Appl Microbiol Biotechnol (2011) 89:1255–1264
DOI 10.1007/s00253-010-3076-3
BIOENERGY AND BIOFUELS
Ethanol production from biodiesel-derived crude glycerol
by newly isolated Kluyvera cryocrescens
Won Jae Choi & Maria Regina Hartono &
Weng Heng Chan & Suan Siong Yeo
Received: 19 November 2010 / Revised: 14 December 2010 / Accepted: 14 December 2010 / Published online: 7 January 2011
# Springer-Verlag 2011
Abstract The rapidly expanding market for biodiesel has
increased the supply and reduced the cost of glycerol,
making it an attractive sustainable feed stock for the fuel
and chemical industry. Glycerol-based biorefinery is the
microbial fermentation of crude glycerol to produce fuels
and chemicals. A major challenge is to obtain microbes
tolerant to inhibitors such as salts and organic solvents
present in crude glycerol. Microbial screening was attemp-
ted to isolate novel strain capable of growing on crude
glycerol as a sole carbon source. The newly isolated
bacteria, identified as nonpathogenic Kluyvera cryocrescens
S26 could convert biodiesel-derived crude glycerol to
ethanol with high yield and productivity. The supplemen-
tation of nutrients such as yeast extract resulted in
distinguished enhancement in cell growth as well as ethanol
productivity under anaerobic condition. When glycerol
fermentation is performed under microaerobic condition,
there is also a remarkable improvement in cell growth,
ethanol productivity and yield, compared with those under
strict anaerobic condition. In batch fermentation under
microaerobic condition, K. cryocrescens S26 produced
27 g/l of ethanol from crude glycerol with high molar yield
of 80% and productivity of 0.61 g/l/h.
Keywords Crude glycerol . Ethanol . Fermentation .
Kluyvera cryocrescens
W. J. Choi (*) : M. R. Hartono : W. H. Chan : S. S. Yeo
Institute of Chemical and Engineering Sciences,
Agency for Science, Technology and Research (A*STAR),
1 Pesek Road,
Jurong Island, Singapore
e-mail: choi_won_jae@ices.a-star.edu.sg
Introduction
The rapidly expanding market for biodiesel is remarkably
altering the cost and availability of glycerol. In general,
approximately 10 lb of crude glycerol are formed for every
100 lb of biodiesel produced (da Silva et al. 2009; Johnson
and Taconi 2007; Karinen and Krause 2006; Pagliaro et al.
2007). Bioethanol process also generates glycerol as a
byproduct up to 4–10% of the sugar feedstock (Nissen et al.
2000; Temudo et al. 2008) and 17% (w/w) of ethanol
produced (Brumm and Hebeda 1998; Munene et al. 2002).
Crude glycerol has thus been widely recognized as an
attractive sustainable resource for oil and chemical indus-
tries. There have been several attempts to explore anaerobic
microbial assimilation of glycerol using reconstructed
microbial systems via microbial screening or metabolic
pathway engineering. As a result, fuels such as ethanol,
hydrogen and biogas as well as chemicals including 1,3-
propanediol, organic acids and some other high-value
products were found to be produced by microbial fermen-
tation of glycerol (Choi 2008; da Silva et al. 2009). The
newly isolated bacteria, Zobellella denitrificans produced
poly(3-hydroxybutyrate) (Ibrahim and Steinbüchel 2010).
The yeast Yarrowia lipolytica successfully converts glycer-
ol via phosphorylation pathway into valuable products such
as single cell oil and citric acid (Papanikolaou et al. 2008;
Makri et al. 2010). The engineered Escherichia coli
exhibited efficient utilization of glycerol for the production
of D-lactic acid (Mazumdar et al. 2010) and succinic acid
(Blankschien et al. 2010). In most commercial applications,
the quality of the glycerol must be high with acceptable
purity that is completely different those out of biodiesel
plants. With respect to fuels and chemicals, it is important
to employ cheap and readily available raw material. The
use of crude glycerol eliminates the need to refine it into
1256
pure form and thus allows fermentation process to be
economically feasible. Currently, biodiesel is often made
from the triglycerides, or fat, found in vegetable oils,
generating crude glycerol with glycerol content from 60%
to 80% (w/w). This low-grade glycerol also contains water,
salts, other organic materials including residual methanol
and free fatty acids and each component varies widely in
contents depending on multiple feedstocks such as rape-
seed, canola, palm, and soybean. Special concern needs to
be taken on remaining methanol as well as sodium or
potassium salt among these impurities because they are
known to be inhibitory to cell growth for fermentation
(Petitdemange et al. 1995). There have been several
attempts to utilize biodiesel-derived crude glycerol as
fermentation feedstock for the production of 1,3-propane-
diol (González-Pajuelo et al. 2004; Mu et al. 2006;
Papanikolaou et al. 2004), succinic acid (Scholten et al.
