Ethanol production from syngas by Clostridium strain P11 using corn steep

liquor as a nutrient replacement to yeast extract

Prasanth Maddipati, Hasan K. Atiyeh

, Danielle D. Bellmer, Raymond L. Huhnke

Department of Biosystems and Agricultural Engineering, Oklahoma State University, Stillwater, OK 74078, USA
a r t i c l e i n f o
Article history:
Received 20 December 2010
Received in revised form 15 March 2011
Accepted 16 March 2011
Available online 21 March 2011
Keywords:
Clostridium
Corn steep liquor
Ethanol
Syngas fermentation
Yeast extract
a b s t r a c t
The feasibility of replacing yeast extract (YE) by corn steep liquor (CSL), a low cost nutrient source, for
syngas fermentation to produce ethanol using Clostridium strain P11 was investigated. About 32% more
ethanol (1.7 g L
1
) was produced with 20 g L
1
CSL media in 250-mL bottle fermentations compared to
media with 1 g L
1
YE after 360 h. Maximum ethanol concentrations after 360 h of fermentation in a
7.5-L fermentor with 10 and 20 g L
1
CSL media were 8.6 and 9.6 g L
1
, respectively, which represent
57% and 60% of the theoretical ethanol yields from CO. Only about 6.1 g L
1
of ethanol was obtained in
the medium with 1 g L
1
YE after 360 h, which represents 53% of the theoretical ethanol yield from
CO. The use of CSL also enhanced butanol production by sevenfold compared to YE in bottle fermenta-
tions. These results demonstrate that CSL can replace YE as the primary medium component and signif-
icantly enhance ethanol production by Clostridium strain P11.
2011 Elsevier Ltd. All rights reserved.
1. Introduction
The dominant biofuel in the US is ethanol made via yeast-based
fermentation of corn starch, which is a well-developed technology.
Ethanol made via fermentation of synthesis gas (syngas) produced
from gasication of non-edible feedstocks such as biomass and
municipal solid wastes is relatively a new technology. Syngas,
primarily containing CO, CO
2
, and H
2
, can be fermented to ethanol
and acetic acid by acetogenic organisms such as Clostridium ljung-
dahlii (Klasson et al., 1992; Phillips et al., 1994; Younesi et al.,
2005), Clostridium autoethanogenum (Abrini et al., 1994), Clostrid-
ium carboxidivorans P7 (Ahmed and Lewis, 2007; Rajagopalan
et al., 2002), and Clostridium strain P11 (Huhnke et al., 2010;
Saxena, 2008). These microorganisms utilize the reductive acetyl-
CoA pathway, also known as the WoodLjungdahl pathway, for
the synthesis of acetyl-CoA, conservation of energy and growth
and production of acetic acid and ethanol (Wood et al., 1986).
The overall reactions for ethanol and acetic acid production from
H
2
and CO are (Vega et al., 1989):
6CO 3H
2
O !C
2
H
5
OH4CO
2
1
6H
2
2CO
2
!C
2
H
5
OH3H
2
O 2
4CO2H
2
O !CH
3
COOH2CO
2
3
2CO
2
4H
2
!CH
3
COOH2H
2
O 4
When equal moles of CO and H
2
are supplied, theoretically two-
thirds of the carbon from CO can be converted to ethanol and the
remaining carbon is accounted for in CO
2
production as seen by
combining Eqs. (1) and (2). However, all the carbon supplied in
the form of CO theoretically can be converted to acetic acid by
combining Eqs. (3) and (4).
Butanol production has also been reported from CO (Shen et al.,
1999; Worden et al., 1991) or a mixture of CO, CO
2
and H
2
(Babu
et al., 2010; Datar et al., 2004; Liou et al., 2005; Rajagopalan
et al., 2002) by only three bacteria. Butyribacterium methylotrophi-
cum was reported to produce between 0.3 and 1.4 g L
1
butanol
from pure CO (Shen et al., 1999; Worden et al., 1991). However,
Clostridium strain P11 produced 0.3 g L
1
butanol from a simulated
syngas mixture (Babu et al., 2010) and C. carboxidivorans produced
1.3 g L
1
butanol from syngas derived from cellulosic feedstock
(Rajagopalan et al., 2002). The overall reactions for butanol produc-
tion from H
2
and CO are (Rajagopalan et al., 2002):
12CO 5H
2
O !C
4
H
9
OH8CO
2
5
12H
2
4CO
2
!C
4
H
9
OH7H
2
O 6
A recent literature review on biomass-derived syngas
fermentation into biofuel describes the factors that affect syngas
0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.03.047

