Microaerobic Conversion of Glycerol to Ethanol in Escherichia coli

Matthew S. Wong, Mai Li, Ryan W. Black, Thao Q. Le, Sharon Puthli, Paul Campbell, Daniel J. Monticello
Glycos Biotechnologies, Houston, Texas, USA

Glycerol has become a desirable feedstock for the production of fuels and chemicals due to its availability and low price, but
many barriers to commercialization remain. Previous investigators have made significant improvements in the yield of ethanol
from glycerol. We have developed a fermentation process for the efficient microaerobic conversion of glycerol to ethanol by
Escherichia coli that presents solutions to several other barriers to commercialization: rate, titer, specific productivity, use of
inducers, use of antibiotics, and safety. To increase the rate, titer, and specific productivity to commercially relevant levels, we
constructed a plasmid that overexpressed glycerol uptake genes dhaKLM, gldA, and glpK, as well as the ethanol pathway gene
adhE. To eliminate the cost of inducers and antibiotics from the fermentation, we used the adhE and icd promoters from E. coli
in our plasmid, and we implemented glycerol addiction to retain the plasmid. To address the safety issue of off-gas flammability,
we optimized the fermentation process with reduced-oxygen sparge gas to ensure that the off-gas remained nonflammable.
These advances represent significant progress toward the commercialization of an E. coli-based glycerol-to-ethanol process.

C oncerns about energy security, environmental damage, and

sustainability have stimulated interest in developing non-pe-
troleum-based sources of fuel. Biodiesel is one of the most com-
mon renewable transportation fuels, and its production has
grown immensely in the last 2 decades (1). This has created a
surplus of crude glycerol, because the transesterification process
produces about 1 kg of crude glycerol for every 10 kg of biodiesel
produced. This cheap, highly reduced carbon source can be used
in microbial fermentations.
Considerable research effort has been focused on converting
glycerol into useful chemicals (2). Although there are many or-
ganisms capable of metabolizing glycerol, we are most interested
in industrial microbes that are amenable to genetic manipulations
for the application of rational metabolic-engineering strategies.
We chose to work with Escherichia coli, which has been shown to
metabolize glycerol under anaerobic and microaerobic conditions
(35). Previous studies have demonstrated the efficient conver-
sion of glycerol into fuels and chemicals such as ethanol (6), 1,3-
propanediol (7), 1,2-propanediol (8), poly(3-hydroxybutyrates)
(PHBs) (9), succinic acid (10), and lactic acid (11, 12).
We chose ethanol as our product due to its potential as a bio-
fuel and feedstock for the production of oxygenated fuels (13).
Previous investigators have proven the concept of converting glyc-
erol to ethanol in a microaerobic environment (6, 14, 15). With
the deletion of specific by-product pathways, they have achieved
ethanol yields from glycerol as high as 95% (6) and 97% (15) of the
maximum theoretical yield. These yields are high enough for a
commercial process, but we need to overcome many other barri-
ers to commercialize the process. Yazdani and Gonzalez showed
that simultaneous overexpression of dhaKLM and gldA increased
glycerol utilization and ethanol synthesis (6). In the work pre-
sented here, we used the foundation created by the previous stud-
ies to engineer a strain and a process for the conversion of crude
glycerol into ethanol. We implemented genetic and process ma-
nipulations to increase the rate, titer, and specific productivity,
avoid the use of antibiotics and inducers, and avoid a flammable
H2-O2 mixture in the reactor headspace. These advances remove
several significant barriers to the commercialization of a glycerol-
to-ethanol fermentation process.
MATERIALS AND METHODS
Bacterial strains, plasmids, and oligonucleotides. All Escherichia coli
strains, along with plasmids used in this work, are listed in Table 1. For
oligonucleotides used in this work, see Table S1 in the supplemental
material.
DNA manipulations and mutant construction. Unless otherwise in-
dicated, all DNA procedures followed standard protocols and specific
recommendations from manufacturers. E. coli knockout mutants were
constructed by the suicide vector method (16). E. coli genes were ampli-
fied from genomic DNA by using AccuPrime Pfx DNA polymerase (Life
Technologies, Invitrogen) and cloned into vectors by using the Quick
Ligation kit (New England BioLabs Inc., Ipswich, MA) or In-Fusion HD
cloning kit (Clontech Laboratories Inc., Mountain View, CA).
Growth media and culture conditions. The MOPS [3-(N-morpholi-
no)propanesulfonic acid] minimal medium designed by Neidhardt et al.
(17) was supplemented with 30 mM NH4Cl, 3.96 mM Na2HPO4 in place
of 1.32 mM K2HPO4, 5 mM (NH4)2SO4, and 1 M sodium selenite. This
medium was used during the preliminary fermentations.
Unless otherwise specified, the minimal medium used for fermenta-
tions was MM29, which was made in the following manner. To 1 liter of
water, we added 0.66 g (NH4)2SO4, 1.2 g Na2HPO4, 3 g NH4Cl, 0.25 g
K2SO4, and 1 ml of micronutrient solution. The mixture was sterilized by
autoclaving. After the mixture cooled to room temperature, 10 ml of
divalent-cation solution was added to the medium to complete MM29.
The micronutrient solution consisted of 0.173 g sodium selenite, 0.004 g
(NH4)6Mo7O24 H2O, 0.025 g H3BO3, 0.007 g CoCl2 6H2O, 0.003 g
CuSO4 5H2O, 0.016 g MnCl2 4H2O, and 0.003 g ZnSO4 7H2O dis-
solved in 1 liter of water. The pH of the micronutrient solution was ad-
justed to 3.0 with 3 M HCl to fully dissolve the chemicals. The divalent-
cation solution was made by adding the following chemicals to 1 liter of
Received 20 November 2013 Accepted 23 February 2014
Published ahead of print 28 February 2014
Editor: R. M. Kelly
Address correspondence to Daniel J. Monticello, dmonticello@glycosbio.com.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AEM.03863-13.
Copyright 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.03863-13

