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33 (2008) 963 – 973
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ijhydene
Characteristics of a new photosynthetic bacterial strain
for hydrogen production and its application
in wastewater treatment
Yongzhen Taoa,b, Yanling Hea, Yongqiang Wub, Fanghua Liub, Xinfeng Lib,
Wenming Zongb, Zhihua Zhoub,
a
School of Life Science and Technology, Xi’an Jiaotong University, Xian ning Road, Xi’an 710049, PR China
Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, PR China
b
art i cle info
ab st rac t
Article history:
The optimal conditions for ZX-5, a newly isolated PNS bacterial strain, to produce hydrogen
Received 7 February 2007
and the potential for it to treat wastewater were investigated. The strain could grow and
Received in revised form
produce hydrogen at pH 5.5–9.5, and it was able to adjust the pH value to about 7 during
9 September 2007
photo-fermentation by itself. ZX-5 could use 22 tested carbon sources for growth, and 15 of
Accepted 27 November 2007
these carbon sources to produce hydrogen. The hydrogen conversion efficiencies of ZX-5
Available online 4 January 2008
from succinate, lactate, butyrate, malate, acetate, pyruvic acid, propionate, D-mannitol and
Keywords:
Purple non-sulfur (PNS) bacteria
Rhodobacter sphaeroides
Photo-fermentation
Hydrogen production
COD removal efficiency
Substrate conversion efficiency
glucose were 89.7, 81.5, 71.5, 78.8, 69.0, 72.6, 61.9, 64.5, and 52.6%, respectively. The highest
hydrogen-producing rate of 118 ml/l h was observed when butyrate was used as a carbon
source. Relatively high efficiencies for reducing chemical oxygen demand (480%) and
hydrogen production were achieved when ZX-5 was used for photo-fermentation of
succinate wastewater, or effluents from dark fermentation of wastewater from a fuel
ethanol manufacturer or kitchen waste. The concentration of total fatty acids was o0:001%
after photo-fermentation by ZX-5. The above results suggest that ZX-5 would possess the
potential for hydrogen production while treating wastewater.
& 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights
reserved.
1.
Introduction
Worldwide industrial development during the past century
has greatly improved the quality of life. However, it has also
brought problems, such as energy shortage and environmental pollution. For example, more than 200 million tons of
wastewater are produced annually by the food and biofermentation industries in China. For wastewater, much of
which is composed of organic acids, sugars, and starch, the
average chemical oxygen demand (COD) is as high as
2000 mg/l. To avoid pollution, a large sums of money are
invested to treat or dispose of the wastewater, and meanwhile, a large amount of biomass in the wastewater is wasted.
Methods for producing hydrogen by anaerobic fermentation
of wastewater (dark fermentation) have been developed [1–5],
which have the dual solution of producing clean energy and
treating wastewater. However, organic acids are usually coproduced during the process of dark anaerobic fermentation
[6–8], and most anaerobic bacteria cannot utilize these acids
as carbon sources [6–9]. Therefore, not only are the organic
acids from wastewater still left, but a much larger quantity of
organic acids also accumulate during the dark-fermentation
Corresponding author. Tel.: +86 21 54924050; fax: +86 21 54924049.
E-mail address: [email protected] (Z. Zhou).
0360-3199/$ - see front matter & 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2007.11.021
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process, and further treatment is needed to make use of the
organic acids and to produce more hydrogen [10–12]. Recently,
more and more studies have indicated that photosynthetic
purple non-sulfur (PNS) bacteria can completely convert most
organic acids to hydrogen and carbon dioxide under illumination, and photo-fermentation is the best complement to
dark anaerobic fermentation for both hydrogen production
and wastewater treatment [13–15].
A variety of carbon sources can be utilized for growth by
PNS bacteria [1,14,16]. However, only a portion of these
sources is suitable for producing hydrogen. Most wastewater
has a complex carbohydrate composition. Therefore, dark
fermentation is necessary to decompose these carbohydrates
before highly efficient hydrogen production through photofermentation by PNS. Butyrate, acetate, and propionate are
the main fatty acid components in the effluents undergoing
dark anaerobic fermentation. The hydrogen yield of the same
carbon source (substrate conversion efficiency) is found to
vary greatly among different PNS species, or even among
different strains of the same species [17–20]. Thus, it is very
important to isolate PNS bacterial strains that are capable of
using a wide range of organic acids to produce hydrogen with
high efficiency, including butyrate, acetate, and propionate,
which are produced in most anaerobic fermentation.
In this study, we isolated several PNS strains, among which
one strain could convert a wide range of organic acids into
hydrogen at high efficiency. Its possible application in
utilizing wastewater to produce hydrogen and reduce COD
of wastewater was also tested.
33 (2008) 963 – 973
2:5 ml 10 Taq reaction buffer, and 0:25ml Taq polymerase
(5 U=ml). The template DNA was amplified by using the
following temperature profile: 95 1C for 5 min for the initial
PCR activation, followed by 35 cycles at 95 1C for 30 s, 60 1C for
30 s, and 72 1C for 1 min 30 s, and then extended at 72 1C for
10 min, ending at 4 1C. The amplified 16S rRNA genes were
then cloned with a T-vector and sequenced. All the sequences
of the 16S rRNA genes of the isolated strains were compared
with those available in GenBank using the BLAST program.
2.2.
Hydrogen yield of isolated PNS strains utilizing
different organic substrates
Batch tests on the hydrogen yield of the isolated PNS bacterial
strains by photo-fermentation of different organic substrates
were carried out in 38-ml anaerobic tubes. RCVB medium
with 7-mM L-glutamic acid as a nitrogen source and various
organic substrates as carbon sources was used for hydrogen
production. Malate, acetate, lactate, pyruvic acid, butyrate,
propionate, succinate, caproate, valeric acid, isovaleric acid,
isobutyric acid, vanillic acid, benzoic acid, glucose, fructose,
sucrose, xylose, arabinose, cellobiose, maltose, D-mannitol
and ethanol at different concentrations were tested as carbon
sources for hydrogen production.
