Biohydrogen production from beet molasses by sequential

dark and photofermentation

Ebru O

zgu r
a,
*, Astrid E. Mars
b
, Begu m Peksel
c
, Annemarie Louwerse
b
, Meral Yu cel
c
,
Ufuk Gu ndu z
c
, Pieternel A.M. Claassen
b
,
_
Inci Erog lu
a
a
Middle East Technical University, Department of Chemical Engineering, 06531, Ankara, Turkey
b
Wageningen UR, Agrotechnology & Food Sciences Group, Wageningen UR, P.O. Box 17, 6700 AA Wageningen, The Netherlands
c
Middle East Technical University, Department of Biology, 06531, Ankara, Turkey
a r t i c l e i n f o
Article history:
Received 19 August 2009
Received in revised form
27 October 2009
Accepted 28 October 2009
Available online 20 November 2009
Keywords:
Biohydrogen
Dark fermentation
Photofermentation
Molasses
a b s t r a c t
Biological hydrogen production using renewable resources is a promising possibility to
generate hydrogen in a sustainable way. In this study, a sequential dark and photo-
fermentation has been employed for biohydrogen production using sugar beet molasses as
a feedstock. An extreme thermophile Caldicellulosiruptor saccharolyticus was used for the
dark fermentation, and several photosynthetic bacteria (Rhodobacter capsulatus wild type,
R. capsulatus hup

mutant, and Rhodopseudomonas palustris) were used for the photo-

fermentation. C. saccharolyticus was grown in a pH-controlled bioreactor, in batch mode, on
molasses with an initial sucrose concentration of 15 g/L. The inuence of additions of NH
4

and yeast extract on sucrose consumption and hydrogen production was determined. The
highest hydrogen yield (4.2 mol of H
2
/mol sucrose) and maximum volumetric productivity
(7.1 mmol H
2
/L
c
.h) were obtained in the absence of NH
4

. The efuent of the dark

fermentation containing no NH
4

was fed to a photobioreactor, and hydrogen production

was monitored under continuous illumination, in batch mode. Productivity and yield were
improved by dilution of the dark fermentor efuent (DFE) and the additions of buffer, iron-
citrate and sodium molybdate. The highest hydrogen yield (58% of the theoretical hydrogen
yield of the consumed organic acids) and productivity (1.37 mmol H
2
/L
c
.h) were attained
using the hup

mutant of R. capsulatus. The overall hydrogen yield from sucrose increased

from the maximum of 4.2 mol H
2
/mol sucrose in dark fermentation to 13.7 mol H
2
/mol
sucrose (corresponding to 57% of the theoretical yield of 24 mol of H
2
/mole of sucrose) by
sequential dark and photofermentation.
2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Today, the energy supply of the worlds population mostly
depends on limited resources of fossil fuels. The utilization of
fossil fuels causes global climate change due to emissions of
greenhouse gasses during their combustion. Hydrogenhas the
potential to be a clean energy carrier, provided that it is
produced in a renewable way, since it only produces water
upon combustion. Among the different technologies
employed, fermentative biohydrogen production offers an
opportunity to utilize renewable resources, including agri-
cultural waste products, which facilitates waste recycling.
Sequential dark and photofermentation of organic
compounds is a promising method of producing renewable
hydrogen. During dark fermentation, sugars are converted to
H
2
, CO
2
and short-chain organic acids with a theoretical
* Corresponding author. Tel.: 90 312 2102696; fax: 90 312 2102600.
E-mail address: bebru@metu.edu.tr (E. O

