimization of a mixed bacte-
rial culture rather than previous pure culture study by (Gaddy and
Clausen, 1992), (iv) Moreover, mixed culture fermentation is cost
effective approach than pure culture.
3.2. Optimizing operational parameters for maximizing ethanol
production
3.2.1. Effect of pH on cell growth and ethanol production
pH is a critical parameter to obtain optimal microbial activity in
the culture media. The extracellular pH directly influences the
intracellular pH, membrane potential, proton motive force, and
consequently substrate utilization and product profile (Devi
et al., 2010). There was an increase in ethanol production and cell
biomass as pH increased from 5.0 to 6.0 (Fig. 2a). The maximum
cell biomass (0.14 g L1), ethanol (1.48 g L1) and acetic acid and
(0.6 g L1) production by mixed culture TERI SA1 was observed
when initial pH of the medium was 6.0. There is a narrow range
of pH for every organism, in which the cells are metabolically
active. A change in pH of environment can lead to death of the cells
and consequently results in loss of biological activity. Therefore,
the metabolite production decreased gradually as the pH increased
beyond 6.0, confirming it as the optimum pH value for the selected
44
0.16
Ethanol (g L-1)
0.12
0.08
0.04
0 0
SD
CD
Ethanol
Fig. 1. Acetic acid and ethanol production pattern of the enrichment cultures. The experiment was performed in duplicate and the data points are average of the
duplicate ± standard deviation (less than 5% of average).
mixed culture (Fig. 2a). Similar pH range has also been reported for
syngas fermenting bacterial strains Clostridium autoethanogenum,
Butyribacterium methylotropphicum (Abrini et al., 1994) and (Shen
et al., 1999). In addition, Rajagopalan et al. (2002) observed maxi-
mum ethanol concentration of 0.56 g L1 with C. carboxidivorans P7
at similar pH range. pH optimization studies for the selected mixed
culture led to increased ethanol production in comparison to the
previously reported in literature by (Rajagopalan et al., 2002).
3.2.2. Effect of temperature on cell growth and ethanol production
Temperature changes have significant effect upon microbial
growth and activity. In most cases, the temperature of the culture
media is decided based on the specific microorganism. The fermen-
tation temperature not only affects substrate utilization, growth
rate and membrane lipid composition of the acetogens, but also
gas substrate availability because gas solubility increases with
decreasing temperature (Munasinghe and Khanal, 2010;
Kundiyana et al., 2011). Acetic acid and ethanol was desirable
product from syngas fermentation (Fig. 2b). As shown in Fig. 2b
the maximum amount of cell biomass (0.17 g L1), ethanol
(1.48 g L1) and acetic acid (0.6 g L1) produced by mixed culture
TERI SA1 was at 37 °C. High temperature did not favor syngas uti-
lization, as there was no cell growth, ethanol and acetic acid pro-
duction at 50 °C, confirming the mesophilic nature of selected
mixed culture TERI SA1. In case of mesophilic syngas fermenting
bacterial strains, higher temperature above 37 °C affects the
growth, as carbon monoxide (CO) and hydrogen (H2) components
of syngas have a decreased solubility with increase in temperature
(Munasinghe and Khanal, 2010). There was very less difference
between the cell growth as well as product formation efficiency
of the mixed culture at temperature 30 °C and 37 °C (Fig. 2b). As
the optimum temperature for mesophilic acetogens are between
30 °C and 40 °C, while thermophilic acetogens grow best between
55 °C and 60 °C (Daniell et al., 2012). These results are efficient
than the previous findings of (Abrini et al., 1994) for C. autoethano-