Anaerobic tubes were sealed with rubber stoppers and
illuminated by tungsten lamps and kept at 30 1 C controlled by air-condition; 60-ml syringes were used to collect
and measure gas yield.
2.3.
Hydrogen yield of the isolated PNS strains under
various photo-fermentation conditions
2.
Materials and methods
2.1.
Isolation and identification of hydrogen-producing
PNS strains
Hydrogen-producing PNS bacterial strains were isolated from
wastewater ponds in the suburbs of Shanghai through
cultures, which were enriched with RCVB medium [21] in
anaerobic tubes, under illuminations of 3500–4000 lux at
30 1 C. After enriching four times, the microflora was
spread on RCVB agar plates and incubated under anaerobic
conditions, using anaerobic Jar HP1 with a gas generating kit
(BR38; Becton Dickinson, Franklin Lakes, NJ). Single colonies
were selected and re-streaked three times, so that pure
cultures were obtained.
The cell morphology of the isolated strains was observed
under scanning electron microscopy (SEM), and the structure
of their internal photosynthetic membranes was observed
under transmission electron microscopy (TEM). Utilization of
carbon sources and other factors for growth were also
analyzed.
The genome DNA isolated from PNS strains was extracted
and purified as described by Choi et al. [22]. The PCR
amplification of 16S rRNA gene (Escherichia coli positions
8–1540) was processed using a pair of bacterial universal
primers (P0-GAGAGTTTGATCC, P6-CTACGGCTACCTTGTTACGA) [23]. Each 25 ml PCR reaction solution contained a final
concentration of the following reagents: 10 ng purified
genomic DNA, 1 ml 20 pM of each primer, 0:5 ml 10 mM dNTP,
Hydrogen production at various initial pH values and
illumination conditions was tested in standard RCVB medium
with 30-mM DL-malic acid as the carbon source and 7-mM
L-glutamic acid as the nitrogen source. The effects of different
nitrogen sources on hydrogen production were estimated
in modified RCVB medium with 30-mM DL-malic acid as a
carbon source and the tested nitrogen sources [L-glutamate,
(NH4)2SO4, ethanolamine] at different concentrations.
All the above tests were carried out in 38-ml anaerobic
tubes, which were sealed with rubber stoppers, illuminated
by tungsten lamps, and kept at 30 1 C controlled by aircondition; 60-ml syringes were used to collect and measure
gas yield.
2.4.
Hydrogen production from wastewater using the
isolated PNS bacterial strains
Wastewater was directly collected from the factory or the
effluent of dark fermentation of waste biomass. Characteristics of the isolated PNS strains for hydrogen production by
photo-fermentation of wastewater were evaluated by batch
tests in a 6-l photobioreactor. Succinate wastewater was
collected from a factory producing succinate (Shen Ren,
Shanghai, China), in which the main composition of the
COD was succinate. Fermentation effluent B was from the
wastewater of a fuel ethanol manufacturer (Tianguan Group,
Henan, China), and fermentation effluent C came from
kitchen waste, in both of which the main fatty acids were
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butyrate and acetate. The batch tests were carried out in
modified RCVB medium in which 30-mM malate was replaced
by wastewater or fermentation effluents as the carbon source,
and the initial pH value was adjusted to 7.0.
The 6-l glass bioreactor jacketed with circulatory water
connected to a water bath to keep the fermentation temperature at 30 1 C, agitated by a magnetic stirrer at 400 rpm,
and illuminated by four tungsten lamps from different
directions (light intensity about 4000 lux). The hydrogen in
the produced biogas, from which CO2 was absorbed by 10-M
NaOH solution, was collected by the water replacement
method.
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perature was kept at 25 1C, and an aqueous solution of 0.005 M
H2SO4 was used for elution at 0.8 ml/min.
2.6.
Data analyses
The yield of gas produced in all of the batch fermentations
was corrected under normal conditions (30 1C, 0.1 MPa).
Hydrogen yield was calculated according to the biogas yield
and hydrogen composition in collected gas. All the data
shown are the average results from at least three experiments.
3.
2.5.
33 (2008) 963 – 973
Results and discussion
Analytical measurements
Cell concentration was measured by a spectrophotometer
(Unic UV280S, Shanghai, China) at 660 nm. Cell dry weight
(g/l) was determined by centrifugation of 10-ml cell suspension, washing the pellet once in distilled water and drying in a
vacuum oven. The composition of gas production (mainly H2
and CO2 ) was determined with a gas chromatograph (Techcomp GC7900, Shanghai, China) equipped with a thermal
conductivity detector (TCD). A 5-m stainless column was
packed with an acidic ethyl acetate (AE) (transformer oil
analysis column). Nitrogen was used as the carrier gas at a
flow rate of 20 ml/min. The operation temperature of the
injector, column, and detector, were 80, 80, and 130 1C,
respectively. Electric current was 85 mA.
The composition of volatile fatty acids (VFA) in fermentation effluents was analyzed by gas chromatography (Shimadzu, Kyoto, Japan) using a flame ionization detector (FID) and a
30-m FFAP capillary column. The temperatures of the injector
and detector were 250 and 260 1C, respectively, while the
column was held at 80 1C for 2 min, heated to 200 1C at
5 1C/min, and maintained at 200 1C for 1 min. The carrier gas
was nitrogen at a flow rate of 70 ml/min.
The concentration of succinate, malic acid, and lactate was
analyzed at 210 nm by HPLC (1100 series; Agilent Technologies, Palo Alto, CA) equipped with a 4.6 ð5 mmÞ 250 mm
Purospher STAR C18 column after filtering the sample
through a 0:45mm disposable filter unit. The column tem-
3.1.
Isolation and identification of hydrogen-producing
PNS bacterial strains
Four PNS strains capable of producing hydrogen, designated
as ZX-2, ZX-3, ZX-4, and ZX-5, were isolated through
enrichment cultures. These strains could grow under dark
aerobic conditions and illuminated anaerobic conditions.