Corresponding author. Address: Department of Biosystems and Agricultural

Engineering, 227 Ag Hall, Oklahoma State University, Stillwater, OK 74078, USA.
Tel.: +1 405 744 8397; fax: +1 405 744 6059.
E-mail address: hasan.atiyeh@okstate.edu (H.K. Atiyeh).
Bioresource Technology 102 (2011) 64946501
Contents lists available at ScienceDirect
Bioresource Technology
j our nal homepage: www. el sevi er . com/ l ocat e/ bi or t ech
fermentation, which include type of biocatalyst and growth media,
mass transfer, reactor conguration and operating conditions
(Munasinghe and Khanal, 2010).
Composition of the fermentation medium plays an important
role in cell and product yields. The development of a low-cost fer-
mentation medium alternative containing all essential nutrients
required for cell growth and product formation would reduce the
overall cost of the fermentation process. Standard mediumfor Clos-
tridium strain P11 is composed of yeast extract, vitamins, minerals,
trace metals and reducing agent (Saxena and Tanner, 2010). Apart
from the reducing agent, yeast extract (YE) is the most expensive
component. Some inexpensive nutrients that could replace YE are
corn steep liquor (CSL), hydrolyzed cotton seed our, hydrolyzed
soy our and ethanol stillage (Witjitra et al., 1996).
CSL is a major by-product fromthe cornwet milling industry. It is
rich in vitamins, minerals, amino acids and proteins (Azeredo et al.,
2006; Kadamand Newman, 1997). It has been used as growth med-
ium for mannitol production by Lactobacillus intermedius (Racine
and Saha, 2007), and solvent productionby Saccharomyces cerevisiae
(Kadam and Newman, 1997), Clostridium beijerinckii (Parekh et al.,
1999) and Zymomonas mobilis (Lawford and Rousseau, 1997). The
cost of bacto-yeast extract from Difco Laboratories (Detroit, MI) is
$157.49 kg
1
(Racine and Saha, 2007), while the cost of CSL on an
industrial scale is $0.07 kg
1
(Lawford and Rousseau, 1997). As per
2010, the industrial price of spray dried YE is $9.2 kg
1
, while the
cost of industrial CSL is $0.18 kg
1
. The incorporation of CSL at
20 g L
1
in the fermentation medium resulted in elimination of a
few growth factors from standard Clostridium strain P11 medium
(Saxena, 2008). However, this study did not investigate growth
and product kinetics of strain P11 in CSL media. In addition, tests
were done in 160-mL serumbottles with 10 mL of mediumand only
CO was fed to strain P11, which was different from the syngas
composition that potentially will be used for ethanol production
on a large scale.
Corn steep liquor (CSL) is selected in the present study as an
alternative to yeast extract (YE) for syngas fermentation because
it is rich in nutrients and lower in cost compared to YE. Accord-
ingly, the main objective of this study was to determine whether
CSL is a potential alternative nutrient source to YE in syngas fer-
mentation medium and to evaluate growth and product kinetics
of Clostridium strain P11 using CSL as compared to YE.
2. Methods
2.1. Microorganism
Clostridium strain P11 (ATCC PTA-7826) provided by Dr. Ralph
Tanner, University of Oklahoma, was maintained on standard yeast
extract medium. Clostridium strain P11 is highly sensitive to O
2
;
and hence, the fermentation was performed under strict anaerobic
conditions. In order to reduce the lag phase and also to ensure that
viable cells were inoculated into the medium, cells from the stock
inoculum were passaged twice prior to inoculation. Cell passaging
and syngas fermentation were performed in 250-L serum bottles
with 100 mL working volume. After cell passaging, the subculture
at 10% (v/v) was transferred to the fermentation serum bottles
and the 7.5-L fermentor to follow syngas fermentation.
2.2. Fermentation media
Three media formulations developed by Saxena (2008) were
used (Table 1). The standard medium contained yeast extract, min-
erals, trace metals, vitamins, morpholinoethanesulfonic acid (MES),
resazurin and cysteine sulde. The second medium (10 g L
1
CSL)
had the same components as the standard medium except yeast
extract was replaced withCSL. The third medium(20 g L
1
CSL) only
contained trace metals, MES, resazurin, ammonium chloride and
cysteine sulde. Yeast extract (Difco Laboratories, Detroit, MI) and
CSL (SigmaAldrich, St. Louis, MO) were used as complex nitrogen
and nutrient sources in the fermentation media. The compositions
of minerals, vitamins and trace metal stock solutions are shown in
Table 2. Unless stated, all media components were purchased from
SigmaAldrich (St. Louis, MO).
In order to produce consistent results, CSL from the same batch
was used throughout the study. CSL contained about 50% solids.
Before the addition of CSL into the fermentation medium, the sol-
