Journal of Biotechnology 85 (2001) 25 – 33
www.elsevier.com/locate/jbiotec
Acetate as a carbon source for hydrogen production by
photosynthetic bacteria
Maria J. Barbosa *, Jorge M.S. Rocha, Johannes Tramper, René H. Wijffels
Food and Bioprocess Engineering Group, Wageningen Uni6ersity, P.O. Box 8129, 6700 EV Wageningen, The Netherlands
Received 15 March 2000; received in revised form 11 September 2000; accepted 25 September 2000
Abstract
Hydrogen is a clean energy alternative to fossil fuels. Photosynthetic bacteria produce hydrogen from organic
compounds by an anaerobic light-dependent electron transfer process. In the present study hydrogen production by
three photosynthetic bacterial strains (Rhodopseudomonas sp., Rhodopseudomonas palustris and a non-identified
strain), from four different short-chain organic acids (lactate, malate, acetate and butyrate) was investigated. The
effect of light intensity on hydrogen production was also studied by supplying two different light intensities, using
acetate as the electron donor. Hydrogen production rates and light efficiencies were compared. Rhodopseudomonas sp.
produced the highest volume of H2. This strain reached a maximum H2 production rate of 25 ml H2 l − 1 h − 1, under
a light intensity of 680 mmol photons m − 2 s − 1, and a maximum light efficiency of 6.2% under a light intensity of 43
mmol photons m − 2 s − 1. Furthermore, a decrease in acetate concentration from 22 to 11 mM resulted in a decrease
in the hydrogen evolved from 214 to 27 ml H2 per vessel. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: Acetate; Hydrogen; Light efficiency; Organic acids; Photosynthetic bacteria
1. Introduction
There are limited reserves of fossil fuels on
Earth, and the combustion of the fuels leads to
serious problems such as global climate changes.
For this reason, much attention is presently being
given to the development of clean, sustainable
energy systems, with the potential to supplement
and even substitute the fossil-fuel-based energy
* Corresponding author. Tel.: +31-317-485096; fax: + 31317-482237.
E-mail address: [email protected] (M.J.
Barbosa).
production. One of the alternatives, as an environmentally acceptable fuel, is molecular hydrogen. Hydrogen is a clean fuel yielding only water
after combustion.
Photosynthetic bacteria can produce hydrogen
at the expense of solar energy and small-chain
organic acids as electron donors. The combination of photosynthetic bacteria with anaerobic
bacteria can provide a system for hydrogen photoproduction from residual carbohydrates, e.g.
from organic wastes (Mao et al., 1986; Sasaki,
1998). In such a system, anaerobic fermentation
of organic wastes produces intermediates like lowmolecular-weights organic acids in a first step,
0168-1656/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 6 5 6 ( 0 0 ) 0 0 3 6 8 - 0
26
M.J. Barbosa et al. / Journal of Biotechnology 85 (2001) 25–33
which are then converted to hydrogen by photosynthetic bacteria at the expense of light energy,
in a second step. The conversion efficiency of light
energy to hydrogen, with the supply of an appropriate carbon source, are the key factors for hydrogen production by biological systems (Hillmer
and Gest, 1977).
The conversion of malate and lactate to hydrogen by photosynthetic bacteria is well documented
(Zürrer and Bachofen, 1979; Kim et al., 1981,
1987; Miyake and Kawamura, 1987; Fascetti and
Todini, 1995; Sasikala et al., 1997; Ike et al.,
1997a,b; Tsygankov et al., 1998). The main products of anaerobic fermentation are acetic and
butyric acids (Segers et al., 1981). Little is known
about the conversion of acetic and butyric acids
to hydrogen by photosynthetic bacteria (Segers
and Verstraete, 1983; Sasaki, 1998). The conversion of these acids would be advantageous in
order to couple energy production with organicwaste treatment. The few works done, in which
hydrogen production rates using different carbon
sources were compared, reported lower hydrogen
production rates with acetate and butyrate than
with malate and/or lactate (Hillmer and Gest,
1977; Kim et al., 1980; Miyake et al., 1984;
Sasaki, 1998).