3276 aem.asm.org Applied and Environmental Microbiology p. 3276 3282 May 2014 Volume 80 Number 10
 Microaerobic Conversion of Glycerol to Ethanol

TABLE 1 Strains and plasmids used in this study

Strain or plasmid Relevant characteristics Reference or source
E. coli strains
ATCC 8739 ATCC, Manassas, VA
GB1 ATCC 8739 frdA This work
GB2 ATCC 8739 frdA pta This work
GB3 ATCC 8739 frdA pta ldhA This work
GB4 ATCC 8739 ackA pta ldhA This work
GB5 ATCC 8739 frdABCD ackA pta ldhA poxB This work
GB6 ATCC 8739 frdABCD ackA pta ldhA poxB gldA glpK This work

Plasmids
pZSKLMgldA E. coli dhaKLM and gldA under the control of PLtetO-1 (oriR SC101; Cmr) 6
pZSadhEpKLMgldA pZSKLMgldA derivative with the E. coli adhE promoter replacing the PLtetO-1 This work
promoter (Cmr, oriR SC101)
pGB1001 pZSadhEpKLMgldA derivative with a ribosomal binding site inserted in front of This work
gldA (Cmr, oriR SC101)
pGB1001.1 Intermediate construct putting the rrnB transcriptional terminator and E. coli This work
adhE operon together in ptrcHisA (Life Technologies, Invitrogen)
pGB1002 pGB1001 derivative with an rrnB transcriptional terminator after the adhEp- This work
dhaKLM-gldA operon and the E. coli adhE operon inserted after the rrnB
terminator (Cmr, oriR SC101)
pGB1003 pGB1002 derivative expressing the glpK gene from E. coli under the control of This work
the E. coli isocitrate dehydrogenase promoter (21) (Cmr, oriR SC101)

