Eng. Life Sci. 2012, 12, No. 1, 57–68
57
Maria Metsoviti1
Spiros Paramithiotis1
Research Article
Eleftherios H. Drosinos1
Maria Galiotou-
Screening of bacterial strains capable of
converting biodiesel-derived raw glycerol into
1,3-propanediol, 2,3-butanediol and ethanol
Panayotou1
George-John E. Nychas1
An-Ping Zeng2
Seraphim Papanikolaou1
1
Department of Food
Science and Technology,
Agricultural University of
Athens, Athens, Greece
2
Institute of Bioprocess and
Biosystems Engineering,
Hamburg University of
Technology (TUHH),
Hamburg, Germany
The ability of bacterial strains to assimilate glycerol derived from biodiesel
facilities to produce metabolic compounds of importance for the food, textile and
chemical industry, such as 1,3-propanediol (PD), 2,3-butanediol (BD) and
ethanol (EtOH), was assessed. The screening of 84 bacterial strains was performed
using glycerol as carbon source. After initial trials, 12 strains were identified
capable of consuming raw glycerol under anaerobic conditions, whereas 5 strains
consumed glycerol under aerobiosis. A plethora of metabolic compounds was
synthesized; in anaerobic batch-bioreactor cultures PD in quantities up to 11.3 g/L
was produced by Clostridium butyricum NRRL B-23495, while the respective value
was 10.1 g/L for a newly isolated Citrobacter freundii. Adaptation of Cl. butyricum
at higher initial glycerol concentration resulted in a PDmax concentration of
32 g/L. BD was produced by a new Enterobacter aerogenes isolate in shake-flask
experiments, under fully aerobic conditions, with a maximum concentration of
22 g/L which was achieved at an initial glycerol quantity of 55 g/L. A new
Klebsiella oxytoca isolate converted waste glycerol into mixtures of PD, BD and
EtOH at various ratios. Finally, another new C. freundii isolate converted waste
glycerol into EtOH in anaerobic batch-bioreactor cultures with constant pH,
achieving a final EtOH concentration of 14.5 g/L, a conversion yield of 0.45 g/g
and a volumetric productivity of 0.7 g/L/h. As a conclusion, the current study
confirmed the utilization of biodiesel-derived raw glycerol as an appropriate
substrate for the production of PD, BD and EtOH by several newly isolated
bacterial strains under different experimental conditions.
Keywords: Biodiesel / 2,3-Butanediol / Ethanol / 1,3-Propanediol / Raw glycerol
Received: April 28, 2011; revised: July 29, 2011; accepted: August 24, 2011
DOI: 10.1002/elsc.201100058
1
Introduction
Biodiesel, defined as principally methyl-esters resulting from
trans-esterification of various natural oils and fats, already
represents an alternative type of fuel for various types of diesel
engines and heating systems. Continuous energetic crisis,
potential exhaustion of conventional fuels and several important
environmental issues imposed (e.g. rise of green-house gases
emission through combustion of fossil fuels, global warming
problem, etc.) have resulted in increasing demands for renewable fuels, the application of which on a large commercial scale
Correspondence: Dr. Seraphim Papanikolaou (spapanik@aua.gr),
Food Bioprocesses, Laboratory of Food Microbiology and Biotechnology, Department of Food Science and Technology, Agricultural
University of Athens, Greece
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
is strongly recommended (e.g. EU directive 2003/30/EC). This
entire situation had as an inevitable event the significant
increment of biodiesel production in the last year [1]. Given
that biodiesel derives from triacylglycerol trans-esterification
yielding both fatty esters and the side-production of glycerol, the
significant expansion of biodiesel has resulted in the generation
of large quantities of glycerol, the principal by-product of this
process is glycerol. Actually, with the production of 10 kg of
biodiesel from various oils, 1 kg of (pure) glycerol becomes
available [1, 2]. This situation has as a result the accumulation
of tremendous quantities of raw glycerol, which hence led to a
remarkable drop to its price into the market volume [3]. Since
the last year, in various countries of Western Europe
(e.g. Germany), crude glycerin water derived from various
biodiesel plants was treated as a typical ‘‘industrial waste-water’’
(with a cost of 0$ per kg – it was, hence, a waste material) being
used directly for biogas production [4].
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58
Eng. Life Sci. 2012, 12, No. 1, 57–68
M. Metsoviti et al.
Due to the impurities it contains (such as methanol, salts,
free-fatty acids, etc.), raw glycerol cannot be utilized in
chemical or pharmaceutical industry without treatment;
however, the high cost of purification makes this application
limited. Therefore, raw glycerol is now considered as an
economical and abundant renewable feedstock for industrial
microbiology, since the last year a constantly increasing
number of reports indicate employment of this residue as a
microbial substrate for the formation of high added-value
products [1, 3, 5]. One of the most promising applications of
raw glycerol is its bioconversion to 1,3-propanediol (PD)
through microbial fermentation. This added-value chemical
compound is mainly utilized as a polymer constituent. It can
be used in plastic industry as a monomer for novel polyester
and biodegradable plastics, which exhibit better product
properties and higher product stability than those produced by
1,2-propanediol or ethylene glycol [6]. Additionally, PD has an
important role in textile and fiber industry, as well as it can
give improved properties for solvents, adhesives, laminates,
resins, detergents and cosmetics [5, 7]. Recently, pure glycerol
has been considered as a potential substrate for the formation
of 2,3-butanediol (BD) [8, 9]. Dehydration of BD yields in the
synthesis of methyl-ethyl-ketone, which is an effective fuel
additive having a higher heat of combustion than ethanol
[10, 11]. Equimolar mixture of ethanol and BD can provide a
combined heating value, so the presence of ethanol does not
affect the usefulness of BD in this application [11, 12].
Furthermore, BD can present various applications in plastics
and solvent production [11]. Additionally, in the last year there
has been interest related with the conversion of glycerol into
ethanol (EtOH), since it was considered that the cost of this
process when using (raw) glycerol as substrate was 40% less
than that of the production of corn-derived sugars [3].