The light efficiency is the other parameter that
determines the feasibility of the biological-hydrogen production process. Only in a few studies
light efficiencies have been reported (Miyake and
Kawamura, 1987; Nakada et al., 1995, 1996; ElShishtawy et al., 1997, 1998; Otsuki et al., 1998;
Yamada et al., 1998). Among these studies, only
Otsuki et al. (1998) used carbon sources other
than malate or lactate (mixture of acetate, propionate and butyrate) and reported a light efficiency
of 0.31%. So far, the highest reported efficiency of
light to hydrogen conversion is 7.9%, with light
from a solar simulator and lactate as electron
donor (Miyake and Kawamura, 1987). However,
this value was obtained at a small-scale experiment and with a low light intensity.
The energy conversion efficiencies and H2 production rates of three different photosynthetic
bacteria under different light intensities and with
different carbon sources are reported on here.
2. Materials and methods
2.1. Photosynthetic bacteria
Rhodopseudomonas sp., culture number HCC
2037 (originally from the Mitsui-Miami collection
with the Mitsui-number 22711), was kindly provided by Dr. Oscar Zaborsky from the University
of Hawaii at Manoa, USA. R. palustris R1 was
kindly provided by Dr. Toshi Otsuki from
Ishikawajima-Harima Heavy Industries Co., Ltd,
Yokohama, Japan. The non-identified photosynthetic strain was kindly provided by Dr Alfons
Stams from the Laboratory of Microbiology, Wageningen University, The Netherlands. This strain
was isolated from the flue gas of anaerobic bioreactors and it will be here referred to as Microbiology strain.
2.2. Growth medium
The strains were grown in an enriched medium
(aSy medium), which was composed of a basal
mineral medium (inorganic salts), 0.1% yeast extract, 10 mM ammonium sulfate and 75 mM
sodium succinate (Miyake et al., 1984). pH was
adjusted to 7.0 with 1 M sodium hydroxide solution. Vitamins were not added, because no differences were found between growth rates (results
not shown). The culture was grown anaerobically
at 30°C in 250 ml rubber-stopper vessels with 6.5
cm diameter and a culture volume of 150 ml.
Argon gas was used to create anaerobic
conditions.
2.3. Hydrogen production media
For each experiment cells were harvested in the
late exponential phase from the growth medium
by centrifugation at 4720× g (Beckman J2-MC)
for 10 min at 25°C and resuspended in hydrogen
production medium (HPM). HPM was prepared
with the basal mineral medium plus nitrogen and
carbon source. pH was adjusted to 7.0 with 1 M
sodium hydroxide solution. Hydrogen production
experiments were carried out under the same conditions and in the same vessels used for growth.
All the experiments were done in batch operation.
M.J. Barbosa et al. / Journal of Biotechnology 85 (2001) 25–33
Sodium glutamate was used as nitrogen source
and different carbon sources were tested at different initial concentrations (lactate 50 mM+
sodium glutamate 10 mM, malate 15
mM +sodium glutamate 2 mM, butyrate 27
Efficiency(%)=
mM +sodium
mM +sodium
mM +sodium
mM +sodium
The efficiency of light energy conversion to
hydrogen was calculated using Eq. (1) (Hall et
al., 1995; Markov et al., 1996) and by assuming
that all the incident light is absorbed.
Combustion enthalpy of hydrogen × Hydrogen production rate
Absorbed light energy×100
glutamate 0.8 mM, acetate 22
glutamate 0.8 mM, acetate 11
glutamate 0.8 mM, acetate 6
glutamate 0.8 mM).
2.4. Light intensity
Two halogen/tungsten lamps were placed on
opposite sides of the culture vessels. Both culture growth and hydrogen production phases
were done at the same light intensity for each of
the light intensities studied (40 and 600 mmol
photons m − 2 s − 1). Light intensity was measured
at the center of the vessel with a 2-p PAR (Photosynthetic Active Radiation, 400 – 700 nm) sensor made by IMAG-DLO (Wageningen, The
Netherlands).
