Aerobic utilization of crude glycerol by recombinant Escherichia coli for

simultaneous production of poly 3-hydroxybutyrate and bioethanol

Pramod Shah,
1
Feng-Shen Chiu,
1
and John Chi-Wei Lan
1, 2,
*
Graduate School of Biotechnology and Bioengineering, Yuan Ze University, 135 Yuan-Tung Road, Chung-Li, Taoyuan 320, Taiwan
1
and Bio-renery and Bioprocess Engineering
Laboratory, Department of Chemical Engineering and Materials Science, Yuan Ze University, 135 Yuan-Tung Road, Chung-Li, Taoyuan 320, Taiwan
2
Received 15 March 2013; accepted 30 August 2013
Available online 17 October 2013
Crude glycerol, an inevitable byproduct during biodiesel production, is emerging as a potential feedstock for
fermentation, due to its availability and a reasonable price. Biological utilization of abundant crude glycerol to several
value added products is contemporary research area with benecial features. Solving the problem of proper disposal and
raising economic viability of biodiesel industries. Several researches have been directed toward the production of
numerous products by using Escherichia coli, an ideal organism for heterologous expression of various foreign proteins.
In this fashion, recombinant E. coli strains were constructed for the simultaneous production of poly 3-hydroxybutyrate
(P3HB) and bioethanol from crude glycerol. The incorporation of aldehyde reductase (Alrd) and aldehyde dehydrogenase
(AldH) in recombinant strain showed 2-fold increment in crude glycerol utilization under aerobic condition. Moreover,
these two enzymes introduced an alternative pathway leading toward the potential production of bioethanol which was
more than redox-balancing steps. Acetate was accumulated as an intermediate product. Subsequently, acetate was
utilized as substrate in the second pathway, which directly converted acetyl-CoA to P3HB. This strategy demonstrated a
potential production manner of bioethanol as an extracellular product and P3HB as water insoluble inclusion bodies
inside E. coli. The maximum production of bioethanol and P3HB in the recombinant strain was 0.8 g L
L1
(17.4 mmol L
L1
)
and 30.2% (w/w dry cell weight), respectively, which were higher than the parental strain.
2013, The Society for Biotechnology, Japan. All rights reserved.
[Key words: Escherichia coli; Aerobic fermentation; Crude glycerol; Bioethanol; Poly 3-hydroxybutyrate; Aldehyde reductase; Aldehyde
dehydrogenase]
The alarming rate of consumption and combustion of fossil fuels
to support global energy demand is continuously causing depletion
of its limited stocks and severe impact on health and environment
(1). Therefore, increasing enthusiasm is focused toward the gen-
eration of alternative energy sources. Utilization of biomass for
biofuel production has created unexceptional opportunities in
dealing with increasing demand and escalating prices of fossil fuel.
Biofuel are renewable energy with rather high efciency and its
sustainability and biodegradability contribute to the reduction of
carbon dioxide emission and other harmful gases during combus-
tion (2,3).
The production capacity and market values of biodiesel and
bioethanol are gaining lots of attention than other categories of
biofuels (4). The appropriate method applied for the production of
biodiesel is a homogeneous base-catalyze trans-esterication of
triglycerides existing in vegetable oils, animal fats, or waste cooking
oils with methanol (5). This process separates fatty acids moiety
from triglycerides resulting in the generation of an inevitable
byproduct, glycerol. One mole of glycerol is produce with the pro-
duction of every 3 mol of methyl esters (i.e., biodiesel), resulting in
10% by weight (wt %) of the total crude glycerol produce (6). In
recent years, the mandatory demand of biodiesel has ooded the
market with excessive crude glycerol. As a result, the price of crude
glycerol has drastically plummeted enforcing burden on economic
viability in the biodiesel industry. Crude glycerol alone account for
13e14 % of the total production costs (7). As a consequence of it,
several biodiesel companies need governmental aids for supporting
its operation (4).
Biodiesel companies are seeking new alternative for this crude
glycerol to higher-value added products. Biological utilization of
crude glycerol serves as a feedstock in various fermentation pro-
cesses with the production of several value-added products such as
1,3-propanediol (PDO), dihydroxy-acetone (DHA), bioethanol,
butanol, propionic acid, succinic acid, and so on (8). Utilization of
crude glycerol in fermentation processes account in reduction of
manufacturing costs and higher yields of products than other sugar
substrates. Among several alternative of value-added products
during the fermentation process, the production of bioethanol is a
redox-balance pathway (9,10).
Utilization of crude glycerol by Escherichia coli, a model organ-
ism in bioindustries has long been proved under aerobic condition
with minimal ethanol production. In recent years, anaerobic and
micro-aerobic fermentation of crude glycerol has been well estab-
lished for the higher yield of bioethanol by the construction of
different recombinants E. coli (10e17). A high production of bio-
ethanol metabolically requires a signicant re-engineering of
* Corresponding author at: Bio-renery and Bioprocess Engineering Laboratory,
Department of Chemical Engineering and Materials Science, Yuan Ze University, 135
Yuan-Tung Road, Chung-Li, Taoyuan 320, Taiwan. Tel.: 886 34638800x3550;
fax: 886 34559373.
E-mail address: lanchiwei@saturn.yzu.edu.tw (J.C.-W. Lan).
www.elsevier.com/locate/jbiosc
Journal of Bioscience and Bioengineering
VOL. 117 No. 3, 343e350, 2014
1389-1723/$ e see front matter 2013, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2013.08.018
metabolic pathway to derive bioethanol exclusively from acetyl-
CoA with ATP as the energy source and NADPH as the sources of
reducing equivalents (14,18).
In this work, the knowledge of aerobic fermentation has been
used to engineer E. coli for the effective conversion of crude glycerol
to bioethanol with aldehyde reductase and aldehyde dehydroge-
nase. Aldehyde reductase (Alrd, EC 1.1.1.21) is an NADPH-dependent
oxidoreductase enzyme which catalytically reduces various alde-
hydes and carbonyls, including monosaccharide to corresponding
polyalcohol (19). Aldehyde dehydrogenase (AldH, EC 1.2.1.3), on the
other hand, catalyzes the NADH-dependent oxidoreductase activ-
ity, oxidizing aldehydes to corresponding carboxylic acids (20).
These two enzymes provided alternative pathways for the utiliza-
tion of glycerol through glycolysis and further, established the
conversion of acetate, the end product of glycolysis, to ethanol. It
has been observed that under aerobic condition, acetic acid is major