A large number of prokaryotic microorganisms can anaerobically or aerobically breakdown glycerol, such as Klebsiella
pneumoniae [13–22], Klebsiella oxytoca [13, 14, 23], Citrobacter
freundii [13, 16, 24], Enterobacter agglomerans [25], Clostridium butyricum [2, 4, 26–31], Lactobacillus brevis [32],
Lactobacillus collinoides [33], Lactobacillus reuterii [34] and
Pediococcus pentosaceus [35]. The biochemical pathways that
are involved in glycerol assimilation by all the abovementioned microorganisms present noticeable differences.
While strains belonging to Enterobacteriaceae family and to
Clostridium sp. can use glycerol as the sole carbon and energy
source, lactic acid bacteria (LAB) mostly utilize glycerol together with fermentable sugar, e.g. glucose. Recently, glycerol is
used as a final electron acceptor and sugars are needed for the
formation of biomass and ATP. Moreover, strains belonging to
Clostridium sp. can breakdown glycerol only under strictly
anaerobic conditions, whereas enteric group and LAB can
assimilate glycerol under anaerobic and/or aerobic conditions.
In most cases, glycerol is assimilated under anaerobic
conditions via two parallel metabolic pathways encoded by the
dha regulon: through the oxidative pathway glycerol is dehydrogenated to dihydroxyacetone (DHA) by the enzyme
glycerol dehydrogenase, which after phosphorylation is
converted into DHA-phosphate. Further, via pyruvate it can be
converted to various end-products, such as acetic acid, lactic
acid, EtOH, etc., the formation of which is a strain-dependent
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
process. Through the reductive pathway, glycerol is dehydrated
to 3-hydroxypropionaldehyde by the enzyme glycerol
dehydratase, which is then reduced to PD by the enzyme
1,3-glycerol oxidoreductase [5, 7, 36, 37]. Recently, glycerol
bioconversion under micro-aerobic conditions through the
glycerol kinase pathway [17, 18, 21, 23, 35] has been reported.
The current investigation was related with an extensive
screening of several newly isolated non-pathogenic bacteria
that could potentially breakdown raw glycerol, waste
discharged after biodiesel production process. It was the
aim of the study to obtain bacteria able to assimilate raw
glycerol under anaerobic or aerobic conditions, in order to
produce PD, BD and EtOH, and finally to improve both
glycerol uptake rate and product formation in batch-bioreactor experiments.
2
Materials and methods
2.1
Microorganisms
The strains used throughout this study are shown in Table 1.
Strains have been isolated from various foodstuffs and have
been identified and characterized in the Department of Food
Science and Technology [38–41] and have been deposited in
the culture collection of this Department. Cl. butyricum NRRL
B-23495 was kindly provided by the NRRL culture collection
(Peoria, USA). Long-term storage took place at 801C in
Tryptic Soy Broth, supplemented with 20% glycerol (Sigma
Chemical, St. Louis, MO, USA). Before experimental use, all
strains were sub-cultured twice in Tryptic Soy Broth and
incubated at the optimum temperature for 24 h.
2.2
2.2.1
Culture conditions
Preliminary trials in 50-mL conical flasks
Preliminary assessment of glycerol assimilation was performed
as follows: overnight 12-h bacterial culture was used to
inoculate (5%, v/v, of inoculum) 50-mL conical flasks,
containing 2071 mL of either Screening Medium 1 [per L:
analytical-grade glucose (purity 99%) 5 g; pure glycerol 15 g;
peptone 5 g; meat extract 5 g; yeast extract 2.5 g; K2HPO4 2 g;
CH3COONa 5 g; MgSO4 0.41 g and MnSO4 0.05 g; pH 7.0] or,
in the case of Clostridium, Screening Medium 2 [per L: pure
glycerol 20 g; NH4Cl 2 g; KCl 0.75 g; NaH2PO4 1.38 g; Na2SO4
0.28 g; citric acid 0.42 g; yeast extract 1.0 g; MgCl2 6H2O
0.26 g; CaCl2 H2O 2.9 mg; 1.0 mL Fe solution; 2.0 mL trace
element solution; pH 6.8]. The composition of the Fe solution
per L was: FeSO4 7H2O 5 g; HCl (37%) 4 mL. Trace element
solution consisted per L: ZnC12 70 mg; MnCl2 4H2O 0.1 g;
H3BO3 60 mg; CoC12 2H2O 0.2 g; CuC12 2H2O 20 mg;
NiC12 6H2O 25 mg; Na2MoO4 2H2O 35 mg; HC1 (37%)
0.9 mL [42]. Flasks were incubated in an orbital shaker (LabLine, IL, USA) at an agitation rate of 15075 rpm and incubation temperature of T 5 301C for strains belonging to
Bacillus sp., whereas at an agitation rate of 8075 rpm and
T 5 351C for Clostridium and T 5 301C for enterobacteria and
LAB, according to Homann et al. [13] and Biebl et al. [16]. In
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Eng. Life Sci. 2012, 12, No. 1, 57–68
Screening bacterial strains capable of fermenting raw glycerol
59
Table 1. Bacterial strains used in the present study
Strain
Glycerol and glucoseco-substrates
Glycerol as a sole carbon source
Growth
Glycerol consumption
Glycerol consumption
PD production
BD production
Anaerobic conditions
Clostridium butyricum NRRL B-23495
Klebsiella oxytoca FMCC-197
Citrobacter freundii FMCC-207
Citrobacter freundii FMCC-8
Citrobacter freundii FMCC-B 294 (VK-19)
Citrobacter farmeri FMCC-5
Citrobacter farmeri FMCC-7
Enterobacter aerogenes FMCC-9
Enterobacter aerogenes FMCC-10
Enterobacter ludwigii FMCC-204
Enterobacter sp. FMCC-208
Pantoea dispersa FMCC-200
Lactobacillus brevis (15 strains)
Leuconostoc mesenteroides (9 strains)
Pediococcus pentosaceus (34 strains)
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
–
–
–
1
1
1
1
1
1
1
1
1
Wa
1
Wa
n
n
n
1
1
Wp
1
1
–
–
–
–
–
1
–
n
n
n
–
1
1
–
–
–
–
1
1
–
1
–
n
n
n
Aerobic conditions
Bacillus licheniformis FMCC-91
Bacillus licheniformis FMCC-92
Bacillus licheniformis FMCC-98
Bacillus altitudinis FMCC-102
Bacillus subtilis FMCC-206
Bacillus sp. (9 strains)
1
1
1
1
1
1
–
1
1
1
1
–
n
1
1
1
1
n
n
–
–
–
–
n
n
–
–
Wp
–
n
1: positive result; –: negative result; Wa: weak assimilation (assimilationo2.0 g/L); Wp: weak production (productiono0.5 g/L); n: not tested.
