ARTICLE IN PRESS

Metabolic Engineering 10 (2008) 340351

Contents lists available at ScienceDirect

Metabolic Engineering
journal homepage: www.elsevier.com/locate/ymben

Engineering Escherichia coli for the efcient conversion of glycerol to ethanol

and co-products
Syed Shams Yazdani a, Ramon Gonzalez a,b,
a
b

Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, USA
Department of Bioengineering, Rice University, Houston, TX, USA

a r t i c l e in f o

a b s t r a c t

Article history:
Received 13 January 2008
Accepted 13 August 2008
Available online 9 September 2008

Given its availability, low prices, and high degree of reduction, glycerol has become an ideal feedstock
for producing reduced compounds via anaerobic fermentation. We recently identied environmental
conditions enabling the fermentative metabolism of glycerol in E. coli, along with the pathways and
mechanisms mediating this metabolic process. In this work, we used the knowledge base created in
previous studies to engineer E. coli for the efcient conversion of crude glycerol to ethanol. Our strategy
capitalized on the high degree of reduction of carbon in glycerol, thus enabling the production of not
only ethanol but also co-products hydrogen and formate. Two strains were created for the coproduction of ethanolhydrogen and ethanolformate: SY03 and SY04, respectively. High ethanol yields
were achieved in both strains by minimizing the synthesis of by-products succinate and acetate through
mutations that inactivated fumarate reductase (DfrdA) and phosphate acetyltransferase (Dpta),
respectively. Strain SY04, which produced ethanolformate, also contained a mutation that inactivated
formatehydrogen lyase (DfdhF), thus preventing the conversion of formate to CO2 and H2. High rates of
glycerol utilization and product synthesis were achieved by simultaneous overexpression of glycerol
dehydrogenase (gldA) and dihydroxyacetone kinase (dhaKLM), which are the enzymes responsible for
the conversion of glycerol to glycolytic intermediate dihydroxyacetone phosphate. The resulting strains,
SY03 (pZSKLMgldA) and SY04 (pZSKLMgldA), produced ethanolhydrogen and ethanolformate from
unrened glycerol at yields exceeding 95% of the theoretical maximum and specic rates in the order of
1530 mmol/gcell/h. These yields and productivities are superior to those reported for the conversion of
glycerol to ethanolH2 or ethanolformate by other organisms and equivalent to those achieved in the
production of ethanol from sugars using E. coli.
& 2008 Elsevier Inc. All rights reserved.

Keywords:
Metabolic engineering
Glycerol fermentation
Biofuels and biochemicals
Escherichia coli
Ethanol
Hydrogen
Formic acid

1. Introduction
Glycerol has become an inexpensive and abundant carbon
source due to its generation as inevitable by-product of biodiesel
fuel production. With every 100 lbs of biodiesel produced by the
transesterication of vegetable oils or animal fats, 10 lbs of crude
glycerol are generated. The tremendous growth of the biodiesel
industry has created a glycerol surplus that resulted in a dramatic
decrease in crude glycerol prices (Yazdani and Gonzalez, 2007 and
references cited therein). This decrease in prices poses a problem
for the glycerol-producing and -rening industries, and the
economic viability of the biodiesel industry itself has been greatly
affected (McCoy, 2006, 2005). The conversion of low-priced

 Corresponding author at: Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main Street, MS-362, Houston, TX 77005, USA.
Fax: +1713 348 5478.
E-mail address: Ramon.Gonzalez@rice.edu (R. Gonzalez).

1096-7176/$ - see front matter & 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymben.2008.08.005

glycerol streams to higher value products has been proposed as

a path to economic viability for the biofuels industry (Yazdani and
Gonzalez, 2007). Such technologies could be readily integrated
into existing biodiesel facilities, thus establishing true bioreneries and revolutionizing the biodiesel industry by dramatically
improving its economics. While availability and low prices make
glycerol an attractive carbon source for fermentation processes,
there is yet another advantage in using this compound: fuels and
reduced chemicals can be produced from glycerol at yields higher
than those obtained from common sugars (Yazdani and Gonzalez,
2007). The latter is possible because the degree of reduction per
carbon, k (Nielsen et al., 2003), of glycerol is signicantly higher
(C3H8O3: k 4.67) than that of sugars such as glucose (C6H12O6:
k 4) or xylose (C5H10O5: k 4). To fully realize the aforementioned advantages, the use of anaerobic fermentation is highly
desirable.
While many microorganisms are able to metabolize glycerol
in the presence of external electron acceptors (respiratory
metabolism), few are able to do so fermentatively (i.e., in the

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S. Shams Yazdani, R. Gonzalez / Metabolic Engineering 10 (2008) 340351

E. coli can metabolize glycerol in a fermentative manner (Gonzalez

et al., 2008; Murarka et al., 2008; Dharmadi et al., 2006). We
identied environmental conditions that enable this metabolic
process, along with the pathways and mechanisms responsible for
it (Gonzalez et al., 2008) (Fig. 1). In the work reported here we
used the knowledge base created by our previous studies to
engineer E. coli for the efcient conversion of crude glycerol into
ethanol. Our strategy took advantage of the high degree of
reduction of carbon in glycerol, thus enabling the production of
not only ethanol but also the co-products hydrogen and formate.
Product yields and productivities in these strains were superior to
those reported for the conversion of glycerol to ethanolH2 or
ethanolformate (Jarvis et al., 1997; Ito et al., 2005; Sakai and
Yagishita, 2007) and similar to those reported for the production
of ethanol from sugars using engineered E. coli strains (e.g.,
Underwood et al., 2002).

absence of electron acceptors). Until recently, the fermentative

metabolism of glycerol had been reported in species of the genera
Klebsiella, Citrobacter, Enterobacter, Clostridium, Lactobacillus,
Bacillus, Propionibacterium, and Anaerobiospirillum (Yazdani and
Gonzalez, 2007 and references cited therein). However, the
potential for using these organisms at the industrial level could be
limited due to issues that include pathogenicity, the need for strict
anaerobic conditions and supplementation with rich nutrients, and
unavailability of the genetic tools and physiological knowledge
necessary for their effective manipulation. The use of microbes such
as Escherichia coli, an organism very amenable to industrial
applications, could help overcome the aforementioned problems.
Although it was long thought that the metabolism of glycerol
in E. coli required the presence of external electron acceptors
(Booth, 2005; Bouvet et al., 1994, 1995; Lin, 1976; Quastel et al.,
1925; Quastel and Stephenson, 1925), we recently discovered that
OH

ATP

NADH

OH Glycerol (42/ )
3

HO

GldA
(gldA)

Biomass (43/10)

NADH

OH Dihydroxyacetone

HO

Phosphoenolpyruvate

DHAK
(dhaKLM)