Cells of the four strains are all gram-negative, red, spherical
(size, 0:9921:31 0:520:7 mm) (Fig. 1a), in which the internal
photosynthetic membrane is vesicular-type (Fig. 1b). No polar
growth or budding fission were observed, but binary fission
was detected (Fig. 1a). The four strains were also characterized by their absorption at 478 and 513 nm from carotenoid
and at 590, 801, and 852 nm from bacteriochlorophyll a. By
addition of three kinds of vitamins (biotin, nicotinic acid, and
thiamine), they could grow by utilizing different carbon
sources, including mannitol, sodium citrate, tartrate, and
ethanol, but were unable to use sodium thiosulfate. The
characteristics of the four isolated strains were similar to
those of Rhodobacter sphaeroides [24].
The sequences of the 16s rRNA gene (1500 bp) of the four
isolated strains were determined and submitted to NBCI for
searching the closest matches by BLAST. The 16S rDNA
sequences of the four isolated strains, ZX-5, ZX-2, ZX-3, and
ZX-4 (high similarity of 99–100% between each of them), had
the highest similarity of 99.7% to a culture of R. sphaeroides.
The result of 16S rDNA sequence alignment further indicated
Fig. 1 – (a) SEM image of the cells of isolated strains; (b) TEM image of the cellular structure of isolated strain.
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that the isolated strains were R. sphaeroides. The sequence of
16S rDNA gene of ZX-5 was submitted to GenBank database
(Accession no. EU123535).
3.2.
Differential hydrogen-producing ability of the isolated
strains
The hydrogen-producing ability of the four strains was
compared by using succinate, malate, acetate, and butyrate
as carbon sources (Table 1). All four strains grew well and
produced hydrogen using succinate and malate. However,
stains ZX-4 and ZX-2 were not able to utilize acetate for
growth and hydrogen production, and ZX-4 was also unable
to utilize butyrate. Strain ZX-5 showed the highest hydrogen
conversion efficiency from the four tested organic acids,
suggesting its potential application for biological hydrogen
production.
3.3.
Effect of illumination intensity on photo-fermentation
hydrogen production of strain ZX-5
In photo-fermentation, the efficiency of hydrogen production
by PNS bacteria was influenced by the illumination intensity;
the maximum hydrogen production rate and substrate
conversion efficiency of strain ZX-5 increased when the
illumination intensity was raised from 1500 to 5000 lux, but
decreased at above 5000 lux (Table 2). The increase likely
occurred because the higher illumination intensity provided
more ATP and reductive power by the photosynthetic system,
which is necessary for hydrogen production [25,26]. However,
light saturation might have occurred when the photosyn-
33 (2008) 963 – 973
thetic system supplied excess ATP and Fdred compared to the
capacity of nitrogenase [27]. The activity of nitrogenase and
hydrogen production decrease when light saturation occurs.
Zhu et al. [13] reported that hydrogen production is inhibited
in their photo-fermentation system when the illumination
intensity is greater than 1:0 kw=m2 . In this study, the
illumination intensity of 4000–5000 lux was the best choice
for strain ZX-5 to produce hydrogen, which is similar to that
of other reported hydrogen-producing PNS strains [28–32].
3.4.
Effect of the initial pH of the medium on hydrogen
production by ZX-5
An initial pH of 7.0 has been suggested in several studies as
the best value for both cell growth and hydrogen production
by R. sphaeroides and Rhodobacter capsulatus [15,33,34]. In this
study, we tested the effects of eight initial pH values, 5.0, 5.5,
6.0, 7.0, 8.0, 9.0, 9.5, and 10.0, on hydrogen production and cell
growth of strain ZX-5. The results indicate that pH 7.0 might
be the best choice for ZX-5 cell growth (Table 3). However, no
significant differences in the cumulative and maximum rates
of evolved hydrogen were observed among pH 6.0, 7.0, 8.0,
and 9.0 (Table 3), suggesting that the initial pH value within
the range 6.0–9.0 might not affect hydrogen production of
ZX-5 too greatly. ZX-5 showed potential application for the
treatment of wastewater and conversion of waste biomass
into hydrogen by displaying a wide pH range suitable for
hydrogen production.
A noteworthy phenomenon is that the final pH values
reached about 7.0 after ZX-5 began to grow and produce
hydrogen, even when the initial pH values differed within the
Table 1 – Characteristics of four isolated PNS bacterial strains for their hydrogen production by photo-fermentation of four
organic acids
Different carbon
source
Different
strains
H2 content in
biogas (%)
Total H2
evolved
(ml/35 ml)
Maximum H2
production
rate (ml/l h)
Substrate
conversion
efficiency (%)
OD (660 nm)
Succinate (50 mM)
ZX-5
ZX-2
ZX-3
ZX-4
96
93
92
90
223 10
172 8
188 7
79 5
94
102
69
38
81.40
62.40
68.60
28.80
2.90
2.65
2.55
2.32
Malate (30 mM)
ZX-5
ZX-2
ZX-3
ZX-4
87
88
87
80
123 4
114 10
101 8
17 2
92
96
88
22
78.90
73.60
64.80
10.90
2.29
2.60
2.56
1.55
Acetate (35 mM)
ZX-5
ZX-2
ZX-3
ZX-4
87
0
84
0
86 4
0
76 2
0
90
0
88
0
69.00
0
63.20
0
2.69
0.22
2.91
0.15
Butyrate (50 mM)
ZX-5
ZX-2
ZX-3
ZX-4
94
85
80
0
324 15
28 2
20 3
0
110
18
15
0
74.60
6.45
4.61
0
3.01
2.55
2.35
0
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Table 2 – Effects of illumination intensity on hydrogen production of strain ZX-5 by photo-fermentation of malate
Light
intensity
(lux)
7000
5000
4000
3500
2500
1500
Total H2 evolved
(ml/35 ml)
Total H2
evolved (ml/l)
Maximum H2 evolution
rate (ml/l h)
Substrate conversion
efficiency (%)
OD
(660 nm)
116 10
120 8
118 5
101 7
110 6
45 2
3068
3157
3105
2671
2894
1184
84.57
87.04
88.94
84.47
62.72
38.25
69.24
71.25
70.52
60.66
65.72
26.89
2.80
2.65
2.57
2.22
2.18
1.98
Table 3 – Effects of initial pH values on hydrogen production of strain ZX-5 by photo-fermentation
Initial pH
value
5.0
5.5
6.0
7.0
8.0
9.0
9.5
10.0
Total H2 evolved
(ml/35 ml)
Maximum H2 evolution
rate (ml/l h)
Substrate conversion
efficiency (%)
Final pH
value
OD
(660 nm)
0
104 10
123 4
128 8
122 5
124 4
80 5
0
0
85
90
92
93
88
60
0
0
67.53
78.87
82.60
79.22
80.00
51.60
0
5.35
7.06
7.05
7.08
7.06
7.09
7.00
9.40
0.10
1.91
1.98
2.30
2.00
2.01
1.65
0.20
strains. The mechanism of the pH self-adjustment by ZX-5
warrants further study.
