i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 1 6 1 7 e1 1 6 2 4

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Coproduction of hydrogen and butanol by

Clostridium acetobutylicum with the biofilm
immobilized on porous particulate carriers

Jingyun Liu a, Wencan Zhou a, Senqing Fan a,**, Boya Qiu a,

Yuyang Wang a, Zeyi Xiao a,*, Xiaoyu Tang b, Wenguo Wang b,
Shizhao Jian a, Yangmei Qin a
a
School of Chemical Engineering, Sichuan University, 610065, Chengdu, China
b
Biogas Institute of Ministry of Agriculture, Chengdu, 610041, China

article info abstract

Article history: The porous particulate carriers of activated carbon, bagasse and brick were used for Clos-
Received 22 January 2019 tridium acetobutylicum immobilization for coproduction of hydrogen and butanol. The dense
Received in revised form microbial population was growing on the carrier surface with the biofilms formed during
11 March 2019 fermentation. The homogeneous array of the microbial cells on the surface looks some
Accepted 14 March 2019 interesting behaviors. The cells have the ability to shuttle between holes in bagasse. Higher
Available online 5 April 2019 efficiency of cell immobilization could be achieved accordingly. The cell concentration
during immobilized fermentation was about one order magnitude higher than that during
Keywords: free cell fermentation. Enhanced fermentation for hydrogen and butanol has been ach-
Clostridium acetobutylicum immobili- ieved during the immobilized fermentation. The highest yield of hydrogen was 1.81 mol/
zation mol when brick was used as immobilization carrier, while the highest butanol yield of
Porous particulate carriers 0.22 g/g was achieved during fermentation with bagasse as immobilization carrier.
Hydrogen Hydrogen productivity and butanol productivity were up to 403.2 ml/L/h and 0.44 g/L/h,
Butanol respectively. Hydrogen and butanol production behaved differently in organic and inor-
Fermentation ganic carrier materials.
© 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

the requirement of mild environmental conditions [2]. As a

Introduction
Hydrogen is known as an ideal renewable energy because of
its high energy content (140 MJ/kg) and lack of carbon emis-
sions from combustion [1]. Biohydrogen production by
fermentation is one of the alternatives to conventional
hydrogen production methods (such as steam reforming,
partial oxidation, electrolysis and gasification) with regard to
typical carbon neutral biofuel for transportation, biobutanol is
regarded as a good gasoline additive and considered as a
renewable fuel with a potential for a significant reduction of
greenhouse gas emissions. Compared with ethanol, butanol is
not sensitive to water, less corrosiveness, lower volatility, less
flammability and reduced toxicity to physical exposure [3].
Butanol can be used in existing motor engines and vehicular
infrastructures without mechanical tailoring [4]. It can be also