zgu r).
Avai l abl e at www. sci encedi r ect . com
j our nal homepage: www. el sevi er . com/ l ocat e/ he
i nt e r na t i o na l j o ur na l o f hy d r og e n e ne r gy 3 5 ( 2 0 1 0 ) 5 1 1 5 1 7
0360-3199/$ see front matter 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2009.10.094
maximumhydrogen yield of 4 mol of H
2
/mole of hexose sugar,
when all sugars are fermented to acetate, CO
2
and H
2
. In
practice, the hydrogen yields are lower due to the formationof
biomass, and more reduced alternative fermentationproducts
like lactate, ethanol, and butyrate. The hydrogen yields are
generally higher when thermophiles are applied due to more
favorable thermodynamics for hydrogen production at
elevated temperatures [13]. The efuent of the dark
fermentation is used as the substrate for photosynthetic
bacteria during the second photofermentative step, in which
short-chain organic acids are assimilated to H
2
when light is
present, producing maximally 4 and 6 mol of H
2
per mole of
acetate and lactate, respectively. Because of this, the
combined two-step process has theoretical maximum
hydrogen yield of 12 mol of H
2
/mole of hexose sugar. This
process is being developed within the EU 6th framework
project HYVOLUTION [4].
Studies with pure sugars showed that the overall yield of
H
2
can be improved by combining the dark fermentation with
the photofermentation [57]. The yields achieved by two-step
processes on pure sugars range from 2.4 to 6.3 mol H
2
/mol
glucose [8]. However, studies from real feedstocks are rather
limited [9,10]. In the present study, the overall hydrogen yield
was improved to 13.7 mol of H
2
/mol of sucrose by the
sequential operation of dark fermentation and photo-
fermentation using sugar beet molasses as feedstock. Sugar
beet molasses is a by-product of the sugar industry that is
obtained as a thick syrup during the crystallization of sucrose.
It contains a high amount of sucrose (ca. 50%), and is rich in
organic nitrogen, vitamins and salt that may support bacterial
growth. It has been described as a suitable feedstock for dark
fermentations under mesophilic conditions [1115]. In this
study, an efcient H
2
production from sugar beet molasses by
sequential operation of dark and photofermentation was
aimed. The dark fermentation was operated under extreme
thermophilic conditions. Caldicellulosiruptor saccharolyticus
was chosen as the hydrogen producing bacterium since the
hydrogenyields that are obtained with this species are usually
very high [16,17]. Several photosynthetic bacteria (Rhodobacter
capsulatus wild type, R. capsulatus hup

mutant, and Rhodop-

seudomonas palustris) were applied during the photo-
fermentative step. The effects of additions of buffer and
nutrients on the hydrogen productivity and yield were
investigated.
2. Materials and methods
2.1. Microorganisms and culture conditions
C. saccharolyticus DSM 8903 was obtained from the Deutsche
Sammlung von Microorganismen und Zellkulturen (DSMZ). It
was grownovernight in small serumasks onsucrose at 70

C.
The growth medium contained per liter: 0.3 g KH
2
PO
4
, 0.3 g
K
2
HPO
4
, 2.5 mg FeCl
3
$6H
2
O, 1 ml SL-10 trace element solution
(www.dsmz.de), 0.5 mg resazurin, 0.4 g MgCl
2
$6H
2
O, 0.75 g
cysteine-HCl, 4 g sucrose, 0.45 g NH
4
Cl, 0.5 g YE (Duchefa, The
Netherlands), and 50 mMof 3-(N-morpholino)propanesulfonic
acid (pH 7.0). The fermentation was started by inoculating
with 10% of a pre-culture into molasses media.
R. capsulatus (DSM1710) and R. palustris (DSM127) were
obtained from Deutsche Sammlung von Mikroorganismen
und Zellkulturen GmbH, Germany. The mutant strain of
R. capsulatus (YO3) lacking uptake hydrogenase gene (hup

)
was obtained previously in our laboratory [18]. Bacteria were
activated in modied SB medium [19] containing acetate
(20 mM) and glutamate (10 mM) as carbon and nitrogen
sources, and 20 mM of potassium phosphate buffer, pH 6.4.
Ten percent inoculation of grown culture was carried out into
H
2
production media of molasses DFE.
2.2. Dark fermentation
Dark fermentation was performed in a pH-controlled jacketed
2-L bioreactor (Applikon, The Netherlands) with a working
volume of 1 L at 72 1

C. The medium contained per Litre:
0.3 g KH
2
PO
4
, 0.3 g K
2
HPO
4
, 2.5 mg FeCl
3
$6H
2
O, 1 ml SL-10 trace
element solution (www.dsmz.de), 0.5 mg resazurin, 0.4 g
MgCl
2
$6H
2
O, 0.75 g cysteine-HCl, and 27.5 g of molasses,
which contained 15 g of sucrose. When applicable, 0.9 g NH
4
Cl
and 1 g of YE (Duchefa, The Netherlands) were added. The pH
was maintained at 6.9 0.1 (measured at room temperature)
by the automatic addition of 2 M of NaOH. The fermentation
was started by inoculating with 10% of a pre-culture that was
grown overnight in small serum asks on sucrose at 70

C.
Immediately after inoculation, the cultures were stirred at
100 rpm and sparged with nitrogen at 1 l/h. When the
hydrogen concentration in the off-gas became >0.11%, the
stirring speed was increased to 350 rpm, and sparging with
nitrogen was increased to 7 l/h.
2.3. Photofermentation
Glass bottles with 55 ml liquid volume were used for photo-
biological hydrogen production. The hydrogen gas produced
by the bacteria was collected from the top by a thin hollow
tube into a graded glass cylinder initially lled with water
which was replaced by the hydrogen produced during the
process. The photobioreactors were maintained at 3033