Anaerobic and aerobic growth on glucose/glycerol mixtures or glycerol utilized as a substrate in 50-mL flasks
order to achieve anaerobic conditions, the medium was
sparged with N2 before autoclave and flasks were sealed with a
rubber lead.
2.2.2
Duran flasks experiments
Strains able to assimilate pure glycerol were cultivated in a
medium containing glycerol as a main carbon source at an
initial (Gly0) concentration of 20 g/L (in the medium there is
also peptone, meat extract and yeast extract that also contain
some carbon quantities). Biodiesel-derived waste glycerol
(provided by the ‘‘ADM Industry’’, Hamburg, Germany) was
used as a carbon source, replacing pure glycerol and glucose,
whereas no further modifications in the above-mentioned
screening media were applied. The purity of the raw glycerol
used was 81% w/w and the impurities included 11–12% w/w
water, 5–6% w/w potassium salts, 1% w/w free-fatty acids and
less than 0.2% w/w methanol. All experiments were carried out
in 1-L Duran flasks containing 80075 mL of the growth
medium, inoculated with 5% pre-culture. Flasks were incubated in the orbital shaker at an agitation rate of 8075 rpm,
and T 5 301C for enterobacteria and T 5 371C for Cl. butyricum. pH was adjusted to 7.0 before autoclaving and remained
un-controlled during the fermentation. Finally, to achieve
anaerobic conditions the medium was sparged with N2 before
autoclaving.
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.2.3
Aerobic shake-flask cultures
Bacillus strains as well as Enterobacter aerogenes strain FMCC10 were aerobically cultivated in 250-mL conical flasks,
containing 5071 mL of the same growth medium, inoculated
with 1%, v/v, of a 20-h pre-culture. Flasks were incubated in
the orbital shaker at an agitation rate of 15075 rpm and
T 5 301C. The pH was adjusted to value 7.0 before autoclaving
and remained un-controlled during the fermentation.
2.2.4
Bioreactor experiments
To further assess the potentiality of bacterial growth on
raw glycerol under anaerobic conditions, batch-bioreactor
fermentations were carried out in a 1.2-L bioreactor
(New Brunswick Scientific, USA), in which the working
volume was adjusted to 0.9 L. The composition of the growth
media was the same as in the Duran-flask experiments.
Cultures were inoculated with 5%, v/v, of a 20-h pre-culture
inoculum. Agitation was performed at 15075 rpm, pH was
maintained at value 7.0 for enterobacteria and 6.8 for Cl.
butyricum and was controlled by automatic addition of 5 M
NaOH. Temperature conditions were the same as in Duran
flasks experiments (T 5 371C for Cl. butyricum and 301C for
the enterobacteria). Continuous gassing with N2 at flow rate of
0.1 LPM provided anaerobic conditions throughout the
fermentations [4, 43].
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60
M. Metsoviti et al.
2.3
Analytical methods
Cell concentration (X, g/L) was determined through a linear
equation of cell dry weight (90751C until constant weight)
and optical density (OD) at 650 nm (Hitachi U-2000 Spectrophotometer, Japan). Cells were collected by centrifugation
(9000 g/15 min, 91C) in a Hettich Universal 320-R
(Germany) centrifuge and washed twice with distilled water. In
the aerobic shake-flask experiments, dissolved oxygen (DO)
concentration was determined by a selective electrode (OXI 96,
B-SET, Germany). Before harvesting, the shaker was stopped
and the probe was placed into the flask. Then, the shaker was
switched on and the measurement was taken after DO equilibration (usually within 10 min). pH value was measured with
a selective pH-meter (Jenway 3020, UK). Finally, concentrations of glycerol, glucose and organic acids were determined
with high-performance liquid chromatography (HPLC)
analysis (Waters 600E) with an Aminex HPX-87 H
(300 mm 7.8 mm, Bio-Rad, USA) column coupled to a
differential refractometer (RI Waters 410) and a UV detector
(Waters 486). Operating conditions were as follows: sample
volume 20 mL; mobile phase 0.005 M H2SO4; flow rate 0.6
mL/min; column temperature T 5 651C. All data presented are
the average of two independent experiments performed under
the same culture conditions.
3
Results
3.1
Initial trials for glycerol assimilation
Initially, all strains were cultured with glucose and glycerol as
co-substrates in the optimum temperature conditions, in order
to investigate glycerol breakdown by prokaryotic microorganisms. As it is shown in Table 1, among the screened
strains all grew on the mixture of glucose and glycerol, in no
one of the lactic acid bacteria screened glycerol underwent
assimilation after 24 h of culture. In contrast, in most cases,
glucose concentration had been significantly reduced. Similarly, nine strains of Bacillus sp. did not show glycerol uptake,
whereas all strains of the family Enterobacteriaceae and also
the strains B. licheniformis, B. altitudinis, B. subtilis and
Cl. butyricum NRRL B-23495 showed simultaneous breakdown
of glucose and glycerol. In a second approach, these strains
were further tested using pure glycerol as a carbon source in
order to select strains that assimilate glycerol and at the same
time produce PD and/or BD. Six strains produced PD and six
strains BD after 24 h of culture.