3.5.
Effect of different nitrogen sources on photofermentation hydrogen production by strain ZX-5
Fig. 2 – Time-course profile of pH values in media during
photo-fermentation process.
range 5.5–9.5 (Table 3). The changes in pH value during photofermentation indicated that it took different times for the
fermentation solutions to reach a stable pH of about 7.0 when
the initial values were different. When initial pH values were
8.0, 6.0, and 5.5, it took about 32 h for fermentation solutions
to reach pH 7.0 (Fig. 2); for an initial pH of 9.0, it took 40 h, and
for an initial pH of 9.5, 48 h. The pH value remained relatively
stable when the initial value was 7.0. Little pH variation and
hardly any cell growth were found at initial pH values of 5.0
and 10.0. It seems that when the initial pH value was 5.5–9.5,
ZX-5 cells had the ability to adjust the pH of the fermentation
solution to about 7.0. To our knowledge, no similar results
have been reported for other hydrogen-producing PNS
Hydrogen production by PNS bacteria through photo-fermentation is catalyzed by nitrogenase, and the activity and gene
expression of nitrogenase are both inhibited by NHþ
4 [13,35,36]
Thus, the efficiency of hydrogen production may be greatly
affected by nitrogen sources and their concentration [14,17].
In our study, we compared the photo-fermentational hydrogen production of ZX-5 with L-glutamate, (NH4)2SO4, or
ethanolamine used as a nitrogen source. ZX-5 could not
produce hydrogen or grow well without adding a nitrogen
source (Table 4). The OD values, reflecting cell growth of ZX-5,
increased with the concentration of the three different
nitrogen sources, and no inhibitory effect of nitrogen sources
on cell growth was observed. In contrast, photo-fermentational hydrogen production decreased as the concentration of
(NH4)2SO4 or ethanolamine increased, as it did for L-glutamate
at a concentration above 7 mM. Hydrogen production was
inhibited by high nitrogen concentration and completely
inhibited by more than 7-mM (NH4)2SO4. For ZX-5, 7-mM
L-glutamate was the best nitrogen source for hydrogen
production, and 2 mM ethanolamine was the second-best
choice. L-glutamate at 7 mM is also the preferred nitrogen
source for most of the reported hydrogen-producing PNS
bacterial strains [14,37]. However, R. capsulatus B100 and its
mutant ST410 produced more hydrogen using 3.5-mM
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Table 4 – Effects of different nitrogen sources on hydrogen production from photo-fermentation by strain ZX-5
Different
concentration (mM)
Total H2
(ml/35 ml)
Conversion efficiency of
substrate (%)
OD (660 nm)
3.5
5.0
7.0
10.0
15.0
80 7
95 5
123 2
66 5
37 5
51.28
60.80
78.73
42.30
23.68
1.54
1.98
2.35
2.25
2.82
(NH4)2SO4
2.0
3.5
5.0
7.0
72 2
25 1
17 2
00
56.47
16.00
11.05
0
1.30
2.19
2.39
2.57
Ethanolamine
2.0
3.5
5.0
10.0
97 2
82 3
77 2
62 1
62.08
52.48
49.35
39.74
1.15
1.79
1.89
2.25
0
0
0
0.31
Different
nitrogen source
L-Glutamate
Control
ethanolamine as a nitrogen source than they did using
L-glutamate [17].
3.6.
Effects of different carbon sources and their
concentrations on hydrogen production of strain ZX-5 during
photo-fermentation
The characteristics of hydrogen production by strain ZX-5
were tested by photo-fermentation of 22 different carbon
sources at different concentrations, including 11 organic
acids, seven carbohydrates, two kinds of ethanol, and two
aromatic acids. ZX-5 was able to use all of the tested carbon
sources for cell growth (Table 5), and use 15 of these to
produce hydrogen. Among the 11 organic acids, nine could be
used to produce hydrogen, of which the conversion efficiencies of acetate, succinate, malate, lactate, butyrate, and
pyruvic acid to hydrogen gas were near or over 70%; that of
propionate was over 60% (Table 5).
For all of the above hydrogen-producing organic acids used
as a carbon source, hydrogen gas was first detected at 9–10 h
after ZX-5 was inoculated into the medium (Fig. 3). For
succinate and butyrate, hydrogen production lasted for more
than 140 h, but for the other carbon sources, hydrogen
production usually finished within 90 h (Fig. 3). Cumulative
hydrogen production, OD value, hydrogen production rate,
and substrate conversion efficiency increased with the
concentration of organic acid, up to a threshold. However,
after the concentration reached the threshold, the substrate
conversion efficiency decreased. The threshold, the most
suitable concentration for the highest conversion efficiency
from carbon sources to hydrogen, varied with different
organic acids (Table 5). For malate, although conversion
efficiency decreased after concentrations reached the threshold (Fig. 4a), the cumulative hydrogen production still
increased slightly (Fig. 4b), as did the OD value. Similar
results were also observed for succinate and acetate (data not
shown). However, for butyrate, when the concentration was
more than 50 mM (the threshold), not only did the substrate
conversion efficiency decrease sharply, but the cumulative
hydrogen production, OD value, and hydrogen production
rate also decreased greatly (Fig. 4a and b). And the same
results were also found for lactate (data not shown). Inhibition of hydrogen production was obviously detected with high
concentrations of butyrate and lactate. A similar phenomenon was also observed in other studies [38,39]. The inhibition
of hydrogen production by organic acids at high concentrations might result from its toxicity or inhibition of cell growth.