product which exits as acetate ion (21). Accumulation of acetate is
toxic to organism thus a specic approach was considered for the
conversion of acetyl-CoA to poly 3-hydroxybutyrate (P3HB), a
second value-added product. These pathways eliminated the
accumulation of acetate.
Poly-hydroxyalkanoates (PHAs) are another value-added prod-
uct gaining lots of attention as an ingredient for thermoplastics.
With its biodegradable nature and a variety of other material
properties, they are suitable for several applications (22,23). PHAs
are carbon and energy reserve accumulated in many bacteria as
intracellular inclusions under excess carbon reserve or unfavorable
condition. These intracellular inclusions enhance the tness of
bacteria and also contribute to redox-balance (24). P3HB is a well
known member of PHAs family which has been widely studied.
However, the production of P3HB in large scale is limited to sub-
strates cost (25). Therefore, the use of crude glycerol for this pur-
pose can be an interesting opportunity. Synthesis of P3HB from
glycerol as carbon source has been evaluated in several microbial
strains along with different fermentation strategy (26,27). Ralstonia
eutropha, a well known organism for the production of PHAs con-
tains phaCAB operon for the conversion of acetyl-CoA to P3HB. It
has been established that R. eutropha can accumulate more than
80% of P3HB content (28). The recombinant E. coli constructed by
harboring phaCAB operon from R. eutropha (phaCAB
RE
) has been
shown to accumulate 67% of P3HB content, using 2e4% glucose as
carbon source (29). In the meantime, bacterial hemoglobin gene
(vgb gene) from Vitreoscilla hemoglobin has been noted to enhance
cell density, protein production and bioremediation by increasing
oxygen availability in the culture (30). The co-expression of pha-
CAB
RE
and vgb genes in E. coli was revealed to enhance P3HB pro-
duction (31).
The aim of the current study was to generate desirable products
from crude glycerol through alternate pathways provided by two
plasmids. Our attempt resulted in simultaneous production of an
extracellular and an intracellular product, bioethanol and P3HB,
respectively.
MATERIALS AND METHODS
Bacteria strains and plasmid used The wild-type strains, R. eutropha H16
and E. coli BL21(lDE3), were kindly provided by Bioresource Collection and Research
Center (BCRC), Taiwan. E. coli BL21(lDE3) was used as a host for high level expres-
sion of cloned genes. It contained T7 RNA-polymerase gene in a lysogenic and
inducible form. This strain is characterized by two protein decomposition enzyme
genes lon and ompT that has been articially damage, making it suitable for the use
of enzymes as a foreign gene expression (32). All strains used in this study along
with plasmids and primers required for the construction of recombinant strains
are listed in Table 1. The molecular biology methods applied here are according to
the manufacturers protocols.
Construction of single and double plasmid Plasmid pBAD33 was kindly
provided by Prof. Soo Po-Chi (Tzu Chi University, Taiwan). This plasmid contained
P
BAD
promoter of araBAD (arabinose) operon and its regulatory gene, araC, followed
by multiple cloning site (MCS2) and M13 intragenic region with chloramphenicol-
resistance (cmR) gene, resulting in a high level expression system (33). Polymerase
Chain Reaction (PCR) was performed for the amplication of Alrd and AldH from
genomic DNA of R. eutropha H16 on Select BioProducts (Edison, NJ, USA). The
reagents used were purchased from New England Biolabs (Ipswich, MA, USA) and
Promega Corporation (Fitchburg, WI, USA). Expression vector pARD33 was
constructed as shown in Fig. 2A, by cloning Alrd and AldH genes in cloning vector
pBAD33 with restriction sites of kpnI/SalI and SalI/HindIII, respectively.
The plasmid, pBHB2, was kindly provided by the Yuan Ze PHA team at Yuan Ze
University (Taiwan) as mentioned in Table 1. This plasmid contains P3HB synthesis
genes phaCAB
RE
, from R. eutropha H16 and a bacterial hemoglobin gene (vgb gene)
from V. hemoglobin with ampicillin-resistance gene (AmpR). The three main en-
zymes that has beenwell established for the conversion of acetyl-CoAto the P3HB in
R. eutropha are: phaA (b-ketothiolase; EC 2.3.1.9), phaB (NADPH-dependent ace-
toacetyl-CoA reductase; EC 1.1.1.36) and phaC (PHA polymerase; EC 2.3.1) (28). For
this plasmid, Isopropyl b-D-1-thiogalactopyranoside (IPTG) acted as an inducer for
protein regulation and was purchased from SigmaeAldrich Corporation (St. Louis,
MO, USA). The expression vectors pARD33 and pBHB2 were transformed into E. coli
BL21(lDE3) individually for the construction of recombinant strains with single
plasmid: E. coli BL21_pARD33 and E. coli BL21_pBHB2, respectively (as shown in
Fig. 2B and C). The size of amplied Alrd and AldH genes, plasmids pBAD33, pARD33
and pBHB2 were conrmed by running 0.8% agarose gel, stained with ethidium
bromide and visualized under UV light in Dolphin-Doc Plus (Wealtec Corp., NV,
USA). The E. coli BL21_pARD33-BHB2 was constructed with transformation of two
plasmids (pBHB2 and pARD33) one after another in single host, E. coli BL21(lDE3) as
shown in Fig. 2D. The constructed recombinant strains were subsequently cultured
and applied for further study.
Culture medium The LuriaeBertani (LB) medium was used to prepare LB
broth and LB plates. LB broth contained: 10.0 g L
1
of tryptone, 5.0 g L
1
of yeast
extract and 10.0 g L
1
of NaCl, whereas LB plates contained ingredient of LB broth
with addition of 15.0 g L
1
of agar. Appropriate antibiotics were used at following
concentrations: 50 mg ml
1
chloramphenicol (Cm) and 100 mg ml
1
Ampicillin
(Amp) obtained from the SigmaeAldrich.
The crude glycerol used in this study was provided by Greatec Green Energy
Company, a local biodiesel plant in Taiwan. Greatec Green Energy produces biodiesel
from rened waste cooking oil using sodium methoxide as catalyst. The crude
glycerol was composed of 80% crude glycerol, 11% water, 9% metal ions and other
impurities. All trials were conducted using the same batch of crude glycerol. The
minimal dene medium used in this study contained 30 g L
1
crude glycerol,
6.70 g L
1
of Na
2
HPO
4
-7H
2
O, 2.50 g L
1
of (NH
4
)
2
SO
4
, 1.50 g L
1
of KH
2
PO
4
, 0.20 g L
1
of MgSO
4
-7H
2
O, 10.00 mg L
1
of CaCl
2
and 5.0 ml of trace metal solution (6.84 g L
1
of H
3
BO
3
, 6.00 g L
1
of Na
2
EDTA, 0.86 g L
1
of MnCl
2
-4H
2
O, 0.29 g L
1
of FeCl
3
-6H
2
O,
0.06 g L
1
of ZnCl
2
, 0.026 g L
1
of CoCl
2
-6H
2
O and 0.002 g L
1
of CuSO
4
-5H
2
O) and
2 g L
1
of yeast extract. Most of the chemicals were obtained fromSigmaeAldrich. To
prevent reaction between chemicals; trace metal solution, crude glycerol, yeast
extract and salts were autoclaved separately for 15 min at 121