2009), β-carotene (Mantzouridou et al. 2008), docosahex-
aenoic acid (Pyle et al. 2008), and human erythropoietin
(Çelik et al. 2008).
In the area of ethanol production from glycerol, yeast
Pachysolen tannophilus could previously convert glycerol
to ethanol at 4 g/l with a yield of 0.4 mol/mol glycerol
during aerobic growth (Maleszka et al. 1982). Ethanol and
formic acid were produced during anaerobic glycerol
fermentation by mixed cultures (Temudo et al. 2008). The
synthesis of ethanol and 1,2-propanediol from glycerol was
also reported by use of Paenibacillus macerans (Gupta et
al. 2009). This bacterial strain could produce ethanol and
1,2-propanediol as major metabolites at 3 and 0.8 g/l,
respectively, during anaerobic fermentation with 10 g/l of
pure glycerol.
Recently, there have been intensive efforts for the
efficient conversion of glycerol to ethanol via metabolic
pathway engineering of E. coli by minimizing the synthesis
of byproducts. E. coli has been reconstructed in its
metabolic processes and tested to convert glycerol into
ethanol through an anaerobic fermentation process
(Dharmadi et al. 2006; Murarka et al. 2008). The
engineered E. coli strain produced 21 g/l of ethanol from
60 g/l of pure glycerol with the volumetric productivity of
0.216 g/l/h under microaerobic condition (Durnin et al.
2009). Furthermore, E. coli strain was designed based on
elementary mode analysis for efficient conversion of
glycerol to ethanol with 90% of the theoretical ethanol yield
(Trinh and Srienc 2009). The E. coli mutant strain expressing
alcohol–acetaldehyde dehydrogenase could grow on glycerol
under oxygen-limited conditions and exhibited ethanol
production (16 g/l) with volumetric productivity of 0.34
g/l/h (Nikel et al. 2010). Saccharomyces cerevisiae was also
metabolically engineered to improve ethanol production
from glycerol. By simultaneous overexpression of glycerol
dehydrogenase, dihydroxyacetone kinase and glycerol up-
Appl Microbiol Biotechnol (2011) 89:1255–1264
take protein, the overall ethanol production was enhanced by
3.4-fold and reached at 2.4 g/l (Yu et al. 2010). There have
been several attempts to produce ethanol using crude
glycerol as feedstock. Enterobacter aerogenes HU-101,
new isolate from methanogenic sludge could anaerobically
convert crude glycerol obtained from biodiesel wastes to
ethanol with a yield of 0.85 mol/mol glycerol (Ito et al.
2005). Ethanol was produced along with hydrogen by E.
aerogenes NBRC 12010 from biodiesel waste glycerol in a
bioelectrochemical reactor with thionine under anaerobic
condition (Sakai and Yagishita 2007). The high level ethanol
(10 g/l) was also produced by engineered E. coli from 22
g/l of crude glycerol with productivity of 0.09 g/l/h under
anaerobic condition (Yazdani and Gonzalez 2008).
Glycerol-based biorefinery is the microbial fermentation
processes using inexpensive and readily available glycerol
as the raw material to produce fuels and chemicals. A major
challenge in fermentation of the low-grade crude glycerol is
to obtain microbial strains tolerant to undesirable inhibitory
components such as salts and organic solvents that present
in crude glycerol. In this study, novel microorganism was
isolated from soil samples. Based on its ability to utilize
crude glycerol as a sole carbon source, it was employed as a
biocatalyst for the production of fuel ethanol.
Materials and methods
Crude glycerol
Crude glycerol was obtained from palm oil-based biodiesel
plant in Malaysia operated by Vance Bioenergy. The
composition of these crude glycerols is summarized in
Table 1, according to company’s specification. Crude
glycerol was used as carbon source in culture medium
without purification.
Isolation of crude glycerol-utilizing microorganism
from soil
Soil samples were collected at various locations in
Singapore. The surface soil was removed and the layer
Table 1 The composition of crude glycerol derived from palm-oil
based biodiesel plant
Component Conc. % (w/v)
Glycerol 80–85
Nonglycerol organics 2
Methanol 0.5
Salts 5–6
Water 10
Appl Microbiol Biotechnol (2011) 89:1255–1264
underneath is collected. Water was added to the sample and
vortexed. The mixture then allowed to be settled for about
10 min and liquid phase was spread on agar plate
containing RG minimal medium supplemented with crude
glycerol (25 g/l) as a sole carbon source. The RG medium
contains (per liter of water) yeast extract 2.0 g, K2HPO4
1.55 g, NaH2PO4 0.85 g, (NH4)2SO4 2.0 g, and 5 ml of
trace element solution (composed of EDTA 1.0 g/l,
CaCl2∙2H2O 0.1 g/l, FeSO4⋅7H2O 0.5 g/l, ZnSO4⋅7H2O
0.2 g/l, CuSO4∙5H2O 0.02 g/l, CoCl2⋅6H2O 0.04 g/l,
MnCl 2 ⋅4H 2 O 0.1 g/l, Na 2 MoO 4 ⋅2H 2 O 0.02 g/l,
MgCl2⋅6H2O 10 g/l). Agar plates were incubated at room
temperature for 3 days under anaerobic condition provided