ids from crude CSL were removed by centrifugation at 13,000 rpm
for 10 min. Only the liquid portion of the crude CSL was used in the
syngas fermentation experiments to avoid interference of solids
with measurement of P11 growth. The presence or removal of
solids from CSL did not affect ethanol and acetate formation by
strain P11 (Saxena, 2008). In addition, removal of solids from CSL
did not affect the nutritional requirements of Zymomonas ethanol
Table 1
Composition of media formulations used for syngas fermentation (Saxena, 2008).
Standard yeast extract CSL
a
CSL
a
1 g L
1
10 g L
1
20 g L
1
Yeast extract (g) 1
Corn steep liquor (g) 10 20
Minerals (mL) 30 30
Trace metals (mL) 10 10 10
Vitamins (mL) 10 10
MES
b
(g) 10 10 10
0.1% Resazurin (mL) 1 1 1
Ammonium chloride (g) 1.25
4% Cysteine sulde (mL) 10 10 2.5
Total volume (L) 1 1 1
a
CSL = corn steep liquor.
b
MES = morpholinoethanesulfonic acid.
Table 2
Composition of trace metal, vitamin and mineral stock
solutions (Huhnke et al., 2010).
Trace metal stock solution g L
1
Cobalt chloride 0.20
Ferrous ammonium sulfate 0.80
Manganese sulfate 1.00
Nickel chloride 0.20
Nitrilotriacetic acid 2.00
Sodium molybdate 0.02
Sodium selenate 0.10
Sodium tungstate 0.20
Zinc sulfate 1.00
Mineral stock solution g L
1
Ammonium chloride 100
Calcium chloride 4
Magnesium sulfate 20
Potassium chloride 10
Potassium phosphate 10
Sodium chloride 80
Vitamin stock solution mg L
1
p-(4)-Aminobenzoic acid 5
d-Biotin 2
Calcium pantothenate 5
Folic acid 2
MESNA
a
10
Nicotinic acid 5
Pyridoxine 10
Riboavin 5
Thiamine 5
Thioctic acid 5
Vitamin B12 5
a
MESNA = mercaptoethanesulfonic acid.
P. Maddipati et al. / Bioresource Technology 102 (2011) 64946501 6495
fermentations (Lawford and Rousseau, 1997). The liquid portion of
crude CSL contained 75 g L
1
of total sugars (43 g L
1
maltose,
11 g L
1
glucose, 5 g L
1
xylose, 6 g L
1
galactose, 7 g L
1
arabinose
and 3 g L
1
mannose). The initial total sugar concentrations in the
10 and 20 g L
1
CSL were 0.6 and 1.2 g L
1
, respectively. Resazurin
solution (0.1%) was added into the medium as a redox indicator.
Morpholinoethanesulfonic acid (MES) was added as a biological
buffer to prevent excessive uctuations in the pH during fermenta-
tion. The initial pH of the medium was adjusted to 6.1 using 2 N
KOH before inoculation.
2.3. Syngas
Commercial syngas with gas composition similar to producer
gas generated from our gasication facility was used. The syngas
was composed of 20% CO, 15% CO
2
, 5% H
2
and 60% N
2
by volume.
2.4. Fermentation in 250-mL serum bottles
Syngas fermentations were conducted in 250-mL serum bottles
(Wheaton, NJ) with 100 mL working volume containing the three
different media described in Table 1. All media components except
reducing agent were mixed in deionized water and pH was ad-
justed to 6.1 using 2 N KOH. The media were boiled and then
purged with N
2
for 3 min to remove O
2
. The serum bottles were
sealed using gas impermeable butyl rubber septum-type stoppers
and aluminum crimp seals. One milliliter of 4% cysteine sulde
solution was added to each bottle to scavenge any remaining dis-
solved O
2
. The fermentation media were autoclaved (Primus Ster-
ilizer Co., Inc., Omaha, NE) at 121 C and 205 kPa for 20 min.
The medium was inoculated with 10% (v/v) of Clostridium strain
P11. Sterile 0.2 lm PTFE (polytetrauoroethylene) membrane l-
ters (VWR International, West Chester, PA) were used in the inlet
line that fed syngas to strain P11 at 239 kPa. The bottles were
placed vertically on an orbital shaker (Innova 2100, New Bruns-
wick Scientic, Edison, NJ) at 150 rpm and 37 C. Liquid samples
were withdrawn every 24 h from the bottles under aseptic condi-
tions to measure optical density at 660 nm, pH and acetic acid
and solvent concentrations. Gas in the head space in each bottle
was replaced every 24 h with fresh syngas at 239 kPa. All studies
were performed in triplicate.
2.5. Fermentation in 7.5-L fermentor
A 7.5-L Bioo 110 (New Brunswick Scientic Co., Edison, NJ)
with a 3 L working volume was used. Agitation was provided by
three six-blade Rushton turbine impellers of 59 mm diameter
located at an equal distance on a top-driven impeller shaft. Four
bafes were symmetrically arranged to avoid vortex formation of
liquid media and improve mixing. A microsparger (New Brunswick
Scientic Co., Edison, NJ) with a pore size of 1015 lm made of 316
SS material was used for gas sparging. Syngas owing out of the
fermentor passed through a condenser cooled at 5 C using a refrig-
erated circulator (1156 D, VWR International, West Chester, PA).
Condensed vapor was returned to the fermentor. Uncondensed
vapor from the condenser with the syngas was bubbled through a