C while antibiotics
were sterile ltered.
TABLE 1. Microbial strains, plasmids and vectors used in this study.
Strain/Plasmid/Primer Description Source
Strain
R. eutropha H16 Wild-type BCRC
a
-13036
E. coli BL21 Wild-type BCRC
a
-51878
E. coli BL21_
pARD33-BHB2
pARD33 and pBHB2 This study
Plasmids
pGEM-T easy Cloning vector Promega
pBAD33 Low level expression
vector
Prof. Su Boqi (Tzu Chi
University)
pGEM-T easy_alrd pGEM-T easy :: alrdRe This study
pGEM-T easy_aldH pGEM-T easy :: aldHRe This study
pARD33 pBAD33 :: alrd/aldH This study
pBHB2 pBluescript II KS ::
phaCAB
RE
The Yuan Ze PHA team
Primers
Alrd primer
Forward GAGGTACCAAGGAGGA
AATGAAGCAAGTCAC
This study
Reverse GAGTCGACAGCTCAAA
GCATTTCCAG
This study
AldH primer
Forward AGTCGACAAGGAGAAT
ATGCGAGAAGTCCC
CGA
This study
Reverse GAAAGCTTTCAACGCAG
AAGCAGGCGCAA
This study
a
BCRC: Bioresource Collection and Research Center (Taiwan).
344 SHAH ET AL. J. BIOSCI. BIOENG.,
Culture condition Each recombinant strain was grown from one loop-full
stock culture, streaking on LB plates with appropriate selection marker and incu-
bated for overnight at 37