3.2
3.2.1
Flasks experiments without pH control
Anaerobic Duran flask cultures
Twelve strains able to assimilate pure glycerol were cultivated
in Duran flask cultures with raw glycerol utilized as a carbon
source, at Gly0 adjusted at 20 g/L. Kinetics were performed, the
maximum duration of which was 48 h, while sampling took
place at 4-h intervals for all trials. As given in Table 2, duration
of the fermentation, glycerol consumption, Xmax, final pH and
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eng. Life Sci. 2012, 12, No. 1, 57–68
product formation varied according to each strain. More
precisely, maximum glycerol assimilation (17.9 g/L) occurred
by the C. freundii strain FMCC-207 after 48 h. The majority of
the strains showed consumption between 12.1 and 17.0 g/L,
while only four strains assimilated less than 5.0 g/L. Additionally, biomass production expressed in g/L fluctuated from
0.4 up to 2.1 g/L, irrespective of glycerol consumption and
product formation. Concerning end-product formation,
remarkable differences in relation to the strains implicated in
glycerol consumption were observed; in all but one cases PD
was formed even in negligible quantities, while for three of the
tested strains PD was the predominant metabolite. Maximum
production of 9.1 g/L was obtained by Cl. butyricum strain
NRRL B-23495 after 24 h, with the highest conversion yield of
PD produced per glycerol consumed (YPD/Gly) 0.54 g/g. In
contrast to the Clostridium strain, enterobacteria exhibited
completely different end-product profiles, compared not only
with Clostridium strain but also with strains of the same genus.
Only two of the three strains of C. freundii, namely FMCC-8
and FMCC-B 294 (VK-19), produced PD as the main product,
while slight quantities of acetic acid and lactic acid were
accumulated in the medium. Moreover, YPD/Gly values were
0.26 and 0.36 g/g, respectively, which were lower values,
compared with that obtained from Cl. butyricum. In the case of
C. freundii strain FMCC-207, the two main metabolic products
EtOH and BD were formed in comparable concentrations, 5.9
and 4.3 g/L, respectively, resulting in similar conversion yields.
Finally, concerning fermentations by strains of the genus
Enterobacter, the end-products synthesized were predominantly BD and EtOH, while low quantities of PD were formed.
BD production varied from 1.5 up to 3.0 g/L and conversion
yield between 0.12 and 0.27 g/g. In the case of the cultures of
E. aerogenes strains, BD and ethanol were produced almost in
the ratio 1:1.
3.2.2
Aerobic conditions
One strain of E. aerogenes (namely FMCC-10) and the strain
B. altitudinis FMCC-102 were tested in cultures with raw
glycerol utilized as a carbon source at Gly0 5 20 g/L, in shakeflask experiments under aerobic conditions. In the kinetic
studies performed, the maximum duration was 48 h. This
selection was performed since B. altitudinis had been revealed
capable to consume glycerol and produce some quantities of
BD (Table 1), whereas as far as E. aerogenes FMCC-10 was
concerned, it was desirable to investigate the possibility of
improvement of both Gly consumption and BD production
under aerobic conditions. Concerning B. altitudinis FMCC-102
(Table 2), kinetic analysis showed that after 48 h, the microorganism consumed 9.0 g/L of glycerol producing only
0.7 g/L of BD. It is interesting to indicate that under these
experimental conditions, the carbon flow was mainly channeled towards biomass formation, the highest concentration of
which was remarkable (Xmax 5 3.6 g/L), with conversion yield
of biomass produced per glycerol consumed value (YX/Gly)
being 0.40 g/g. Moreover, the fermentation performance of
E. aerogenes strain FMCC-10 was significantly improved in the
shake-flask aerated cultures (Table 2). As depicted in Fig. 1A,
under aerobic conditions glycerol assimilation as well as BD
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& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
6.4
6.4
6.5
5.8
6.0
6.5
6.4
6.7
6.7
6.6
6.5
6.7
6.2
6.7
pHf
1.270.3
–
9.170.7
1.670.2
–
3.970.4
5.870.6
–
–
Wp
Wp
–
Wp
–
Concentration
(g/L)
PD
–
–
0.54
0.10
–
0.26
0.36
–
–
–
–
–
–
–
Yield
(g/g)
5.070.5
0.770.1
–
4.270.4
4.370.4
–
–
–
–
1.570.2
2.070.2
–
3.070.3
–
Concentration
(g/L)
BD
0.23
0.08
–
0.27
0.24
–
–
–
–
0.12
0.14
–
0.18
–
Yield
(g/g)
1.070.1
–
–
4.870.4
5.970.5
–
–
–
–
1.270.2
1.670.2
–
6.670.5
–
Concentration
(g/L)
EtOH
0.05
–
–
0.30
0.30
–
–
–
–
0.10
0.11
–
0.40
–
Yield
(g/g)
–
–
0.570.1
–
–
0.470.1
0.570.1
0.970.1
1.070.1
–
–
0.670.1
–
0.670.1
Concentration
(g/L)
Acetic acid
–
–
0.03
–
–
0.08
0.08
0.22
0.25
–
–
0.27
–
0.27
Yield
(g/g)
All experimental points presented are mean values from duplicate experiments performed by using different inocula; Wp: weak production (productiono0.5 g/L); Xmax: maximum biomass production;
pHf: final pH of the substrate.
2.170.20
3.670.33
Xmax
(g/L)
Aerobic conditions, cultures in 250-mL shake flasks
E. aerogenes FMCC-10
24
21.971.0
B. altitudinis FMCC-102
48
9.170.9
Glycerol consumed
(g/L)
0.670.06
1.370.11
1.170.10
0.570.05
0.570.05
0.870.08
0.870.08
1.270.10
1.670.10
0.470.04
1.270.11
0.470.04
Time
(h)
Anaerobic conditions, cultures on 1-L Duran flasks
Cl. butyricum NRRL B-23495
24
17.070.4
Kl. oxytoca FMCC-197
24
15.570.8
C. freundii FMCC-207
48
17.970.5
C. freundii FMCC-8
32
14.970.7
C. freundii FMCC-B 294 (VK-19)
32
16.170.9
C. farmeri FMCC-5
48
4.170.2
C. farmeri FMCC-7
48
3.970.3
E. aerogenes FMCC-9
24
12.170.5
E. aerogenes FMCC-10
24
14.270.6
E. ludwigii FMCC-204
48
2.270.2
Enterobacter sp. FMCC-208
48
16.770.6
P. dispersa FMCC-200
48
2.170.2
Strain
Table 2. Growth parameters, final products concentrations based on the kinetic data and conversion yields in fermentations glycerol at initial concentration 20 g/L, under conditions of
un-controlled pH (utilization of raw glycerol as a carbon source)
Eng. Life Sci. 2012, 12, No. 1, 57–68
Screening bacterial strains capable of fermenting raw glycerol
61
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25
DO (%, v/v)
Gly (g/L)
B
120
80
15
60
10
40
5
20
0
0
5
10
15
Time (h)
20
25
60
50
100
20
X (g/L)
BD (g/L)
EtOH (g/L)
0
25
20
40
15
30
10
20
5
10
0
0
10
20
30
40
Time (h)
50
Biomass (X, g/L), 2,3-Butanediol (BD, g/L),
Ethanol (EtOH, g/L)
X (g/L), anaerobic conditions
Gly (g/L), anaerobic conditions
BD (g/L), anaerobic conditions
X (g/L), aerobic conditions
Gly (g/L), aerobic conditions
BD (g/L), aerobic conditions
DO (%, v/v)
Biomass (X, g/L), Glycerol (Gly, g/L),
2,3-Butanediol (BD, g/L)