genum which produced up to 0.32 g L1 ethanol at similar temper-
ature range.
3.2.3. Effect of agitation speed on cell growth and ethanol production
Agitation plays a very important role in anaerobic fermenta-
tions utilizing gaseous substrates because gases such as CO, CO2
and H2 are less soluble in water and mass transfer of these sub-
strates to the cells are an important factor in reactor performance
(Kapic et al., 2006). The maximum amount of ethanol (1.47 g L1)
and acetic acid (0.85 g L1) production achieved by selected mixed
culture TERI SA1 was at agitation of 100 rpm (Fig. 2c). Cell growth
A. Singla et al. / Bioresource Technology 172 (2014) 41–49
Enrichment study
1.6
Acetic acid (g L-1)
1.2
0.8
0.4
S3
HS
S10
S25
T01
T08A
T08
T03
T05
T08b
IT2
TM9A
MT55
TERI SA1
Enrichment cultures
Acec acid
and metabolite production decreased gradually as the agitation
speed was further increased to 150 and 200 rpm (Fig. 2c). Cell lysis
of the mixed culture TERI SA1 was observed at high agitation rates.
The drop in ethanol and acetic acid formation at 200 rpm followed
by 150 rpm could possibly be due to the shear sensitivity and CO
toxicity of mixed culture TERI SA1 to higher agitation rates at high
syngas pressure (Gaddy et al., 2003; Ramachandriya, 2006).
Enzymes such as CODH and hydrogenase involved in the acetyl-
CoA pathway could be affected due to the shear and the presence
of excess CO also inhibits growth by inhibiting the CO-dehydroge-
nase. Moreover, presence of excess CO unfortunately results in
poor H2 conversion which eventually causes cell lysis (Gaddy
et al., 2003; Ramachandriya, 2006). Ramachandriya (2006)
observed that ethanol concentration decreased by 50% when P11
cells were agitated at 250 rpm (0.9 g L1) as compared to
150 rpm (1.8 g L1). Similarly, the maximum acetic acid concentra-
tions were 52% lower when cells were agitated at 250 rpm (2 g L1)
than 150 rpm (4.2 g L1).
3.2.4. Effect of syngas pressure on cell growth and ethanol production
One potential bottleneck of syngas bioconversion is the mass
transfer limitation due to the sparingly soluble nature of the sub-
strate. Hence, one way to overcome this limitation is by increasing
the pressure. In batch fermentation, different CO pressures mean
different gaseous substrate concentrations which are directly pro-
portional to the product formation and cell growth (Abubackar
et al., 2012). As the initial syngas pressure in the reactor bottle
headspace was increased there was a linear increase in cell growth,
ethanol and acetic acid production up to 2 kg cm2 (Fig. 2d). How-
ever, as the pressure was increased beyond 2 kg cm2 (up to 2.5
and 3.0 kg cm2) both ethanol and acetic acid production gradually
decreased (Fig. 2d). This can be interpreted from this study that
high syngas pressure (above 2 kg cm2) in the reactor bottle head-
space probably inhibited CO consumption, thus inhibiting the
growth and product formation efficiency of the selected mixed cul-
ture TERI SA1. Maximum amount of ethanol and acetic acid pro-
duction achieved by the mixed culture TERI SA1 at pressure
2 kg cm2 was 1.54 g L1 and 0.9 g L1 respectively which was
comparatively higher than achieved at 0.5 kg cm2 (Fig. 2d). In a
similar study by (Hurst and Lewis, 2010) increase of CO partial
pressure from 0.35 to 2.0 atm. increased the cell concentration
from 0.20 to 1.08 g L1, which represented a 440% improvement.
Klasson et al. (1991), examined that there was a linear relation
between the reaction rate and CO partial pressures up to 1.6 atm.
At CO partial pressure of 2.5 atm. there was a short period of CO
uptake, thereafter the culture failed to utilize the CO. This was
 A. Singla et al. / Bioresource Technology 172 (2014) 41–49 45