2.4.1. Biomass yield
Optical density was measured at 660 nm using
a Beckman DU-640 spectrophotometer. Cell dry
weight was determined by centrifuging 10 ml of
cell suspension at 5000 × g for 10 min (Beckman J2-MC) and then washing the pellet twice
with deionized water and drying at 80°C until
constant weight.
2.5. Hydrogen e6olution/light efficiency
Hydrogen evolution was followed by withdrawing 0.2 ml of the gas phase and analyzing it
by gas chromatography (Chrompack CP9001).
The packed column with molsieve 13X was
maintained at 50°C. The thermal conductivity
detector (TCD) was set at 130°C. Argon was
employed as carrier gas with a flow rate of 20
ml min − 1.
27
(1)
2.6. Organic-acid analysis
To determine the residual acetate concentration in the culture medium, 1.5 ml of cell suspension was centrifuged at 9200× g for 10 min
(Beckman GS-15R) and the supernatant was analyzed for organic acids by gas chromatography
(Hewlett Packard 5890A), after acidification to
pH 2 with formic acid 3%. The packed column
(10% Fluorad 431 on Supelco-port 100–120
mesh) was maintained at 130°C. The flame ionization detector (FID) was set at 280°C. Nitrogen, saturated with formic acid, was used as
carrier gas with a flow rate of 40 ml min − 1.
3. Results and discussion
3.1. Carbon source
Four different carbon sources were used with
the photosynthetic bacteria, Rhodopseudomonas
sp., R. palustris and Microbiology strain. Lactate
and malate were chosen, as they are the most
widely used carbon sources for H2 production
and, thus, can be used as a reference. The initial
concentrations were taken from literature as those
required for optimal H2 production (Sasikala et
al., 1997; Eroglu et al., 1999). Acetate and butyrate were chosen as models of substrates of
interest, as they are dominant products of anaerobic-fermentation processes (Segers et al., 1981).
Due to the lack of knowledge on the optimal
initial concentration of such substrates for H2
production, the initial concentrations were chosen
to be within the range of concentrations previously used by Sasaki (1998) and Otsuki et al.
(1998). The strains were selected by their potential
28
Carbon source (Mm)
Strain
Rhodopseudomonas sp.
H2 yield (%)
H2 evolved (ml H2 vessel−1)
Maximum H2 production rate
(ml H2 1−1 h−1)
Maximum light efficiency (%)
a
No H2 production.
Microbiology strain
R. palustris
Lactate
(50)
Malate
(15)
Butyrate
(27)
Acetate
(22)
Lactate
(50)
Malate
(15)
Butyrate
(27)a
Acetate
(22)
Lactate
(50)
Malate
(15)
Butyrate
(27)
Acetate
(22)
9.6
139
10.7
6.6
29
1.1
8.4
100
7.6
72.8
269
25.2
12.6
183
9.1
36.0
158
5.8
–
–
–
14.8
56
2.2
14.4
204
7.9
36.7
156
6.0
0.3
4
0.2
35.3
134
5.3
0.4
0.0
0.3
0.9
0.5
0.3
–
0.1
0.3
0.3
0.0
0.2
M.J. Barbosa et al. / Journal of Biotechnology 85 (2001) 25–33
Table 1
Influence of the carbon source on H2 production by Rhodopseudomonas sp., R. palustris and Microbiology strain. Light intensities were 680, 480 and 575 mmol photons m−2 s−1, respectively, for each of the strains; sodium
glutamate was the nitrogen source
M.J. Barbosa et al. / Journal of Biotechnology 85 (2001) 25–33
ability to convert acetate (Kim et al., 1981; Otsuki
et al., 1998). Lactate was preferably used by R.