The above results also indicated that ZX-5 possessed varied
tolerance to the inhibition from different organic acids.
Numerous studies have reported on hydrogen production
by different PNS bacterial strains using malate, succinate,
butyrate, and acetate [16,30,32,40,41]. ZX-5 could produce
hydrogen from acetate with a relatively high substrate
conversion efficiency of 69%, but a little less than that of
ST410, which has a mutation in the hydrogenase gene from
R. capsulatus B100 [17] and a Rhodopseudomonas strain (72.8%)
[40] (Table 6). However, compared to the reported PNS strains,
ZX-5 could produce more hydrogen much more quickly from
malate, succinate, lactate, and butyrate, demonstrating the
highest substrate conversion efficiency and hydrogen production rate (Table 6). Thus far, only a few reports involving
hydrogen production from propionate are available, in which
R. capsulatus strain produced hydrogen from a mixture of
acetate and propionate at a conversion efficiency of 40–45%
[41]. The composition of organic acids in most wastewater is
complex, and those from anaerobic fermentation are mainly
composed of acetate, butyrate, lactate, and propionate
[18,42,43]. As ZX-5 could produce hydrogen from the aforementioned acids at a relatively high efficiency, it is likely that
ZX-5 has an advantage in converting the organic acids in
wastewaters from anaerobic fermentation into hydrogen.
Among the seven tested carbohydrates, five could be used
by ZX-5 to produce hydrogen, with conversion efficiencies of
6.4–52.5% (Table 5). The conversion efficiency and hydrogen
production rate of all carbohydrates were much lower in
comparison to those of the tested organic acids. A few other
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33 (2008) 963 – 973
Table 5 – Optimal concentration of different carbon sources for growth and hydrogen production of strain ZX-5 during
photo-fermentation
Carbon source
Optical
concentration
(mM)
Total H2
evolved
(ml/35 ml)
Maximum
H2 evolved
rate (ml/l)
Conversion
efficiency (%)
OD (660 nm)
Organic acid
Malate
Acetate
Succinate
Lactate
Butyrate
Pyruvic acid
Propionate
Valeric acid
Caproate
Isovaleric acid
Isobutyric acid
30
35
50
50
50
50
70
70
70
30
30
123 10
86 4
272 6
212 2
279 50
170 12
238 12
110 5
0
0
56 4
92
90
108
103
118
110
112
25
0
0
20
78.85
69.00
89.71
81.50
71.50
72.65
61.90
12.93
0
0
21.70
2.29
1.62
2.44
2.42
4.12
1.66
2.45
2.85
0.89
2.25
2.06
Carbohydrate
Glucose
Xylose
Fructose
Arabinose
Cellobiose
Maltose
Sucrose
15
20
25
30
7
7
7
82 3
64 4
69 6
0
0
10 1
27 1
75
70
61
0
0
5
40
52.56
34.30
24.64
0
0
6.37
17.20
2.12
2.58
2.41
2.58
2.63
2.10
2.05
Others
Ethanol
D-mannitol
Benzoic acid
Vanillic acid
20
20
30
10
0
130
0
0
0
75
0
0
0
64.50
0
0
1.87
2.95
2.34
1.23
Fig. 3 – Time course of hydrogen production during photo-fermentation by strain ZX-5 utilizing different carbon sources at
their most suitable concentration (35 ml working volume).
reports involving hydrogen production by photo-fermentation of carbohydrates also showed much lower efficiency
(Table 6), except that mutant ST410 could convert glucose to
hydrogen at an efficiency of 54.2%, which was close to that of
ZX-5. It seems impractical for PNS bacteria to directly use
most carbohydrates as hydrogen-producing carbon sources,
which is why more attention has been paid recently to a twostep hydrogen production process; in this process, dark
fermentation is first employed to convert different carbohydrates into hydrogen and organic acids, and then PNS bacteria
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I N T E R N AT 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
Fig. 4 – (a) Effects of butyrate and malate concentration on
substrate conversion efficiency of strain ZX-5; (b) Effects of
butyrate and malate concentration on cumulative hydrogen
evolved and cell growth (OD at 660 nm) of strain ZX-5.
are introduced to completely convert the organic acids into
hydrogen and CO2 [11,12,18,44,45].
ZX-5 was not able to utilize ethanol, benzoic acid or vanillic
acid to produce hydrogen, but was able to use D-mannitol with
relatively high conversion efficiency (64%). A few studies
involved in hydrogen production from ethanol and some
aromatic acids by photo-fermentation [46–48], in which hydrogen production at very low rate was detected when 2-mM
benzoic acid was used as carbon source [46], but no hydrogen
production from photo-fermentation was found even when
ethanol was mixed with acetate as carbon source [47].
3.7.
Application of strain ZX-5 to hydrogen production
and treatment of wastewater
The COD of succinate wastewater A was measured to be
40.2 g/l, and it was mainly composed of succinate. After the
wastewater was diluted five times to ensure the succinate
concentration was below the threshold point (50 mM), the
diluted wastewater was mixed with basic RCVB medium
(without a carbon source) to be used to produce hydrogen.
The result indicated that 25 l of hydrogen per liter of original
wastewater could be produced by ZX-5 (Table 7), and the
average H2 production rate was 55 ml H2/l h and H2 content in
biogas reach to 94%. During photo-fermentation, the COD
33 (2008) 963 – 973
decreased greatly with a removal efficiency of 87.5% being
achieved (Table 7), no succinate was detected in the final
fermentation effluent by HPLC.