water: 40 g MgCl2 6H2O, 0.3 g FeSO4 7H2O, and 7 g CaCl2 2H2O. The
divalent-cation solution was filter sterilized before use.
Chloramphenicol (40 mg/liter) was added when it was appropriate.
Chemicals were obtained from Sigma-Aldrich Co. (St. Louis, MO). Tech-
nical-grade glycerol (99.7% purity) was obtained from KIC Chemicals
(New Paltz, NY). Crude glycerol was obtained from REG (Ames, IA). The
crude glycerin had the following reported composition (wt/vol): 83%
glycerol, 11% water, and 6% ash (most of which was in the form of
NaCl); minor constituents included free fatty acids (0.31%) and methanol
(0.05%). No other MONG (matter organic nonglycerol) was detected in
this sample. Experiments using crude glycerol directly substituted crude
glycerol for technical-grade glycerol.
Bioreactor cultivation. Bioreactor cultivations were carried out in
one of three bioreactor systems. Preliminary fermentations used 0.75-liter
glass jar reactors from Wards Science (Rochester, NY). Batch cultures
were grown in a 0.5-liter working volume with the agitation accomplished
by a stir bar at the bottom of the vessel. The agitation was set to provide the
desired oxygen transfer coefficient (kLa). The fermentor was sparged with
air, and the pH was controlled at 6.3 by automatic addition of 5 M NaOH.
Temperature was controlled at 37C.
Bioreactor cultivations were also carried out in 7-liter stirred-tank
reactors equipped with two Rushton impellers on the stirrer shaft (Biostat
A Plus; Sartorius North America, Edgewood, NY). Batch cultures were
grown in a 4-liter working volume with the agitation set to provide the
desired kLa. The fermentor was sparged with air or a 5% O2-95% N2 gas
mixture, and the pH was controlled at 6.3 by automatic addition of 9%
NH4OH (wt/vol). Temperature was controlled at 37C. Fed-batch cul-
tures were fed by manual injections of a feed solution consisting of 1,000
g/liter glycerol in water.
The final bioreactor cultivations were carried out in 0.75-liter stirred- FIG 1 Main fermentative pathways involved in the microaerobic fermenta-
tank reactors equipped with two Rushton impellers on the stirrer shaft tion of glycerol in E. coli. Relevant genes and generation/consumption of redox
(Biostat Q Plus; Sartorius North America, Edgewood, NY). Batch cultures equivalents are included. Glycerol dissimilation under microaerobic condi-
tions proceeds through both the dhaKLM-gldA and glpK-glpD branches. Eth-
were grown in a 0.5-liter working volume with the agitation set to provide
anol, lactate, succinate, and acetate are the main products of glycerol fermen-
the desired kLa. The fermentor was sparged with a 5% O2-95% N2 gas tation. Genetic modifications are indicated by a plus symbol () for plasmid
mixture, and the pH was controlled at 6.3 by automatic addition of 9% overexpressions (dhaKLM, gldA, glpK, and adhE) and by a circle with a slash
NH4OH (wt/vol). Temperature was controlled at 37C. Fed-batch cul- through it for chromosomal knockouts (KO) (frdABCD, ackA, pta, ldhA, poxB,
tures were fed by manual injections of a feed solution consisting of 1,000 gldA, and glpK). DHA, dihydroxyacetone; G3P, glyceraldehyde 3-phosphate;
g/liter glycerol in water. The cultures were fed so that the glycerol concen- PEP, phosphoenolpyruvate; Pyr, pyruvate; Ac-CoA, acetyl coenzyme A.