palustris and Microbiology strain. The highest
conversion yield (72.8%), hydrogen evolved (269 ml
H2 per vessel), hydrogen production rate (25 ml H2
l − 1 h − 1) and light efficiency (0.9%) were achieved
by Rhodopseudomonas sp. with acetate as the carbon source (Table 1). Initial time was determined
by inoculation. So, the lag phase prior to H2
production start-up is included in the time of the
overall experiment. The conversion yields were
evaluated as the percentage of the maximum theoretical production, resulting from complete conversion of the substrate to H2 and CO2, according to
the equations:
Lactate: C3H6O3 +3H2O “6H2 +3CO2
(2)
Malate: C4H6O5 +3H2O “6H2 +4CO2
(3)
Butyrate: C4H8O2 +6H2O “10H2 +4CO2
(4)
Acetate: C2H4O2 + 2H2O “4H2 +2CO2
(5)
Rhodopseudomonas sp. showed a high capability
to convert acetate to hydrogen. However, light
efficiency still needs to be improved in order to
attain a cost-effective process.
3.2. Light intensity
Two different light intensities were used for each
of the three photosynthetic bacteria, with acetate as
carbon source. Table 2 shows the effect of light
intensity on H2 production yield, H2 evolved,
29
maximum H2 production rate and maximum light
efficiency.
H2 production by photosynthetic bacteria under
a low light intensity (ca. 40 mmol m − 2 s − 1) lead to
an increase in light efficiency and a decrease in H2
yield, total volume of H2 evolved and H2 production rate (Table 2). These results are in agreement
with previous studies (Miyake and Kawamura,
1987; Nakada et al., 1995; Yamada et al., 1998). A
lower acetate consumption rate when a low light
intensity is supplied can also be observed in Fig. 1.
At a certain biomass concentration and light
intensity, depending on the strain, light can become
a limiting factor due to self shading and light
absorption by the cells close to the illuminated
surface. In Fig. 2, it can be observed that cells
grown under the highest light intensity reached a
higher biomass concentration. At a certain time,
cells grown under the lowest light intensity started
to grow slower than those cultivated under high
light intensity (Fig. 2). By comparing cells cultivated under the two light intensities, in both cases
H2 production rates started to decrease when
acetate was still available, sooner for high light
intensity (due to the faster increase of biomass
concentration) and later for low light intensity (Fig.
1a and Fig. 2a). These results suggest that light
became a growth limiting factor and a relationship
between cell concentration, hydrogen production
rate and light intensity can be expected.
On the other hand, light efficiency increased by
lowering light intensity (Table 2). Nakada et al.
(1995) also observed that a decrease in light intensity from 720 to 22 W M − 2 leads to an increase in
light efficiency from 0.5 to 1.8%. With Rhodopseu-
Table 2
Effect of light intensity on H2 production by Rhodopseudomonas sp., R. palustris and Microbiology strain; acetate (22 mM) and
sodium glutamate (0.8 mM) were the carbon and nitrogen source, respectively
Strain
Rhodopseudomonas sp.
LightIntensity (mmol photons m−2 s−1)
H2 yield (%)
H2 evolved (ml H2 vessel−1)
Maximum H2 production rate (ml H2 1−1 h−1)
Maximum light efficiency (%)
a
Mean values (n =2) 9 S.D.
680
72.8
269
25.2
0.9
43a
50.4 9 1.8
204 914
10.2 90.9
6.2 90.0
Microbiology strain
R. palustris
480
14.8
56
2.2
0.1
38
2.6
10
0.4
0.2
573
35.3
134
5.3
0.2
37
17.4
67
2.6
1.7
30
M.J. Barbosa et al. / Journal of Biotechnology 85 (2001) 25–33
Fig. 1. Effect of light intensity on H2 production rate and on
acetate consumption by, (a) Rhodopseudomonas sp., (b) R.
palustris, (c) Microbiology strain. (, ): high light intensities
(680, 480 and 575 mmol photons m − 2 s − 1, respectively, for the
strains), (, ): low light intensity (ca. 40 mmol photons m − 2
s − 1 for each strain). (, ): residual acetate concentration, (
,
): H2 production rate. Acetate (22 mM) and sodium glutamate
(0.8 mM) were, respectively, the carbon and nitrogen sources.