water trap lled with 150 mL deionized water maintained at
10 C using chilled water. The water trap captured ethanol that
was not condensed in the condenser. The amount of ethanol col-
lected in the water trap was included in the total ethanol produced
after 360 days of syngas fermentation. BioCommand software was
used for data acquisition, monitoring and controlling the fermentor.
All media components in Table 1 except cysteine sulde were
added into the fermentor to a total volume of 3 L. The pH of the
media was adjusted to 6.1 before sterilization using 5 N KOH.
The fermentor containing the medium was autoclaved at 121 C
and 205 kPa for 20 min. The medium was allowed to cool to room
temperature and was then purged with N
2
at 0.2 L min
1
for 24 h
to remove any dissolved O
2
before the start of the fermentation.
Then, syngas (20% CO, 15% CO
2
, 5% H
2
and 60% N
2
by volume)
was piped to the fermentor at 0.15 L min
1
and cysteine sulde
solution was added as per Table 1. The medium was then inocu-
lated with 10% (v/v) of strain P11 cells. The pressure inside the fer-
mentor was controlled at 143 kPa using a back pressure regulator.
The temperature in the fermentor was maintained at 37 C and the
agitation speed was set at 150 rpm. Antifoam B Emulsion
(SigmaAldrich, Saint Louis, MO) at a concentration of 10% (v/v)
was used to control foam formation inside the fermentor. About
0.2 mL of sterilized antifoam solution was added when the height
of the foam in the fermentor was two inches. Gas samples were
withdrawn from the head space periodically to determine gas com-
position. Liquid samples were collected to measure optical density
at 660 nm, pH and acetic acid and solvent concentrations.
2.6. Analytical procedures
2.6.1. Cell concentration
Cell concentration was determined using a UVVis spectropho-
tometer (Varian Inc., Palo Alto, CA) at 660 nm. Samples with OD
values above 0.4 were diluted so that the OD was within the
linear range of the calibration curve (cell mass, g L
1
= 0.396
OD 0.0521) as established using the dry weight method
(Panneerselvam, 2009).
2.6.2. Solvent analysis
Liquid samples were centrifuged at 13,000 rpm for 13 min. The
supernatant was ltered through 0.45-lm nylon membrane lters
(VWR International, West Chester, PA). Ethanol, acetic acid and
butanol concentrations were analyzed using a gas chromatograph
(GC), (Agilent Technologies, Wilmington, DE). A PoraPak QS 80/
100 (Alltech, Deereld, IL) packed column connected to a ame
ionization detector (FID) was used. The GC was operated under iso-
thermal conditions with the oven temperature set at 210 C. He-
lium was the carrier gas with a ow rate of 0.025 L min
1
. The
resulting chromatograms were analyzed using ChemStation data
analysis software.
2.6.3. Gas analysis
A GC equipped with a thermal conductivity detector (TCD) was
used for gas analysis. A capillary column, Carboxen 1010 PLOT
(Supelco, Bellefonte, PA), was used to detect CO, CO
2
, H
2
and N
2
. Ar-
gon was used as a carrier gas in the GC with an initial gas ow rate
of 0.4 mL min
1
for the rst 12 min, and then it was increased at a
rate of 0.1 mL min
1
until reaching 0.8 mL min
1
. Oven tempera-
ture was set at 32 C for 12 min, after which the temperature
was increased at a rate of 30 C per min until it reached 236 C.
The temperatures of the column inlet and detector were set at
200 and 230 C, respectively. Resulting chromatograms were ana-
lyzed using ChemStation data analysis software. The GC was cali-
brated regularly by injecting samples with different volumes of
known gas concentrations. Error in CO, CO
2
, H
2
and N
2
measure-
ments was below 5%.
2.6.4. Oxidationreduction potential
The oxidationreduction potential (ORP) was measured using a
redox probe with an Ag/AgCl reference electrode (InPro3250/325/
PT100, Mettler Toledo, Columbus, OH) connected to a multi-
parameter M400 transmitter (Mettler Toledo, Columbus, OH).
The redox potential value from Ag/AgCl reference electrode was
calculated vs. standard hydrogen electrode (SHE) by the addition
of +222.4 mV to the value obtained by the Ag/AgCl electrode
(Kakiuchi et al., 2007).
6496 P. Maddipati et al. / Bioresource Technology 102 (2011) 64946501
2.6.5. Sugar analysis
The sugar content of the liquid portion of crude CSL was mea-
sured using high pressure liquid chromatography (HPLC) (Agilent
1100 series, Wilmington, DE) with refractive index detector. The
column used was Aminex HPX 87P (Bio-Rad, Sunnyvale, CA,
USA), which was operated at 85 C with de-ionized water as the
mobile phase pumped at 0.6 mL min
1
for 30 min per sample.
2.6.6. Statistical analysis
Analysis of variance (ANOVA) was determined using the GLM
procedure of SAS Release 9.2 (Cary, NC). A Dunnetts test (Dunnett,