C in
an incubator. The illumination was provided by 100 W tung-
sten lamp, adjusted to provide a uniform light intensity of
150200 W/m
2
at the surface of the reactor. The initial pH of
the medium was 6.7.
Dark fermentor efuent (DFE) was centrifuged and steril-
ized by autoclaving prior to use, to remove contaminants and
any colloidal materials that may interfere with light penetra-
tion. Several adjustments were carried out to optimize the
conditions for photofermentative hydrogen production from
DFE: the organic acid concentration of the DFE was decreased
3, 5 and 10 times by diluting the DFE with sterile distilled
water, 20 mM of potassium phosphate buffer (pH 6.4) was
added to the DFE to maintain the pH in a proper range for
photofermentation, and iron (Fe-Citrate, 0.1 mM) and molyb-
denum (Na
2
MoO
4
.2H
2
O, 0.16 mM) were added.
2.4. Analytical methods
The concentrations of H
2
and CO
2
in the headspace of the dark
fermentor culture, and the concentrations of sucrose, ethanol,
and organic acids in the culture supernatant were determined
i nt e r na t i ona l j o ur na l o f hy d r o g e n e ne r g y 3 5 ( 2 0 1 0 ) 5 1 1 5 1 7 512
as previously described [15]. The cell density in the dark
fermentor was determined by the micro-biuret assay with
bovine serum albumin as a standard [20]. For this, cells were
harvested by centrifugation and washed prior to protein
analysis. The molecular weight of C. saccharolyticus was
previously determined to be 24.6 g (mol C)
1
[15]. The cell dry
weight of C. saccharolyticus is similar to the protein content as
determined with the micro-biuret assay (unpublished data).
The total carbon and total nitrogen contents of the DFE of
molasses were analyzed spectrophotometrically (DR/2400,
Hach-Lange, Germany). A detailed elemental composition of
the efuent was determined by atomic absorption spectros-
copy (Philips, PU9200X). The evolved hydrogen during photo-
fermentation was measured by gas chromatography (Agilent
Technologies 6890N) equipped with a thermal conductivity
detector and a Supelco Carboxen 1000 column (60/80 mesh).
The oven, injector and detector temperatures were 140, 160
and 170

C, respectively. Argon was used as a carrier gas at
a ow rate of 25 ml/min. The organic acid concentrations in
the culture medium of the photofermentation were analyzed
by HPLC on a MetaCarb 87H column (300 7.8 mm, by Varian
ProStar). The biomass concentration in the photofermentor
was determined by measuring the optical density at 660 nm
with a visible spectrophotometer (Shimadzu UV-1201V). An
optical density of 1 at 660 nm corresponds to a cell density of
0.55 g dry weight/L.
The molar yield of the photofermentation was calculated
as the percent of the ratio of moles of hydrogen that was
produced to the theoretical moles of hydrogen that can be
produced fromutilized organic acids (acetate and lactate). The
stoichiometric equations for hydrogen production from
acetate and lactate are given below:
Acetate : C
2
H
4
O
2
2H
2
O/4H
2
2CO
2
(1)
Lactate : C
3
H
6
O
3
3H
2
O/6H
2
3CO
2
(2)
The light conversion efciency was determined as the ratio
of the total energy value of the hydrogen that has been
obtained to the total energy input to the photobioreactor by
light radiation. It is calculated by the following equation:
h% 33:6 r
H2
V
H2
100=I A t (3)
where V
H2
is the volume of produced H
2
in l, r
H2
is the density
of the produced hydrogen gas in g/L, I is the light intensity in
W/m
2
, A is the irradiated area in m
2
and t is the duration of
hydrogen production in hours [21].
3. Results and discussion
3.1. Dark fermentation
C. saccharolyticus was grown in pH-controlled bioreactors on
molasses with an initial sucrose concentration of 15 g/L. The
inuence of additions of NH
4

and YE on sucrose consumption

and hydrogen production was determined. When NH
4

was
present in the growth medium, the specic hydrogen
production rate was 20 mmol of H
2
/g protein.h at the start of
exponential production of hydrogen. The consumption of
sucrose and production of hydrogen were largely completed
within 11.5 day (Fig. 1A, B). Only a limited amount of NH
4

was
consumed when YE was added to the culture medium, but
this increased to 33% when YE was omitted (Table 1), sug-
gesting that C. saccharolyticus mainly uses YE as nitrogen
source when available. The addition of YE is necessary to
obtain good growth of C. saccharolyticus on pure sugars.
However, the fermentation patterns on molasses were similar
in the presence and absence of YE (Fig. 1A,B), indicating that
molasses can eliminate the need for expensive YE in the
fermentation medium of C. saccharolyticus.
When NH
4

was omitted from the medium, over 2 days

were required to complete the fermentations (Fig. 1C, D).
When YE was present, the specic hydrogen production rate
at the start of exponential production of hydrogen was equal
to the rates observed during the fermentations with NH
4