A
Eng. Life Sci. 2012, 12, No. 1, 57–68
M. Metsoviti et al.
Glycerol (Gly, g/L)
62
0
60
Figure 1. Kinetics of glycerol (Gly – g/L) consumption, biomass (X – g/L) formation, 2,3-butanediol (BD – g/L) and ethanol (EtOH – g/L)
production and dissolved oxygen (DO – %, v/v) concentration during growth of Enterobacter aerogenes FMCC-10 on raw glycerol, under
aerobic and anaerobic conditions. (A) Culture conditions: initial glycerol concentration 20 g/L, T 5 301C, growth on flasks, initial pH
7.070.2. Each point is the mean value of two independent measurements. (B) Culture conditions: initial glycerol concentration 55 g/L,
T 5 301C, growth on flasks, initial pH 7.070.2, DO415%, v/v for all culture phases. Each point is the mean value of two independent
measurements.
production was significantly improved for the E. aerogenes
strain, compared with the experiments under anaerobic
conditions. BD and PD production increased from 2.0 up to
5.0 g/L and from 0.5 up to 1.2 g/L, respectively, while EtOH
production was not influenced by oxygen conditions and was
formed in the same amounts in both cases. As it was previously
stated, in both trials (B. altitudinis and E. aerogenes), cultures
were performed under fully aerobic conditions since for all
growth steps of the strains, DO concentration was higher than
15%, v/v (see the case of DO kinetics for the culture of
E. aerogenes in Fig. 1A). By taking into consideration that BD
production under fully aerobic conditions is a quite interesting
result [11], in order to further examine the prospective of a
higher glycerol assimilation as well as a more interesting BDmax
production achieved, a culture at higher Gly0 concentration
(55 g/L) was performed, and the strain under investigation,
after 48 h of culture, produced 22 g/L of BD with a yield of
BD produced per glycerol consumed (YBD/Gly) of 0.40 g/g
(Fig. 1B). As in the previous trials, the above-mentioned
experiment was performed under fully aerobic conditions
(DO415%, v/v, for all culture steps).
3.3
Batch-bioreactor experiments performed in
constant pH
From the above-mentioned analysis, five among the screened
strains that were revealed capable to present remarkable
growth, substrate assimilation and product formation on raw
glycerol-based media in Duran flask anaerobic experiments
were selected and were further tested in batch-bioreactor
cultures. These trials were done in order to investigate the
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
formation of products under constant pH value 7.0 and
anaerobic conditions imposed by continuously gassing the
medium with 0.1 LPM of N2 during the fermentation. The
obtained results are given in Table 3. In particular, 10.1 g/L of
PD were produced by C. freundii strain FMCC-B 294 (VK-19),
giving the highest volumetric productivity, namely 1.1 g/L/h.
Glycerol was completely and rapidly consumed (after 10 h) and
PD production was accompanied by the formation of low
quantities of acetic, lactic and formic acid (1.8, 1.8 and 0.9 g/L,
respectively). Moreover, by taking into account that PD
production can be influenced by various environmental factors
like the pH value of the culture medium [7, 44], a second
batch-bioreactor experiment was performed in which the pH
value of the medium was maintained at 6.0. As it is shown in
Fig. 2, the reduction of the pH resulted in an insignificant
decline of PD production from 10.1 to 9.6 g/L. However,
change in the pH value of the medium increased the
fermentation period from 10 to 24 h, presumably due to higher
lag time observed in the latter case (see Fig. 2). On the other
hand, Cl. butyricum exhibited the highest PD production
among all tested strains with PDmax concentration of 11.3 g/L
after 17 h of culture, which corresponded to a yield YPD/Gly
value of 0.58 g/g and a productivity of 0.66 g/L/h. Additionally,
small quantities of acetic, lactic and butyric acid were formed
(0.5, 1.7 and 3.1 g/L, respectively). Taking into consideration
that this microorganism was found to be the most suitable
among all tested strains with regard to its potentiality of
producing PD, in order to enhance the PD production from
raw glycerol, a batch-bioreactor culture of Cl. butyricum at
higher Gly0 concentration (55 g/L) was performed; indeed,
despite the relatively elevated Gly0 concentration employed,
the strain produced 32.3 g/L of PD with a YPD/Gly yield of
http://www.els-journal.com
–
0.38
0.67
–
0.33
–
0.30
0.45
–
0.22
X (g / L), pH=7
PD (g / L), pH=7
X (g / L), pH=6
PD (g / L), pH=6
Gly (g / L), pH=7
Gly (g / L), pH=6
Glycerol (Gly,g / L)
All experimental points presented are mean values from duplicate experiments performed by using different inocula. Xmax: maximum biomass production
–
4.670.3
8.070.8
–
4.670.3
–
0.06
–
–
0.04
0.58
0.21
–
0.48
–
17
12
12
10
14
Cl. butyricum NRRL B-23495
K. oxytoca FMCC-197
C. freundii FMCC-207
C. freundii FMCC-B 294 (VK-19)
E. aerogenes FMCC-10
19.670.8
18.370.7
17.970.7
21.170.8
20.770.6
0.770.06
2.270.20
2.770.30
1.970.17
2.870.30
11.370.6
3.870.4
–
10.170.6
–
0.66
0.32
–
1.01
–
–
1.170.2
–
–
0.870.1
–
0.09
–
–
0.04
(g/L)
Productivity
(g/L/h)
Yield
(g/g)
(g/L)
Yield
(g/g)
(g/L)
Productivity
(g/L/h)
BD
PD
Xmax
(g/L)
Glycerol consumed
(g/L)
Time
(h)
12
10
20
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
63
8
15
6
10
4
5
2
0
0
5
10
15
Time (h)
20
Biomass (X, g / L), 1,3-Propanediol (PD, g / L)
Productivity
(g/L/h)
Yield
(g/g)
EtOH
Screening bacterial strains capable of fermenting raw glycerol
25
Strain
Table 3. Fermentation of glycerol at initial concentration 20 g/L in 1.2-L bioreactor, under anaerobic conditions (continuous sparging with N2 at 0.1 LPM) and conditions of constant pH
(value 6.8 for Cl. butyricum and 7.0 for eneterobacteria) (utilization of raw glycerol as a carbon source)
Eng. Life Sci. 2012, 12, No. 1, 57–68
0
25
Figure 2. Kinetics of glycerol (Gly – g/L) consumption, biomass
(X – g/L) and 1,3-propanediol (PD – g/L) production during
growth of Citrobacter freundii FMCC-B 294 (VK-19) on raw
glycerol in batch-bioreactor experiments. Culture conditions:
initial glycerol concentration 20 g/L, T 5 301C, pH 7.070.2
(filled symbols) and pH 6.070.2 (open symbols), growth on
1.2-L bioreactor. Each point is the mean value of two independent measurements.