1955) at 95% condence level was used to determine the statistical
signicance of the fermentation parameters (pH, cell mass, etha-
nol, acetic acid and butanol) between the three different media
used in 250-mL fermentation bottles. Signicance level was tested
at p = 0.05.
3. Results and discussion
3.1. Fermentation in 250 mL serum bottles
3.1.1. Cell growth and pH proles
Cell mass concentrations of Clostridium strain P11 increased
during the rst 48 h in the media that contained 1 g L
1
YE and
10 g L
1
CSL (Fig. 1). However, cells were in the exponential phase
in the rst 24 h in the medium with 20 g L
1
CSL. No lag phase was
observed in any of the media used. In addition, cells entered the
stationary phase in all media after 48 h. Minor changes in cell mass
concentrations were observed during the stationary phase until
360 h. The specic growth rates of strain P11 in 1 g L
1
YE,
10 g L
1
CSL and 20 g L
1
CSL media were 0.059, 0.049 and
0.076 h
1
, respectively. The specic growth rate in the medium
with 20 g L
1
CSL was 29% and 55% higher than in 1 g L
1
YE and
10 g L
1
CSL media, respectively. Though the specic growth rate
of strain P11 in the 1 g L
1
YE medium was 20% higher than in
the 10 g L
1
CSL medium, about 84% more cells were produced in
the 10 g L
1
CSL medium (Fig. 1). This could be due to the presence
of additional nutrients such as sugars, water soluble vitamins, ami-
no acids, minerals and trace metals in CSL (Liggett and Kofer,
1948). Statistical analysis indicated that the cell mass concentra-
tions in 10 and 20 g L
1
CSL media between 24 and 360 h were sig-
nicantly higher than in the 1 g L
1
YE medium, conrming that
nutrients present in CSL signicantly enhanced cell growth
(p < 0.05). Also, cell mass concentrations in 10 g L
1
CSL between
24 and 264 h were signicantly lower than in the 20 g L
1
CSL
medium (p < 0.05). However, the difference in cell mass concentra-
tion between the two CSL media after 264 h of fermentation was
insignicant (p > 0.05).
The pH of the YE and CSL media decreased during cell growth
due to the production of acetic acid (Fig. 1). The pH in the 1 g L
1
YE medium decreased from 6.1 to 4.8 at 168 h, while the pH in
the 10 g L
1
CSL medium decreased from 6.0 to 5.1 at 240 h, at
which time the acetic acid concentrations in these media reached
a maximum (Fig. 2). Subsequently, an increase in pH to 5.1 and
5.3 was observed after 360 h in the 1 g L
1
YE and 10 g L
1
CSL
media, respectively. This correlated with a decrease in acetic acid
concentration. The lowest pH value in the 20 g L
1
CSL medium
was 5.4 after 48 of fermentation, after which the pH started to in-
crease due to acetic acid consumption during syngas fermentation
(Fig. 2). The pH in the 20 g L
1
CSL mediumwas signicantly higher
than in 1 g L
1
YE and 10 g L
1
CSL media after 72 h (p < 0.05). Also,
the pH in the 10 g L
1
CSL medium was signicantly higher com-
pared to 1 g L
1
YE medium between 72 and 264 h (p < 0.05).
3.1.2. Product proles
Acetic acid and ethanol were the major products formed during
syngas fermentation in both YE and CSL media (Fig. 2). Acetic acid
is a primary metabolite, and its production is associated with cell
growth. The production of acetic acid was higher during the
growth phase and gradually decreased when the cells entered
the stationary phase. A maximum acetic acid concentration of
2.6 g L
1
was reached at 144 h in the 1 g L
1
YE medium (Fig. 2).
Acetic acid concentration then decreased to 2.3 g L
1
at the end
of the fermentation. Acetic acid concentrations were signicantly
lower between 96 and 168 h and from 96 to 360 h in the 10 and
20 g L
1
CSL media, respectively, compared to 1 g L
1
YE medium
(p < 0.05). The difference in acetic acid concentrations between
10 g L
1
CSL and 1 g L
1
YE media was insignicant after 192 h
(p > 0.05).
Consumption of acetic acid by strain P11 was observed at later
stages of fermentation in the three media. More acetic acid was
consumed in the 20 g L
1
CSL medium compared to the other
two media. Acetic acid consumption was associated with produc-
tion of ethanol. This was also observed in other syngas fermenta-
tion studies using Clostridium strain P11 (Frankman, 2009;
Panneerselvam et al., 2010; Saxena, 2008) and C. carboxidivorans
P7 (Hurst and Lewis, 2010).
Ethanol production started in the stationary phase (Fig. 2). The
rate of ethanol production increased in the early stationary phase
and decreased with the decline in cell concentration. The nal eth-
anol concentration in the 20 g L
1
CSL medium was 1.7 g L
1
,
which was 31% and 21% higher than in 1 g L
1
YE and 10 g L
1
CSL media, respectively. In addition, the amount of ethanol
0.0
0.1
1.0
4.5
5.0
5.5
6.0
6.5
7.0
0 48 96 144 192 240 288 336 384
C
e
l
l