Pyruvate

Dihydroxyacetone HO
Phosphate

OH
O
P

OH

OH
OH

O
P

NADH

ATP

HO

341

Phosphoenolpyruvate

OO

CO2+ NADH
PYK (pykF)
ATP
O

O
OH

OH

HO
OH
Pyruvate
O
H
PFL
(pflB)

HO
O
FRD (frdABCD)
Fumarate

OH

NADH/H2

O
Succinate (3)
Succinate

Formate (2)
FHL
(fdhF, hycB-I)
H2

CO2

CoA
H
Acetyl-Coenzyme-A

H2

NADH

CO2
ADH PTA
(adhE) (pta)

ADH
(adhE )
OH
Ethanol (6)

NADH

O
H
Acetaldehyde

O
P

OH
OH
Acetyl-phosphate
O

ACK
(ackA)

O
OH

ATP

Acetate (4)

Fig. 1. Main fermentative pathways involved in the anaerobic fermentation of glycerol in E. coli (Murarka et al., 2008; Gonzalez et al., 2008). Relevant genes and
corresponding enzymes are included. Glycerol dissimilation in the absence of electron acceptors is mediated by glycerol dehydrogenase and dihydroxyacetone kinase
(Gonzalez et al., 2008). Ethanol, succinate, acetate, and formate are the main products of the fermentative utilization of glycerol (Dharmadi et al., 2006). Proposed genetic
modications are illustrated by thicker lines (overexpression of gldA and dhaKLM) or double bars (disruption of frdA, pta, and fdhF). Broken lines illustrate multiple steps.
Substrates, products, and biomass are boxed. Abbreviations: ADH, acetaldehyde/alcohol dehydrogenase; ACK, acetate kinase; DHAK, dihydroxyacetone kinase; FHL, formate
hydrogen-lyase; FRD, fumarate reductase; GldA, glycerol dehydrogenase; PFL, pyruvate formate-lyase; PTA, phosphate acetyltransferase; PYK, pyruvate kinase.

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S. Shams Yazdani, R. Gonzalez / Metabolic Engineering 10 (2008) 340351

2. Materials and methods

2.1. Strains, plasmids, and genetic methods
Wild-type K12 E. coli strain MG1655 was obtained from the
University of Wisconsin E. coli Genome Project (www.genome.
wisc.edu) (Kang et al., 2004) and used as the host to implement
the proposed metabolic engineering strategies. All resulting
strains, along with primers and plasmids used in their construction, are listed in Table 1.
Gene knockouts were introduced in MG1655 and its derivatives by P1 phage transduction as described elsewhere (Miller,
1972). Single gene knock out mutants from the National
BioResource Project (NIG, Japan) (Baba et al., 2006) were used
as donors of specic mutations. Various dilutions of P1 phage
were mixed with the donor cells at exponential growth phase and
tryptone top agar (1% tryptone, 0.5% NaCl, 0.65% Agar, 5 mM
CaCl2) and overlaid on Luria-Bertani broth (LB) agar plates
containing 5 mM CaCl2. Plates showing many isolated plaques
were used to collect phage lysates by adding 2 ml P1 adsorption
media (LB+5 mM CaCl2) and incubating overnight at 4 1C. The
phage lysate was further shaken with chloroform to remove
bacterial cell background and used to reinfect the donor cells to
enrich for the mutation. The enriched phages were mixed with the
recipient strain at a multiplicity of infection (MOI) of 0.5 and
incubated in 1XA media (10.5 g/L K2HPO4, 4.5 g/L KH2PO4, 1 g/L
(NH4)2SO4, and 0.5 g/L sodium citrate.2H2O) for 2 h. Cells were
centrifuged and plated on LB plates supplemented with appropriate antibiotics. The plates were incubated overnight at room
temperature and then shifted to 30 1C and further grown for 12
days until colonies appeared. Colonies were puried on fresh LB
plates, incubated at 42 1C to cure the remaining lysogens, and
further grown at 37 1C. To eliminate the kanamycin resistance
marker, the mutants were transformed with pCP20 (Datsenko and

Wanner, 2000), a temperature sensitive plasmid expressing

ippase (FLP) recombinase. FLP expressed from this plasmid
removed the Km region from the FRT::Km::FRT site, leaving one
FRT site behind. pCP20 was then removed by growing the cells at
42 1C. All mutations created in the host cells were conrmed by
polymerase chain reaction using the verication primers listed
in Table 1. The disruption of multiple genes in a common host was
achieved by sequentially implementing the procedure described
above. This sequential procedure, in turn, could lead to chromosomal rearrangements resulting from FLP-promoted recombination events between FRT sites at different loci (Datsenko and
Wanner, 2000). To verify that these events did not occur in strains
with multiple gene disruptions, each strain was rechecked in
all disruption sites by PCR every time a new mutation was
introduced.
The construction of plasmids used in this study is illustrated in
Fig. 2. The control plasmid pZSblank was constructed by digesting
pZSKLcf (Bachler et al., 2005) with KpnI and BamHI restriction
endonucleases, blunting overhangs with T4 DNA polymerase, and
ligating the vector backbone with T4 DNA ligase. The expression
vector pZSgldA was constructed as follows. The coding region of
the gldA gene was PCR amplied using genomic DNA of E. coli
MG1655 as template and c1-gldA primers (Table 1). The
restriction enzyme sites KpnI and PstI were introduced through
the forward and reverse primers, respectively, to facilitate cloning
of the PCR product in the expression vector pZSKLM (Bachler et al.,
2005). The amplied product (1.1 kB) was digested with KpnI and
PstI and used for ligation at the corresponding sites of the pZSKLM
plasmid. The expression vector pZSKLMGldA was constructed by
amplifying the gldA gene along with its ribosome binding site
from the genomic DNA of E. coli MG1655 using c2-gldA primers
(Table 1). The amplied product was digested with PstI and MluI
and cloned at the corresponding sites of pZSKLM, downstream of
the dhaKLM gene. The PCR was performed using Pfu turbo DNA

Table 1
Strains, plasmids, and primers used in this study
Strain/plasmid/primer

Description/Genotype/Sequence

Source

Strains
MG1655
YD01
SY01
SY02
SY03
SY04

F- l- ilvG- rfb-50 rph-1

MG1655, DfdhF::FRT-tet-FRT; deletion mutant for fdhF gene in MG1655 strain
MG1655, DfrdA::FRT-tet-FRT; deletion mutant for frdA gene in MG1655 strain
MG1655, DfdhF::FRT DfrdA::FRT-tet-FRT; deletion mutant for frdA gene in YD01 strain
MG1655, DfrdA::FRT Dpta::FRT-tet-FRT; deletion mutant for pta gene in SY01 strain
MG1655, DfdhF::FRT DfrdA::FRT Dpta::FRT-tet-FRT; deletion mutant for pta gene in SY02 strain