by anaerobic chamber (Anaerogen, Oxoid, UK). Isolated
strains through agar plate culture were subsequently
transferred to liquid culture containing the same RG
medium with crude glycerol (25 g/l) for measurement of
growth and metabolite analysis. Liquid culture was con-
ducted at 30°C for 3 days in a screw-capped bottle (100 ml
size) with gas inlet and outlet port for continuous supply of
either CO2 or N2 in order to provide anaerobic condition.
Screening of ethanol producing microorganism
The liquid culture broth of crude glycerol-utilizing strains
was analyzed by HPLC to quantify its metabolites
including ethanol and other organic acids. The culture was
centrifuged for 10 min at 10,000×g, 4°C to remove cells
and the supernatant was analyzed by HPLC. HPLC was
performed on a Shimadzu Prominence system equipped
with UV and refractive index detector. Aqueous sulfuric
acid solution (5 mM) as a mobile phase was pumped at the
rate of 0.6 ml/min. Samples were injected (1 μl) and
metabolites were analyzed by using a Aminex® HPX-87 H
column (7.8 mm×30 cm, Bio-Rad, US) at oven temperature
of 42°C. The commercially available glycerol, ethanol, and
other organic acids were employed as a standard for
quantitative analysis. The mixture of ten standards includ-
ing L-lactic acid, succinic acid, acetic acid, formic acid,
propionic acid, glycerol, 1,3-propanediol, 1,2-propanediol,
2,3-butanediol, and ethanol was injected under this condi-
tion and each metabolite was successfully analyzed without
peak overlapping.
Measurement of substrate and product inhibition
Growth inhibition by substrate glycerol and product ethanol
was measured in anaerobic cultures performed in a screw-
capped bottle (100 ml size) containing RG medium (20 ml).
For substrate inhibition test, each bottle was supplemented
with different amounts of crude glycerol. For product
inhibition, crude glycerol (25 g/l) was added into each
bottle, followed by addition of different amounts of ethanol.
1257
Culture was initiated by inoculating 1 ml cells obtained in
early exponential growth phase and incubated at 30°C
under anaerobic condition. Growth was monitored by
optical density (OD) measurement at 600 nm. The
maximum specific growth rate (μ, in h−1) on different
concentrations of glycerol or ethanol was determined from
the slope of the least square regression lines of the
logarithm of OD versus time.
Fermentation of crude glycerol
The seed culture (50 ml) with RG medium containing crude
glycerol (25 g/l) was prepared by incubation at 30°C for
3 days in a screw-capped bottle (100 ml size) with gas inlet
and outlet port for continuous supply of N2 in order to
provide anaerobic condition. The batch culture was carried
out in a 2.5 l stirring bioreactor (BIOSTAT A Plus,
Sartorious, Germany) with a working volume of 1 l
consisting of RG medium supplemented with crude
glycerol as a sole carbon source. Temperature and agitation
speed were maintained at 30°C and 500 rpm, respectively.
The pH was controlled at 7.0 by adding either 2 N of HCl
or NaOH. Samples were taken periodically from fermentor,
filtrated and analyzed by HPLC as mentioned above. For
anaerobic fermentation, nitrogen gas was first supplied to
fermentor in order to remove oxygen present in culture
medium. Gas line was then closed and the fermentation was
initiated by inoculating seed culture. The exit gas was
collected in tightly sealed plastic bag, followed by GC
analysis. Hydrogen and carbon dioxide were analyzed with
a Shimadzu GC-2014 gas chromatograph equipped with a
thermal conductivity detector. The temperatures of column
and detector were 80°C and 110°C, respectively. Nitrogen
was used as a carrier gas. For microaerobic culture, gas
mixture composed of air and nitrogen was used to obtain a
specified volumetric transfer coefficient (kLa in min−1) for
each experiment. Air was mixed in advance with nitrogen
at different volume ratios (0%, 25%, 50%, 75%, and 100%)
in a gas mixer and resulting gas mixture was sparged into
fermentor at the fixed rate of 0.25 l/min. The agitation rate
was fixed to 500 rpm. The kLa was determined by using the
nonsteady-state method (Shuler and Kargi 2002). The
fermentor containing culture medium was initially sparged
with nitrogen to completely remove oxygen. Then, the
mixture of air and nitrogen with various volume ratios was
introduced into the fermentor through gas mixer. The
percentage of dissolved oxygen (DO) can be monitored
by using a DO probe. The increase in the DO was recorded
and the slope of a plot of ln (100%—DO) versus time (in
min) was used to calculate kLa. In order to investigate the
effect of oxygen on ethanol production, kLa was varied by
using different ratio between air and nitrogen in inlet gas.
Each kLa represents the oxygen supply at different levels.
1258 Appl Microbiol Biotechnol (2011) 89:1255–1264