m
a
s
s

(
g

L
-
1
)
Time (h)
p
H
Fig. 1. Cell mass (solid symbols) and pH (open symbols) proles using Clostridium
strain P11 in 250-mL serum bottles: (s) 1 g L
1
YE medium, (h) 10 g L
1
CSL
medium, (D) 20 g L
1
CSL medium (n = 3).
0.0
1.0
2.0
3.0
4.0
0.0
1.0
2.0
3.0
0 48 96 144 192 240 288 336 384
E
t
h
a
n
o
l

(
g
/
L
)
Time (h)
A
c
e
t
i
c
a
c
i
d

(
g
/
L
)
Fig. 2. Acetic acid (solid symbols) and ethanol (open symbols) proles using
Clostridium strain P11 in 250-mL serum bottles: (s) 1 g L
1
YE medium, (h) 10 g L
1
CSL medium, (D) 20 g L
1
CSL medium (n = 3).
P. Maddipati et al. / Bioresource Technology 102 (2011) 64946501 6497
produced in the 20 g L
1
CSL medium was signicantly higher
compared to 1 g L
1
YE and 10 g L
1
CSL media from the beginning
of the fermentation until 240 h (p < 0.05). Higher ethanol produc-
tion in 20 g L
1
CSL can also be due to the presence of more nutri-
ents compared to 1 g L
1
YE and 10 g L
1
CSL media. The difference
in ethanol concentrations between the 1 g L
1
YE and 10 g L
1
CSL
media was statistically insignicant during the entire fermentation
(p > 0.05). Ethanol productivities of Clostridium strain P11 were 3.6,
4.0 and 4.7 mg L
1
h
1
in 1 g L
1
YE, 10 g L
1
CSL and 20 g L
1
CSL
media, respectively.
Butanol was also produced during syngas fermentation in YE
and CSL media (Fig. 3). Butanol production started after 24 and
72 h in the 10 and 20 g L
1
CSL media, respectively. After 96 h,
butanol was observed in the 1 g L
1
YE medium. The nal concen-
trations of butanol were below 0.1 and 0.6 g L
1
in the YE and CSL
media, respectively. Statistical analysis indicated that CSL has sig-
nicant effect on butanol production by strain P11. Over sevenfold
more butanol was produced in the 10 and 20 g L
1
CSL media com-
pared to the 1 g L
1
YE medium, which was statistically signicant
(p < 0.05). The difference in butanol concentrations in the 10 and
20 g L
1
CSL media after 360 h was insignicant (p > 0.05). The
amount of butanol produced by Clostridium strain P11 in 20 g L
1
CSL medium using 20% CO, 15% CO
2
, 5% H
2
and 60% N
2
gas mixture
was two times lower than with C. carboxidivorans using 25% CO,
15% CO
2
and 60% N
2
gas mixture (Rajagopalan et al., 2002) and B.
methylotrophicum using pure CO (Worden et al., 1991), respec-
tively. The compositions of the gas mixture and media used have
a large effect on the amount of solvents produced during
fermentation.
3.2. Fermentation in 7.5-L fermentor
After studying the growth and product kinetics of Clostridium
strain P11 in 250-mL serum bottles, it was necessary to scale-up
the process to better understand and evaluate the kinetics in a pro-
totype bench top stirred tank reactor. Therefore, syngas fermenta-
tions by Clostridium strain P11 with the three media compositions
used in bottle fermentations were investigated in a 7.5-L stirred
tank reactor. Fermentation runs were not conducted in triplicate
due to limited time and available resources.
3.2.1. Cell growth and pH proles
The growth and pH proles of strain P11 in 1 g L
1
YE, 10 g L
1
CSL and 20 g L
1
CSL media are shown in Fig. 4. Similar fermenta-
tion proles were noticed in all three media. There was a short lag
phase followed by an exponential phase. The cell concentrations in
all three media increased exponentially during the rst 48 h of
fermentation. The specic growth rates of strain P11 were 0.055,
0.061 and 0.075 h
1
in the 1 g L
1
YE, 10 g L
1
CSL and 20 g L
1
CSL media, respectively (Table 3)
.
The specic growth rates of C.
ljungdahlii (Phillips et al., 1994), and Clostridium strain P11 (Frank-
man, 2009) in yeast extract medium were reported as 0.06 and
0.0491 h
1
, respectively. These specic growth rates are lower
than those with strain P11 in the 20 g L
1
CSL medium.
Similar to the results obtained in bottle fermentations, more
cells were produced in media that contained CSL. The maximum
cell concentrations in 1 g L
1
YE, 10 g L
1
CSL and 20 g L
1
CSL
media were 0.53, 0.55 and 0.74 g L
1
, respectively. About 39%
and 35% more cells were produced in the 20 g L
1
CSL medium
compared to 1 g L
1
YE and 10 g L
1
CSL media, respectively. This
conrms the advantage of using CSL in the fermentation medium
compared to YE. The drastic decrease in cell concentration in
10 g L
1
CSL medium after 264 h was due to foam formation, which
caused cells to stick onto the wall of the fermentor. Foam forma-
tion in the medium was due to the proteins present in the medium
and proteins released from dead cells (Doran, 2006). In the present
study, the cell mass yield (g cells produced per g CO consumed)
calculated at the maximum cell mass concentrations in 1 g L
1
YE medium was about 15% higher than in both CSL media (Table
3). This is due to the consumption of more CO by strain P11 at
the times at which the maximum cell mass concentrations were
reached in 10 g L
1
CSL (192 h) and 20 g L
1
CSL media (240 h)
compared to 1 g L
1
YE medium (168 h) as shown in Figs. 4 and 6a.
The pH proles for the fermentation in the three media used
were generally similar. The pH of the 1 g L
1
YE and 10 g L
1
CSL
media decreased from 6 to 4.7 during cell growth in the rst
72 h due to the production of acetic acid (Fig. 4). However, the
pH in the 20 g L
1
CSL medium decreased from 6 to 4.7 after
0.0
0.2
0.4
0.6
0.8
0 48 96 144 192 240 288 336 384
Time (h)
B
u
t
a
n
o
l

(
g
/
L
)
Fig. 3. Butanol proles using Clostridium strain P11 in 250-mL serum bottles: (s)
1 g L
1
YE medium, (h) 10 g L
1
CSL medium, (D) 20 g L
1
CSL medium (n = 3).
0.0
0.1
1.0
4.5
5.0
5.5
6.0
6.5
7.0
0 48 96 144 192 240 288 336 384
C
e
l
l