pH optimization
(a)

Cell biomass (g L-1)

1.8 0.16

AA & EtOH. (g L-1)

1.5
0.12
1.2
0.9 0.08
0.6
0.04
0.3
0 0
5 6 7 8
Initial media pH
Ethanol Acec acid Cell biomass

Temperature optimization
(b)
1.8 0.2

L-1)
AA & EtOH. ( g L-1)

1.5 0.16

Cell biomass (g
1.2
0.12
0.9
0.08
0.6
0.3 0.04
0 0
30 37 50
Incubation temperature (°C)
Ethanol Acec acid Cell biomass

(c) 1.8
Agitation speed optimization
0.2

Cell biomass (g L-1)

AA & EtOH. (g L-1)

1.5 0.16
1.2
0.12
0.9
0.08
0.6
0.3 0.04

0 0
100 150 200
Agitation speed (rpm)
Ethanol Acec acid Cell biomass

Syngas pressure optimization

(d)
1.8 0.2
Cell biomass (g L-1)

1.5 0.16
AA & EtOH. (g L-1)

1.2
0.12
0.9
0.08
0.6
0.3 0.04
0 0
0.5 1 1.5 2 2.5 3
Syngas Pressure (kg cm-2)
Ethanol Acec acid Cell biomass
Fig. 2. (a) Optimization of initial media pH, (b) Incubation temperature, (c) Agitation speed and (d) Initial syngas pressure in the reactor headspace for cell biomass, acetic
acid and ethanol production by syngas fermenting mixed culture TERI SA1. The experiment was performed in duplicate and the data points are average of the
duplicate ± standard deviation (less than 5% of average).
46 A. Singla et al. / Bioresource Technology 172 (2014) 41–49

probably due to the insufficient cell concentration which could not
keep the reaction at the mass transfer limited stage and caused
toxicity for the microbe. It was suggested that high CO partial
pressure could be employed after a sufficient cell concentration
was achieved (Klasson et al., 1991). Younesi et al. (2005) also
studied the effect of various initial total pressures of syngas
(0.8–1.8 atm.) in the batch culture of C. ljungdahlii.
3.3. Semi-continuous fermentation studies for enhanced ethanol
production by the selected mixed culture TERI SA1
3.3.1. Cell growth and pH profiles
Cell mass concentrations of mixed culture TERI SA1 increased
during the first 144 h (Fig. 3a). However, cells were in the lag phase
in the first 24 h. After 24 h cells entered the exponential phase and
there was increase in cell mass concentration until 144 h. In addi-
tion, cells entered the stationary phase after 144 h. The maximum
cell mass concentration of 0.2 g L1 was observed at 144 h. Minor
changes in cell mass concentrations were observed during the sta-
tionary phase until 216 h. The pH of the culture media decreased
during cell growth due to the production of acetic acid (Fig. 3a).
The pH decreased from 6.0 to 4.6 until 192 h, at which time the
acetic acid concentrations in medium reached a maximum
(Fig. 3a). Subsequently, a minor change in pH was observed after
192 h.
3.3.2. Product profiles
Acetic acid and ethanol were the major products formed during
syngas fermentation in culture media (Fig. 3b). Acetic acid is a pri-
mary metabolite, and its production is associated with cell growth.
The production of acetic acid was higher during the growth phase
and gradually increased till the cells entered the stationary phase.
A maximum acetic acid concentration of 1.67 g L1 was reached at
192 h (Fig. 3b). Acetic acid concentration then decreased to
1.3 g L1 at the end of the fermentation. Acetic acid concentrations
were comparatively lower between 120 and 168 h. Consumption of
acetic acid by mixed culture TERI SA1 was observed at later stages
of fermentation. Acetic acid consumption was associated with pro-
duction of ethanol after 192 h of fermentation. This was also
observed in other syngas fermentation studies using Clostridium
strain P11 (Panneerselvam et al., 2010) and C. carboxidivorans P7
(Hurst and Lewis, 2010). Ethanol production started in the expo-
nential phase (Fig. 3b). The rate of ethanol production increased
in the early exponential phase and then slightly decreased till
the cell entered in stationary phase. During stationary phase the
ethanol concentration again start increasing and decreased with
the decline in cell concentration. This trend in ethanol production
was due to the effect of syngas pressure in reactor headspace
(Hurst and Lewis, 2010). The maximum ethanol concentration
was 2.3 g L1 at 72 h, which was 49% higher than achieved during
batch fermentation conditions (Fig. 3b). Higher ethanol production
in semi-continuous fermentation mode could possibly be due to
the sparging of fresh syngas after every 24 h as compared to batch
fermentation (sparged and pressurized with syngas just in the
beginning of fermentation).
3.4. Scale up studies using mixed culture TERI SA1 for increased
ethanol production
3.4.1. Cell growth and pH profiles
The growth and pH profiles of mixed culture TERI SA1 is shown
in Fig. 4a. There was a short lag Phase (24 h) followed by an