The COD of fermentation effluent B, whose source was the
dark fermentation of wastewater from a fuel ethanol
manufacturer, was 38.9 g/l, in which the main fatty acids
were 5.4 g/l acetate and 6.5 g/l butyrate. ZX-5 could produce
20 l of hydrogen per liter of original wastewater while using
the dilution (threefold) of fermentation effluents B as a
carbon source, and the average H2 production rate was
48 ml H2/l h and H2 content in biogas reached to 90%. The
original COD was reduced from 38.9 to 5.99 g/l, with a COD
removal efficiency of 84.6% (Table 7). In the final fermentation
effluents no acetate but 0.0005-g/l butyrate was detected.
Butyrate at 7.7 g/l and acetate at 3.4 g/l were the main
organic acids in fermentation effluent C from dark fermentation of kitchen waste (18 g-TS of waste per liter fermentation
effluent). ZX-5 used the diluted effluent C (twofold) as a
carbon source to produce hydrogen, and the cumulative
hydrogen production was 9 l per liter of original effluent, the
average H2 production rate was 45 ml H2/l h and H2 content in
biogas 89%. The COD removal efficiency for the fermentation
effluent C was 80% (Table 7). The total fatty acid concentration
in the above effluents was o0:001% after photo-fermentation.
PNS bacteria were also used with tofu wastewater, olive mill
wastewater, or the effluent from dark fermentation of olive
mill wastewater in other studies (Table 7). A hydrogen yield of
1.9 l per liter tofu wastewater and a COD removal efficiency of
41% were observed when the original COD of the tofu
wastewater was 27.4 g/l [13]; a hydrogen yield of 2.0 l H2 per
liter wastewater and a COD removal efficiency of 52% were
observed when the original wastewater (original COD: 42.0 g/l)
from a olive mill was diluted twofold [11]; a hydrogen yield of
13.9 l H2 per liter original wastewater and a COD removal
efficiency of 35% were estimated when the original olive
wastewater was diluted 50-fold [49]. Compared to the PNS
bacterial strains used in those studies, ZX-5 showed higher
efficiency in hydrogen production and COD removal from
wastewater or dark fermentation effluents.
PNS bacteria could produce hydrogen directly at high
efficiency using wastewaters that were mainly composed of
organic acids; for example, ZX-5 could produce 25 l of
hydrogen per liter of original succinate wastewater in this
study. However, for most wastewater, its components were
complex, including many kinds of carbohydrate. Most PNS
bacteria, including ZX-5, could only produce a small amount
of hydrogen when such types of wastewater were used (data
not shown). However, more hydrogen can be obtained from
the wastewater by photo-fermentation if the wastewater is
pretreated by dark fermentation or another acidification
process. For example, Eroğlu et al. [11] found that hydrogen
production increased three to fourfold from olive mill wastewater after pretreating the original wastewater using dark
fermentation or clay process.
4.
Conclusions
Four hydrogen-producing PNS bacterial strains identified as
R. sphaeroides were isolated from different wastewaters.
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33 (2008) 963 – 973
Table 6 – Substrate conversion efficiency and rate of hydrogen production of photo-fermentation by different PSN bacterial
strains
Carbon
source
Organism
Concentration
(mM)
Conversion
efficiency (%)
Maximum H2 rate
(ml H2/l h)
Process
Reference
Acetate
Rhodopseudomonas.sp
R. palustris P4
R. capsulata
R. capsulata
R. capsulatus B100
R. capsulatus ST410
ZX-5
22
22
60
30
30
30
35
72.8
60–70
27.5
32.6
53.0
84.0
69.0
25.2
NA
0.8
45.0
NA
NA
90.0
Batch
Batch
Batch
Batch
Batch
Batch
Batch
[40]
[30]
[1]
[14]
[17]
[17]
This study
Lactate
Rhodopseudomonas.sp
R. palustris
R. sphaeroides RV
ZX-5
50
50
100
50
9.6
12.6
80.0
81.2
10.7
9.1
62.5
103.0
Batch
Batch
CSTR
Batch
[40]
[40]
[50]
This study
Butyrate
Rhodopseudomonas.sp
R. capsulata
R. capsulata
ZX-5
27
11
Mix-acid
50
8.4
37.0
40–45
71.5
7.6
1.3
65.0
118.0
Batch
Batch
Batch
Batch
[40]
[1]
[41]
This study
Malate
Rhodopseudomonas.sp
R. palustris
R. sphaeroides
O.U.001
R. capsulatus B100
R. capsulatus ST410
ZX-5
15
15
7.5
6.6
36.0
35–45
1.1
5.8
5.0
Batch
Batch
Batch
[40]
[40]
[51]
30
30
35
46.0
73.0
78.9
90.0
130.0
92.0
Batch
Batch
Batch
[17]
[17]
This study
R. sulfidophilum
ZX-5
50
50
65.0
89.7
26.6
108.0
Batch
Batch
[52]
This study
R. capsulatus B100
R. capsulatus ST410
R. sphaeroides
mutant
ZX-5
30
30
20
33.8
54.0
24.0
NA
NA
12.6
Batch
Batch
Batch
[17]
[17]
[53]
15
52.6
75.0
Batch
This study
R. capsulatus B100
R. capsulatus ST410
R. capsulatus Z1
ZX-5
30
30
30
7
7.6
12.2
6.0
17.2
NA
NA
NA
40.0
Batch
Batch
Batch
Batch
[17]
[17]
[54]
This study
Succinate
Glucose
Sucrose
NA: not available.
Table 7 – Yield and rate of hydrogen production from wastewater and COD removal efficiency by photo-fermentation
Waste
water
Organism
Original
COD (g/l)
Dilute
fold
Maximum H2
rate (ml/l h)
H2 yield
H2 l/l ww
COD
removed (%)
Reference
A
B
C
Olive ww
Tofu ww
Olive ww
R.s zx-5
R.s zx-5
R.s zx-5
R.s.O.U.001
R.s. RV
R.s.O.U.001
40.15
38.85
18.50
1.10
27.44
42.00
5
3
2
50
0
2
55
48
45
NA
NA
NA
25.0
20.0
9.0
13.9
1.9
2.0
87.55
84.55
80.00
35.00
41.00
52.00
This study
This study
This study
[49]
[13]
[11]
A: succinate wastewater; B: effluents from dark fermentation of wastewater from a fuel ethanol manufacturer; C: fermentation effluent from
dark fermentation of kitchen waste; Tofu ww: Tofu wastewater; Olive ww: wastewater from olive mill.