C. Single colony fromplate was used to inoculate 10 mL LB

broth with specied selection marker and was incubated in a rotary shaker
(200 rpm) at 37

C for 24 h. Batch cultures were conducted in 500 mL shake ask
with 100 mL working volume. A pre-culture (3% v/v) were used to inoculate 100 ml
of minimal dene medium and incubated in rotary shaker with a speed of 200 rpm
at 37

C for 48e72 h. Samples were drawn periodically from the post inoculation at
regular intervals of time.
Assay of expression enzymes The expression of Alrd and AldH proteins
were analyzed on a 12% sodium dodecyl sulfate poly-acrylamide gel electrophoresis
(SDS-PAGE) in a vertical slab gel apparatus, followed by Coomassie Brilliant Blue
staining. The protein concentrations in the supernatant and cell pellet extract after
sonication of wet cell-pallets were determined by using the Bradford method (34)
with bovine serum albumin (BSA) as a standard.
The enzyme activities were calculated from the linear slope of increasing
absorption of NAD

/NADP

reduced to NADH/NADPH (mM mL

1
) at 340 nm,
respectively. The Alrd activity was performed in a 1 mL mixture at 37

C by using
4.67 mM D,L-glyceraldehyde as substrate in a buffering solution comprised of
0.25 M sodium phosphate, 0.38 M ammonium sulfate, 0.11 mM NADPH and
0.1 mM EDTA at pH 6.8 (35). The activity of AldH was performed in a 1 mL
mixture containing 50 mM sodium phosphate buffering solution at pH 7.5,
50e100 mL of crude cell extract or 10e15 mg of puried enzyme, 0.5 mM NAD

and 1 mM D,L-glyceraldehyde as substrate (36). One unit of enzyme activity is

dened as the amount of protein that produces 1 pmol NADH or NADPH per
minute.
Analytical methods Biomass was measured by measuring dry cell weight
which in turn was correlated with optical density. Cell growth was monitored by
measuring the optical density of culture at 600 nm, on UltraSpec 2100 pro
UVeVisible spectrophotometer (GE Healthcare, Wauwatosa, WI, USA). In this case,
original mediumwas used as the blank. Three consecutive samples were drawn and
analyzed each time for the accuracy of work.
The concentration of bioethanol and residual crude glycerol along with acetate
was analyzed on a LC-2000 plus series high performance liquid chromatography
(HPLC) systems (Jasco-Analytical Instruments, Japan) equipped with an ion
exclusion column ICEeIONe300 (Transgenomic Labs, Omaha, NE, USA) operated
at 60

C. Firstly, the samples were ltered through 0.45 mm of HEPA lters
(Billerica, MA, USA) and 10 ml was auto-injected to the HPLC. The refractive index
(RI-2031 plus) detector at 35

C was employed to analyze these samples. The
samples were eluted at 0.4 mL min
1
with 5 mM sulfuric acid (0.0085 N H
2
SO
4
)
prepared in HPLC grade water. Data were processed and analyzed using the
ChromNAV software.
P3HB was recovered from the dry cell mass through solvent extraction and
the P3HB content (wt %) was expressed as percentage of P3HB mass in dry cell
mass. Briey, 20 mg of P3HB-containing cell dry weight was mix with 2 mL of
chloroform and 2 mL of methanol solution containing sulfuric acid (1 mL of
H
2
SO
4
and 99 mL of methanol). The solution was incubated at 100

C for 15 h,
and then 1 mL of 1 M NaCl was added to facilitate phase separation. After re-
covery of organic (chloroform) phase, 20 mL of gas chromatograph internal
standard (GCIS) was added, and 300 mL of bottom organic layer was collected as
sample. For analysis, 0.5 mL of sample was injected into a Shimadzu gas chro-
matograph GC-2014 system (Shimadzu Scientic Instruments, Columbia, MD,
USA) equipped with a ame ionization detector (GC-FID) and a Phenomenex
Zebron ZB-5 capillary column (30 m 0.25 mm 0.25 mm). Oven temperature
was held at 100