(a)
6.5 0.25
Cell biomass (g L-1)
6 0.2

5.5 0.15
pH

5 0.1

4.5 0.05

4 0
24 48 72 96 120 144 168 192 216
Time (h)
pH Cell biomass

(b) 3 1.8
L-1)

2.5 1.5
Ethanol (g L-1)

Acetic acid (g

2 1.2
1.5 0.9
1 0.6
0.5 0.3
0 0
24 48 72 96 120 144 168 192 216
Time (h)
EtOH. AA
Fig. 3. (a) Cell growth and pH profiles and (b) ethanol and acetic acid production at different time intervals by mixed culture TERI SA1 under semi-continuous fermentation
conditions. The experiment was performed in duplicate and the data points are average of the duplicate ± standard deviation (less than 5% of average).
 A. Singla et al. / Bioresource Technology 172 (2014) 41–49 47

(a)
6.5 0.2

Cell biomass (g L-1)

6 0.16

5.5 0.12

pH 5 0.08

4.5 0.04

4 0
24 48 72 96 120 144 168 192 216
Time (h)
pH cell biomass

EtOH. & AA Production (g L-1)

(b)
12 2.5
CO & H2 utilization (mM)

10 2
8
1.5
6
1
4
2 0.5
0 0
0
24

48

72

96

120

144

168

192

216
24, rs

48, rs

72, rs

96, rs

120, rs

144, rs

168, rs

192, rs
Time (h)
EtOH. AA CO H₂
Fig. 4. (a) Change in pH and cell biomass concentration, (b) Ethanol and acetic acid production and CO and H2 consumption at different time intervals by mixed culture TERI
SA1 under semi-continuous fermentation conditions in 1 L reactor bottles. The experiment was performed in duplicate and the data points are average of the
duplicate ± standard deviation (less than 5% of average).

exponential phase. The cell concentration increased exponentially because 1 L reactor bottle could not tolerate high syngas pressure
during the first 72 h of fermentation. The maximum cell biomass of 2 kg cm2. It was reported that ethanol production by C. carbox-
concentration of 0.17 g L1 was achieved at 144 h. The pH of idivorans P7 was non-growth related when the partial pressure of
culture media decreased from 6 to 5.1 during cell growth in the CO in the headspace was below 106 kPa (Hurst and Lewis, 2010).
first 72 h due to the production of acetic acid (Fig. 4a). However, However, ethanol formation was growth associated with C. carbox-
the pH of the medium decreased from 6 to 4.7 after 72 h, during idivorans P7 when the partial pressure of CO in the headspace was
which cells were still growing. Then, the pH of culture media above 106 kPa. In the present study, the rate of ethanol production
slightly decreased after 120 h due to stationary phase of bacterial was high in the exponential phase and decreased with time espe-
cells (Fig. 4a). cially with the increase in cell biomass concentration. After 216 h
of fermentation, ethanol in 1 L reactor bottle was 2.0 g L1
3.4.2. Gas consumption and product profiles (Fig. 4b). Both CO and H2 were utilized by mixed culture TERI
Acetic acid and ethanol production with mixed culture TERI SA1 SA1 for growth, acetic acid and ethanol production. The total moles
was observed during the acidogenic and solventogenic phases of CO and H2 consumed by mixed culture TERI SA1 during syngas
respectively. Acetic acid concentration increased in the first 72 h fermentation is shown in Fig. 4b. About 28% and 3% of the total
and further increased till 192 h (Fig. 4b). Acetic acid formation by CO and H2 moles were consumed by mixed culture TERI SA1 during
mixed culture TERI SA1 was growth related, which resulted a the first 144 h of fermentation for mostly growth and acetic acid
decrease in the pH of the fermentation medium (Fig. 4b). The max- production respectively. In the acetyl-CoA pathway, both H2 and
imum acetic acid concentration was 1.9 g L1 at 192 h. After the CO serve as an energy and electron source for cell growth and
concentration of acetic acid reached a maximum, mixed culture product formation (Ragsdale, 2004). Significant amounts of carbon
TERI SA1 started to consume it. About 8.4% of the acetic acid was from CO can be converted to cell biomass and ethanol if H2 is uti-
consumed between 192 and 216 h (Fig. 4b). The ethanol produc- lized as an electron source. In contrast, high concentration of CO
tion was observed during exponential phase which decreased after was found to inhibit hydrogenase enzyme and thus reduce the
the stationary phase. The slight increase in ethanol production was uptake of H2 in non-CO fermenting organisms (Bennett et al.,
observed during stationary phase when the pH was around 4.7 2000). This could explain the decrease in H2 consumption during
after 120 h (Fig. 4a and b). Ethanol formation by mixed culture TERI the stationary phase with mixed culture TERI SA1 (Fig. 4b). Trends
SA1 in 1L reactor bottle fermentation was growth as well as non- in CO consumption profile were similar to ethanol profiles (Fig. 4b).
growth associated depending on the actual syngas pressure in Ethanol production started after 24 h of the fermentation. For eth-
the reactor bottle headspace. The syngas pressure during 1 L reac- anol production and cell maintenance between 24 and 216 h,
tor bottles fermentation study was maintained at 1.0 kg cm2 approximately 34% and 2% of the total moles of CO were consumed
48 A. Singla et al. / Bioresource Technology 172 (2014) 41–49