Microbial identification analysis was carried out by the neighbor-joining method

Genomic DNA extraction, PCR and sequencing of 16 S
rRNA gene were conducted by the general procedures
described elsewhere. The 16 S rRNA gene was amplified
from genomic DNA by PCR using the bacterial primers.
The sequences of the primers used for amplification were
5′-AGAGTTTGATCATGGCTCAG-3′ (forward) and
5′ AAGGAGGTGATCCAGCCGCA-3′ (reverse),
corresponding to positions 8–27 and 1,544–1,525, respec-
tively, of the E. coli 16 S rRNA sequence. The PCR
product was purified using a Wizard PCR DNA purification
system (Promega, US) and sequenced using an ABI PRISM
310 genetic analyzer by first base Pte Ltd, Singapore.
Results
Screening of microorganisms producing ethanol from crude
glycerol
Among about 600 soil samples tested, 57 microbial strains
were isolated based on its ability to grow on crude glycerol
as a sole carbon source under anaerobic condition. Through
metabolite analysis for these candidates by HPLC, nine
strains were found to produce ethanol as major metabolite
under anaerobic condition (Table 2). Obviously, there is in
most cases no formation of 1,3-propanediol that is a
common product during anaerobic fermentation of glycerol.
Strain S26 was finally selected for further investigation
based on its highest conversion yield for ethanol formation.
The 16 S rRNA gene sequence (1,030 bases, GenBank
accession number, HQ285932) was compared with the
sequence data in GenBank database by using the BLAST
algorithm. The BLAST analysis on sequence similarity
indicated that the closest relatives of the strain S26 were
Kluyvera cryocrescens 169 (99.4%), K. cryocrescens
WAB1904 (99.4%), E. aerogenes BAB-223 (99.3%), E.
aerogenes T2 (99.3%), Kluyvera ascorbata 4105 (99.3%),
and K. cryocrescens 4701 (99.3%). The phylogenetic
using the PHYLIP program package, version 3.68 (Depart-
ment of Genome Sciences and Department of Biology,
University of Washington) as shown in Fig. 1. On the basis
of sequence similarity as well as phylogenetic analysis, it
was concluded that newly isolated bacterial strain S26
belongs to K. cryocrescens. The K. cryocrescens S26 was
deposited in American Type Culture Collection with
accession number of PTA-10600.
Anaerobic fermentation of crude glycerol by K.
cryocrescens S26
A time profile of the anaerobic fermentation of crude
glycerol by K. cryocrescens S26 was demonstrated in Fig. 2
with cell growth and product formation. The fermentation
was carried out in a RG minimal medium supplemented
with 25 g/l of crude glycerol (80%, w/v). The glycerol
content in the medium is equivalent to 20 g/l. The
fermentation was monitored by quantitative analysis of
samples taken at various times. Glycerol was almost
completely consumed within 136 h of active growth,
resulting in a maximum biomass concentration of 1.0 g
dry cell weight/l. Ethanol was accumulated as a major
product at the rate of 0.052 g/l/h with molar yield of 84.8%
per consumed glycerol. Formic acid was primarily gener-
ated as byproduct and negligible amount of lactic and
succinic acid was observed. There were no other organic
acids such as acetic acid and no 1,2- or 1,3-propanediol
detected in the culture broth. In a gas phase, H2 and CO2
were generated gradually, resulting in final cumulative
amount of 95 and 90 mmol at 136 h, respectively. Carbon
recovery at the end of bioreactor operation was calculated
as 0.94 based on initial amount of glycerol, quantitative
analysis of each product, biomass and CO2 evolution. The
elementary composition of biomass was taken as C4H7O2N,
correspond to a molecular weight of 101 g/mol (Herbert et
al. 1971). The high carbon recovery in products along with
the synthesis of reduced compound, ethanol as a major
product reflects fermentative nature of the culture.