(Table 1). C. saccharolyticus was also able to grow without NH

4

and without YE, Apparently, molasses contains sufcient

nutrients to support growth in the absence of NH
4

and YE,
although hardly any hydrogen was produced and growth was
very slow (Fig. 1D; Table 1).
C
0 1 2 3
time (days)
0 1 2 3
time (days)
0.0
0.3
0.6
c
e
l
l

d
e
n
s
i
t
y
(
g

p
r
o
t
e
i
n
/
L
)
c
e
l
l

d
e
n
s
i
t
y
(
g

p
r
o
t
e
i
n
/
L
)
50
100
150
200
m
m
o
l
/
L
0.0
0.3
0.6
0.9
0.0
0.3
0.6
c
e
l
l

d
e
n
s
i
t
y
(
g

p
r
o
t
e
i
n
/
L
)
c
e
l
l

d
e
n
s
i
t
y
(
g

p
r
o
t
e
i
n
/
L
)
0.0
0.3
0.6
0.9
+ YE + NH
4
+

YE

NH
4
+

YE + NH
4
+
+ YE

NH
4
+
0
50
100
150
m
m
o
l
/
L
0
50
100
150
200
m
m
o
l
/
L
0
50
100
150
m
m
o
l
/
L
0
A
D
B
Fig. 1 Fermentation proles of batch cultivations of C. saccharolyticus in pH-controlled bioreactors on molasses (15 g/L
sucrose) in the presence and absence of NH
4
D
and YE. The production of H
2
(B), acetate (C), lactate (-), and cell biomass (A),
and the consumption of sucrose (>) are indicated.
i nt e r na t i o na l j o ur na l o f hy d r og e n e ne r gy 3 5 ( 2 0 1 0 ) 5 1 1 5 1 7 513
Large amounts of lactate were produced besides acetate
during all batch fermentations, which reduced the molar
hydrogen yields to 1055% of the theoretical maximum of
8 mol of H
2
per mol of sucrose. Furthermore, low amounts of
ethanol were observed in some of the cultures (Table 1), but
other organic acids, like butyrate or succinate were not
detected. The highest hydrogen yield (4.2 mol of H
2
/mol
sucrose) and maximum hydrogen productivity (7.1 mmol of
H
2
/L
c
.h) were obtained with the fermentation on molasses
with YE without NH
4

(Table 1). Previously, a very high

hydrogen yield of 3.3 mol of H
2
/mol hexose was obtained with
batch cultivations of C. saccharolyticus on hydrolysate of the
lignocellulosic energy crop Miscanthus at a similar initial sugar
concentration of 14 g/L [16]. The hydrogen yield and produc-
tivity of the dark fermentation with Miscanthus hydrolysate
were considerably higher than those with molasses since the
formation of large amounts of lactate was not observed with
Miscanthus hydrolysate.
3.2. Photofermentation
3.2.1. Suitability of the DFE of molasses for
photofermentation
The ammonium content of the DFE is especially important,
because NH
4

is the inhibitor of the nitrogenase enzyme of

purple non-sulphur bacteria [2225]. No hydrogen production
could be observed from dark fermentor efuents containing
more than 2 mM ammonium (unpublished data). The best
performing dark fermentation on molasses was done without
NH
4

and the DFE of this fermentation is therefore very suit-

able for photofermentative hydrogen production.
The composition of the DFE of molasses was determined,
and compared with the composition of the dened SB
mediumused in our previous studies for hydrogen production
(given in Table 2) [26]. The DFE contained almost ten times
more total carbon and nitrogen than the dened SB medium,
which might require dilution of the efuent since high organic
acid concentrations decreases the yield [27]. The total nitrogen
content was high, despite the absence of NH
4

, due to nitrog-
enous compounds (like amino acids) that are present in the
molasses itself. The carbon to nitrogen ratio, which is
important for efcient hydrogen production, was almost the
same as in SB medium. Some of the elements (like Mn, Zn, Ni,
and Cu) were higher in the DFE of molasses. However, the
amount of iron, which is crucial for nitrogenase activity, was
low in the efuent. Molybdenum, another important cofactor
of the nitrogenases of PNS bacteria, was even undetectable
within the precision of the instrument.
3.2.2. Effect of dilution on hydrogen production by
R. capsulatus on molasses efuent
Hydrogen production was not observed from DFE without
buffering, as the pH increased up to 10.0. Since negatively
charged organic acids are taken into the cell during photo-
fermentation, cells restore their membrane potential by the
efux of negatively charged OH

ions, causing increase in pH.