Kang et al., 2004

This study
This study
This study
This study
This study

E. coli dhaKLM gene under control of PLtetO-1 (tetR, oriR SC101*, cat)
C. freundii dhaKL gene under control of PLtetO-1 (tetR, oriR SC101*, cat)
E. coli gldA gene under control of PLtetO-1 (tetR, oriR SC101*, cat)
E. coli dhaKLM and gldA genes under control of PLtetO-1 (tetR, oriR SC101*, cat)
Blank plasmid created by removing C. freundii dhaKL fragment from pZSKLcf and self-ligating the plasmid (tetR,
oriR SC101*, cat)

Bachler et al., 2005

Bachler et al., 2005
This study
This study
This study

cgtaatatcagggaatgaccc
gggcaaagaatgtcaaaaacaa
accctgaagtacgtggctg
gcaccacctcaattttcagg
gctgttttgtaacccgcc
gcagcgcaaagctgcgg
agtcacggtaccatggaccgcattattcaatcac
gatcgtctgcagttattcccactcttgcaggaaac
gacactgcagaggagcaattatggaccgca
caagctacgcgtttattcccactcttgcagga

This study

Plasmids
pZSKLM
pZSKLcf
pZSGldA
pZSKLMGldA
pZSblank
Primersa
v-fdhF
v-frdA
v-pta
c1-gldA
c2-glda

This study
This study
This study
This study

a
v indicates the primer sequences (5 to 3) that were used for verication purposes during the creation of disruption mutants by phage transduction. c indicates
the primers that were used for cloning purposes, c1 to clone gldA alone (pZSGldA) and c2 to clone gldA along with dhaKLM (pZSKLMGldA). The forward sequence follows
the reverse sequence in each case. Genes or operons deleted or cloned are apparent from primer names.

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343

Fig. 2. Diagram illustrating the construction of plasmids used in this study. Plasmids pZSKLcf and pZSKLM have been described elsewhere (Bachler et al., 2005).

polymerase (Stratagene, CA, USA) under standard conditions

described by the supplier. The ligated products described above
were used to transform E. coli DH5aT1 (Invitrogen, CA, USA).
Positive clones were screened by plasmid isolation and restriction
digestion.
Standard recombinant DNA procedures were used for gene
cloning, plasmid isolation, and electroporation. Manufacturer
protocols and standard methods (Miller, 1972; Sambrook et al.,
1989) were followed for DNA purication (Qiagen, CA, USA),
restriction endonuclease digestion (New England Biolabs, MA,
USA), and DNA amplication (Stratagene, CA, USA and Invitrogen,
CA, USA). The strains were kept in 32.5% glycerol stocks at 80 1C.
Plates were prepared using LB medium containing 1.5% agar, and
appropriate antibiotics were included at the following concentrations: 34 mg/ml chloramphenicol and 50 mg/ml kanamycin.

2.2. Culture medium and cultivation conditions

The minimal medium (MM) designed by Neidhardt et al.
(1974) supplemented with 2 g/L tryptone (Difco, USA), 5 mM
sodium selenite, 1.32 mM Na2HPO4 in place of K2HPO4, and
varying concentrations of glycerol was used unless otherwise
specied. Morpholino-propanesulfonic (MOPS) acid was only
included in the inoculum preparation phase or in experiments
conducted in tubes (see below). Antibiotics and inducer (anhydrotetracycline) were included when appropriate. Chemicals
were obtained from Fisher Scientic (Pittsburgh, PA) and Sigma-

Aldrich Co. (St Louis, MO). Experiments using crude glycerol

obtained in the production of biodiesel were conducted in the
medium described above. The crude glycerol, kindly provided by
FutureFuel Chemical Company (Batesville, AR), had the following
composition (w/v): 84% glycerol, 5% salts, and 0.02% methanol.
Fermentations were conducted in a SixFors multi-fermentation
system (Infors HT, Bottmingen, Switzerland) with six 500-ml
working volume vessels and independent control of temperature
(37 1C), pH, stirrer speed (200 rpm), and dissolved oxygen (not
controlled) (Dharmadi et al., 2006). The system is fully equipped
and computer controlled using manufacturer IRIS NT software.
Each vessel was tted with a condenser operated with a 2 1C
cooling methanolwater supply to minimize evaporation. Anaerobic conditions were maintained by sparging the medium with
ultrahigh purity argon (Matheson, Tri-Gas, Houston, TX) at
0.01 L/min. In some experiments (specied in each case), air was
ushed through the headspace at 0.01 L/min to create a microaerobic environment. Operation under these conditions resulted
in decreasing dissolved oxygen concentrations that reached
undetectable levels after 1 h of cultivation (i.e., undetectable
dissolved oxygen concentrations during almost the entire course
of the fermentation). Experiments in tubes were conducted using
17-ml Hungate tubes (Bellco Glass, Inc., Vineland, NJ), which were
modied by piercing the septa with two luer lock needles, one for
oxygen-free argon sparging (20 G  8 in, Hamilton Company-USA,
Reno, NV) and one for gas efux (20 G  2 in, Hamilton CompanyUSA, Reno, NV). The working volume of these modied Hungate
tubes was 10 ml.

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Prior to use, the cultures (stored as glycerol stocks at 80 1C)

were streaked onto LB plates and incubated overnight at 37 1C. A
single colony was used to inoculate 17.5-ml Hungate tubes
completely lled with the medium (MM supplemented with
10 g/L tryptone, 5 g/L yeast extract, and 10 g/L glycerol). The tubes
were incubated at 37 1C until an OD550 of 0.4 was reached. An
appropriate volume of this actively growing pre-culture was
centrifuged, and the pellet was washed and used to inoculate
350 ml of medium in each fermenter, with a target initial optical
density of 0.05 at 550 nm.

Dihydroxyacetone was added to a concentration of 1 mM and the

decrease in absorbance was followed at 340 nm. All spectrophotometric measurements were conducted in a BioMate 5
spectrophotometer (Thermo Scientic, MA, USA). The linearity of
reactions (protein concentration and time) was established for all
assays and the nonenzymatic rates were subtracted from the
observed initial reaction rates. Enzyme activities are reported as
mmol of substrate/min/mg of cell protein and represent averages
for at least three cell preparations.
2.5. Calculation of fermentation parameters