C. Both injector and detector temperatures were set at 220

C.
Nitrogen was used as the carrier gas and diphenyl ether was used as internal
standard. Data were processed and analyzed by using Peak-ABC software. The
FIG. 1. Metabolic pathways involved in glycerol metabolism in Escherichia coli. Abbreviations: Alrd, aldehyde reductase (EC 1.1.1.21); AldH, aldehyde dehydrogenase (EC 1.2.1.3);
phaA, b-ketothiolase (EC 2.3.1.9), phaB, NADPH-dependent acetoacetyl-CoA reductase (EC 1.1.1.36) and phaC, PHA polymerase (EC 2.3.1).
VOL. 117, 2014 SIMULTANEOUS REFINERY OF CHEMICALS FROM GLYCEROL 345
total P3HB content, yield of biomass and products were calculated according to
the following equations:
P3HB content w=w gram of P3HB=gram of total biomass produced 100
(1)
Yieldof biomass : Y
x=s
gramof biomass produced=gramof crudeglycerol consumed
(2)
Yield of product : Y
p=x
gramof product produced=gramof dry cell weight DCW
(3)
Substrate consumption rate : d
s=t
total substrate consumed mass=volume=
time interval
(4)
RESULTS AND DISCUSSION
Effect of pBAD33 plasmid on crude glycerol utilization The
role of plasmid pBAD33 was marked in the strain E. coli
BL21_pBAD33. Fig. 3A and Table 2 depicted the results in terms of
cell growth and crude glycerol utilization, respectively. The E. coli
BL21(lDE3), which was employed as control, showed better
growth than E. coli BL21_pBAD33. However, crude glycerol
consumption was noted to be similar in both strains, i.e., around
50%. This result proved that cloning vector had no signicant
effect upon cell growth and crude glycerol utilization.
Impact of Alrd and AldH upon crude glycerol
consumption With the conrmation of ineffective
FIG. 2. Construction and transformation of plasmids. (A) Cloning of AldH and Alrd to plasmid pBAD33 for the construction of pARD33. (BeD) Transformation of the constructed
plasmids in Escherichia coli BL21(lDE3).
346 SHAH ET AL. J. BIOSCI. BIOENG.,
phenomenon on crude glycerol utilization from original vector,
pBAD33, the effect of expression vector, pARD33 containing Alrd
and AldH was investigated in E. coli BL21_pARD33. Fig. 3A
demonstrated that the prole of cell growth of E. coli
BL21_pARD33 was similar to that of E. coli BL21(lDE3). However,
the crude glycerol utilization was nearly 2-fold higher in E. coli
BL21_pARD33 (Table 2) than the latter. The residual crude
glycerol concentrations for E. coli BL21_pARD33 and E. coli
BL21(lDE3) were noted to be 1.13 and 12.54 g L
1
, respectively.
The comparisons among different recombinant strains are
summarized in Table 2. The results indicated that the presence
of Alrd and AldH dramatically improved in crude glycerol
utilization. Fig. 1 shows an alternative pathway introduced by
Alrd and AldH without occluding the original pathway of crude
glycerol metabolism. The Alrd catalyzes the oxidation of crude
glycerol to D-glyceraldehyde, governing the release of NADPH
(35). However, AldH catalyzes the NADH-dependent conversion
of D-glyceraldehyde to D-glycerate with reduction of NAD