Uyar et al. [28] have compared the photofermentative
hydrogen production from volatile fatty acids present in dark
fermentation efuents. The organic acids such as malate,
propionate, lactate and butyrate required low buffering
(3.5 mM potassium phosphate), however, acetate containing
medium required 6 times higher amounts of buffer in order to
keep pHat a favorable range for hydrogenproduction. The low
buffering capacity of DFE of molasses is not sufcient for
hydrogen production. Therefore, 20 mM of potassium
Table 1 Fermentation data of batch cultivations of C. saccharolyticus in pH-controlled bioreactors on molasses (15 g/L
sucrose) in the presence and absence of NH
4
D
and YE.
Fermentation NH
4

YE Max Q
H
2
a
mmol H
2
/
(l.h)
q
H
2
b
mmol H
2
/
(g protein.h)
Molar yield (mol/mol sucrose) C-balance Sucrose
consumption
%
NH
4

consumption
%
H
2
CO
2
Acetate Lactate Ethanol
A 6.8 20 2.0 1.5 1.1 2.1 0.3 0.93 97 5
B 4.0 20 2.0 1.4 0.9 2.0 0.3 0.86 97 33
C 7.1 20 4.2 2.1 1.9 1.5 0.0 0.91 94 n.a.
c
D 0.9 3 0.9 0.8 0.7 2.5 0.1 0.88 79 n.a.
c
a Max Q
H2
: maximum volumetric hydrogen productivity.
b q
H2
: specic hydrogen productivity after 6 h of fermentation.
c n.a.: not applicable.
Table 2 The composition of the DFE of molasses
compared with synthetic SB medium.
DFE of Molasses SB Medium
Total N (M) 0.025 0.002
Total C (M) 0.85 0.070
NH
4

N 0 0
C/N molar ratio 34 35
Fe (mM) 0.035 0.1
Mn (mM) 10.5 0.51
Zn (mM) 23.5 0.51
Ni (mM) 3.4 0.084
Mg (mM) 3.65 2
Ca (mM) 1.34 0.3
Co (mM) 3.05 0.84
Cu (mM) 4.7 0.12
Mo (mM) 0 0.16
Acetate (mM) 86 30
Lactate (mM) 69
Sucrose (mM) 2.7
Glutamate(mM) n.a.
a
2
Biotin (ug/L) n.a 15
Thiamin (ug/L) n.a 250
Niacin(ug/L) n.a 250
a n.a.: not available.
i nt e r na t i ona l j o ur na l o f hy d r o g e n e ne r g y 3 5 ( 2 0 1 0 ) 5 1 1 5 1 7 514
phosphate buffer (pH 6.4) was added to the DFE, which
maintained the pH in a tolerable range (6.87.4) for hydrogen
production by PNS bacteria (Fig. 2B). As the total amounts of
carbon and nitrogen of the efuent were high compared to the
dened SB medium (Table 2), the efuent was diluted 10, 5, 3
and 2 times with sterile distilled water to determine the
optimum initial organic acid concentration for efcient
hydrogen production. The hydrogen production on undiluted
DFE was nil (data not shown). Hydrogen production and
growth were observed with all dilutions during photo-
fermentation with the wild type strain of R. capsulatus (DSM
1710), and acetate and lactate were completely consumed
(Fig. 2). The best hydrogen productivity and yield were
obtained with 3 times diluted DFE, with 67 mmol/L
c
of
cumulative hydrogen produced at the end of 5 days of incu-
bation (Table 3, Fig. 2A).
3.2.3. Effect of additions of Fe and Mo on hydrogen production
by different PNS bacterial strains on molasses efuent
Fe and Mo are two essential cofactors for the nitrogenase
activity of photosynthetic purple non-sulphur bacteria. It was
previously reported that additions of Mo and Fe stimulated the
hydrogen production by Rhodobacter sphaeroides, with
optimum concentrations of 0.1 mM of Fe-citrate and 0.16 mM
6
6,5
7
7,5
8
p
H
0
10
20
30
40
50
60
70
80
90
0 1 2 3 4 5 6 7
H
2

(
m
m
o
l
/
L
c
)
Time (days)
0 1 2 3 4 5 6 7
Time (days)
0 1 2 3 4 5 6 7
Time (days)
0 1 2 3 4 5 6 7
Time (days)
0
5
10
15
20
25
30
L
a
c
t
a
t
e