2.3. Analytical methods

Optical density was measured at 550 nm and used as an
estimate of cell mass (1 OD 0.34 g dry weight/L). After
centrifugation, the supernatant was stored at 20 1C for HPLC
(high performance liquid chromatography) analysis. Glycerol,
organic acids, ethanol, and hydrogen were quantied as previously described (Dharmadi et al., 2006; Dharmadi and Gonzalez,
2005). The transfer of oxygen in microaerobic cultures was
characterized by estimating the volumetric oxygen transfer rate
(NO2 in mg O2/L/h) as NO2 kL aC  C L . kL is the oxygen
transport coefcient (cm/h), a is the gasliquid interfacial area
(cm2/cm3), kLa is the volumetric oxygen transfer coefcient (h1),
C* is the saturated dissolved oxygen concentration (mg/L), and CL
is the actual dissolved oxygen concentration in the liquid (mg/L)
(Shuler and Kargi, 2002). The volumetric oxygen transfer
coefcient (kLa) was estimated by the static gassing out method
(Shuler and Kargi, 2002). The oxygen transfer rate, which, under
oxygen limiting conditions, also represents the volumetric oxygen
uptake rate, was then calculated as described above. In these
calculations, C* was assumed to be 8 mg/L.
2.4. Enzyme activities
The activity of glycerol dehydrogenase in the oxidation of
glycerol was measured as described by Truniger and Boos (1994).
Cells from anaerobic cultures (OD550 of 0.7) were harvested by
centrifugation (2 min, 10,000  g), washed twice with 9 g/L NaCl,
and stored as cell pellets at 20 1C. For glycerol dehydrogenase
assays, the cells were resuspended in 0.2 ml of the appropriate
buffer (depending on the pH of the assay: see below) and
permeabilized by vortex mixing with chloroform (Osman et al.,
1987; Tao et al., 2001). Activity towards glycerol was assayed by
measuring the change in absorbance at 340 nm and 25 1C in a 1 ml
reaction mixture containing 2 mM MgCl2, 500 mM NAD+, 100 mM
glycerol, 30 ml crude cell extract, and 100 mM of the appropriate
buffer according to the pH of the assay (Truniger and Boos, 1994).
To study the effect of pH on glycerol dehydrogenase activity, the
following buffers were used (pH in parenthesis): sodium phosphate (pH 68), potassium carbonate (pH 9.5), and sodium citrate
(pH 56). Dihydroxyacetone kinase activity was assayed using the
method reported by Kornberg and Reeves (1972) with minor
modications. Cells from anaerobic cultures (OD550 of 0.7) were
harvested by centrifugation (2 min, 10,000  g), washed with
decryptication buffer (0.1 M sodiumpotassium phosphate, pH
7.2, and 5 mM MgCl2) and stored as cell pellets at 20 1C. For
enzyme assays, cells were resuspended in decryptication buffer
to obtain 1 mg dry cell weight/ml. A portion of the ice-cold cell
suspension was placed in a test tube and vigorously mixed for
1 min; 0.2 volume of tolueneethanol (1:9, v/v) was added. The
assay was conducted in a 1 ml reaction mixture containing 1 mM
phosphoenolpyruvate trisodium, 0.1 mM NADH, 2 U lactate dehydrogenase, and the tolueneethanol treated cells (50 mg dry
weight). The assay mixture was incubated at 30 1C for 15 min.

Data for cell growth, glycerol consumption, and product

synthesis were used to calculate volumetric rates/productivities
(mmol/L/h) for each 12-h interval. Specic rates (mmol/gcell/h)
were calculated by dividing the volumetric rates by the time
average concentration of cells over the same period of time.
Growth and product yields (mmol/mmol of glycerol) were
calculated as the amount of cell mass or product synthesized
per amount of glycerol consumed once the cultures reached the
stationary phase. In the above calculations, we used an average
molecular weight of 24.7, which corresponds to an average cell of
a molecular formula CH1.9O0.5N0.2 (Nielsen et al., 2003).

3. Results and discussion

3.1. Metabolic engineering strategy to efciently convert glycerol into
ethanol and co-products hydrogen and formate
Improvement of yield and rates are two of the most anticipated
outcomes of a metabolic engineering effort. In this study, we
focused on the improvement of these two parameters during the
conversion of glycerol to ethanol and co-products hydrogen and
formate. In the following we briey describe our engineering
strategies.
Yield of ethanol and co-products: the fermentation of glycerol by
E. coli results in a product mixture containing predominantly
ethanol but also acetate, succinate, and minor amounts of formate
(Dharmadi et al., 2006). The synthesis of ethanol is preferred
over other fermentation products because it represents a redoxbalanced pathway that produces ATP via substrate-level phosphorylation: one molecule of ATP is generated per each molecule
of glycerol converted to ethanol (Fig. 1). Since ethanol was
considered the target product in this study, succinate and acetate
become competing by-products that would eventually decrease
the ethanol yield by diverting carbon to their formation (Fig. 1).
Our strategy to minimize the generation of these by-products
involves blocking the metabolic pathways responsible for their
synthesis (Fig. 1). Unlike succinate and acetate, formate represents
a potential co-product that could enhance the value of the
fermentation process. Note that glycerol could be converted into
ethanol plus either hydrogen or formate (Fig. 1). The signicant
differences in the properties of ethanol and formate (boiling point,
ionization constant, etc.) would facilitate their separation. An
alternative approach, also explored in this work, is the complete
conversion of formate to carbon dioxide and hydrogen, a process
that would yield another important biofuelH2. Our study used
these two approaches, namely: (i) co-production of ethanol and
H2, and (ii) co-production of ethanol and formate (Fig. 1). The
maximum theoretical yield in both cases is 1 mol of ethanol plus
1 mol of either formate or hydrogen per each mol of glycerol
utilized.
Rates of glycerol utilization and synthesis of ethanol and coproducts: we previously reported that a two-enzyme pathway is

ARTICLE IN PRESS
S. Shams Yazdani, R. Gonzalez / Metabolic Engineering 10 (2008) 340351

Product yield (g/g glycerol)

0.6
0.5
0.4
0.3
0.2
0.1
0
Cells Succinate Acetate Ethanol

Formate

12

0.8

10
0.6
8
0.4

6
4

0.2
2
0

3.2. Improving the yield of ethanol and co-products

hydrogen and formate
As described in the previous section, the elimination of
competing by-products succinic and acetic acids is desirable. For
this purpose, we disrupted the genes encoding fumarate reductase
(frdA) and phosphotransacetylase (pta), two key enzymes in the
synthesis of succinate and acetate, respectively (Sawers and Clark,
2004) (Fig. 1). A double mutant of these two genes, strain SY03,
produced negligible amounts of succinic and acetic acids, and,
when cultivated at acidic conditions (pH 6.3), converted all
formate to H2 and CO2 (Fig. 3A). Strain SY03 produced almost
equimolar amounts of ethanol and hydrogen at yields that
approached the maximum theoretical yield (Fig. 3A and Table 2;
the maximum theoretical yield is 1 mol each of H2 and ethanol per
mol of glycerol fermented: Fig. 1). The only other organism
reported to convert glycerol into ethanol and hydrogen is
E. aerogenes mutant HU-101 (Ito et al., 2005). Under culture
conditions similar to those used here for strain SY03 (i.e., medium
supplemented with 10 g/L pure glycerol), E. aerogenes HU-101
produced a mixture of products containing (in mol of product per
mol of glycerol fermented): 0.89 H2, 0.86 ethanol, 0.14 lactate,
0.09 acetate, 0.16 1,3-propanediol (1,3-PDO), and 0.12 formate (Ito
et al., 2005). The distribution of carbon among six different
fermentation products clearly limited the ethanol and hydrogen
yields and would pose signicant problems for the separation of
ethanol from this mixture.
We engineered a second strain to produce ethanol along with
the co-product formate, which would allow the recovery of all
fermented glycerol in these two products. Since the enzyme
formate hydrogen-lyase (FHL) is responsible for the oxidation of
formate, we blocked FHL by disrupting the gene fdhF, which
encodes the formate dehydrogenase component of FHL. Given the
absolute requirement of fdhF for FHL activity (Bagramyan and
Trchounian, 2003; Bagramyan et al., 2002; Self et al., 2004), its