0.59 g/g after 38 h of culture, while all of the available glycerol
quantity was assimilated by the microorganism (Fig. 3).
As in the Duran flask experiments, Kl. oxytoca growing in
batch-bioreactor cultures produced a mixture of PD, BD and
EtOH, which remained as the predominant synthesized
metabolites (Table 3). Nevertheless, differences in the final
concentrations of the metabolic products were observed in
comparison with the flask experiments; although EtOH
production was more or less unaffected from the different
fermentation configurations applied, PD production was
enhanced from 1.1 to 3.8 g/L, whereas BD production was
considerably reduced from 4.2 to 1.1 g/L comparing the results
from the Duran flask experiments under conditions of uncontrolled pH (Tables 2 and 3). Finally, strains C. freundii
FMCC-207 and E. aerogenes FMCC-10, although consumed
under strictly anaerobic conditions 17.9 and 20.7 g/L of
glycerol, respectively, were mutually unable to convert them
into PD nor BD. In both cases, metabolism shifted towards
EtOH production and small quantities of lactic and formic
acid were observed, as well. Apparently, the fact that pH in the
fermentation medium remained constant and released from
the CO2 metabolism did not remain in the fermentation vessel
resulted in this metabolic shift favoring the accumulation of
EtOH into the medium. EtOHmax concentration 8.0 g/L with
the remarkable conversion yield of EtOH produced per
glycerol consumed (YEtOH/Gly) of 0.45 g/g and the volumetric
productivity of 0.67 g/L/h was recorded with C. freundii strain
FMCC-207. By taking this result into consideration, a batch
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Eng. Life Sci. 2012, 12, No. 1, 57–68
M. Metsoviti et al.
X (g / L)
PD (g / L)
Ace (g / L)
60
18
Lac (g / L)
But (g / L)
35
30
50
Glycerol (Gly, g / L)
25
40
20
30
15
20
10
10
5
0
0
5
10
15
20
25
Time (h)
30
35
0
40
Figure 3. Kinetics of glycerol (Gly – g/L) consumption and
biomass (X – g/L), 1,3-propanediol (PD – g/L), acetic acid (Ace –
g/L), lactic acid (Lac – g/L) and butyric acid (But – g/L)
production during growth of Clostridium butyricum NRRL
B-23495 on raw glycerol in batch-bioreactor experiments. Culture
conditions: initial glycerol concentration 55 g/L, T 5 371C, pH
6.870.2, growth on 1.2-L bioreactor. Each point is the mean
value of two independent measurements.
culture with a higher Gly0 concentration (35 g/L) was
performed, and the strain, after 22 h of culture, produced
14.5 g/L of EtOH with a YEtOH/Gly yield of 0.45 g/g and a
volumetric productivity of 0.69 g/L/h. The global yield YEtOH/
Gly of ethanol synthesized per glycerol consumed for both
fermentations performed (Gly0 5 20 and 35 g/L) by C. freundii
strain FMCC-207, as illustrated by the quantity of EtOH
produced per Gly consumed for all fermentation points, is
0.44 g/g (Fig. 4), indicating the constancy of the conversion
of the above-mentioned process performed by C. freundii
within the range of Gly0 concentrations tested.
4
Discussion
The present study validates the ability of various bacterial
strains to assimilate raw glycerol, waste derived from biodiesel
production plants, and convert it into metabolic compounds
of added-value and industrial importance, such as PD, BD and
EtOH. The principal conversion related to glycerol fermentation under anaerobic conditions refers to the production of
PD, but so far in the majority of the studies performed,
utilization of pure glycerol as a substrate had been employed
(for state-of-the-art reviews see: Willke and Vorlop [5], Zeng
and Biebl [7], Papanikolaou [44]). In the last decade, the idea
of utilizing raw glycerol as a microbial substrate has begun to
develop, due to the constantly increasing production of
biodiesel that resulted in low (or even zero) cost of this residue
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Glyo = 35 g / L
Glyo = 20 g / L
15
Ethanol (EtOH, g / L)
Gly (g / L)
Biomass (X, g/L) and Products formation (g / L)
64
12
9
YEtOH / Gly=0.44 g / g
6
3
0
0
5
10
15
20
25
Glycerol consumed (Gly, g / L)
30
35
Figure 4. Ethanol produced (EtOH – g/L) versus glycerol
consumed (Gly – g/L) for Citrobacter freundii FMCC-207, in
batch-bioreactor experiments. Culture conditions: initial glycerol
concentration 20 and 35 g/L, T 5 301C, pH 7.070.2, growth on
1.2-L bioreactor. Each point is the mean value of two independent measurements.