(
m
M
)
0
10
20
30
40
50
A
c
e
t
a
t
e

(
m
M
)
A
B
C D
Fig. 2 Hydrogen production (A), pH change (B), acetate utilization (C) and lactate utilization (D) by R. capsulatus (DSM1710)
during photofermentation on 10 (C), 5 (-) and 3 (:) times diluted DFE of molasses.
Table 3 The effect of dilution and additions of iron (0.1 mM) and molybdenum (0.16 mM) on hydrogen production yield,
productivity and substrate/light conversionefciency by R. capsulatus wild type, R. capsulatus hup
L
mutant and R. palustris.
20 mM of potassium phosphate buffer (pH 6.4) was added to the DFE of molasses. pH was between 6.8 and 7.6 for all
reactors throughout the experiment.
Bacteria Dilution Fe Mo Max
Biomass
(gdw/L
c
)
Max Q
H
2
a
(mmol/L
c
.h)
q
H
2
b
(mmol/g
biomass.h)
Molar Yield
(% of Theoretical
Max)
Light
Conversion
Efciency (%)
R. capsulatus
(DSM1710)
10 0.58 0.08 0.14 30 0.09
5 0.85 0.38 0.45 18 0.19
3 1.21 0.73 0.60 26 0.42
2 2.29 0.80 0.35 30 0.55
3 1.66 1.10 0.66 39 0.66
3 1.48 0.75 0.50 20 0.59
R. capsulatus
hup

(YO3)
3 1.14 0.72 0.63 34 0.49
3 1.03 1.37 1.33 58 0.83
R. palustris
(DSM127)
3 1.44 0.89 0.62 37 0.53
3 1.59 1.16 0.73 46 0.65
a Max Q
H
2
: Maximum volumetric hydrogen productivity.
b q
H
2
: Specic hydrogen productivity.
i nt e r na t i o na l j o ur na l o f hy d r og e n e ne r gy 3 5 ( 2 0 1 0 ) 5 1 1 5 1 7 515
of molybdenum. Besides, the genes regulating nitrogenase
activity were repressed under Fe and Mo starvation [29,30].
Elemental analysis of DFE revealed low levels of Fe and Mo.
Because of this, both cofactors were added to 3 times diluted
DFE, and the effect on hydrogen production was monitored.
For wild type R. capsulatus, the hydrogen production improved
(80 mmol H
2
/L
c
) after addition of Fe (0.1 mM) (Fig. 3). The
productivity was increased from0.73 mmol/L
c
.h to 1.10 mmol/
L
c
.h, and a yield of 39% (acetate and lactate) was achieved
(Table 3). However, addition of Mo (0.16 mM) resulted in
a signicant decrease of the hydrogen production by wild type
R. capsulatus (Fig. 3). In contrast, the addition of Mo largely
improved the hydrogen production by the hup

strain of
R. capsulatus and by R. palustris. The highest hydrogen
productivity (1.37 mmol/L
c
.h) and yield (58%) were attained
withthe hup

strainof R. capsulatus onDFE containing buffer, Fe

and Mo (Table 3). These parameters were all higher than those
obtained using dened SB medium containing acetate (40 mM)
and lactate (7.5 mM) as carbon sources, and glutamate (2 mM)
as nitrogensource, which resulted ina yield and productivity of
19% and 0.49 mmol/L
c
.h, respectively (unpublished data).
The light conversion efciency is a signicant measure of
the energy utilized, and especially critical in indoor experi-
ments. Light conversion efciency decreased from 0.42 to
0.09% with dilution of the DFE from 3X to 10X (Table 3). The
maximum light conversion efciency was estimated as 0.83
that has been obtained for R. capsulatus hup- after 3X dilution
and addition of Fe and Mo. This value is quite comparable to
the results obtained previously ondened mediumcontaining
acetate and lactate mixtures [27].
4. Conclusion
In this study, the feasibility was shown of the sequential
operation of thermophilic dark fermentation and photo-
fermentation with sugar beet molasses as the feedstock using
batch fermentation systems. The hydrogen yields that were
obtained during the thermophilic dark fermentations on
molasses were considerably lower than the theoretical
maximum due to the formation of large amounts of lactate,
with the highest yield being 4.2 mol of H
2
/mol sucrose. The
efuent of this fermentation did not contain NH
4

and was
therefore a very suitable hydrogen production medium for
photosynthetic purple non-sulphur bacteria, provided that
the efuent was diluted and buffered to keep the pH at
a tolerable range.
The DFE of molasses contained acetate and lactate as the
major carbon sources for photofermentation. Since lactate
was completely consumed by the PNS bacteria, the reducing
equivalents that were consumed for the production of lactate
during the dark fermentation were recovered as hydrogen
during the photofermentation, restoring the overall hydrogen
yield of the process.
The additions of Fe and Mo signicantly improved the
hydrogen production by hup