Cell growth (OD550)

Glycerol utilization (g/L)

responsible for the conversion of glycerol to glycolytic intermediates (Fig. 1) (Gonzalez et al., 2008). These two enzymes convert
glycerol to dihydroxyacteone (DHA) (enzyme glycerol dehydrogenase, GldA, encoded by the gldA gene) and DHA to dihydroxyacetone phosphate (DHAP) (enzyme DHA kinase, DHAK, encoded
by the dhaKLM operon). This trunk pathway, however, appears to
be expressed at low levels, as can be inferred from the slow
kinetics of glycerol fermentation; the fermentation of 8.5 g/L
glycerol took approximately four days (Dharmadi et al., 2006). Our
key metabolic engineering strategy to increase the rate of glycerol
utilization then involves overexpression of the enzymes GldA and
DHAK (Fig. 1), an approach expected to accelerate the conversion
of glycerol to DHAP. Common glycolytic enzymes known to be
expressed at high levels, and therefore able to support highcarbon uxes, catalyze the remaining steps in the conversion of
DHAP to pyruvate (Fig. 1). Two fermentative enzymes, also known
to support high uxes, catalyze the conversion of pyruvate to
acetyl coenzyme-A (acetyl-CoA) and acetyl-CoA to acetaldehyde
to ethanol: pyruvate formate lyase (PFL) and acetaldehyde/alcohol
dehydrogenase (ADH), respectively (Fig. 1). Therefore, overexpression of GldA and DHAK (individually or in combination) should
increase the rate of conversion of glycerol to DHAP, which in turn
would increase the rate of synthesis of ethanol and co-products
from glycerol.
The metabolic engineering strategies described above are
summarized in Fig. 1. In each case, the engineered pathways that
convert glycerol to ethanol and co-products are redox-balanced
and ATP generating, and thus represented viable alternatives for
the functioning of the cells.

345

0.0
0

30

60
Time (h)

90

120

Fig. 3. Performance of strains SY03 and SY04, which were engineered to convert
glycerol into ethanolH2 and ethanolformate, respectively. (A) Biomass, succinate, acetate, ethanol, and formate yields for strains SY03 (white bars) and SY04
(solid bars). Experiments were conducted in fully controlled fermenters at pH 6.3
(SY03) or 7.5 (SY04). Data represent the average of three measurements for
stationary phase cultures from B. (B) Kinetics of cell growth (closed symbols)
and glycerol utilization (open symbols) for strains SY03 (triangles) and SY04
(squares).

disruption was envisioned to be more effective than that of the

hydrogenase components. fdhF disruption was implemented in a
genetic background containing the frdA and pta mutations. This
triple mutant is referred to as SY04. Strain SY04 produced
exclusively ethanol and formate at yields 9296 of the theoretical
maximum: 0.92 mol of ethanol and 0.96 mol of formate per mol of
glycerol fermented (Fig. 3A and Table 2). To the best of our
knowledge, there has been only one report of the co-production of
ethanolformate from glycerol. Ethanol and formate were the two
main products of glycerol fermentation by a K. planticola strain
isolated from the rumen of red deer (Jarvis et al., 1997). This
organism produced 39 and 34 mol of ethanol and formate,
respectively, during the fermentation of approximately 70 mol of
glycerol. This translates into yields of 0.56 and 0.49 mol of ethanol
and formate, respectively, per mol of glycerol fermented. Clearly,
the ethanol and formate yields in strain SY04 were superior to
those reported during the fermentation of glycerol by K. planticola.
Although strains SY03 (frdA and pta mutations) and SY04 (frdA,
pta, and fdhF mutations) produced ethanolH2 and ethanol
formate at high yields, fermentations took place at very low rates
(Fig. 3B and Table 2). Therefore, we investigated the feasibility of
improving the rate at which glycerol is utilized and ethanol coproducts synthesized by overexpressing enzymes GldA and DHAK.
Our results are described in the next sections.

ARTICLE IN PRESS

a
Cells were cultivated using the medium and conditions described in the Materials and Methods section. Unless otherwise specied, the cultures were sparged with argon and 10 g/L pure glycerol was used as carbon
source.
b
Carbon recovery accounts for the percentage of carbon recovered in all products and biomass.
c
Maximum specic productivities, in mmol per gram of cells per hour, were calculated by taking into account the interval at which maximum mmol of substrate were consumed and cells and products formed. The
concentration of cells during the given period of time is calculated as time average.
d
Maximum volumetric productivities are reported for the cultivation period at which the maximum mmol of substrate were consumed and cells and products formed per liter per hour.

1.11

0.94
1.70
2.61
2.33
4.65

0.39
0.75
1.80
3.18

3.00
2.97
1.09
0.33
0.78
2.09
3.58
0.91
1.89
2.96
2.53
4.68
3.28
3.41
0.13
0.02
0.09
0.24
0.55
0.14
0.21
0.90
0.64
1.19
0.39
0.55
7.90

8.17
11.16
7.48
6.28
8.84

9.96
8.63
7.88
19.67

18.34
31.96
7.67
7.73
7.84
8.70
16.02
8.16
11.14
6.89
5.63
8.82
15.83
27.18
8.07
8.33
8.96
12.62
15.85
6.16
11.16
8.30
6.13
9.65
20.26
29.54
2.49
0.73
1.45
3.39
5.23
2.41
1.91
6.26
6.28
6.04
4.03
3.79
1.02

1.02
0.91
0.83
0.88
0.96

0.96
0.75
0.85
0.92

0.93
0.88
1.01
0.92
1.00
0.91
1.04
1.02
0.90
0.75
0.82
0.96
0.97
1.02
0.11
0.10
0.10
0.14
0.13
0.10
0.13
0.26
0.26
0.28
0.13
0.09
106
102
96
84
102
103
94
103
97
99
98
100
pH
pH
pH
pH
pH
pH
pH
pH
pH
pH
pH
pH
SY03
SY04
MG1655 (pZSBlank)
MG1655 (pZSKLMgldA)
SY04 (pZSKLMgldA)
SY03 (pZSKLMgldA)
MG1655 (pZSKLMgldA)
MG1655
SY03
SY03 (pZSKLMgldA)
SY04 (pZSKLMgldA)
SY04 (pZSKLMgldA)