* Corresponding author. Sichuan University, No.24 South Section 1, Yihuan Road, 610065 Chengdu, China.
** Corresponding author.
E-mail addresses: fansenqing86@scu.edu.cn (S. Fan), mgch@scu.edu.cn (Z. Xiao).
https://doi.org/10.1016/j.ijhydene.2019.03.099
0360-3199/© 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
11618 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 1 6 1 7 e1 1 6 2 4
transported with the existing pipelines for its low vapor
pressure and high energy value (29.2 MJ/L) [5]. Coproduction of
hydrogen and butanol can be achieved by fermentation with
Clostridium acetobutylicum (C. acetobutylicum) [6,7].
Acidogenesis and solventogenesis phases are performed in
the metabolic pathway of C. acetobutylicum. During acido-
genesis phase, cells could be exponentially grown along with
glucose consumption, acid accumulation, hydrogen produc-
tion and pH decrease. When the pH is decreased to a threshold
value, the metabolism of the cells would be shifted to sol-
ventogenesis [8]. During this phase, butanol is produced with
less hydrogen production or even stopped, and cell growth are
kept in a stationary stage [9]. In general, either hydrogen or
butanol fermentation performance is paid attention to
[10e12]. Compared with the inherent disadvantage “low titer”
of hydrogen or butanol fermentation, coproduction of
hydrogen and butanol by fermentation would be more
competitive [13]. Batch fermentation with suspended cell is
the conventional process for the coproduction of hydrogen
and butanol [12,14,15]. During batch fermentation process,
several shortcomings with lower yield, lower productivity,
lower final product concentration and lower utilization of the
equipments are performed, owing to low concentration of
cells [16]. In general, the productivity and equipments utili-
zation efficiency can be somewhat improved in a Continuous
Stirring Tank Reactor (CSTR). Rapid decrease in cell concen-
tration along with the reduction of hydrogen and butanol
production could be occurred, if the dilution rate is too large
[17].
In recent years, many literatures suggest that the
fermentation with immobilized cells would be superior to
free cell fermentation, and it has received more and more
attention [17e20]. During immobilized fermentation, the
immobilization carriers are able to retain the majority of the
cells in the fermenter so that higher cell concentration and
higher productivity can be achieved [21]. The common
employed immobilization methods are self-granulation, gel
entrapment and surface attachment [17]. Surface attach-
ment is currently the main method for Clostridium ssp.
immobilization for the biofilm formation [22]. The steps of
biofilm formation is followed the orders: 1) the free cells are
contacted with the carriers owing to the movement of cells;
2) the cells contacted with the carriers are attached on the
surface of the carriers owing to the affinity of carriers to the
cells; 3) the attached cells are growing on the surface of the
carries with biofilm formed [23,24]. Compared with gel
entrapment cell immobilization, the biofilm can be directly
contacted with fermentation broth, which reduces greatly
the mass transfer resistance of both substrates and metab-
olites through entrapment materials and broth [25]. It has
been reported that the yield of hydrogen was five times
higher than that of free cell fermentation with one eighth of
reactor volume required, during fermentation with ceramic
balls as cell carriers [17].
Undoubtedly, the carrier materials for biofilm immobili-
zation are dominant or even fatal. The carriers should be
porous, rough, non-toxic to cells, high biomass holding ca-
pacity and no resistant to mass transfer [26]. Activated car-
bon is commonly used for its non-toxic, good adsorption
performance and excellent mechanical properties [27]. It has
good mobility in broth and could capture the cells more
effectively, owing to its lower density. Furthermore, the
activated carbon particles would not be excessive extrusion
when they were piled up. All the surfaces of materials could
be posed in the broth which was beneficial to the evenly
distributed of cells on the materials surface. Sugarcane
bagasse is a cheap local-available material with the advan-
tages of higher porosity and having a good water retention
capacity. Besides, bagasse can be used as substrate for bio-
fuels production after hydrolysis with minimizing waste