May 2014 Volume 80 Number 10 aem.asm.org 3277

Wong et al.
FIG 2 Fermentation product profiles of the wild-type strain and various mu-
tants. Ethanol, succinate, lactate, and acetate titers are shown. Experiments
were conducted in Wards Science 0.75-liter reactors. The cells were grown in
MOPS minimal medium at pH 6.3 and a kLa of 2.4/h for 72 h.
tration remained above 0 g/liter. Each feed bolus raised the reactor glyc-
erol concentration by only 10 to 15 g/liter to keep the osmotic shock to a
minimum.
Analytical methods. Optical density was measured at 600 nm
(OD600). Fermentation broth samples were centrifuged, and the superna-
tants were analyzed by high-performance liquid chromatography (HPLC)
for extracellular glycerol, lactate, acetate, succinate, and ethanol. The ion-
exclusion HPLC technique used a Shimadzu Prominence SIL 20 system
(Shimadzu Scientific Instruments, Inc., Columbia, MD) equipped with an
HPX-87H organic acid column (Bio-Rad, Hercules, CA). Operating con-
ditions consisted of a 2.5 mM H2SO4 mobile phase, a column temperature
of 55C, a flow rate of 0.6 ml/min, and an isocratic run mode. The transfer
of oxygen in microaerobic cultures was characterized by the volumetric
oxygen transfer coefficient, kLa (h1). The rate of volumetric oxygen
transfer (dC/dt) is estimated by the equation dC/dt kLa(C* C), where
C is the actual dissolved oxygen concentration in the liquid (mg/liter), kL
is the oxygen transport coefficient (cm/h), a is the gas-liquid interfacial
area (cm2/cm3), and C* is the saturated dissolved oxygen concentration in
the liquid (mg/liter) (18). C* was assumed to be 6.7 mg/liter in our calcu-
lations. We estimated the kLa by the static gassing-out method (18). Off-
gas concentrations of N2, H2, O2, and CO2 were determined by a benchtop
gas analysis system. We used an HPR-20 quartz inert capillary (QIC) from
Hiden Analytical (Warrington, United Kingdom).
RESULTS
Metabolic engineering strategy to increase yield. We first fo-
cused our efforts on reducing the accumulation of by-products in
the glycerol-to-ethanol fermentation. The main fermentative
pathways in E. coli are shown in Fig. 1. Possible modifications to
the E. coli metabolism are also shown in Fig. 1. Intermediate
strains contained subsets of these modifications, and the final pro-
TABLE 2 Metabolic engineering to increase the rate of ethanol production in strain GB5a
Final ethanol
Strain titer (g/liter) Final OD600
GB5 1.0 16.3
GB5/pGB1001 24.0 13.2
GB5/pGB1002 25.7 8.3
a
Experiments were conducted in Wards Science 0.75-liter reactors. The cells were grown in MOPS minimal medium at pH 6.3 and a kLa of 50/h for 72 h.
3278 aem.asm.org
duction strain, GB6/pGB1003, contained all of the modifications.
The genotypes of all strains in this study are listed in Table 1. We
first constructed several deletion strains to determine which dele-
tions or combinations of deletions were desirable. Figure 2 shows
the results from running ATCC 8739 and GB1 through GB5 in the
Wards Science 0.75-liter reactors at a kLa of 2.4/h. The wild-type
ATCC 8739 strain made ethanol as its main product but also accu-
mulated significant amounts of succinate and acetate. The yield of
ATCC 8739 was 0.36 g ethanol/g glycerol (the maximum theoretical
yield is 0.5 g ethanol/g glycerol). In GB1, the deletion of frdA elimi-
nated succinate production, but lactate was increased. In GB2, acetate
and lactate remained as by-products after deletion of frdA and pta. In
GB3, deletions of frdA, pta, and ldhA left acetate as the lone by-prod-
uct. GB4 showed that the addition of the ackA deletion reduced ace-
tate to its lowest levels. The final GB5 strain had all of the deletions
and had a yield of 0.43 g ethanol/g glycerol, which was in line with
results from previous studies (6, 14, 15). The deletions in GB5 in-
creased the yield but disrupted metabolism such that the rate was
reduced from 0.20 g/liter/h ethanol in the wild type to 0.14 g/liter/h
ethanol in GB5. This led us to target selected genes for overexpression
to increase the ethanol production rate.
Metabolic engineering strategy to increase rate. Our next
goal was to increase the ethanol production rate as much as pos-
sible. The decreased specific glycerol consumption rate was a large
factor in the decreased ethanol production rate. Cell growth was
actually greater in GB5 (final OD600 6.7) than in ATCC 8739
(final OD600 4.3), possibly due to the minimization of by-prod-
uct toxicity and the reduction in carbon loss to by-products. How-
ever, the specific glycerol consumption rate in GB5 (0.06 g/liter/
h/OD) was much lower than the rate in ATCC 8739 (0.13 g/liter/
h/OD). We constructed pGB1001 to overexpress dhaKLM and
gldA (Table 1) and tested GB5/pGB1001 against GB5 at a kLa of
50/h (Table 2). The kLa was increased from previous experiments
to allow for faster cell growth. As the baseline strain, GB5 grew well
but had a low specific glycerol consumption rate and little ethanol
production (Table 2). The addition of pGB1001 to GB5 led to
large increases in final ethanol titer, ethanol yield, specific glycerol
consumption rate, and specific ethanol production rate. To ex-
plore the possibility of adhE being a bottleneck, we constructed
pGB1002, which overexpressed dhaKLM, gldA, and adhE. GB5/
pGB1002 experienced a reduction in cell growth that was likely
caused by a greater plasmid/protein burden (Table 2). The ethanol
titer and yield were unchanged. However, the specific ethanol pro-
duction rate showed a 70% increase due to the overexpression of
adhE. We were satisfied at this point with the improvements in
titer, yield, and specific ethanol production rate, and so we shifted
our focus to the flammability problem caused by the H2 content of
the off-gas.
Elimination of off-gas flammability by reducing O2 in inlet
gas. According to published data, gas mixtures with hydrogen in
Specific glycerol Specific ethanol
Yield consumption production rate
(g ethanol/g glycerol) rate (g/liter/h/OD) (g/liter/h/OD)
0.02 0.04 0.001
0.44 0.06 0.025
0.47 0.10 0.043
Applied and Environmental Microbiology
 Microaerobic Conversion of Glycerol to Ethanol