[1, 5, 7, 44, 45]. Meanwhile, few studies have focused on the
production of PD from biodiesel-derived raw glycerol and all
of them concern principally bacteria of the species Cl. butyricum and Kl. pneumoniae [1, 2, 4, 27, 30, 31, 46–53]. Even
fewer studies utilize glycerol as a substrate for the production
of BD or EtOH as the principal compounds of the cellular
metabolism [8, 9, 16, 25, 54–57]. To the best of our knowledge,
there has been no other reports demonstrating BD production
as the main product from raw glycerol, while equally, this is the
first report that deals with the production of EtOH from
glycerol as a principal metabolic compound by C. freundii so
far. Under this view, 83 new bacterial strains derived from
various foodstuffs and Cl. butyricum NRRL B-23495 were
screened in order to select the microbial candidates with
maximum glycerol assimilation and product formation for
further investigations. Twelve strains consumed raw glycerol
under anaerobiosis and three under aerobiosis and intraspecies differences as regards their metabolic profiles were
observed. The most promising bacteria in terms of PD
formation were found to be Cl. butyricum NRLL B-23495 and
C. freundii FMCC-B 294 (VK-19), while Kl. oxytoca FMCC197, C. freundii FMCC-207 and E. aerogenes FMCC-10 formed
quantities of BD and EtOH. Some discrepancies related with
the ‘‘tolerance’’ of microbial strains against the impurities of
raw glycerol feedstock have appeared in the literature; Mu et al.
[50] reported that Kl. pneumoniae DSM 2026 was able to
convert 20 g/L of glycerol into 8.4 g/L of PD when raw glycerol
was utilized as a substrate, instead of 9.4 g/L when using pure
glycerol. On the other hand, Jun et al. [51] indicated better
performances of Kl. pneumoniae DSM 4799 on raw glycerol
compared with pure one. However, Petitdemange et al. [27]
reported that several Cl. butyricum strains obtained from
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Eng. Life Sci. 2012, 12, No. 1, 57–68
various bacterial culture collections were unable to grow on
raw glycerol, while among the ten wild strains isolated from a
stagnant pond only four grew well on this residue. In the
present study, most of the strains capable of consuming
glycerol could present satisfactory consumption also of the unpurified glycerol residue; on the other hand, fermentations at
Gly0 5 55 g/L (in the case of E. aerogenes and Cl. butyricum
strains) that were successfully accomplished revealed the
feasibility of using raw glycerol at relatively high initial
concentrations, suggesting that impurities found in the waste
did not have significant negative effect upon the microbial
metabolism.
As it was observed through the present study, the most
important factors that influenced the fermentation of raw
glycerol and the product formation were the pH conditions of
the medium, the N2 sparging (meaning the removal from the
medium of CO2 – and potentially of H2 – that are produced
through the bacterial anaerobic metabolism and could
potentially have negative effect upon the cellular metabolism
[14, 15]) and the strain specificity; thus, significant variations
were observed related with the strains implicated in the present
study. As reported by Biebl et al. [16] for the strain
Kl. pneumoniae, maximum BD formation was obtained during
fermentations with natural acidification cultivation conditions.
Similarly, in the present investigation, it was found that
cultivations without pH control were proved to be favorable
for BD production by strains C. freundii FMCC-207 and
E. aerogenes FMCC-10. In contrast to BD production, PD
biosynthesis was significantly improved by conditions of
constant pH and N2 sparging into the medium, indicating that
these parameters can remarkably affect the microbial metabolism towards different products or different ratios among
the same products.
Although the majority of reports in the international literature refer to glycerol fermentation by bacteria under anaerobic
conditions, a limited number of publications deal with bacterial
glycerol fermentation under aerobic or micro-aerobic conditions
[8, 9, 17, 18, 21, 23]. In most of the cases, the principal metabolic compound is that of PD. The PDmax concentrations
achieved in the current investigation ranged between 10.1 g/L
(case of C. freundii) and 32.3 g/L (case of Cl. butyricum). PDmax
concentrations reported in the literature principally refer to
anaerobic batch or fed-batch cultures, and quantities ranging in
most of the cases between 40 and 90 g/L (values higher than
those presented in the current investigation) can be obtained
under optimized conditions (for state-of-the-art reviews dealing
with the biotechnological PD production see: Willke and Vorlop
[5], Papanikolaou [44], Zeng and Sabra [58]). For instance,
Himmi et al. [28] were able to produce a PDmax quantity of
65.4 g/L by using Cl. butyricum strain CNCM 1211 in batchbioreactor cultures using raw glycerol, while the respective value
for Cl. butyricum strain E5 in fed-batch culture on raw glycerol
was 65.6 g/L [27]. Mu et al. [50] reported PDmax values of 61.9
and 53.0 g/L by Kl. pneumoniae DSM 2026 when this microorganism was cultivated in fed-batch experiments using pure
and raw glycerol, respectively, as carbon sources. Hirschmann
et al. [49] performed fed-batch cultures of the strain Clostridium
sp. IK 124 and achieved PDmax values of 87.7 and 80.1 g/L when
pure and raw glycerol, respectively, were used as carbon sources.
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Screening bacterial strains capable of fermenting raw glycerol
65
Reimann and Biebl [42] reported PDmax values of 57.0 and
70.5 g/L by the strains Cl. butyricum DSM 5431 and Cl. butyricum mutant 2-2, respectively, during growth on pure glycerol in
fed-batch experiments. Barbirato et al. [46] indicated a PDmax
quantity of 31.2 g/L during growth of C. freundii strain ATCC
8090 on batch cultures using pure glycerol as a substrate, while
the respective value for C. freundii DSM 30040 was 41.1 g/L
when growth was performed on two-stage continuous culture
with pure glycerol utilized as a substrate [24].