generation [28,29]. If bricks made from clay can be used as
the support material, the microbes would live in a more
comfortable environment, since C. acetobutylicum is origi-
nally a kind of soil-microorganism [30]. Furthermore, brick
contains many kinds of cations (Fe3þ, Al3þ, Ca2þ and so on)
which could be linked with the negative-charged cell mem-
brane. In this case, a more stable bioflim structure would be
formed on the carrier surface. The Fe3þ in the brick is an
important element during cell metabolism, which would
enhance the activity of some key enzymes in the cells [31,32].
In this study, C. acetobutylicum was immobilized on the sur-
face of activated carbon, bagasse and brick during fermen-
tation. The fermentation performance with suspended cell
was compared with that with immobilized cell. Besides, the
coproduction performance of hydrogen and butanol during
fermentation has been also explored.
Material and methods
Microorganisms and medium
C. acetobutylicum strain CICC 8012 was purchased from China
Center of Industrial Culture Collection (Beijing, China) in the
form of freeze-dried cells. Through three rounds of pure
cultivation, the strain was suitable for fermentation. The
medium for culture and fermentation contained: glucose
27e35 g/L, yeast extraction 3 g/L, phosphate buffer (KH2PO4
0.5 g/L, K2HPO4 0.5 g/L, ammonium acetate 2.2 g/L), vitamins
(para-amino-benzoic acid 1 mg/L, thiamin 1 mg/L, biotin
0.01 mg/L), mineral salts (MgSO4$7H2O 0.2 g/L, MnSO4$H2O
0.01 g/L, FeSO4$7H2O 0.01 g/L, NaCl 0.01 g/L). Glucose and yeast
extraction were sterilized by autoclaving at 121  C for 20 min.
Phosphate buffer, vitamins and mineral salts were filter-
sterilized through 0.45 mm sterile membrane.
Carrier materials
Activated carbon, bagasse and brick were the carrier materials
for immobilization. Activated carbon with particle size of
3e4 mm was purchased from a supplier (Chengdu Cybernaut,
China). Bagasse was prepared by squeezing fresh sugar-cane
and cut into small and uniform size below 10 mm. Brick was
picked up from construction waste arbitrarily, and then
smashed with the hammer, and screened the material for
particles with size 2e4 mm. All of the carrier materials were
washed by running water and deionized water, dried in the
oven overnight and then sterilized at 121  C for 20 min. The
loading amount of both activated carbon and brick were 67 g/
L, while for bagasse it was 12.5 g/L.
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 1 6 1 7 e1 1 6 2 4
Experiment procedure
There were totally forty conical flasks applied during each
fermentation process. Four of them were 250 mL with the
addition of 150 mL medium for measurement of gas produc-
tion. The gas sample used to analyze the hydrogen and carbon
dioxide content was collected with a gas sampling bag. The
gas amount was measured by drained water. The other 36
conical flasks were 50 mL, which were filled with 40 mL me-
dium. After inoculation with a ratio of 10% (v/v), conical flasks
were incubated at 37  C in a water bath. Two 50 mL conical
flasks were picked out every 4 h to determined glucose con-
centration, cell concentration and butanol concentration. At
the same time, the gas sample and drained water were also
harvested for further analyzing.
Assay
The glucose concentration in the broth was detected with
DNS methods [33]. Cell concentration in the suspended broth
was determined with the spectrophotometer [34].The rela-
tionship between cell concentration and optical density
can be expressed by a linear function (Cell
concentration ¼ 0.1842  optical density at 540 nm þ 0.001).
The mass of cells immobilized on the carrier materials were
measured according to the mass difference of the dry carrier
materials before and after fermentation. The pH was
measured off-line using a pH-meter.
The proportion of the hydrogen produced in the fermen-
tation was analyzed with a gas chromatograph (GC-9790II,
Zhejiang Fuli, China) equipped with a thermal conductivity
detector and a 2 m stainless column TDX-01. The carrier gas
was N2 with flow rate of 24 mL/min. The temperature of the
column oven was set at 90  C, and the temperature for both
the injection port and the detector was set at 100  C. For
measurement of CO2, hydrogen was chosen as the carrier gas
with flow rate of 40 mL/min, and the other settings were the
same as hydrogen measurement. The concentrations of