Table 2 Newly isolated strains

producing ethanol from crude Strain Biomass (g/l)a Ethanol (mM) 13PDO (mM) LA (mM) AA (mM) Conv (%)b
glycerol
S26 1.2 88 0 2.1 0 94
S29 1.1 73 0 12 5 85
S39 1.4 85 15 10 7 72
S257 1.6 80 0 8 10 49
Metabolic products, 1,3-pro-
panediol (13PDO ), L-lactic acid S280 1.2 87 4.3 15 9 53
(LA), and acetic acid (AA) were S344 1.1 65 0 18 5 70
analyzed by HPLC S362 1.3 82 0 9 13 79
a
Dry cell weight S233 1.1 75 0 22 10 65
b
Ethanol produced/glycerol S76 1.2 63 0 25 18 60
consumed (mole/mole)
Appl Microbiol Biotechnol (2011) 89:1255–1264 1259

Fig. 1 Phylogenetic tree of

newly isolated S26 and related
organisms based on 16 S rRNA
sequences (GenBank accession
number, HQ285932)

37

42

100
65
62

15

41
25

55

44

10

Substrate and product inhibition

250 1.2
The effect of crude glycerol and ethanol on the growth of
K. cryocrescens S26 is presented in Fig. 3. The specific
1
growth rate was gradually decreased when glycerol was
200
Glycerol and products (mM)

added at level up to 100 g/l. The growth inhibition effect

was more evident when the medium contained more than
Dry cell weight (g/l)

0.8
150 100 g/l of glycerol, leading to significantly suppressed cell
0.6 growth. Beside inhibitory effect derived from glycerol
100
itself, at such a high level of nonrefined crude glycerol,
0.4 fermentation broth contains other impurities like sodium or
potassium salts and heavy metals in concentrations that
50 might severely interfere with cell growth. The product,
0.2
ethanol is generally known to toxic for microbial growth.
0 0 The growth of K. cryocrescens S26 was also hindered in
0 20 40 60 80 100 120 140 the presence of ethanol, resulting in approximately 80%
Time (h)
reduction in specific growth rate at ethanol level of 50 g/l.
Fig. 2 The time profile for anaerobic fermentation of crude glycerol
by K. cryocrescens S26. Symbols represents glycerol (empty square), Effect of nutrients on cell growth and ethanol synthesis
ethanol (filled square), biomass (filled triangle), formic acid (filled
diamond), L-lactic acid (empty diamond), succinic acid (empty
triangle), H2 (empty circle), and CO2 (multiplication symbol). H2 Some nutritional components present in complex nutrient
and CO2 were expressed as accumulative amounts in mmol sources can be essential for cell growth and metabolite
1260 Appl Microbiol Biotechnol (2011) 89:1255–1264

1.4
A extract, polypeptone and typtone resulted in improvement
Specific growth rate (1/h)

1.2
in cell growth and ethanol production, compared with
1
culture without nutrient addition. Among nutrients tested,
0.8
yeast extract exhibited more than twofold enhancement in
0.6
ethanol productivity as well as biomass yield. Since yeast
0.4
extract is known to contain nitrogen and carbohydrate, it
0.2
may be utilized as carbon source to synthesized ethanol
0
25 50 75 100 125 150
or biomass. Experiment in the same medium with yeast
Crude glycerol (g/l)
extract lacking glycerol resulted in biomass concentration
of 0.89 g dry cell weight/l (74% of control culture).
1.4 However, it is noteworthy that there was negligible
B
Specific growth rate (1/h)

1.2
1
0.8
0.6
0.4
0.2
0
0 10 20 30
Fig. 3 Growth inhibition of K. cryocrescens S26 by substrate (crude
glycerol, a) and product (ethanol, b). For substrate inhibition, cell
growth was measured under anaerobic condition with RG medium
supplemented with different amounts of initial crude glycerol (25–
150 g/l). For ethanol inhibition test, cells were cultured under
anaerobic condition using RG medium containing 25 g/l crude
glycerol with ethanol initially added at different levels (0–100 g/l)
production. The effect of several nutrients on cell growth
and ethanol production was investigated (Fig. 4). K.
cryocrescens S26 was cultivated under anaerobic condi-
tion at 30°C for 1 day in liquid RG medium (total volume
of 50 ml) containing crude glycerol (25 g/l) as a sole
carbon source, supplemented with various nutrients. Yeast
12
Dry cell weight (g/l), EtOH (g/l)
production of ethanol (0.22 g/l), acetic acid (0.57 g/l)
and succinic acid (0.56 g/l). Therefore, it is concluded
that ethanol was exclusively synthesized from glycerol
rather than from yeast extract.
Effect of dissolved oxygen on ethanol fermentation by K.
cryocrescens S26
40 50 60 70 80 90 100
Ethanol (g/l) In general, glycerol fermentation has been conducted under
anaerobic condition (in the absence of electron acceptors).
The anaerobic fermentation of glycerol could be an
excellent platform but as shown in Fig. 4, it requires
expensive nutrients such as yeast extract and tryptone for
biomass synthesis. Glycerol fermentation in the presence of
oxygen is considered as a means of eliminating the need for
rich nutrients. The use of low amounts of oxygen-enabled
redox balance while preserving the ability to synthesize
reduced products. Experiments were performed in on-line
controlled fermentation system. K. cryocrescens S26 was
cultivated in the liquid culture medium (total volume, 1 l)
with 50 g/l of crude glycerol. To optimize ethanol
production, the volumetric transfer coefficient, kLa was
varied by using different ratio between air and nitrogen in
inlet gas while other conditions such as agitation speed
(500 rpm), gas flow rate (0.25 l/min) supplied to bioreactor,
90 temperature (30°C), and pH 7.0 were kept constant (Table 3).