to
NADH (37,38). The aerobic condition was important for the
reduction of NADP

/NAD

to NADPH/NADH, respectively. The

accumulated D-glycerate enters the glycolysis pathway for the
generation of pyruvate and later acetyl-CoA under aerobic
condition. This alternative pathway furnished with the
improvement of crude glycerol utilization in E. coli BL21_pARD33.
Effects of Alrd and AldH in strains harboring single and
double plasmid The presence of Alrd and AldH showed a 2-
fold increment in crude glycerol consumption by E. coli
BL21_pARD33 harboring the single plasmid (Table 2). The E. coli
BL21_pARD33-BHB2 harboring two plasmids showed similar
results in term of crude glycerol utilization and total crude
glycerol consumption rate with E. coli BL21_pARD33. After 48 h,
the crude glycerol consumption was reported to be 96.1% and
98.2% (w/w) in E. coli BL21_pARD33 and E. coli BL21_pARD33-
BHB2, respectively. Nevertheless, higher biomass was observed in
E. coli BL21_pARD33-BHB2 due to the elimination of the toxic
effect of acetate and conversion of acetyl-CoA to P3HB which was
redox-balancing pathways. Presence of vgb gene was another
reason for the production of higher biomass which increases the
oxygen availability in the batch culture (30,31).
The recombinant strains harboring the plasmid pBHB2, i.e.,
E. coli BL21_pBHB2 and E. coli BL21_pARD33-BHB2 were distin-
guished in terms of biomass and crude glycerol utilization. Higher
biomass and crude glycerol utilization were observed in E. coli
BL21_pARD33-BHB2 whereas only 59.73% of crude glycerol was
utilized by E. coli BL21_pBHB2. Higher crude glycerol utilization
(98.2 %) was the reason for the higher biomass production in E. coli
BL21_pARD33-BHB2. This result demonstrated that Alrd and AldH
had beneciary action in terms of crude glycerol consumption
(Table 2).
Acetic acid as a competitive by-product Under aerobic
condition, instead of oxygen availability, the production of acetate
is dependent on the rate of crude glycerol utilization and biomass
production (21). The clear explanation for the maximum
consumption of crude glycerol in E. coli BL21_pARD33 and other
recombinant strains were enlightened through HPLC analysis.
Acetate is toxic to E. coli in higher concentration, thus its
accumulation retarded the cell growth and hampered the
consumption of crude glycerol. Continuous accumulation of
acetate was observed in E. coli BL21(lDE3) for 24 h with
stationary value thereafter (Fig. 3C) this retarded the further
growth. In E. coli BL21_pARD33, high accumulation of acetate was
observed at 9th hour of culture presumably due to overfeeding of
abundant crude glycerol facilitated with Alrd and AldH.
Declination in the acetate concentration was noted between 9
and 24 h, which resulted in acceleration of bioethanol production
through Alrd and AldH (Fig. 3B). The lowest value of acetate was
observed at 24 h. By nullifying the toxicity of acetic acid, E. coli
BL21_pARD33 was able to further utilize crude glycerol. After
24 h, a slight increment in the accumulation of acetate was
noticed with respect to the limitations in metabolic pathways and
also oxygen availability. This resulted in no further increment in
biomass without occluding bioethanol production.
FIG. 3. Outcomes of crude glycerol utilization by E. coli strains in terms of dry cell
weight, ethanol production and acetate accumulation: (A) cell growth prole in term
of dry cell weight, (B) bioethanol production at different time periods, and (C) pro-
duction of acetic acid during cell culture.
VOL. 117, 2014 SIMULTANEOUS REFINERY OF CHEMICALS FROM GLYCEROL 347
Signicantly, lower accumulation of acetate was observed in
E. coli BL21_pBHB2 as an overow mechanism which exceeded the
transfer rate of acetyl-CoA to P3HB. In E. coli BL21_pARD33-BHB2,
negligible amount of acetate was noted due to well balanced
metabolic pathways for the conversion of acetyl-CoA to P3HB and
generation of bioethanol from over accumulation of acetate. This
resulted in higher conversion of crude glycerol in term of biomass,
bioethanol production and P3HB accumulation.
Bioethanol as an extracellular product The bioconversion
of crude glycerol leads to the diversity of metabolic products due to
the complexity of metabolismin micro-organisms (10). In reference
to Fig. 1, the acetyl-CoA produced in glycolysis pathway, proceed
through two metabolic routes. Firstly, acetyl-CoA acted as
substrate in tricarboxylic acid cycle (TCA cycle) for the generation
of energy to support cell functions and growth. The second
pathways led to the conversion of acetyl-CoA to acetate, catalyzed
by acetyl-CoA synthetase. The reversed pathway was catalyzed by
acetyl-CoA hydrolase which converted acetate to acetyl-CoA (39).
The production of bioethanol from acetate or acetyl-CoA was the
redox-balancing pathways that regenerated necessary equivalents
of NAD