acetone, butane and ethanol were also analyzed with a gas
chromatograph equipped a flame ionization detector and a
30 m FFAP capillary column (i.d.0.32 mm), with N2 as the
carrier gas. The oven temperature was increased from 60  C to
180  C at a rate of 20  C/min using a temperature program. The
temperatures of the injector and the detector were set at
180  C and 210  C, respectively.
Results and discussion
Biofilm cell immobilization
The microstructure of the particulate carriers (activated car-
bon, sugarcane bagasse and brick) used in the study was
illustrated in Fig. 1 (a), (c), (e). It could be seen that all of them
were porous materials and the surfaces were rough enough
for cell attachment. The diameter of the holes for activated
carbon was 700e900 nm. The honeycomb structure at cross
section and film-like tissue were exhibited in bagasse. A
hexagonal column and hollow structure in longitudinal sec-
tion of sugarcane bagasse were exhibited, which was
11619
segmented in longitudinal direction just like the outside
appearance of sugar cane. The space inside bagasse could
provide large surface area for cells attachment. Brick surface
was bumpiness and gully-like, and these uneven structures
could provide irregular holes. From Fig. 1(b), (d), (f), it could be
seen that a dense microbial population was attached on the
carrier surface with the biofilms formed, after the carriers
were loaded in the broth. Thickness of the biofilm on the
activated carbon surface was approximate 3.14 mm, as shown
in Fig. 1(g). It has been observed that the immobilized cell
concentration was much higher than suspended cell con-
centration, as shown in Fig. 2. It was reported that biofilm has
a complex three-dimensional structure that created an in-
ternal protective environment for bacterial cells [35]. Cells in
biofilm exhibited enhanced tolerance to adverse environ-
mental stress conditions [36]. These properties could promote
proliferation and metabolism of the cells in biofilms.
It has been observed that some of the cells were moving
through the holes of the sugarcane bagasse, as shown in
Fig. 1(h). This migration implied that cells had the ability to
shuttle among holes in bagasse and grew in the holes. The
homogeneous array of the microbial cells on the surface looks
some interesting behaviors. In order to make full use of the
space of the carrier surface, the cells could be orderly arranged
spontaneously on the surface. The arrangement of cell-
pioneers has already formed a pattern which was domi-
nated by the nature of the attachment surface, so the suc-
cessors have to array at the same way as that of the pioneers,
with obvious orientation behavior performed [22,37]. The
suspended cell concentration and immobilized cell were
measured for the judgment of immobilization efficiencies of
carrier materials. As shown in Fig. 2, the suspended cell con-
centration in broth was gauged no distinct difference for
immobilization and free cell fermentation. This implied that
the immobilization materials did not affect free cell growth in
broth probably. The amount of cell attached on the material
surface was much higher than free cell in broth, which means
that enhanced fermentation may be achieved with immobi-
lization. All of the three carrier materials performed better
immobilization efficiency especially for activated carbon and
bagasse.
Fermentation kinetics
The time course profiles of fermentation performances during
experiments were shown in Fig. 3. It could be seen that the
immobilized fermentation presented much higher cell con-
centration and much shorter lagging time than the free cell
fermentation, as shown in Fig. 3(a). There was almost no
lagging phase in the case of fermentation with bagasse and
brick used as carriers. Higher cell concentration during the
biofilm immobilized fermentation elevated utilization rate of
glucose as shown in Fig. 3(b). The glucose concentration
decreased by near zero at 28 h for bagasse and brick, while the
knee point for free cell fermentation appeared at 52 h. The
glucose consumption began immediately and decreased lin-
early until the stable stage in immobilization fermentations.
While in the free cell fermentation, a long lag phase emerged
prior to rapid utilization of glucose. The higher cell concen-
tration also accelerated the accumulation of hydrogen and
11620 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 1 6 1 7 e1 1 6 2 4