R. capsulatus and R. palustris. The

best hydrogen production was attained with hup

R. capsulatus
on 3 times diluted molasses efuent supplemented with
adequate amounts of buffer, Fe and Mo.
When the hup

strain of R. capsulatus was used during

photofermentation, an overall hydrogen yield of the two-step
fermentation process of 13.7 mol of H
2
/mol of sucrose was
obtained, which corresponds to 57% of the theoretical yield of
24 mol of H
2
/mole of sucrose. Further investigation of
sequential dark and photofermentation for hydrogen
production on molasses should be based on the continuous
operations.
Acknowledgements
This study was nancially supported by the Commission of
the European Communities, Sixth Framework Programme,
Priority 6, Sustainable Energy Systems (019825 HYVOLUTION).
r e f e r e n c e s
[1] de Vrije T, Claassen PAM. Dark hydrogen fermentations. In:
Reith JH, Wijffels RH, Barten H, editors. Bio-methane & bio-
hydrogen. Status and perspectives of biological methane
and hydrogen production. The Hague: Smiet Offset; 2003.
p. 10323.
[2] Hallenbeck PC. Fundamentals of the fermentative
production of hydrogen. Water Sci Technol 2005;52:219.
[3] Kengen SWM, Goorissen HP, Verhaart M, van Niel EWJ,
Claassen PAM, Stams AJM. Biological hydrogen production
by anaerobic microorganisms. In: Soetaert W, Vandamme EJ,
editors. Biofuels. John Wiley and Sons; 2009.
[4] Claassen PAM, de Vrije T. Non-thermal production of pure
hydrogen from biomass: HYVOLUTION. Int J Hydrogen
Energy 2006;31:141623.
[5] Nath K, Kumar A, Das D. Hydrogen production by Rhodobacter
sphaeroides strain O.U.001 using spent media of Enterobacter
cloacae DM11. Appl Microbiol Biotechnol 2005;68:53341.
[6] Tao Y, Chen Y, Wu Y, He Y, Zhou Z. High hydrogen yield from
a two-step process of dark and photofermentation of
sucrose. Int J Hydrogen Energy 2007;32:2006.
0
20
40
60
80
100
140
120
160
0 1 2 3 4 5 6 7 8 9
Time (days)
H
2

(
m
m
o
l
/
L
c
)
Fig. 3 Effect of iron (0.1 mM) and Mo (0.16 mM) addition on
H
2
production by PNS bacteria on modied molasses
efuent. The efuent was diluted 3 times and all reactors
contained 20 mM of KP
i
buffer at pH 6.4. H
2
production by
(D)wild type R. capsulatus with Fe addition, (:)wild type R.
capsulatus with Fe and Mo addition, (B)hup
L
mutant of R.
capsulatus with Fe addition, (C)hup
L
mutant of R.
capsulatus with Fe and Mo addition,(,)R. palustris with Fe
addition,(-)R. palustris with Fe and Mo addition.
i nt e r na t i ona l j o ur na l o f hy d r o g e n e ne r g y 3 5 ( 2 0 1 0 ) 5 1 1 5 1 7 516
[7] Nath K, Muthukumar M, Kumar A, Das D. Kinetics of two-
stage fermentation process for the production of hydrogen.
Int J Hydrogen Energy 2008;33:1195203.
[8] Liu BF, Ren NQ, Ding FXJ, Zheng GX, Guo WQ, Xu JF, et al.
Hydrogen production by immobilized R. faecalis RLD-53 using
soluble metabolites from ethanol fermentation bacteria E.
harbinense B49. Bioresour Technol 2009;100:271923.
[9] Uyar B, Schumacher M, Gebicki J, Modigell M.
Photoproduction of hydrogen by Rhodobacter capsulatus from
thermophilic fermentation efuent. Bioprocess Biosyst Eng
2009;32:6036.
[10] Su H, Cheng J, Zhou J, Song W, Cen K. Improving hydrogen
production of dark and photo fermentation. Int J Hydrogen
Energy 2009;34:17806.
[11] Li JZ, Zheng GC, He JG, Chang S, Qin Z. Hydrogen-producing
capability of anaerobic activated sludge in three types of
fermentations in a continuous stirred-tank reactor.
Biotechnol Adv 2009;27:5737.
[12] Guo WQ, Ren NQ, Wang XJ, Xiang WS, Meng ZH, Ding J, et al.
Biohydrogen production from ethanol-type fermentation of
molasses in an expanded granular sludge bed (EGSB) reactor.
Int J Hydrogen Energy 2008;33:49818.
[13] Ren N, Li J, Li B, Wang Y, Liu S. Biohydrogen production from
molasses by anaerobic fermentation with a pilot-scale
bioreactor system. Int J Hydrogen Energy 2006;31:214757.
[14] Ren NQ, Xing DF, Rittmann BE, Zhao LH, Xie TH, Zhao X.
Microbial community structure of ethanol type
fermentation in bio-hydrogen production. Environ
Microbiol 2007;9:111225.
[15] Aceves-Lara CA, Latrille E, Bufere P, Bernet N, Steyer JP.
Experimental determination by principal component
analysis of a reaction pathway of biohydrogen production by
anaerobic fermentation. Chem Eng Process 2008;47:196875.
[16] de Vrije T, Mars AE, Budde MAW, Lai MH, Dijkema C, de
Waard P, et al. Glycolytic pathway and hydrogen yield
studies of the extreme thermophile Caldicellulosiruptor
saccharolyticus. Appl Microbiol Biotechnol 2007;74:135867.
[17] de Vrije T, Bakker RR, Budde MAW, Lai MH, Mars AE,
Claassen PAM. Efcient hydrogen production from the
lignocellulosic energy crop Miscanthus by the extreme
thermophilic bacteria Caldicellulosiruptor saccharolyticus and
Thermotoga neapolitana. Biotechnol Biofuels 2009;2:12. doi:10.
1186/1754-6834-2-12.
[18] O