the concentration range between 4% and 75% (vol/vol) are po-

tentially flammable if oxygen is present at a concentration of ap-
proximately 5% (vol/vol) or more (19). In order to avoid this
situation, we tested the use of inlet gas with 5% O2 and 95% N2.
We cultivated GB5/pGB1002 in 7-liter bioreactors at a kLa of 16/h,
which was the optimal kLa for this strain (data not shown). kLa
values higher than 16/h resulted in excess acetate production. We
also switched from using technical-grade glycerol to using crude
REG glycerin for the remaining fermentations in this study, since
this material more closely resembles the actual industrial feed-
stock for the fermentation. We had found that there was no per-
formance difference between fermentations run with technical-
grade glycerol and those employing crude REG glycerin (data not
shown). The off-gas composition time course for GB5/pGB1002
sparged with air is shown in Fig. 3A, and the ethanol production
time course is shown in Fig. 3C. The oxygen concentration stayed
above 4% for the entire fermentation, and the hydrogen concen-
tration rose very quickly after a short lag phase to reside in the
flammable range for nearly the whole fermentation. When the 5%
O2-95% N2 sparge gas was used, the off-gas stayed out of the
flammability envelope throughout the whole fermentation (Fig.
3B). The off-gas oxygen concentration at 0 h was 3.4%, and the
concentration dropped below 1% at 20 h. The hydrogen concen-
tration rose as normal, but it was rendered safe by the low oxygen
concentration. Figure 3C shows that the two types of sparge gas
gave similar ethanol production rates. The fermentation pro-
duced 0.52 g/liter/h ethanol with air as the inlet gas and 0.57 g/li-
ter/h ethanol with 5% O2 as the inlet gas.
Implementation of plasmid addiction. The use of antibiotics
to reduce contamination and select for bacteria with plasmids is
not economically feasible in a commercial-scale process (20). To
address this issue, we implemented a plasmid addiction system
based on glycerol addiction. Our goal was to construct an addicted
strain to match the ethanol time course (Fig. 4) and fermentation
performance parameters (Table 3) of GB5/pGB1002. Note that
the fermentation process had undergone some optimization com-
pared to the GB5/pGB1002 fermentation described in Table 2. We
constructed GB6, which has the gldA and glpK genes deleted, ren-
dering GB6 unable to grow on glycerol as its sole carbon source
under both aerobic and anaerobic conditions (data not shown).
The results for the combination of GB6 and pGB1002 show that
ethanol production was nearly eliminated (Fig. 4) and that every
performance parameter was significantly reduced (Table 3). The
overexpression of dhaKLM and gldA was not enough to fully com-
plement the deletions of gldA and glpK. We hypothesized that
under the microaerobic fermentation conditions, both gldA and
glpK needed to be expressed to fully complement the chromo- FIG 3 Changing the sparge gas composition to avoid off-gas flammability. (A)
somal deletions in GB6. We proceeded to construct plasmid Off-gas composition profile time course with air as sparge gas. Nitrogen and
pGB1003, which expressed glpK under the control of the Picd pro- trace components are not shown. (B) Off-gas composition profile time course
moter in addition to dhaKLM and gldA. The combination of GB6 with the 5% O2 gas mixture as sparge gas. Nitrogen and trace components are
and pGB1003 recovered the ethanol production time course that not shown. (C) Comparison of ethanol production profiles for fermentations
using sparged air and those using sparged 5% O2 gas. Experiments were con-
was seen in GB5/pGB1002 (Fig. 4) after we increased the kLa to ducted in Sartorius Biostat A Plus 7-liter reactors. GB5/pGB1002 was grown in
60/h from 16/h. However, the specific glycerol consumption rate MM29 minimal medium at pH 6.3 and a kLa of 16/h for 72 h.
and specific ethanol production rate of GB6/pGB1003 were infe-
rior to those of GB5/pGB1002 (Table 3). This forced us to grow
the fermentation to a higher OD600, which in turn decreased the was the optimal oxygen transfer rate. We ran the fermentation in
yield. Our next step was to optimize the process to improve the fed-batch mode (the feeding schedule is described in Materials
performance of the glycerol-addicted strain. and Methods). The glycerol concentration, time course, and
Process optimization for maximum performance. We tested growth curve (final OD600 19.3) are shown in Fig. 5A. This
several different aeration conditions and found that a kLa of 95/h process resulted in an improved ethanol titer (46.5 g/liter) and