Fig. 1 e SEM of carriers' microstructure and biofilm morphology: Original microstructure: (a) activated carbon; (c) bagasse; (e)
brick. Attached biofilm on the surface: (b) activated carbon; (d) bagasse; (f) brick. Biofilm thickness: (g) activated carbon, Cells
moving through the wall: (h) bagasse.

butanol. As shown in Fig. 3(c) and (d), hydrogen and butanol
production were all terminated earlier in the immobilization
fermentations than in the free cell fermentation. For the
hydrogen generation, the terminal moment was at 44 h, 28 h
and 24 h for carrier material, activated carbon, bagasse and
brick, respectively. The corresponding accumulative amount
of hydrogen is 6.55 L/L, 4.77 L/L and 6.07 L/L, respectively.
While in the free cell fermentation, hydrogen emission was
terminated at 56 h with a production of 5.25 L/L. As for butanol
production in Fig. 3(d), the accumulative butanol
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 1 6 1 7 e1 1 6 2 4 11621

after lying under a relative long lag time (almost 32 h) and

Fig. 2 e The suspended cell concentration and immobilized
cell concentration during the experiments.
concentration was 3.83 g/L, 5.80 g/L and 4.92 g/L, at 44 h, 36 h
and 32 h, for activated carbon, bagasse and brick, respectively.
While for the free cell fermentation, butanol production began
accumulated up 5.66 g/L at 52 h.
As principal kinetic parameter of fermentation, cell growth
rate and cell specific growth rate were extracted from the time
course profile of cell growth (Fig. 3(a)). The cell growth rates
and corresponding specific cell growth rates of the fermen-
tations were shown in Table 1. It could be seen that the cell
growth rates and corresponding specific cell growth rates
during fermentation with immobilized cell were both higher
than those during fermentation with suspended cell. The
highest the cell growth rates and specific cell growth rates
could be achieved during fermentation with bagasse as cell
immobilization carrier. The glucose consumption rates were
also calculated based on time course profiles in Fig. 3(b), which
were 0.74 g/L/h, 0.82 g/L/h and 0.85 g/L/h, with activated car-
bon, bagasse and brick used as carriers, respectively. During
free cell fermentation, the glucose consumption rate was
0.67 g/L/h. It suggests that the cell biofilm immobilization had
enhanced the fermentation. As shown in Fig. 3(a), although