80
10 This enabled precise control of the oxygen transfer rate
70
in a bioreactor. As the kLa value was increased up to
EtOH Yield (%)

critical level, i.e., 0.1 min−1, there were enhanced perform-

8 60
50
6 ances in ethanol production rate, yield, and cell growth as
40
4 30
shown in Table 3. When glycerol fermentation is performed
20
in the presence of limited oxygen (i.e., microaerobic
2
10
condition, kLa value between 0 and 0.1 min−1), there is a
0 0 remarkable enhancement in cell growth, ethanol produc-
Cont YE PP TR CSL tivity and yield, compared with those under strict
Fig. 4 Effect of nutrients supplementation on the cell growth (white
anaerobic condition. In case of higher oxygen concentra-
bar) and ethanol production (black bar). Each culture was performed tion (kLa value between 0.1 and 0.2 min−1), cell growth
under anaerobic condition with RG medium containing crude glycerol continued to increase but ethanol yield was decreased
(25 g/l) supplemented with yeast extract (YE), polypeptone (PP), substantially due to the formation of byproduct such as
tryptone (TR) or corn steep liquor (CSL) at 10 g/l each. Control (cont)
culture was performed using RG medium without nutrient supple-
lactic acid and acetic acid. Ethanol synthesis was also
mentation. Ethanol yield (hatched bar) was expressed as molar yield depressed less than that obtained in anaerobic condition,
of ethanol per total glycerol added resulting in lower productivity.
Appl Microbiol Biotechnol (2011) 89:1255–1264 1261

Table 3 Fermentation of crude glycerol (50 g/l) by K. cryocrescens S26 in the presence of oxygen

kLa (min−1)a Biomass (g/l)b Ethanol (mM) SA (mM) LA (mM) FA (mM) AA (mM) Y (%)c PD (g/l/h)d

0 1.33 272 6.64 0.8 14.8 0.5 58 0.066

0.05 2.98 310 6.85 9.5 15 1.1 66 0.75
0.10 3.46 398 7.2 16 16 4.2 84 0.92
0.15 4.42 225 8.9 48 14 9.5 48 0.58
0.20 4.55 217 10.5 50.7 15 11.3 46 0.55

Metabolic products, succinic acid (SA), L-lactic acid (LA), formic acid (FA), and acetic acid (AA) were analyzed by HPLC. Hydrogen and carbon
dioxide were not determined
a
The volumetric transfer coefficient (kLa) varied by using air and nitrogen mixture as inlet gas. Air was mixed in advance with nitrogen at different volume
ratios (0%, 25%, 50%, 75%, and 100%) in a gas mixer and resulting gas mixture was sparged into fermentor at the fixed rate of 0.25 l/min. For each
experiment, the fermentor operation condition was kept constant at 500 rpm, 30 °C, and pH 7.0
b
Dry cell weight
c
Molar yield of ethanol per total glycerol added initially
d
Ethanol production rate

Production of ethanol from crude glycerol by K. biodiesel plant with distinguished high production rate of
cryocrescens S26 under microaerobic condition 0.61 g/l/h (Fig. 5).

Glycerol fermentation was conducted to optimize initial

glycerol level. K. cryocrescens S26 was cultivated in on-
line controlled fermentation system with the liquid RG
medium (total volume 1 l) containing different amounts of
crude glycerol. In order to provide microaerobic condition,
gas was supplied to bioreactor at the rate of 0.25 l/min,
including air and nitrogen with 50% each (i.e., kLa,
0.1 min−1). Fermentation was carried out at 30°C until
ethanol concentration reached maximum level. Ethanol
production was increased as the amount of glycerol
increased up to 90 g/l as described in Table 4. There was
no significant byproduct formation in all experiments. As
described in Fig. 2a, cells were severely inhibited by
glycerol at higher level of glycerol, resulting in reduced cell
growth as well as depression in ethanol synthesis. Produc-
tion of ethanol was performed under microaerobic condi-
tion with the liquid RG medium containing crude glycerol
of 90 g/l. The newly isolated, K. cryocrescens produced
27 g/l of ethanol from crude glycerol directly obtained from
Discussion
The glycerol-based biorefinery so far has not been
practically applicable in chemical industry due to the lack
of efficient biocatalysts. There have been few reports on
microbial conversion of glycerol to ethanol by use of wild
type strains. P. tannophilus, P. macerans, and E. aerogenes
have only been known to ferment glycerol to produce
ethanol as a major metabolite. Most works on ethanol
production were recently done by genetically modified E.
coli mainly using refined glycerol as raw material. The
engineered E. coli was reconstructed by addition of
fermentative pathways such as propanediol pathways that
served as electron acceptors. Glycerol has been utilized as
carbon source for many microorganisms, particularly in
anaerobes (Chen et al. 2003; Gonzalez et al. 2008). Under
anaerobic condition, it can be either oxidized to dihydroxy-
acetone or dehydrated to 3-hydroxypropionaldehyde. Dihy-