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Wong et al.
FIG 4 Implementation of plasmid addiction. Data for GB5/pGB1002, GB6/
pGB1002, and GB6/pGB1003 are shown. Experiments for GB5/pGB1002 and
GB6/pGB1002 were conducted in Sartorius Biostat A Plus 7-liter reactors.
GB5/pGB1002 and GB6/pGB1002 were grown in MM29 minimal medium at
pH 6.3 and a kLa of 16/h for 72 h. The experiment for GB6/pGB1003 was
conducted in Sartorius Biostat Q Plus 0.75-liter reactors. GB6/pGB1003 was
grown in MM29 minimal medium at pH 6.3 and a kLa of 60/h for 72 h.
small amounts of by-products (Fig. 5B). The process optimization
also improved the other performance parameters for GB6/
pGB1003 shown in Table 3. The ethanol yield was 0.44 g ethanol/g
glycerol, the specific glycerol consumption rate was 0.12 g/liter/h/
OD, and the specific ethanol production rate was 0.053 g/liter/h/
OD. The dissolved-oxygen profile showed that microaerobic con-
ditions were reached within about 5 min of the inoculation (see
Fig. S1 in the supplemental material). The culture stayed mi-
croaerobic for the remainder of the 45-h fermentation. The redox
potential leveled off about 10 h into the fermentation at 390
mV and then slowly rose for the rest of the fermentation (see Fig.
S1). The off-gas composition remained outside the H2-O2 flam-
mability envelope for the entire fermentation (Fig. 5C). This ex-
periment showed that we were able to increase yield, rate, and titer
of GB6/pGB1003 while maintaining an inert off-gas and a lack of
inducer/antibiotics.
DISCUSSION
Several recent publications have explored the microaerobic fer-
mentation of glycerol to ethanol (3, 6, 14, 15). They provided the
proof of concept and data showing many avenues for greatly im-
proving rate and yield. Although the yields were commercially
relevant, many other aspects of the strains and fermentation pro-
cesses were not suited for commercial application. We undertook
TABLE 3 Implementation of plasmid addictiona
Final ethanol
Strain titer (g/liter) Final OD600
GB5/pGB1002 40.8 8.5
GB6/pGB1002 5.4 3.7
GB6/pGB1003 41.9 16.9
a
Experiments for GB5/pGB1002 and GB6/pGB1002 were conducted in Sartorius Biostat A Plus 7-liter reactors. GB5/pGB1002 and GB6/pGB1002 were grown in MM29 minimal
medium at pH 6.3 and a kLa of 16/h for 72 h. The experiment for GB6/pGB1003 was conducted in Sartorius Biostat Q Plus 0.75-liter reactors. GB6/pGB1003 was grown in MM29
minimal medium at pH 6.3 and a kLa of 60/h for 72 h.
3280 aem.asm.org
the current work to extend their results for commercial applica-
tion.
The first step toward increasing the ethanol production rate
was to increase the rate of glycerol dissimilation. Yazdani and
Gonzalez (6) showed that overexpression of dhaKLM and gldA
under the control of the PLtetO-1 promoter increased the specific
glycerol consumption rate by 1.4-fold. We found that overexpres-
sion of dhaKLM and gldA (with an additional ribosomal binding
site in front of gldA) under the control of the adhE promoter
increased the specific glycerol consumption rate by 1.5-fold before
accounting for plasmid burden. The metabolic profiling of gene
deletion mutants by Durnin et al. indicated that both respiratory
(GlpK-GlpD) and fermentative (GldA-DHAK) pathways play a
significant role in the conversion of glycerol to the glycolytic in-
termediate dihydroxyacetone (3). We confirmed the importance
of the role of GlpK-GlpD in microaerobic glycerol metabolism.
During the implementation of plasmid addiction, we deleted both
gldA and glpK from GB5 to create GB6. When only dhaKLM and
gldA were complemented in GB6/pGB1002, the specific glycerol
consumption decreased approximately 2-fold relative to that of
GB5/pGB1002. Through overexpression of both dhaKLM-gldA
and glpK in GB6/pGB1003, we nearly recovered the original spe-
cific glycerol consumption rate seen in GB5/pGB1002. We did not
expect to fully recover the original specific glycerol consumption
rate, due to the chromosomal deletions in GB6. We hypothesize
that overexpression of both glycerol dissimilation branches in-
stead of just dhaKLM-gldA had a 3-fold effect: (i) the absolute
amount of glycerol dissimilation enzymes was increased and
therefore increased flux from glycerol to dihydroxyacetone phos-
phate (DHAP), (ii) the chromosomal gldA and glpK deletions in
GB6 were fully complemented, and (iii) the generation of reduc-
ing equivalents, the use of cofactors, and the production of inter-
mediates such as dihydroxyacetone and glycerol 3-phosphate
were more like the wild-type situation. Since NADH was being
created faster due to dhaKLM-gldA and glpK overexpression, we
overexpressed adhE to help recycle the NADH and maintain redox
balance. Overexpressing adhE improved the rate and yield by pull-
ing more carbon toward ethanol instead of by-products.
The amount of oxygen provided to the culture has a profound
effect on substrate consumption, growth rate, and product mix
due to its role as the terminal electron acceptor for the recycling of
reducing equivalents. Yazdani and Gonzalez found the use of mi-
croaerobic conditions (kLa 0.5/h) instead of anaerobic condi-
tions greatly improved specific and volumetric glycerol utilization
(6). Trinh and Srienc found that the maximum ethanol yield (97%
of the theoretical maximum) for their system occurred at a kLa of
9/h (15). More oxygen transfer led to more carbon flux toward
biomass. We found that a kLa of 95/h with the 5% O2 gas was the
Specific glycerol Specific ethanol
Yield consumption production rate
(g ethanol/g glycerol) rate (g/liter/h/OD) (g/liter/h/OD)
0.44 0.15 0.067
0.28 0.07 0.020
0.37 0.09 0.034
Applied and Environmental Microbiology