6.3
7.5
7.5
7.5
7.5
6.3
6.3
6.0, air in headspace
6.0, air in headspace
6.0, air in headspace
7.5, 10 g/L crude glycerol
7.5, 20 g/L crude glycerol

Ethanol
Glycerol
H2
Formate
Ethanol

1.10
0.33
0.83
2.06
3.58
0.94
1.70
2.48
2.22
4.65
3.39
3.52

H2
Ethanol
Glycerol
Cells
H2
Cells
Cells

Formate

Max specic productivity (mmoles/g cells/h)c

Yield (mmole product/mmole glycerol)
Carbon
recoveryb
Conditiona
Strains

Table 2
Fermentation parameters for cell growth, glycerol utilization, and product synthesis

Formate

S. Shams Yazdani, R. Gonzalez / Metabolic Engineering 10 (2008) 340351

Max volumetric productivity (mmoles/L/h)d

346

3.3. Improving the rate of glycerol utilization: effect of

overexpressing glycerol dehydrogenase and dihydroxyacetone
kinase activities
To assess the effect of overexpressing glycerol dehydrogenase
(GldA, encoded by the gene gldA) and dihydroxyacetone kinase
(DHAK, encoded by the operon dhaKLM) on the kinetics of glycerol
fermentation, we used the following plasmids that express these
two enzymes individually and in combination: pZSgldA (expressing glycerol dehydrogenase), pZSKLM (expressing dihydroxyacetone kinase), and pZSKLMgldA (expressing both glycerol
dehydrogenase and dihydroxyacetone kinase). Plasmid pZSblank
was used as a control and it contains the backbone of the
plasmids described above (see Fig. 2, Table 1, and Materials and
Methods section for details about plasmids and their construction). We conducted a functional characterization of each of these
constructs by measuring the changes in GldA and DHAK activities
in response to different concentrations of inducer anhydrotetracycline (Fig. 4). These experiments were carried out in Hungate
tubes under anaerobic conditions (see Materials and Methods). The
reported values represent the average for 48-h cultures of strain
MG1655 transformed with each of the above plasmids. We
observed a 10- to 20-fold increase in the activity of GldA,
depending on the construct and inducer concentration (Fig. 4A).
Overexpression of the dhaKLM operon, on the other hand, resulted
in up to a 5- to 6-fold increase in PEP-dependent DHAK activities
(Fig. 4B). These results demonstrate the tunability of GldA and
DHAK activities using the above constructs.
The studies described above also provided an initial assessment on how cell growth and glycerol fermentation are affected
by the overexpression of GldA and DHAK. We observed a 3.4-fold
increase in the amount of glycerol fermented by 48-h Hungate
tube cultures when these enzymes were simultaneously overexpressed from plasmid pZSKLMgldA (Fig. 5). However, individual
overexpression of either GldA or DHAK did not have a signicant
effect on glycerol fermentation (Fig. 5). We speculate that this
behavior was due to the low activities of GldA and DHAK in wildtype E. coli: 6.6 and 1.7 mmol/g cell/h, respectively. In fact, these
activities are very similar to the rate at which glycerol is utilized
by MG1655 (5.6 mmol/g cell/h) and therefore might be a limiting
factor in glycerol metabolism. As an alternative explanation, the
individual overexpression of these enzymes could lead to a
metabolic imbalance, which in turn would result in the accumulation of toxic intermediates. However, the 1D 1H-NMR (nuclear
magnetic resonance) spectra of the extracellular media of cultures
overexpressing GldA and DHAK, individually or in combination,
did not show evidence of DHA or methylglyoxal accumulation
(data not shown). These are likely intermediates that could be
generated by the imbalanced metabolism of glycerol (Booth,
2005). Therefore, the lack of changes in glycolytic uxes upon the
individual overexpression of either GldA or DHAK does not appear
to be due to the creation of a metabolic imbalance, but rather to
the low activities of both enzymes, which in turn required their
simultaneous overexpression. Our ndings suggest a metabolic
control structure in which GldA and DHAK share the control of the
glycolytic ux during fermentative utilization of glycerol.
To further assess the impact of GldA and DHAK overexpression,
we conducted fermentations in a more controlled environment
(fully controlled fermenters). Fig. 6 shows complete proles for
strains MG1655 (pZSblank) and MG1655 (pZSKLMgldA). The
advantages of GldA and DHAK overexpression are very evident.
While a culture of strain MG1655 (pZSKLMgldA) consumed
almost 8 g/L of glycerol when reaching stationary phase at 60 h,
the reference strain MG1655 (pZSblank) only consumed 6.5 g/L
glycerol over a period of 120 h (Fig. 6). This translates into a 2.7fold increase in the volumetric rate of glycerol consumption:

ARTICLE IN PRESS
S. Shams Yazdani, R. Gonzalez / Metabolic Engineering 10 (2008) 340351

347

GldA activity
(mol glycerol/mg protein/min)

1.0

0.8

0.6

0.4

0.2

0.0
0

25

50

75

100

ng/ml ng/ml ng/ml ng/ml ng/ml

25

50

75

100

ng/ml ng/ml ng/ml ng/ml ng/ml

pZSgldA

pZSKLMgldA

DHAK activity
(mol DHA/mg protein/min)

0.3

0.2

0.1

0.0
0

25

50

75

25

50

75

100

ng/ml

ng/ml

ng/ml

ng/ml

ng/ml

ng/ml

ng/ml

ng/ml

ng/ml

pZSKLM

pZSKLMgldA

Fig. 4. Functional characterization of constructs overexpressing enzymes glycerol dehydrogenase (GldA) and dihydroxyacetone kinase (DHAK). Effect of inducer
concentration (anhydrotetracycline) on GldA (A) and DHAK (B) activities is shown. Overexpression of GldA and DHAK individually (pZSgldA and pZSKLM) and in
combination (pZSKLMgldA) was evaluated. Experiments were conducted in Hungate tubes under anaerobic conditions and using specied concentrations of
anhydrotetracycline. Reported values are the average of three samples from 48-h cultures of MG1655 transformed with the specied plasmid. Overexpressed enzymes are
apparent from plasmid name.