and NADP

. In E. coli BL21(lDE3) the production of

bioethanol from acetyl-CoA is governed by two enzymes:
acetaldehyde dehydrogenase and alcohol dehydrogenase (40).
Under aerobic condition, the presence of oxygen inhibits the
pathways for the conversion of bioethanol. As a results, 0.10 g L
1
bioethanol was only produced in E. coli BL21(lDE3) culture
(Fig. 3B). However, the presence of Alrd and AldH provided
secondary metabolic pathway for the utilization of excess acetate
to bioethanol. Here, AldH catalyzed the reduction of acetate to
acetaldehyde (37), which was further catalyzed to bioethanol by
Alrd.
The higher metabolismof crude glycerol by E. coli BL21_pARD33
was due to further utilization of intermediate acetate to bioethanol
with the help of Alrd and AldH. Fig. 3C shows the amount of acetic
acid accumulated in E. coli BL21_pARD33 culture (0.57 g L
1
) which
was nearly 4-fold lower than that of E. coli BL21(lDE3) (2.14 g L
1
).
Moreover, the high production of bioethanol was noted in the re-
combinant strains harboring ethanol producing enzymes Alrd and
AldH, i.e., E. coli BL21_pARD33 and E. coli BL21_pARD33-BHB2 with
the maximum concentration of 0.65 g L
1
and 0.8 g L
1
, respec-
tively, at 48 h (Fig. 3B).
The total production of bioethanol was nearly 8-fold higher in
E. coli BL21_pARD33-BHB2 than that of E. coli BL21(lDE3) which
has been summarized in Table 2. The yield of bioethanol was
comparatively low in E. coli BL21_pBAD33 and E. coli BL21(lDE3)
(0.02 g g
1
DCW). Notably, no bioethanol production was observed
in E. coli BL21_pBHB2 due to the lack of Alrd and AldH. Never-
theless, acetyl-CoA was presumably employed for P3HB produc-
tion that balanced the redox pathway in E. coli BL21_pBHB2. The
production of bioethanol was not associated with the growth in
biomass, as production of bioethanol was even observed in the
stationary phase where biomass concentration remained constant
(Fig. 3B).
Intracellular production of P3HB The P3HB production was
accompanied through acetyl-CoA with a sequential enzymatic
pathway of phaCAB
RE
operon. In order to eliminate the over accu-
mulation of acetate from crude glycerol digestion in E. coli
BL21_pARD33, the second metabolic pathway was introduced
through plasmid pBHB2 in E. coli BL21_pARD33-BHB2 as
mentioned in materials and methods section. The phaCAB operon
eliminated the production of acetate by the conversion of acetyl-
CoA to P3HB. The total P3HB content accumulated in E. coli
BL21_pBHB2 and E. coli BL21_pARD33-BHB2 were analyzed to be
15.6% and 30.2% (w/w), respectively (Table 2). The P3HB yield was
enhanced by 2-fold from 0.15 to 0.30 g g
1
DCW with total P3HB
concentration from 0.87 to 1.95 in E. coli BL21_pARD33-pBHB2
than E. coli BL21_pBHB2.
Anaerobic utilization of crude glycerol and products
formed In accordance with the results shown in Table 2, very
slow rate of glycerol utilization and biomass production were
demonstrated under anaerobic condition (Table 3). The anaerobic
cultivation conditions were similar to aerobic condition, despite
of purging nitrogen gas for the purpose of anaerobic
circumstance. Under this circumstance, the growths of strains
without Alrd and AldH were lowered due to imbalance of the
redox potential. In E. coli BL21(lDE3), the bioethanol production
was noted to be 5-fold higher than that of aerobic condition. This
result was in accordance with the several references
(9,11,14,15,17) showing that anaerobic condition was more
appropriate for bioethanol production from glycerol. Under
anaerobic condition, no signicant improvement in the yield of
bioethanol production was noted in the strains harboring Alrd
and AldH. Anaerobically, higher biomass was observed in E. coli
BL21_pARD33-BHB2 than other strains under same condition.
Moreover, 2-fold higher bioethanol production was noted in
TABLE 2. Summarization of results observed during aerobic utilization of crude glycerol by E. coli strains.
Strain DCW
(g L
1
) X
Max
m
EtOH concn.
(g L
1
) p1
PHB cont.
(w/w)
PHB concn.
(g L
1
) p2
Consumption of crude
glycerol(s) cont. (w/w)
Y
x/s
(g g
1
)
Y
p1/x
(g g
1
)
Y
p2/x
(g g
1
)
d
s/t
(g L
1
h
1
)
E. coli (WT) 4.16 0.10 55.13 0.27 0.02 0.32
E. coli (pBAD33) 3.71 0.09 52.91 0.25 0.02 0.31
E. coli (pARD33) 4.92 0.65 96.1 0.18 0.13 0.58
E. coli (pBHB2) 5.58 0.00 15.6 0.87 59.73 0.31 0.00 0.15 0.37
E. coli (pARD33-BHB2) 6.47 0.80 30.2 1.95 98.2 0.23 0.12 0.30 0.60
En dash (e) denotes no data found; whereas X, p1, p2 and s denote dry cell weight (DCW), ethanol production, P3HB production and substrate, respectively. Y denotes yields
whereas d
s/t
is total substrate consumption rate.
TABLE 3. Summarization of results obtained under anaerobic condition in E. coli strains.
Strain DCW
(g L
1
) X
Max
m
EtOH concn.
(g L
1
) p1
PHB cont.
(w/w)
PHB concn.
(g L
1
) P2
Consumption of crude
glycerol(s) cont. (w/w)
Y
x/s
(g g
1
)
Y
p1/x
(g g
1
)
Y
p2/x
(g g
1
)
d
s/t
(g L
1
h
1
)
E. coli (WT) 0.37 0.48 e e 4.19 0.28 1.30 e 0.03
E. coli (pARD33) 0.41 0.53 e e 5.71 0.22 1.29 e 0.04
E. coli (pBHB2 0.26 0.17 e e 2.65 0.31 0.65 e 0.02
E. coli (pARD33-
BHB2)
0.80 0.42 31.5 0.17 7.42 0.35 0.53 0.27 0.05