FIG 5 Process data from optimized fermentation. Data for GB6/pGB1003 are
shown. (A) Glycerol time course profile for optimized fermentation. (B) Me-
tabolite time course profiles for optimized fermentation. The time course pro-
files for ethanol, acetate, succinate, and lactate are shown. (C) Off-gas compo-
sition profile time course with 5% O2 gas mixture as the inlet gas. Nitrogen and
trace components are not shown. The experiment for GB6/pGB1003 was con-
ducted in Sartorius Biostat Q Plus 0.75-liter reactors. GB6/pGB1003 was
grown in MM29 minimal medium at pH 6.3 and a kLa of 95/h for 45 h.
optimum oxygen transfer rate with our system. The dissolved ox-
ygen dropped to microaerobic levels within 5 min, but we were
able to track the redox potential to observe further changes in the
fermentation broth. The most productive portion of the fermen-
May 2014 Volume 80 Number 10
Microaerobic Conversion of Glycerol to Ethanol
tation occurred when the redox potential was near 350 to 390
mV. We hypothesize that the reduced nature of the broth coupled
with oxygen limitation encouraged a high rate of ethanol produc-
tion. This situation was termed growth-associated ethanol pro-
duction by Trinh and Srienc (15), because the only way to con-
sume more glycerol was to recycle NADH through ethanol
production, as shown in Fig. 1. As the ethanol toxicity became a
factor later in the fermentation, the dissimilation of glycerol
slowed down and less NADH was produced. This is reflected in the
slow rise of the broths redox potential. In future work, we would
like to run fermentations with variable oxygen transfer so that we
can control the redox potential at a certain set point at which
ethanol production is favored.
In the development of a commercial process, economics must
be seriously considered. The profit margins for commodity chem-
icals such as ethanol are thin, making the use of expensive inducers
infeasible. The blank pZS vector contains a PLtetO-1 promoter that
is induced with anhydrotetracycline. To eliminate the use of an-
hydrotetracycline, we replaced the PLtetO-1 promoter with the
adhE promoter for the expression of dhaKLM-gldA. We hypoth-
esized that the adhE promoter would be strongly activated under
microaerobic conditions. This choice of promoter would allow
the expression of dhaKLM-gldA to be in sync with the expression
of adhE and dhaKLM-gldAs normal role as the fermentative
branch of the glycerol dissimilation pathway. We used the native
adhE promoter in front of the adhE gene on the plasmid. We used
the isocitrate dehydrogenase promoter (Picd) to control the ex-
pression of glpK. This promoter would express glpK strongly dur-
ing the aerobic preculture stages and then reduce glpK expression
during the microaerobic fermentation through the arcA and fnr
gene products (21). The use of these promoters gave us the expres-
sion profiles that we wanted without the need for inducers.
Also in the interest of economics, antibiotics cannot be used in
a commercial process to stabilize plasmids. Plasmid-based expres-
sion systems are often used to produce chemicals, polymers, fuels,
and proteins. In these bioprocesses, plasmids have a huge impact
on the productivity of the process. Plasmid-free cells lead to losses
in decreased productivity and profitability. The use of antibiotics
in industrial fermentations is not feasible due to the cost of the
antibiotic itself and the cost of inactivating and removing the an-
tibiotic from the spent broth. Furthermore, the addition of anti-
biotics does not even guarantee the stability of the plasmid. Chro-
mosomal integration of foreign genes is applicable in some cases,
but important factors such as polar effects and limited gene copy
number are disadvantages (20). Kroll et al. described, as a possible