Fig. 3 e Time course profiles of immobilization fermentations and suspended fermentation. (a) cell concentration; (b)
glucose concentration; (c) hydrogen accumulated production; (d) butanol concentration; (e) pH.
11622 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 1 6 1 7 e1 1 6 2 4
Table 1 e Kinetic parameters of the four fermentations.
Carriers Cell growth Cell specific
rate (g/L/h) growth rate (1/h)
Productivity (mL/L/h)
Free cell 0.013 0.03 93.7
Activated carbon 0.117 0.08 148.8
Bagasse 0.554 0.25 168.5
Brick 0.353 0.24 251.4
the cell concentration for the carrier bagasse was much higher
than for brick, the glucose consumption rate for bagasse was a
little lower than for brick. This might be attributed to the re-
sidual sucrose in the bagasse, or even the fermentable carbon
substances from hydrolysis of cellulose or semi-cellulose in
bagasse. From Fig. 3(c) and (d), the average hydrogen and
butanol productivity, hydrogen and butanol yield were
calculated and were shown in Table 1. It can be seen that the
immobilization fermentation had significative higher
hydrogen production and yield than the free cell fermenta-
tion. The immobilization fermentation for brick achieved
highest hydrogen productivity and yield, and on the other
hand, the immobilization fermentation for bagasse achieved
highest butanol productivity and yield.
Based on those kinetic parameters in Table 1, it could be
concluded that the cell concentration, the carbon source
consumption rate, hydrogen production and butanol produc-
tion had been promoted to some extent during the immobi-
lization fermentation over free cell fermentation. It was
reported that most of the genes (hydGEF, hydA1,hydA2, fhuBDC,
CA_P0141-CA_P0142) for participating the coding of hydroge-
nase, NADH-ferredoxin oxidoreductase and ferredoxin were
up-regulated with cell immobilization during C. acetobutylicum
fermentation [9,38e41]. This would be considered as an
Fig. 4 e Time course profiles of hydrogen and butanol production rates: (a) free cell; (b) activated carbon as carrier; (c) bagasse
as carrier; (d) brick as carrier.
Hydrogen Butanol
Yield (mol/mol) Productivity (g/L/h) Yield (g/g)
1.15 0.109 0.15
1.44 0.084 0.10
1.42 0.161 0.22
1.81 0.130 0.19
explanation for higher hydrogen productivity and yield in the
experiments.
Coproduction of hydrogen and butanol
The productivity time course profiles of hydrogen and butanol
were illustrated in Fig. 4. Of all the fermentations, highest
hydrogen productivity 403.2 mL/L/h and butanol productivity
0.44 g/L/h were achieved, when brick and bagasse used as the
carrier. As shown in Fig. 4(a), the hydrogen production and
butanol production during free cell fermentation were
finished almost in the same duration from 20 h to 60 h, which
indicated the hydrogen production and butanol production
were possibly synchronized. While in the immobilization
fermentations, as shown in Fig. 4(b), (c), (d), the production of
hydrogen and butanol behaved some asynchronous in time
phase and the main production stage of hydrogen came
earlier than butanol. According to the metabolic pathway
described generally [9], hydrogen was indeed generated dur-
ing the acidogenesis in the early stage of fermentation. The
solventogenesis would begin and ABE (acetone, butanol and
ethanol) could start to accumulate when the acids accumu-
lated to a certain value with the pH dropped to about 4.5, as
shown in Fig. 3(d) and (e). Particularly, the microbial cell
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 1 6 1 7 e1 1 6 2 4
Table 2 e The molar ratio of hydrogen and butanol production in four fermentations.
Carriers Hydrogen
production (mol/L)
Free cell 0.235
Activated carbon 0.293
Bagasse 0.213
Brick 0.271
biofilm (as shown in previous Fig. 1(g)) was growing on the
carrier surface and would provide a larger resistance against
extracellular butanol diffusion. The asynchronous behaviors
presented slightly difference for different carriers. For acti-
vated carbon and brick as inorganic materials, butanol emis-
sion was lagging more distinctly than hydrogen emission. As
showed in Table 2, the molar ratio of hydrogen and butanol of
the four fermentations was extracted as 3.067, 5.650, 2.713 and
4.076 in the free cell fermentation and in the activated carbon,
bagasse and brick immobilized fermentation, respectively.
Compared with free cell fermentation, the hydrogen produc-
tion was enhanced when activated carbon and brick used for
immobilizing, while the butanol production was improved
when bagasse was used as the carrier material. It seems that
there was some dissimilarity in hydrogen and butanol pro-
duction between inorganic and organic carrier materials.
It was said that, activated carbon was able to adsorb and
accumulate butanol in its pores, leading to a higher butanol
concentration surround the biofilm. In this case, butanol
production could be inhibited. With mapping the metabolic
pathway of C. acetobutylicum, it had been known well that the
glucose was firstly converted into pyruvate through embden-
meyerhof-parnas pathway (EMP), and then pyruvate was
converted to acetyleCoA by pyruvateeferredoxin oxidore-
ductase (PFOR) with concomitant conversion of oxidized
ferredoxin to its reduced form, FdH2. In acidogenesis, FdH2
was liable to be oxidized by hydrogenase to release hydrogen.
While in solventogenesis, another enzyme, ferredox-
ineNAD(P) reductase, competed with hydrogenase to oxidize
FdH2 and convert NAD(P) to NAD(P)H [42e44]. In other words,
the electrons derived from the phosphoroclastic cleavage of
pyruvate were transferred into hydrogen in the acid-
producing metabolism. In the solvent-producing phase, elec-
trons were transferred into NADPH, which was mainly used in
the butanol production. The residual sucrose in the bagasse
resulted in a higher and quick accumulation of acetic acid and
butyric acid leading to the earlier coming of solventogenesis
when bagasse was used for immobilizing. Consequently, more
electrons derived from the phosphoroclastic cleavage of py-
ruvate could flow into butanol, and hydrogen production
would be declined. For the brick immobilized fermentation,
Fe2O3 contained in brick may be dissolved by the fermentation
broth, and the Fe3þ would have chance to participate in the
generation of hydrogenase and ferredoxin, and improve
further the amount and activity of the two enzymes [45].
Conclusions
Experimental fermentation for coproduction of hydrogen and
butanol has been carried out with attaching C. acetobutylicum
11623
Butanol Hydrogen:Butanol
production (mol/L)
0.076 3.067
0.052 5.650
0.078 2.713
0.067 4.076
on porous particulate carriers of activated carbon, bagasse
and brick to form biofilm. The glucose consumption rate,
microbial amount as well as hydrogen or butanol production
during immobilized cell-fermentation were enhanced more or
less. The results showed the potential of cell reuse according
to the number of attached cells after immobilized fermenta-
tion. And the problem of “cell losing” in CSTR would be solved
to some degree if carriers could participate in CSTR fermen-
tation. The accumulative amount, productivity and yield of
hydrogen and butanol production presented some interesting
features. It seems that there was some dissimilarity in
hydrogen and butanol production between inorganic and
organic carrier materials and the dissimilarity could be a
reference when either hydrogen or butanol was the only
objective product.
Acknowledgements
The present work was supported by the National Nature Sci-
ence Foundation of China (No. 21808144) and the Funda-
mental Research Funds for the Central Universities (No.
20822041B4013).
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