Table 4 Effect of initial crude

glycerol concentration on etha- Initial crude glycerol concentration (g/l)
nol production by K. cryocres-
cens S26 under microaerobic 30 60 90 120
condition
Biomass (g/l) 2.12 4.22 4.85 3.69
Microaerobic condition was
provided by setting the volu- Residual glycerol (g/l) 0 0 0 0
metric transfer coefficient (kLa) Ethanol (g/l) 9.1 18.3 27 15.2
to 0.1 min−1 Ethanol yield (%)a 84 84 80 33
a
Molar yield of ethanol per total Ethanol productivity (g/l/h) 0.89 0.92 0.61 0.27
glycerol added initially
1262
80
Glycerol and products (g/l)
70
60
50
40
30
20
10
0 0
0 10 20 30 40 50
Time (h)
Fig. 5 Production of ethanol from crude glycerol by K. cryocrescens
S26 under microaerobic condition (kLa, 0.1 min−1). Culture was
performed with the liquid RG medium containing crude glycerol of
90 g/l. Symbols represents glycerol (empty square), ethanol (filled
square), biomass (filled triangle), and succinic acid (empty triangle)
droxyacetone can be further metabolized into pyruvate,
which is one of the main metabolic intermediate found in
glycolysis pathway and subsequently converted to various
fermentation products such as lactic acid, acetic acid,
ethanol, and succinic acid. 3-Hydroxypropionaldehyde is
reduced to 1,3-propanediol, which is the most common
product during anaerobic fermentation of glycerol. The
production of 1,3-propanediol is a way to regenerate NAD,
which is used for biomass production and other products
from glycerol. Glycerol can also be assimilated via glycerol
3-phosphate to pyruvate under aerobic condition. Based on
the metabolite production observed in anaerobic culture
(Fig. 2), the newly isolated K. cryocrescens is likely to
follow proposed metabolic pathways as previously de-
scribed (Dharmadi et al. 2006; Durnin et al. 2009) in which
glycerol is mainly converted into biomass and reduced
products like hydrogen and ethanol. The generation of
hydrogen and carbon dioxide under anaerobic condition
reveals the fermentative metabolism of glycerol in K.
cryocrescens. The combined amount of formate (28 mmol)
and hydrogen (95 mmol) is close to total ethanol
(153 mmol) synthesis, indicating that pyruvate is mainly
dissimilated via pyruvate-formate lyase. This result corre-
lated with the fact that enzyme pyruvate dehydrogenase
activity is known to be negligible during the anaerobic
fermentation of glucose (de Graef et al. 1999). In other
species such as Clostridium butyricum, Klebsiella pneumo-
niae, and E. aerogenes, pyruvate can also be converted into
acetyl-CoA along with the regulated formation of reducing
equivalents and hydrogen via pyruvate dehydrogenase
(Zeng et al. 1993). In anaerobic condition, cells can grow
only if external electron acceptors are provided either in the
form of feeding substrate or metabolic pathways that can
consume reducing equivalent NADH. 1,2- or 1,3-Propane-
diol, the product of metabolic pathway that can serve as an
electron sink, was not detected in the fermentation broth of
K. cryocrescens. The excess of reducing equivalents
Appl Microbiol Biotechnol (2011) 89:1255–1264
5
generated during cell growth cannot be consumed by the
ethanol synthesis because its formation is redox-balanced
Dry cell weight (g/l)
4
process. Although there was no external electron acceptor
3
provided in culture medium for K. cryocrescens, it may be
2 derived from crude glycerol containing unidentified impu-
1
rities such as nonglycerol organics.
It was reported that glycerol fermentation in E. coli
required the supplementation of the medium with rich
nutrients in the form of tryptone or yeast extract (Dharmadi
et al. 2006; Durnin et al. 2009; Murarka et al. 2008; Nikel
et al. 2010). Under anaerobic condition, these nutrients were
found to be essential for cell growth and not served as electron
acceptors, enabling to maintain the fermentative nature of
glycerol metabolism for the synthesis of reduced products like
ethanol (Murarka et al. 2008). In K. cryocrescens, yeast
extract showed the highest improvement among nutrients
tested in ethanol productivity as well as biomass yield. Yeast
extract mainly contributed to cell growth but there was no
influence on the production of metabolites such as ethanol.
Apart from being a carbon and nitrogen source, yeast extract
provides a significant amount of vitamins. For instance, it
was found that D-pantothenate was mainly responsible for
enhanced ethanol synthesis from glycerol by E. coli because
it is an important precursor in the acetyl-CoA biosynthetic
pathway of E. coli (Nikel et al. 2010).
It has been believed that glycerol metabolism in E. coli
was limited to respiratory conditions. Recently, availability