m
a
s
s

(
g

L
-
1
)
Time (h)
p
H
Fig. 4. Cell mass (solid symbols) and pH (open symbols) proles using Clostridium
strain P11 in 7.5-L fermentor: (s) 1 g L
1
YE medium, (h) 10 g L
1
CSL medium, (D)
20 g L
1
CSL medium.
Table 3
Fermentation characteristics of Clostridium strain P11 in yeast extract (YE) and corn
steep liquor (CSL) media in 7.5-L fermentor.
Medium
YE CSL CSL
1 g L
1
10 g L
1
20 g L
1
Fermentation time (h) 360 360 360
l
max
(h
1
) 0.055 0.061 0.075
Cell mass yield
a
(g cells g
1
CO consumed) 0.0055 0.0047 0.0048
Acetic acid (g L
1
) 1.5 2.4 3.4
Ethanol (g L
1
) 6.1 8.6 9.6
Ethanol productivity (mg L
1
h
1
) 16.9 23.9 26.7
Ethanol yield from CO (% of theoretical value) 53 57 60
Conversion efciency of CO (%) 11 15 15
Conversion efciency of H
2
(%) 5 7 5
a
Values were calculated at maximum cell mass concentrations.
6498 P. Maddipati et al. / Bioresource Technology 102 (2011) 64946501
120 h, during which cells were still growing. Then, the pH in all
three media slightly increased due to consumption of acetic acid
(Fig. 5). The difference in pH proles for 1 g L
1
YE and 10 g L
1
CSL media was smaller compared to the 20 g L
1
CSL medium after
144 h of fermentation.
3.2.2. Product proles
Similar to bottle fermentations, acetic acid and ethanol produc-
tion in all media with Clostridium strain P11 was observed during
the acidogenic and solventogenic phases, respectively. However
unlike bottle fermentations, no butanol was produced by strain
P11 in the 7.5 L fermentor. The focus of the present study was
not on butanol production. In addition, the results showed that
the type of media and reactor conguration used has a large effect
on strain P11 ability to produce butanol.
Acetic acid concentration increased in the rst 72 h in both
1 g L
1
YE and 10 g L
1
CSL media and during the rst 120 h in
the 20 g L
1
CSL medium (Fig. 5). Acetic acid formation by strain
P11 was growth related, which resulted in a decrease in the pH
of the fermentation medium (Fig. 4). The maximum acetic acid
concentrations in 1 g L
1
YE, 10 g L
1
CSL and 20 g L
1
CSL media
were 3.9, 4.2 and 4.0 g L
1
, respectively. After the concentration
of acetic acid reached a maximum, strain P11 started to consume
it. However, the consumption of acetic acid was the highest in
the 1 g L
1
YE medium (Fig. 5). About 60% and 44% of the acetic
acid was consumed between 72 and 360 h in 1 g L
1
YE and
10 g L
1
CSL media, respectively (Fig. 5). Only 16% of the acetic acid
in the 20 g L
1
CSL medium was consumed between 120 and 360 h.
In bottle fermentations, about 57% of the acetic acid was consumed
in the 20 g L
1
CSL medium compared to 12% in the 1 g L
1
YE
medium (Fig. 2).
The switch to ethanol production was observed during deceler-
ated growth and stationary phases when the pH was around 4.7
(Figs. 2 and 5). Ethanol formation by strain P11 in both bottle fer-
mentation and 7.5-L fermentor was non-growth associated. The
partial pressures of CO in the headspace in bottle fermentations
and 7.5-L fermentor were 48 and 29 kPa, respectively. It was re-
ported that ethanol production by C. carboxidivorans P7 was non-
growth related when the partial pressure of CO in the headspace
was below 106 kPa (Hurst and Lewis, 2010). However, ethanol for-
mation was growth associated with C. carboxidivorans P7 when the
partial pressure of CO in the headspace was above 106 kPa. In the
present study, the rate of ethanol production in all media was high
in the early stationary phase and decreased with time especially
with the decline in cell mass concentration. After 360 h of fermen-
tation, ethanol concentrations in 1 g L
1
YE, 10 g L
1
CSL and
20 g L
1
CSL media were 6.1, 8.6 and 9.6 g L
1
, respectively (Table
3). This includes ethanol in the 7.5-L fermentor and collected in
the water trap after the condenser. The volumetric ethanol produc-
tivities of Clostridium strain P11 in 10 g L
1
CSL and 20 g L
1
CSL
media were 41% and 58% higher than in 1 g L
1
YE medium, respec-
tively (Table 3). About 3 g L
1
of ethanol was formed after 504 h in
a 5-L fermentor using strain P11 and cotton seed extract (CSE) as a
medium component (Kundiyana et al., 2010). In the present study,
ethanol productivities in 10 and 20 g L
1
CSL media (Table 3) were
over fourfold higher than previously reported for CSE media.
Ethanol formation was noticed when the ranges of redox poten-
tial (ORP) in 1 g L
1
YE, 10 g L
1
CSL and 20 g L
1
CSL media were
206 to 118 mV SHE, 150 to 45 mV SHE and 121 to
75 mV SHE, respectively. The ORP range for ethanol production
with strain P11 in cotton seed extract medium was from 240 to
130 mV SHE (Kundiyana et al., 2010).
In the present study, the presence of more nutrients in the
20 g L
1
CSL medium could have contributed to the higher perfor-
mance of strain P11 compared to 1 g L
1
YE medium. The initial to-
tal sugar concentrations in the 10 and 20 g L
1
CSL media were 0.6
and 1.2 g L
1
, respectively. Among the sugars (maltose, glucose, xy-
lose, galactose, arabinose and mannose) in CSL media, only glucose
was utilized by strain P11 after 360 h of syngas fermentation. The
initial glucose concentrations in the 10 and 20 g L
1
CSL media
were 0.12 and 0.32 g L
1
, respectively. Only 10% and 76% of the ini-
tial amounts of glucose in 10 and 20 g L
1
CSL media were con-
sumed after 360 h, respectively. The maximum possible amount
of ethanol production assuming 100% conversion efciency of glu-
cose to ethanol in the 10 and 20 g L
1
CSL media are 0.06 and
0.16 g L
1
, respectively. These amounts of ethanol that can be pro-
duced from the sugars present in CSL are negligible compared to
8.6 and 9.6 g L
1
of ethanol produced from syngas in 10 and
20 g L
1
CSL media, respectively (Table 3). This conrms that prac-
tically all of the ethanol formed in the 7.5-L fermentor with 10 and
20 g L
1
CSL media was produced by strain P11 using syngas.
0.0
0.2
0.4
0.6
0.8
1.0
0.0
2.0
4.0
6.0
0 48 96 144 192 240 288 336 384
C
O

(
m
o
l
)
Time (h)
H
2
(
m
o
l
)
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
0 48 96 144 192 240 288 336 384
C
O
2
(
m
o
l
)
Time (h)
b
a
Fig. 6. (a) Cumulative CO (solid symbols) and H
2
(open symbols) consumed;
(b) cumulative CO
2
produced (open symbols) by Clostridium strain P11 in 7.5-L
fermentor: (s) 1 g L
1
YE medium, (h) 10 g L
1
CSL medium, (D) 20 g L
1
CSL
medium.
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
0.0
1.0
2.0
3.0
4.0
5.0
0 48 96 144 192 240 288 336 384
E
t
h
a
n
o
l