Among these strains, ZX-5 could convert acetate, malate,
succinate, and butyrate into hydrogen with the highest
efficiency. ZX-5 could adjust the pH value to about 7 during
photo-fermentation when the initial pH value was 5.5–9.5.
No significant difference in hydrogen production was detected when the initial pH value was 6–9. The optimized
conditions for ZX-5 to produce hydrogen at high efficiency
were as follows: illumination of 4000–5000 lux, initial pH of
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I N T E R N AT 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
6–9, and 7-mM L-glutamate as a nitrogen source. The optimized
carbon sources for ZX-5 to produce hydrogen were succinate,
lactate, butyrate, malate, acetate, pyruvic acid, propionate,
D-mannitol and glucose. Although ZX-5 could not use ethanol,
aromatic acids and complex carbohydrates to produce hydrogen or produced hydrogen at a relatively low efficiency, ZX-5
was able to use most of the carbon sources for growth. ZX-5
could use kitchen waste and wastewater from fuel ethanol
manufacturing for hydrogen production, after pretreated by
dark fermentation, or directly using succinate wastewater as a
carbon source to produce hydrogen at a relatively high
efficiency, about 500 ml of hydrogen per gram COD, and also
reduce COD with a removal efficiency of over 80%. The above
results demonstrate that PNS strain ZX-5, which could be
acclimatized to a wide range of pH values and use many kinds
of carbon sources for growth and hydrogen production at
relatively high efficiency, possesses potential application in
wastewater treatment and hydrogen production.
Acknowledgments
This study was sponsored by the Shanghai Pujiang Program
(05PJ14106), the Science and Technology Commission of
the Shanghai Municipality (05DZ12034), and 863 Program
(Hi-Tech Research and Development Program of China)
(2006AA05Z105).
R E F E R E N C E S
[1] Fang HHP, Liu H, Zhang T. Phototrophic hydrogen production
from acetate and butyrate in wastewater. Int J Hydrogen
Energy 2005;30:785–93.
[2] van Ginkel SW, Oh SE, Logan BE. Biohydrogen gas production
from food processing and domestic wastewaters. Int J
Hydrogen Energy 2005;30:1535–42.
[3] Hu ZH, Yu HQ. Anaerobic digestion of cattail by rumen
cultures. Waste Manage 2006;26:1222–8.
[4] Yang HJ, Shao P, Lu TM, Shen JQ, Wang DF, Xu ZN, et al.
Continuous bio-hydrogen production from citric acid wastewater via facultative anaerobic bacteria. Int J Hydrogen
Energy 2006;31:1306–13.
[5] Levin DB, Zhu HG, Beland M, Cicek N, Holbein BE. Potential
for hydrogen and methane production from biomass residues in Canada. Bioresource Technol 2007;98:654–60.
[6] Hussy I, Hawkes FR, Dinsdale R, Hawkes DL. Continuous
fermentative hydrogen production from sucrose and sugarbeet. Int J Hydrogen Energy 2005;30(5):471–83.
[7] Liu H, Grot S, Logan BE. Electrochemically assisted microbial
production of hydrogen from acetate. Environ Sci Technol
2005;39:4317–20.
[8] Fang HHP, Li CL, Zhang T. Acidophilic biohydrogen production from rice slurry. Int J Hydrogen Energy 2006;31(6):683–92.
[9] Khanal SK, Chen WH, Sung SW. Biohydrogen production in
continuous-flow reactor using mixed microbial culture.
Water Environ Res 2006;78(2):110–7.
[10] Nath K, Kumar A, Das D. Hydrogen production by Rhodobacter
sphaeroides strain OU001 using spent media of Enterobacter
cloacae strain DM11. Applied Microbiol Biotechnol
2005;68(4):533–41.
[11] Eroğlu E, Eroğlu İ, Gündüz U, Türker L, Yücel M. Biological
hydrogen production from olive mill wastewater with twostage processes. Int J Hydrogen Energy 2006;31:1527–35.
33 (2008) 963 – 973
[12] Tao YZ, Chen Y, Wu YQ, He YL, Zhou ZH. High hydrogen yield
from a two-step process of dark- and photo-fermentation of
sucrose. Int J Hydrogen Energy 2007;32:200–6.
[13] Zhu HG, Suzuki T, Tsygankov AA, Asada Y, Miyake J.
Hydrogen production from tofu wastewater by Rhodobacter
sphaeroides immobilized in agar gels. Int J Hydrogen Energy
1999;24:305–10.
[14] Shi X-Y, Yu H-Q. Optimization of glutamate concentration
and pH for H2 production from volatile fatty acids by
Rhodopseudomonas capsulata. Lett Appl Microbiol
2005;40:401–6.
[15] Zabut B, El-Kahlout K, Yücel M, Gündüz U, Türker L, Eroğlu I.
Hydrogen gas production by combined systems of Rhodobacter sphaeroides O.U.001 and Halobacterium salinarum in a
photobioreactor. Int J Hydrogen Energy 2006;31:1553–62.
[16] Najafpour G, Younesi H, Mohamed AR. A survey on various
carbon sources for biological hydrogen production via the
water–gas reaction using a photosynthetic bacterium Rhodospirillum rubrum. Energy Sources Part a Recovery Util
Environ Effects 2006;28:1013–26.
[17] Ooshima H, Takakuwa S, Katsuda T, Okuda M, Shirasawa T,
Azuma M, et al. Production of hydrogen by a hydrogenasedeficient mutant of Rhodobacter capsulatus. J Ferment Bioeng
1998;85:470–5.
[18] Khanal SK, Chen WH, Li L, Sung SW. Biological hydrogen
production: effects of pH and intermediate products. Int J
Hydrogen Energy 2004;29:1123–31.