0.78 mmol/L/h for MG1655 (pZSblank) and 2.09 g/L/h for MG1655
(pZSKLMgldA) (Table 2). Faster consumption of glycerol was the
result of both faster growth (about 2.3-fold increase in specic
growth rate) and higher specic rate of glycerol consumption (1.4fold increase) (Table 2).
The performance of strain MG1655 (pZSKLMgldA) was superior to that reported for E. coli strains engineered to ferment
glycerol (Zhu et al., 2002). For example, recombinant E. coli strains
expressing glycerol-fermenting pathways from organisms such as
K. pneumoniae required glucose to efciently metabolize glycerol
(i.e., co-fermentation of glycerol and glucose) (Zhu et al., 2002;
Cameron et al., 1998). The rate of glycerol utilization by strain
MG1655 (pZSKLMgldA) compared well with that of glycerol-

fermenting organisms when no external electron acceptors were

provided (Biebl et al., 1999; Bories et al., 2004; Lee et al., 2001;
Sakai and Yagishita, 2007; Yazdani and Gonzalez, 2007 and
references cited therein). The maximum specic (12.6 mmol/g
cell/h) and volumetric (2.09 mmol/L/h) rates of glycerol utilization
were also much higher than those reported for organisms that
convert glycerol to ethanolhydrogen or ethanolformate (Jarvis
et al., 1997; Ito et al., 2005; Sakai and Yagishita, 2007).
Furthermore, using the same carbon basis, the specic rate of
glycerol consumption by MG1655 (pZSKLMgldA) was similar to
that reported for the utilization of sugars by recombinant E. coli
strains optimized for the production of ethanol as the main
fermentation product (Underwood et al., 2002).

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348

S. Shams Yazdani, R. Gonzalez / Metabolic Engineering 10 (2008) 340351

Cell growth (OD550)

1.0

6
0.8

5
4

0.6

0.4

2
0.2

Glycerol consumption (g/L)

1.2

0.0
0 ng/ml 25-100 0 ng/ml 25-100 0 ng/ml 25-100 0 ng/ml
ng/ml
pZSblank

ng/ml

ng/ml

pZSgldA

25
ng/ml

pZSKLM

50

75

100

ng/ml

ng/ml

ng/ml

pZSKLMgldA

10

1.0

0.8

0.6

0.4

0.2

0
0

30

60
Time (h)

90

0.0
120

Fig. 6. Kinetics of glycerol fermentation in response to the simultaneous

overexpression of glycerol dehydrogenase (gldA) and dihydroxyacetone kinase
(dhaKLM). Cell growth (closed symbols) and glycerol utilization (open symbols) are
shown for strains MG1655 (pZSblank) (squares) and MG1655 (pZSKLMgldA)
(diamonds). Experiments were conducted in fully controlled fermenters, at pH 7.5,
and using 100 ng/ml of anhydrotetracycline.

3.4. Integrated biocatalyst to convert glycerol into ethanol

and co-product formate
Given our success on improving the rate of glycerol utilization
through GldA and DHAK overexpression, we evaluated the
performance of an integrated biocatalyst for the co-production
of ethanol and formate: i.e., overexpression of both GldA and
DHAK in strain SY04, the latter a triple mutant containing
disruptions on the genes frdA, pta, and fdhF. Strain SY04
(pZSKLMgldA) exhibited all the desired characteristics of a
biocatalyst to convert glycerol into ethanol and formate: i.e., fast
utilization of glycerol and exclusive synthesis of ethanol and
formate (Fig. 7). Product yields were above 90% of the theoretical
maximum: approximately 1.04 and 0.92 mol of ethanol and
formate, respectively, for each mol of glycerol fermented. The
growth of strain SY04 (pZSKLMgldA) signicantly improved when
compared to SY04. The specic and volumetric growth rates were
3.6- and 5.8-fold higher (Table 2; also compare Fig. 7 and Fig. 3B).
Similar changes were observed in glycerol utilization and product
synthesis: 2-fold and 45-fold increases in specic and volumetric

12

1.2

10

1.0

0.8

0.6

0.4

0.2

Cell growth (OD550)

1.2

Glycerol and products (g/L)

12

Cell growth (OD550)

Glycerol utilization (g/L)

Fig. 5. Effect of expression level of glycerol dehydrogenase (GldA) and dihydroxyacetone kinase (DHAK) on cell growth (bars) and glycerol fermentation (line). Experiments
were conducted in Hungate tubes under anaerobic conditions and initial pH of 7.5. Reported values are the average of three samples from 48-h cultures of MG1655
transformed with the specied plasmid. Coefcients of variation were below 10% in all cases. Average results for the use of different concentrations of inducer are shown for
plasmids pZSblank, pZSKLM, and pZSgldA. Cultures carrying these plasmids did not exhibit signicant changes in cell growth or glycerol utilization at different inducer
concentrations. Overexpressed enzymes are apparent from plasmid name.

0.0

0
0

10

20

30
Time (h)

40

50

60

Fig. 7. Co-production of ethanol and formate by strain SY04 (pZSKLMgldA).

Fermentation prole for cell growth, glycerol utilization, and product accumulation. Data are shown for concentration of cells (OD550: ~), glycerol (&), ethanol
(J), formate (K), and other products (n). Experimental conditions were those
reported in Fig. 6. Coefcients of variation were below 5% in all cases.

rates, respectively. Essentially the same results were obtained

using higher concentrations of glycerol (data not shown). The
performance of strain SY04 (pZSKLMgldA) was clearly superior to
that reported for the conversion of glycerol to ethanol and formate
by K. planticola (Jarvis et al., 1997), a process that resulted in
0.5 mol of product per mol of glycerol fermented and took about
100 h to ferment 70 mM of glycerol. The specic rate of ethanol
production and ethanol yield when using strain SY04
(pZSKLMgldA) were also similar to those achieved with E. coli
strains engineered to convert cellulosic sugars to ethanol (Underwood et al., 2002). In addition to synthesizing ethanol at high
rates, strain SY04 (pZSKLMgldA) also produced formate, which
implies an overall rate of product synthesis per unit of cell mass
twice of that reported for the production of ethanol from sugars.

3.5. Integrated biocatalyst to convert glycerol into ethanol

and co-product hydrogen
Although results from previous sections would indicate that
overexpression of GldA and DHAK in a genetic background

ARTICLE IN PRESS
S. Shams Yazdani, R. Gonzalez / Metabolic Engineering 10 (2008) 340351

12

2.4

10

2.0

1.6

1.2

0.8

0.4

Cell growth (OD550)