ztu rk Y, Yu cel M, Daldal F, Mandac S, Gu ndu z U, Tu rker L,

et al. Hydrogen production by using Rhodobacter capsulatus
mutants with genetically modied electron transfer chains.
Int J Hydrogen Energy 2006;31:154552.
[19] Bieble H, Pfennig N. Isolation of members of the family
Rhodosprillaceae. 1981. In: Starr MP, Stolp H, Tru per HG,
Balows A, Schlegel HG, editors. The prokaryotes, vol. 1. New
York: Springer; 1981. p. 26773.
[20] Goa J. A micro biuret method for protein determination.
Determination of total protein in cerebrospinal uid. Scand J
Clin Lab Invest 1953;5:21822.
[21] Uyar B, Erog lu I, Yu cel M, Gu ndu z U, Tu rker L. Effect of light
intensity, wavelength and illumination protocol on hydrogen
production in photobioreactors. Int J Hydrogen Energy 2007;
32:46707.
[22] Yakunin AF, Hallenbeck PC. Short term regulation of
nitrogenase activity by NH
4

in Rhodobacter capsulatus:
multiple in vivo nitrogenase responses to NH
4

addition.
J Bacteriol 1998;180(23):63925.
[23] Yakunin A, Laurinavichene TV, Tsygankov A, Hallenbeck PC.
The presence of ADP-ribosylated Fe protein of nitrogenase in
Rhodobacter capsulatus is correlated with cellular nitrogen
status. J Bacteriol 2003;181(7):19942000.
[24] Drepper T, Gro S, Yakunin AF, Hallenbeck PC, Masepohl B,
Klipp W. Role of GlnB and GlnK in ammonium control of both
nitrogenase systems in the phototrophic bacterium
Rhodobacter capsulatus. Microbiology 2003;149:220312.
[25] Akko se S, Gu ndu z U, Yu cel M, Erog lu I. Effects of ammonium
ion, acetate and aerobic conditions on hydrogen production
and expression levels of nitrogenase genes in Rhodobacter
sphaeroides O.U.001. Int J Hydrogen Energy 2009;34:881827.
[26] Erog lu I, Tabanog lu A, Gu ndu z U, Erog lu E, Yu cel M.
Hydrogen production by Rhodobacter sphaeroides O.U.001 in
a at plate solar bioreactor. Int J Hydrogen Energy 2008;33:
53141.
[27] O

zgu r E, Uyar B, O

ztu rk Y, Yu cel M, Gu ndu z U, Erog lu I.

Biohydrogen production by Rhodobacter capsulatus on acetate
at uctuating temperatures. Resour Conserv Recy 2009;. doi:
10.1016/j.resconrec.2009.06.002.
[28] Uyar B, Eroglu I, Yu cel M, Gu ndu z U. Photofermentative
hydrogen production from volatile fatty acids present in
dark fermentation efuents. Int J Hydrogen Energy 2008;34:
451723.
[29] Kars G, Gu ndu z U, Yu cel M, Tu rker L, Erog lu
_
I. Hydrogen
production and transcriptional analysis of nifD, nifK and
hupS genes in Rhodobacter sphaeroides O.U.001 grown in
media with different concentrations of molybdenum and
iron. Int J Hydrogen Energy 2006;31:153644.
[30] Zhu H, Fang HHP, Zhang T, Beauddette LA. Effect of ferrous
ion on photo heterotrophic hydrogen production by
Rhodobacter sphaeroides. Int J Hydrogen Energy 2007;32:41128.
i nt e r na t i o na l j o ur na l o f hy d r og e n e ne r gy 3 5 ( 2 0 1 0 ) 5 1 1 5 1 7 517