Table 1
Comparison of ethanol yield by different mesophilic syngas fermenting bacterial strains.

Microbial species Topt (°C) pHopt Yield (g/l) EtOH. Products References
Clostridium ljungdahlii 37 N.A 0.6 Acetate, ethanol Younesi et al. (2005)
Clostridium autoethanogenum 37 5.8–6.0 0.32 Acetate, ethanol Abrini et al. (1994)
Clostridium ljungdahlii 37 4.0–5.0 1.0 Acetate, ethanol Gaddy and Clausen (1992)
Clostridium carboxidivorans P7T 37 5.8–5.9 0.56 Acetate, ethanol, butanol Rajagopalan et al. (2002)
Clostridium ragsdalei 32 6.0 1.89 Acetate, ethanol Kundiyana et al. (2011)
Butyribacterium methylotropphicum 37 6.0 N.A Acetate, ethanol, butanol Shen et al. (1999)
Eubacterium limosum 38–39 7.0–7.2 N.A Acetate, ethanol Chang et al. (2001)
Peptostreptococcus productus 37 7.0 N.A Acetate, ethanol Lorowitz and Bryant (1984)
A. bacchi 37 5.8–7.0 1.7 Acetate, ethanol Liu et al. (2012)
Mixed culture TERI SA1 37 6.0 2.3 Acetate, ethanol This study
Mixed culture 37 8.0 8.0 Acetate, ethanol Liu et al. (2013)

by mixed culture TERI SA1 respectively. This shows that most of
the CO consumed by mixed culture TERI SA1 was utilized for eth-
anol production. The detailed comparison for ethanol yield of
mixed culture TERI SA1 with different syngas fermenting strains
reported from mesophilic origin is also provided in Table 1.
4. Conclusion
Enriched anaerobic mixed culture TERI SA1 was capable of uti-
lizing syngas as carbon source for ethanol production. Optimiza-
tion of operational parameters enhanced ethanol production
from 0.15 g L1 to 1.54 g L1 indicating 927% improvement. Subse-
quent study of semi-continuous and up-scaling parameters further
enhanced ethanol production up to 2.3 g L1 which was consider-
ably higher than 1.89 g L1 with Clostridium ragsdalei (Kundiyana
et al., 2011) but lower than 8 g L1 with reported mixed culture
(Liu et al., 2013), therefore higher volume bio-reactor studies with
selected mixed culture TERI SA1 needs to be undertaken to provide
further insights.
Acknowledgements
The financial support for this research was provided by Depart-
ment of Biotechnology, Ministry of Science and Technology, India.
The authors are thankful to Dr. R.K. Pachauri Director-General,
TERI, for providing us excellent infrastructure support for the
study. The authors also thank Mr. Samarjeet (TERI, India) and Mr.
Shivaji (TERI, India) for their technical assistance and Ms. Sneha
Singh (TERI University, India), Ms. Mohita Sharma (TERI University,
India) and Ms. Anchal priya (TERI University, India) for helpful
discussions.
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