of low level of oxygen enabled redox balance while
maintaining the ability to produce reduced products. The
engineered E. coli strain produced 21 g/l of ethanol from
60 g/l of pure glycerol with the volumetric productivity of
0.216 g/l/h under microaerobic condition (Durnin et al.
2009). K. cryocrescens also metabolized glycerol to
produce ethanol in the presence of limited oxygen. The
supply of limited amount of an electron acceptor such as
oxygen facilitated in glycerol metabolism, enabling redox
balance by consuming the excess reducing equivalents
generated in the course of biomass synthesis. Oxygen could
also eliminate the use of expensive nutritional components
in culture medium. Although the use of oxygen as an
electron acceptor enables redox balance and eliminates the
need for medium supplementation, high level oxygen
would lead to low product yields as most of the carbon is
integrated into cellular mass and converted to carbon
dioxide. The use of microaerobic conditions (i.e., limited
amount of oxygen), on the other hand, has the potential to
eliminate the requirement of rich nutrients while preserving
the capacity to synthesize large amounts of products.
Limitation of oxygen consumption would likely lead to
higher ethanol yield. The level of oxygen available plays a
key role in controlling the competition between the biomass
formation and ethanol production pathways. As demon-
strated in Table 3, K. cryocrescens was able to convert
Appl Microbiol Biotechnol (2011) 89:1255–1264
glycerol to ethanol with an ethanol yield as high as 84% in
a defined medium under oxygen-limiting growth conditions
in which kLa was set as 0.1 min−1. In contrast, it produced
mainly biomass and not ethanol when sufficient oxygen
was supplied at higher kLa. Oxygen supply at high level can
enhance the generation of ATP by reducing NADH, which
is then used for biomass synthesis. Therefore, more carbon
flux is driven to biomass production but less carbon flux for
ethanol synthesis. On the other hand, under microaerobic
condition, limited amount of oxygen managed to convert
NADH generated during cell growth into NAD while
maintaining carbon flux into ethanol synthesis. This ethanol
production pathway is dramatically enhanced in the strain
K. cryocrescens S26. In glycerol metabolism, biomass
production requires NAD for oxidation reactions, resulting
in NADH. Cells can only grow under redox-balanced
condition in which the formed NADH is regenerated to
NAD. The redox balanced condition can be obtained by
synthesizing reduced metabolites such as 1,3-propanediol.
Since E. coli and K. cryocrescens do not have 1,3-propane-
diol pathway, they need another form of NAD regeneration
such as oxygen utilization or even prevent the necessity of
NAD regeneration by not synthesizing biomass but using
complex compounds present in the medium.
There have been several attempts to produce ethanol
from glycerol using microbial fermentation. In most cases,
however, pure glycerol was utilized as substrate (Maleszka
et al. 1982; Dharmadi et al. 2006; Murarka et al. 2008;
Durnin et al. 2009; Gupta et al. 2009) due to the toxicity of
crude glycerol towards microbial strains, especially when
used at high level. There have been only few reports on use
of crude glycerol as fermentation feedstock for ethanol
production. However, the level of crude glycerol used was
not as high as its pure form. The newly isolated K.
cryocrescens produced 27 g/l of ethanol from biodiesel-
derived crude glycerol with much higher production rate of
0.61 g/l/h. It is the first report on the fact that novel
bacteria, K. cryocrescens was isolated from nature and it
was applied to microbial fermentation of crude glycerol in
order to produce ethanol with the highest productivity.
Although bioprocess development for ethanol production
has been almost saturated, there might be a niche market
for glycerol-oriented ethanol as crude glycerol price is
getting decreased and availability is continued to
increase. Considering worldwide surplus of crude glyc-
erol, ethanol fermentation using glycerol as substrate is
promising alternative process to supply fuel ethanol. In
biorefinery process for the production of fuels and
chemicals, the product price is affected mainly by raw
material costs. The introduction of new technologies for
the efficient use of such raw materials is currently being
promoted. The utilization of crude glycerol, for example,
provides great potential.
1263
Acknowledgments This work was supported by Science and
Engineering Research Council of Agency for Science, Technology
and Research (A*STAR), Singapore, grant number ICES/07-173A02.
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