Fig. 2. Effect of light intensity on biomass concentration of the
cultures, (a) Rhodopseudomonas sp., (b) R. palustris, (c) microbiology strain, : High light intensities (680, 480 and 575
mmol photons m − 2 s − 1, respectively, for the strains); , light
intensity (ca. 40 mmol photons m − 2 s − 1 for each strain).
Acetate (22 mM) and sodium glutamate (0.8 mM) were the
carbon and nitrogen sources, respectively.
M.J. Barbosa et al. / Journal of Biotechnology 85 (2001) 25–33
31
domonas sp. and acetate as the carbon source,
light efficiency increased from 0.9 to 6.2% by
changing light intensity from 680 to 40 mmol
photons m − 2 s − 1 (Table 2). The big difference
between light efficiencies when a low and a high
light intensity were supplied might be due to the
fact that at high light intensities not all light is
absorbed. The other reason could be the supply of
energy in excess of the capability of the hydrogen
production enzyme (nitrogenase) when light intensity is high, leading to a low light efficiency. So
far, strong light illumination has shown to be not
effective for hydrogen production, as light efficiency decreases with the increase of light energy
(Miyake and Kawamura, 1987; Nakada et al.,
1995, 1996). Rather, weak light should be distributed evenly to all parts of the reactor. A light
efficiency of 6.2% was achieved by Rhodopseudomonas sp. under a light intensity of 43 mmol
photons m − 2 s − 1. At the best of our knowledge,
this is the best value reported with acetate as
carbon source. The total H2 evolved with high
light intensity, was larger than the one obtained
with low light intensity, for all the strains. However, that difference was rather small for
Rhodopseudomonas sp., when compared with the
other strains.
These results stand for the high potential of
Rhodopseudomonas sp. for H2 production using
acetate as the electron donor. However, optimization of the system and cultivation conditions still
needs to be done.
3.3. Acetate concentration
Fig. 3. Effect of initial acetate concentration on, (a) biomass
concentration, (b) H2 evolved, (c) acetate consumption of
Rhodopseudomonas sp. , acetate 22 mM; , acetate 11 mM;
, acetate 6 mM. Initial sodium glutamate concentration was
0.8 mM and light intensity was approximately 40 mmol photons m − 2 s − 1
The effect of initial acetate concentration on
biomass concentration, H2 evolved and acetate
consumption is shown in Fig. 3. Acetate concentration strongly affected H2 production and
biomass concentration. A decrease of acetate concentration from 22 to 6 mM resulted in a decrease
of hydrogen evolved (214–27 ml H2 per vessel).
At concentrations of 6 and 11 mM, acetate limited H2 production, approximately 50 h after inoculation (Fig. 3). Kim et al. (1980) reported no
changes in the hydrogen evolution rate, when the
concentration of acetate or lactate increased from
10 to 50 mM.
32
M.J. Barbosa et al. / Journal of Biotechnology 85 (2001) 25–33
On the other hand, the results obtained by
Sasikala et al. (1997) showed a clear effect of
malate concentration on H2 production and
biomass yield. The results presented here also
emphasize the need to optimize the concentration of each particular substrate to be employed
in commercial production. Higher acetate concentrations should be considered in future research.
Acknowledgements
The project was sponsored by the Dutch research program, Economy, Ecology and technology (KIEM 98013). Jorge M.S. Rocha thanks
for the post doctoral fellowship PRAXIS XXI/
BDP/18840/98 from Fundação para a Ciência e
Tecnologia, Lisbon, Portugal. The authors thank
Dr Toshi Otsuki, Dr Oscar Zaborsky and Dr
Alfons Stams, respectively, for the supply of the
strains R. palustris R1, Rhodopseudomonas sp.
and the non-identified photosynthetic bacteria.
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