solution to these problems, several plasmid addiction systems
(20). We chose to implement a catabolism-based addiction sys-
tem. It was necessary to delete gldA and glpK for complete plasmid
addiction under microaerobic conditions. During the proof-of-
concept experiments, deletion of only one branch of glycerol dis-
similation resulted in approximately 50% plasmid retention (data
not shown). The plasmid retention of the double-deletion addic-
tion system was 90% (data not shown). Relative to the antibiot-
ic-stabilized system, the plasmid addiction system had similar eth-
anol production after some process parameters were adjusted
(Fig. 5). Therefore, we were able to eliminate the use of antibiotics
without any decrease in process performance.
The glycerol-to-ethanol process creates a significant amount of
hydrogen under microaerobic and acidic conditions through the
enzymes pyruvate formate lyase (encoded by pflB) and formate-
aem.asm.org 3281
Wong et al.
hydrogen lyase. Due to that production of hydrogen, the off-gas
may be flammable and the prevention of accidental explosions
during the process must be a safety consideration. As shown by
Schrder and Molnarne, the lower explosion limit (LEL) for H2 in
air is 3.8% by volume (19). When we used conventional aeration,
our ethanol process routinely surpassed this threshold within a
few hours of starting the fermentation and reached over 25% by
volume at its peak. A common solution for a flammable atmo-
sphere would be to pump an overlay of air into the fermentor
headspace to dilute the H2 to 0.95% by volume or below, to com-
ply with the 4:1 margin of safety below the LEL recommended by
the National Fire Protection Association standard NFPA 86. The
inlet air sparge rate of 0.01 volume/volume/min (vvm) used in the
fermentation shown in Fig. 3A would require a 0.24 vvm air over-
lay, which would incur capital and utility expenses. We would also
incur additional capital expenses to make the fermentor and other
equipment explosion-proof in case of overlay equipment failure.
Furthermore, the effect of a large air overlay on our fermentation
is unknown. These findings led us to explore the possibility of
limiting the inlet gas oxygen concentration to 5%, which is below
the amount of oxygen needed to ignite any amount of hydrogen.
This strategy was inherently safe, and we were able to maintain the
same process performance that we had achieved with air as the
inlet gas. A gas separation system would be required to generate
the 5% O2 inlet gas stream, and this expense was included in our
economic model.
Glycerol offers the advantages of low price and a high degree of
reduction, making it an ideal substrate from which to produce
reduced products. Building on previous demonstrations of mi-
croaerobic glycerol fermentations by other investigators, we have
advanced our glycerol-to-ethanol process toward commercializa-
tion. Our process has commercially relevant productivity, does
not require inducers or antibiotics, and results in an O2-limited
off-gas that is inherently safe. Other issues remain, such as the cost
of the ammonia for pH control and the cost of the gas separation
skid. We will continue to work on several fronts to eliminate or
absorb these costs, including (i) further genetic manipulation or
process optimization to increase ethanol productivity and de-
crease by-product accumulation, (ii) medium optimization to re-
duce nutrient costs, and (iii) optimization of the glycerol feeding
schedule.
ACKNOWLEDGMENTS
All the authors are employees of Glycos Biotechnologies, Inc., which sup-
ported this work.
We thank Ramon Gonzalez at Rice University for his support and for
supplying plasmid pZSKLMgldA.
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