(
g

L
-
1
)
Time (h)
A
c
e
t
i
c
a
c
i
d

(
g

L
-
1
)
Fig. 5. Acetic acid (solid symbols) and ethanol (open symbols) proles using
Clostridium strain P11 in 7.5-L fermentor: (s) 1 g L
1
YE medium, (h) 10 g L
1
CSL
medium, (D) 20 g L
1
CSL medium.
P. Maddipati et al. / Bioresource Technology 102 (2011) 64946501 6499
Ethanol yields in 1 g L
1
YE, 10 g L
1
CSL and 20 g L
1
CSL media
were 53%, 57% and 60% of the theoretical values from CO, respec-
tively (Table 3).
Based on prices of CSL ($0.18 kg
1
) and YE ($9.2 kg
1
) in 2010,
CSL contributed $0.30 L
1
ethanol produced in 20 g L
1
CSL med-
ium at 9.6 g L
1
ethanol, which is fourfold lower than YE contribu-
tion in 1 g L
1
YE medium at 6.1 g L
1
ethanol. The use of 20 g L
1
CSL is currently not cost-effective at the present ethanol titers and
yields from syngas fermentation. However, higher ethanol concen-
tration and yield will make the use of CSL more cost effective.
3.2.3. Gas consumption
Both CO and H
2
were utilized by strain P11 for growth and ace-
tic acid and ethanol production. The cumulative CO and H
2
con-
sumed by strain P11 during syngas fermentation is shown in
Fig. 6a. The total moles of CO consumed by strain P11 in 1 g L
1
YE, 10 g L
1
CSL and 20 g L
1
CSL were 4.3, 5.5 and 5.8 mol, respec-
tively. The total moles of H
2
consumed by strain P11 in 1 g L
1
YE,
10 g L
1
CSL and 20 g L
1
CSL were 0.45, 0.63 and 0.48 mol, respec-
tively. About 39%, 23% and 28% of the total CO was consumed in
1 g L
1
YE, 10 g L
1
CSL and 20 g L
1
CSL media, respectively, dur-
ing the rst 72 h of fermentation for mostly growth and acetic acid
production. During the same fermentation period, about 55%, 61%
and 57% of the total H
2
was consumed in 1 g L
1
YE, 10 g L
1
CSL
and 20 g L
1
CSL media, respectively.
In the acetyl-CoA pathway, both H
2
and CO serve as an energy
and electron source for cell growth and product formation (Rags-
dale, 2004). Signicant amounts of carbon from CO can be con-
verted to cell material and ethanol if H
2
is utilized as an electron
source. The depletion of H
2
in the fermentation medium would re-
duce the amount of carbon available for ethanol production be-
cause a fraction of CO would be utilized for generating reducing
equivalents required (Ahmed and Lewis, 2007). Therefore, increas-
ing the concentration of H
2
in the gas phase would increase the
contribution of CO to ethanol production. In contrast, high concen-
trations of CO were found to inhibit hydrogenase enzyme and thus
reduce the uptake of H
2
in non-CO fermenting organisms (Bennett
et al., 2000; Kim et al., 1984). This could explain the decrease in H
2
consumption during the stationary phase with strain P11 (Fig. 6a).
Trends in CO consumption proles (Fig. 6a) were similar to eth-
anol proles in all media used (Fig. 5). Ethanol production started
after 48 h of the fermentation in all media. For ethanol production
and cell maintenance between 48 and 360 h, approximately 77%,
89% and 85% of the total moles of CO were consumed in 1 g L
1
YE, 10 g L
1
CSL and 20 g L
1
CSL media, respectively. This shows
that most of the CO consumed by strain P11 was utilized for etha-
nol production according to Eq. (1). More CO was consumed in the
20 g L
1
CSL medium compared to 1 g L
1
YE and 10 g L
1
CSL
media (Fig. 6a), which resulted in higher ethanol production in
the 20 g L
1
CSL medium (Fig. 5). The conversion efciencies of
CO and H
2
in all media used were low due to the continuous supply
of syngas at a rate that strain P11 could not utilize (Table 3). To en-
hance the CO and H
2
conversion efciencies, lower syngas ow
rates and/or higher initial cell concentrations in the fermentor
should be used.
The consumption of CO
2
was observed during the growth phase
in the 1 g L
1
YE medium (Fig. 6b). Then, there was no net produc-
tion of CO
2
in this medium. However, there was a net production of
CO
2
in 10 and 20 g L
1
CSL media. Some CO
2
was consumed in 10
and 20 g L
1
CSL medium during late stationary and death phases.
Acetic acid production was observed in late stationary phase and
death phase in 10 and 20 g L
1
CSL media with the consumption
of CO
2
and H
2
via Eq. (4).
It is clear that strain P11 utilized more CO than H
2
for growth
and product formation (Fig. 6a). Over 50% of the total H
2
consumed
in the fermentation was utilized by strain P11 during growth and
acetic acid production. Moreover, small amounts of CO
2
were con-
sumed in the fermentation media with a net CO
2
production espe-
cially in both CSL media (Fig. 6b). The CO
2
and H
2
consumption by
strain P11 could have been used for ethanol or acetic acid produc-
tion according to Eqs. (2) and (4). This is also supported by the cur-
rent results because CO
2
utilization by strain P11 was mostly
associated with acetic acid production (Figs. 5 and 6b).
To make syngas fermentation more efcient, the medium used
should be inexpensive and process parameters such as syngas
composition and feeding rate should be optimized to increase syn-
gas conversion efciency and ethanol productivity.
4. Conclusions
CSL can be used as a lower cost nutrient source in syngas fer-
mentation using Clostridium strain P11 to replace more expensive
YE. High cell density and extended stationary phase were achieved
in the 7.5-L fermentor with 10 and 20 g L
1
CSL media, which re-
sulted in over 40% more ethanol production compared to the
1 g L
1
YE medium. The use of CSL as a nutrient enhanced butanol
production in bottle fermentations. Over sevenfold more butanol
was produced in both CSL media compared to the 1 g L
1
YE
medium.
Acknowledgements
Support for this research was provided by USDA-CSREES Special
Research Grant Award 2008-34417-19201, Oklahoma Agricultural
Experiment Station, and the Oklahoma Bioenergy Center.
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