En dash (e) denotes no data found; whereas X, p1, p2 and s denote dry cell weight (DCW), ethanol production, P3HB production and substrate, respectively. Y denotes yields
whereas d
s/t
is total substrate consumption rate.
348 SHAH ET AL. J. BIOSCI. BIOENG.,
E. coli BL21_pARD33-BHB2 under aerobic condition than anaerobic.
In spite of no bioethanol production in E. coli BL21_pBHB2 under
aerobic condition, slightly bioethanol production was noted
anaerobically. This was for sure to balance the redox-equivalent
Pathways. However no P3HB was accounted because of
signicantly low biomass.
The production of bioethanol under anaerobic culture was lower
than the aerobic culture in the recombinant strains harboring Alrd
and AldH. This showed the beneciary role of Alrd and AldH for the
production of bioethanol aerobically. Improvement of crude glyc-
erol utilization under aerobic condition yielded in higher produc-
tion of bioethanol and P3HB. Total crude glycerol consumption rate
(d
s/t
) was noted to be nearly 10-fold lower in anaerobic than aerobic
condition (Table 2).
Use of crude glycerol over pure glycerol Crude glycerol is a
cheap and abundant material containing unreacted methanol, soap,
salts, water and solid organic materials acquire during biodiesel
production (40). Presence of these impurities in crude glycerol has
been expected to inuence the negative effect in bioconversion
process. Hence, for achieving a higher yield of bioethanol, many
researchers focused on using pure glycerol under anaerobic or
micro-aerobic conditions due to high glycerol content and
negligible amount of impurities. Several grades of rened glycerol
have been commercialized that differ in glycerol content.
Purication of crude glycerol is an expensive process due to no
market value of crude glycerol. Therefore, adopting the process of
purication for crude glycerol or using commercially puried
glycerol for the production of value added products is an
infeasible route in bio-industry (13e18). Table 4 summarizes the
production of bioethanol by different strains of E. coli on pure and
crude glycerol, under different cultivation condition. Under
aerobic condition, the complete utilization of crude glycerol can
be achieved with higher biomass and low bioethanol yield
(12,14). The production of only bioethanol was not a feasible
proposition due to high operational cost and no effective use of
biomass. Hence, the strategy discussed here resulted in the
effectual use of biomass for the accumulation of P3HB.
Awareness to environmental and energy issues have led to the
development of biomass-based bioconversion processes. Produc-
tion of fuels and chemicals from crude glycerol is cost-effective
process which has been evaluated in recent years. E. coli can utilize
crude glycerol for the production of bioethanol under aerobic as
well as anaerobic condition (Table 2) (13,14). Bioethanol is only the
secondary product of the crude glycerol fermentation under any
condition. As a result, the yield of bioethanol production is rela-
tively low than other metabolites. Hence, several modications
have been made with the help of biotechnology and molecular
biology for the improvement of bioethanol production from crude
glycerol. In this work, the natural pathway for crude glycerol uti-
lization was enhanced by the addition of Alrd and AldH, which
further provided the alternative and effective route for bioethanol
production in E. coli BL21_pARD33 (Fig. 1). Under aerobic condition,
acetate was the major product derived through acetyl-CoA. The
phaCAB operon used acetyl-CoA as a substrate for the production of
P3HB which reduced the formation of acetate (28). Higher yield of
bioethanol was observed under aerobic condition in the strains
harboring Alrd and AldH. This result was contradictory with the
general belief that high bioethanol can be only produced under
anaerobic condition. This was probably due to the role of Alrd and
AldH in aerobic condition which provided with addition pathway
for the production of bioethanol and also balanced the redox-
equivalents. The production of bioethanol and P3HB at the same
time facilitated with high degree of value-added products from
crude glycerol under both aerobic and anaerobic condition. How-
ever, higher yields of bioethanol and P3HB were noted under aer-
obic condition along with complete utilization of crude glycerol in
E. coli BL21_pARD33-BHB2 at 48 h of post-culture.
Considering the huge crude glycerol surplus, our nding
established a new model for the conversion of crude glycerol under
aerobic condition by using an E. coli-based platform. The total
production of bioethanol and P3HB content was noted to be
0.8 g L
1
and 30.2% (w/w), respectively, in E. coli BL21_pARD33-
BHB2.
ACKNOWLEDGMENTS
This research was supported by National Science Council
(Taiwan) with funding number 99-2622-E-155-002-CC2 and 101-
2221-E-155-042. We thankfully acknowledge help from Prof.
Shaw-Shan Wang (Distinguished Professor, Department of Chemi-
cal Engineering and Materials Science, Yuan Ze University) for
improving English grammar, verb usage, sentence structure and
general readability of the manuscript revision.
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