In the present study, aerobic conditions enhanced glycerol
assimilation in the case of E. aerogenes strain FMCC-10 and
final consumption was improved to 54% compared with
anaerobic conditions, while BD production was significantly
ameliorated, as well (Table 2). Adaptation of E. aerogenes on
relatively high Gly0 media under aerobic conditions, remarkably ameliorated the BDmax concentration achieved
(BDmax22 g/L, yield YBD/Gly 0.40 g/g). Cheng et al. [18]
reported that final concentration of BD was almost doubled
during aerobic fermentation and similar results were achieved
in our study. To our knowledge, a limited number of investigations deal with BD production as the principal metabolic
compound of glycerol assimilation under fully aerobic conditions, since enhanced BD production is generally performed
under low O2 supply, and as such, the fermentation of BD is
considered as a classical anaerobic (or micro-aerobic)
fermentation (for state-of-the-art reviews see: Celińska and
Grajek [11], Zeng and Sabra [58]). In most of the cases, BD
production is performed by strains belonging to the species
Paenibacillus polymyxa, Serratia marcescens, Kl. pneumoniae
and Kl. oxytoca with glucose being used as a carbon source
[11, 58], whereas in the last year utilization of sugar-based raw
materials (i.e. Jerusalem artichoke tubers) has been considered
as potential substrates with BDmax concentration obtaining
very high final values (e.g. 80.5 g/L) [59]. On the other hand,
BD production through glycerol bioconversion under fully
aerobic conditions is a quite scarce and interesting result, and
was performed in the literature only by the use of pure glycerol
as a substrate; therefore, Petrov and Petrova [8] reported the
production of 49 g/L by using the strain Kl. pneumoniae G31,
whereas optimization of the aeration process and the pH
control resulted in BDmax quantities ranging between 52.6 and
70.0 g/L [9] (quantities, in any case, higher than the current
investigation). However, as in the case of PD formation, the
current investigation aimed to screen a number of newly
isolated non-pathogenic strains towards their potentialities of
consuming biodiesel-derived raw glycerol, and not to produce
excessively high PD and BD quantities at this stage. Nevertheless, the strains Cl. butyricum NRLL B-23495 and E. aerogenes FMCC-10 were revealed to be promising candidates for
PD and BD production, respectively, with a waste feedstock
(raw glycerol) being utilized as a substrate.
A result of importance that was achieved in the current
investigation was that a new C. freundii isolate (strain FMCC207) converted waste glycerol into (almost exclusively) ethanol in
remarkable quantities in bioreactor experiments performed at
constant pH and continuous N2 sparging into the medium. An
EtOHmax quantity of 14.5 g/L with a YEtOH/Gly yield of 0.45 g/g
and a volumetric productivity of 0.69 g/L/h were achieved. These
results are of interest, since a scarce number of reports deal with
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66
the conversion of glycerol into EtOH by bacteria belonging to
the enteric group. For instance, when raw glycerol was utilized as
a substrate, an EtOHmax quantity of 10.0 g/L (volumetric
productivity 0.83 g/L/h) has been reported by E. aerogenes
Hu-101 in batch-bioreactor cultures [56], while the respective
values of Kl. pneumoniae GEM167/pBR-pdc-adh were 25.0 g/L
and 0.78 /L/h in fed-batch bioreactor experiments [54]. It should
be noted that in the last case, a mutant strain (GEM167) was used
(exposure of a wild strain to g-irradiation) and its capabilities
towards ethanol production were further ameliorated by overexpressing Zymomonas mobilis pdc and adhII genes encoding
pyruvate decarboxylase (Pdc) and aldehyde dehydrogenase
(Adh), respectively [54]. Mu et al. [50] reported the production
of ethanol from crude glycerol to a maximum level of 11.9 g/L
(volumetric productivity 0.5 g/L/h). Higher EtOH production
was achieved by using a derivative of Kl. oxytoca M5al when
production of the competitive metabolite (namely lactic acid)
was diminished by deletion of the lactate dehydrogenase gene
wherein the glycerol yield was 19.5 g/L and the volumetric
productivity was 0.56 g/L/h [23]. The maximum concentration
of EtOH from glycerol obtained so far in the literature has been
reported by a newly isolated Kluyvera cryocrescens strain, with
EtOH quantity achieved at 27 g/L. The respective YEtOH/Gly
and volumetric productivity values were 0.40 g/g and 0.61 g/L/
h [55]. The above-indicated results indicate the potentiality of
the newly isolated C. freundii (strain FMCC-207) towards the
conversion of waste glycerol into ethanol. Finally, recently,
Yazdani and Gonzalez [57] demonstrated anaerobic fermentation of glycerol by a genetically engineered Escherichia coli, a
species that had long been considered to be incapable of glycerol
utilization, and production of ethanol from this substrate.
5
Eng. Life Sci. 2012, 12, No. 1, 57–68
M. Metsoviti et al.
Concluding remarks
This study confirmed the utilization of raw glycerol as an
appropriate substrate for the production PD, BD and EtOH by
several bacterial strains under different experimental conditions. It was indicated that end-product profiles and glycerol
assimilation could be influenced by a range of parameters such
as the strain dependency, the pH of the medium, the aerobiosis–anaerobiosis conditions and the sparging (or not) with
N2 (for the anaerobic cultures). To the best of our knowledge,
raw glycerol fermentation by the strains Kl. oxytoca, C. freundii
and E. aerogenes is reported for the first time, as well as the
production of BD as the predominant metabolic product by
the strain E. aerogenes during cultivations on raw glycerol
under fully aerobic conditions. Similarly, this is the first report
in the literature that deals with EtOH production by
C. freundii when raw glycerol is utilized as a substrate. A
Cl. butyricum strain (NRLL B-23495) and an E. aerogenes
strain (FMCC-10) proved to be potential PD and BD producers, respectively, since relatively satisfactory quantities of the
above-mentioned diols were synthesized by these microorganisms. The EtOH production achieved by C. freundii, in
terms of absolutes values (g/L), relative values (g per g of
glycerol consumed) and volumetric productivities value (g/L/
h), is quite satisfactory and comparable with the data reported
in the international literature.
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
This research was financially supported by: (i) The State Scholarship Foundation (Athens – Greece) and DAAD (project
IKYDA ‘‘Development of a novel bioconversion process involving
a defined microbial community’’); (ii) The EU (FP7 Program
‘‘Propanergy – Integrated bioconversion of glycerine into valueadded products and biogas at pilot plant scale’’, Grant number:
212671).
The authors have declared no conflict of interest.
6
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