Fig. 3B and Table 2) and (ii) little improvement of cell growth

upon GldA and DHAK overexpression at acidic conditions (Fig. 8B).
We then evaluated an alternative strategy to improve the rate
of production of ethanolhydrogen, namely the use of microaerobic conditions. Microaerobic conditions were created by
circulating air through the headspace of the fermenters, a
condition that produced detectable amounts of dissolved oxygen
only during the initial hour of the culture (data not shown).
Operation was characterized by a volumetric oxygen transfer
coefcient (kLa) of 0.4970.07 h1. Glycerol utilization under these
conditions still led to the synthesis of reduced compounds ethanol
and succinate as the major products, although signicant amounts
of acetate were also produced (Fig. 9A). The synthesis of succinate
and acetate decreased the ethanol yield to about 0.75 (Table 2). In
terms of rates, 10 g/L of glycerol were completely consumed in
about 60 h (Fig. 9A), which corresponds to an approximately 2fold increase in volumetric rates (Table 2). When grown under
microaerobic conditions and pH 6.0, strain SY03 exhibited a
signicantly higher ethanol yield, but only reached 82% of the
theoretical maximum (Table 2). In addition, SY03 still produced an
amount of succinate equivalent to 10% (w/w) the amount of
ethanol. Simultaneous overexpression of GldA and DHAK in SY03
and its cultivation under the same conditions resulted in the

Glycerol and products (g/L),

Oxygen transfer rate (mg O2/L/h)

containing frdA and pta mutations (i.e., strain SY03 (pZSKLMgldA))

should result in the efcient co-production of ethanol and
hydrogen, growth and glycerol utilization by this strain at pH
6.3 were very inefcient (Fig. 8A). To further investigate this
result, we examined the effect of GldA and DHAK overexpression
in MG1655 at different pHs and found that GldA and DHAK
overexpression under acidic conditions was not as effective as
previously found at alkaline conditions (Fig. 8B). Such behavior
appears to be related to the lower activity of GldA at acid pH
(Fig. 8C). These results suggest that the poor performance of strain
SY03 (pZSKLMgldA) may have resulted from the combined effect
of: (i) negative impact of pta and frdA mutations (strain SY03,

349

0.0
0

10

20

30

40

50

60

70

12

2.4

10

2.0

1.6

1.2

0.8

0.4

Cell growth (OD550)

Glycerol and products (g/L),

Oxygen transfer rate (mg O2/L/h)

Time (h)

0.0

0
0

10

20

30

40

50

Time (h)
Fig. 8. Glycerol dehydrogenase (gldA) and dihydroxyacetone kinase (dhaKLM)
overexpression in SY03 at acidic conditions. (A) Cell growth (closed symbols) and
glycerol utilization (open symbols) in strains SY03 (squares) and SY03
(pZSKLMgldA) (triangles) at pH 6.3. (B) Cell growth for strains MG1655 (pZSblank)
(circles), and MG1655 (pZSKLMgldA) (triangles) at pH 6.3 (open symbols) and 7.5
(closed symbols). (C) Impact of pH on in vitro GldA activities.

Fig. 9. Glycerol utilization and product synthesis under microaerobic and acidic
conditions (air in the headspace and pH 6.0) by strains (A) MG1655 and (B).SY03
(pZSKLMgldA). Microaerobic conditions are characterized by a volumetric oxygen
transfer coefcient (kLa) of 0.4970.07 h1. Data is shown for concentration of cells
(OD550: ~), glycerol (&), ethanol (J), formate (K), succinateacetate (W), and
oxygen transfer rate ().

ARTICLE IN PRESS
350

S. Shams Yazdani, R. Gonzalez / Metabolic Engineering 10 (2008) 340351

complete elimination of by-product succinate, leaving ethanol as

the only product found in the fermentation broth (Fig. 9B).
Ethanol yield was 0.96 mol per mol of glycerol utilized, and
volumetric productivities for glycerol utilization and ethanol
synthesis were about 4.6 mmol/L/h (Table 2). When compared to
strain SY03, this performance represents 1.17- and 2-fold
improvements in yield and productivity, respectively (Table 2).
The high productivities realized in this system were the result of
higher specic rates (Table 2). Although the increase in productivity upon overexpression of GldA and DHAK agreed with our
previous results, the observed higher ethanol yield was unexpected. We speculate that overexpression of DHAK, a phosphoenolpyruvate-dependent enzyme, decreased the availability of
phosphoenolpyruvate, and as a consequence reduced the accumulation of succinate in the medium (Fig. 9B). This, in turn, would
favor the channeling of carbon to the synthesis of ethanol.
3.6. Utilization of crude glycerol

10

1.5

1.2

0.9

0.6

0.3

Cell growth (OD550)

Glycerol and products (g/L)

The use of an industrial medium containing crude glycerol,

which is generated as an inevitable by-product in the biodiesel
industry, is of great relevance for the biocatalysts developed in
this work. Engineered strains performed well when crude glycerol

4. Conclusions
This work demonstrates the feasibility of converting glycerol,
an inevitable byproduct of biodiesel production, to ethanol and
valuable co-products. Using a rational engineering approach
enabled by our previous studies of glycerol fermentation in
E. coli, two microbial platforms for the efcient production of
ethanolhydrogen and ethanolformate were developed. Product
yields and productivities in these strains were superior to those
reported for the conversion of glycerol to ethanolH2 or
ethanolformate and similar to or better than those reported for
the production of ethanol from sugars using engineered E. coli
strains. Additional improvements of these strains in order to
achieve very high-product titers are envisioned through further
increases in GldA and DHAK expression and the use of metabolic
evolution techniques. Metabolic evolution is a well-established
approach that has been successfully implemented in E. coli for the
efcient production of biofuels and other products (Zhang et al.,
2007; Yomano et al., 1998). Process-based improvements, including fed-batch cultivations and high-density cultures, are also
envisioned to further optimize the production of ethanol and coproducts hydrogen and formate.

Acknowledgments

25

1.5

This work was supported by grants from the US National

Science Foundation (CBET-0645188) and the National Research
Initiative of the US Department of Agriculture Cooperative State
Research, Education and Extension Service (2005-35504-16698).
S.S. Yazdani was partially supported by a fellowship from the
Department of Biotechnology, Ministry of Science and Technology,
Government of India. We thank H. Mori and B. Erni for providing
research materials and Y. Dharmadi for construction of the
fdhF mutant.

20

1.2

References

15

0.9

10

0.6

0.3

0.0

0
0

20

40

60

Cell growth (OD550)

Time (h)

Glycerol and products (g/L)

was used as a carbon source. Representative data for the

production of ethanolformate by SY04 (pZSKLMgldA) are shown
in Fig. 10. Glycerol consumption and synthesis of ethanol and
formate were similar to that reported for the consumption of pure
glycerol (compare Fig. 7 and Fig. 10A and fermentation parameters
in Table 2). The use of higher concentrations of glycerol did not
have a negative impact in this process either, as similar results
were observed with the use of 20 g/L of crude glycerol (Fig. 10B
and Table 2).

0.0

0
0

20

40

60
Time (h)

80

100

Fig. 10. Conversion of crude glycerol to ethanol and formate by strain SY04
(pZSKLMgldA). Data are shown for concentration of cells (OD550: ~), glycerol (&),
ethanol (J), formate (K), and other products (W) in fermentations conducted
using crude glycerol at (A) 10 g/L and (B) 20 g/L.

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