[19] Nath K, Das D. Improvement of fermentative hydrogen
production: various approaches. Appl Microbiol Biotechnol
2004;65:520–9.
[20] Hoekema S, Douma RD, Janssen M, Tramper J, Wijffels RH.
Controlling light-use by Rhodobacter capsulatus continuous
cultures in a flat-panel photobioreactor. Biotechnol Bioeng
2006;95:613–26.
[21] Weaver PF, Wall JD, Gest H. Characterization of Rhodopseudomonas capusulata. Arch Microbiol 1975;5:207–16.
[22] Choi HP, Kang HJ, Seo HC, Sung HC. Isolation and identification of photosynthetic bacterium useful for wastewater
treatment. J Microbiol Biotechnol 2002;12:643–8.
[23] Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal
DNA amplification for phylogenetic study. J Bacteriol
1991;173:697–703.
[24] Imhoff JF, Trüper HG, Fenning NP. Rearrangement of the
species and genera of the phototrophic ‘‘purple nonsulfur
bacteria’’. Int J Syst Bacteriol 1984;34:340–3.
[25] Klasson KT, Lundback KMO, Clausen EC, Gaddy JL. Kinetics of
light limited growth and biological hydrogen-production
from carbon-monoxide and water by Rhodospirillum Rubrum.
J Biotechnol 1993;29:177–88.
[26] Kim NJ, Lee JK, Lee CG. Pigment reduction to improve
photosynthetic productivity of Rhodobacter sphaeroides.
J Microbiol Biotechnol 2004;14(3):442–9.
[27] Asada Y, Miyake J. Photobiological hydrogen production.
J Biosci Bioeng 1999;88(1):1–6.
[28] Kondo T, Arakawa M, Hirai T, Wakayama T, Hara M, Miyaye J.
Enhancement of hydrogen production by a photosynthetic
bacterium mutant with reduced pigment. J Biosci Bioeng
2002;93:145–50.
[29] Wakayama T, Miyake J. Light shade bands for the improvement of solar hydrogen production efficiency by Rhodobacter
sphaeroides RV. Int J Hydrogen Energy 2002;27:1495–500.
[30] Oh YK, Seol EH, Kim MS, Park S. Photoproduction of hydrogen
from acetate by a chemoheterotrophic bacterium Rhodopseudomonas palustris P4. Int J Hydrogen Energy 2004;29:1115–21.
[31] Shi X-Y, Yu H-Q. Response surface analysis on the effect of
cell concentration and light intensity on hydrogen production by Rhodopseudomonas capsulata. Process Biochem
2005;40:2475–81.
ARTICLE IN PRESS
I N T E R N AT 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
[32] Kim EJ, Kim JS, Kim MS, Lee JK. Effect of changes in the level
of light harvesting complexes of Rhodobacter sphaeroides on
the photoheterotrophic production of hydrogen. Int J Hydrogen Energy 2006;31:531–8.
[33] Franchi E, Tosi C, Scolla G, DellaPenna G, Rodriguez F, Pedroni
PM. Metabolically engineered Rhodobacter sphaeroides RV strains
for improved biohydrogen photoproduction combined with
disposal of food wastes. Mar Biotechnol 2004;6:552–65.
[34] Kantachote, D, Torpee, S, Umsakul, K. The potential use of
anoxygenic phototrophic bacteria for treating latex rubber sheet
wastewater. Electron J Biotechnol 2005; 8(3), ISSN: 0717-3458.
[35] Zinchenko VV. Genetic regulation of nitrogen assimilation in
photosynthetic bacteria. Russ J Genet 1999;35:1287–301.
[36] Yakunin AF, Hallenbeck PC. Regulation of nitrogenase
activity in Rhodobacter capsulatus under dark microoxic
conditions. Arch Microbiol 2000;173:366–72.
[37] Kern M, Klipp W, Klemme J-H. Increased nitrogenasedependent H2 photoproduction by hup mutants of Rhodospirillum rubrum. Appl Environ Microbiol 1994;60:1768–74.
[38] VanNiel EWJ, Claassen PAM, Stams AJM. Substrate and
product inhibition of hydrogen production by the extreme
thermophile, Caldicellulosiruptor saccharolyticus. Biotechnol
Bioeng 2003;81:255–62.
[39] Zheng XJ, Yu HQ. Inhibitory effects of butyrate on biological
hydrogen production with mixed anaerobic cultures.
J Environ Manage 2005;74:65–70.
[40] Barbosa MJ, Rocha JMS, Tramper J, Wijffels RH. Acetate as a
carbon source for hydrogen production by photosynthetic
bacteria. J Biotechnol 2001;85:25–33.
[41] Shi X-Y, Yu H-Q. Continuous production of hydrogen from
mixed volatile fatty acids with Rhodopseudomonas capsulata.
Int J Hydrogen Energy 2006;31:1641–7.
[42] Lee KS, Lo YC, Lin PJ, Chang JS. Improving biohydrogen
production in a carrier-induced granular sludge bed by
altering physical configuration and agitation pattern of the
bioreactor. Int J Hydrogen Energy 2006;31:1648–57.
[43] Levin DB, Islam R, Cicek N, Sparling R. Hydrogen production
by Clostridium thermocellum 27405 from cellulosic biomass
substrates. Int J Hydrogen Energy 2006;31:1496–503.
33 (2008) 963 – 973
973
[44] Kawaguchi H, Hashimoto K, Hirata K, Miyamoto K. H2
production from algal biomass by a mixed culture of
Rhodobium marinum A-501 and Lactobacillus amylovorus.
J Biosci Bioeng 2001;91:277–82.
[45] Claassen, PAM, Budde, MAW, van Noorden, GE, Hoekema, S,
Hazewinkel JHO, Van Groenestijn, JW, et al., Biological
hydrogen production from agro-food by-products. In: biological hydrogen product proceedings. 2004.
[46] Fissler J, Schirra C, Kohring GW, Giffhorn F. Hydrogenproduction from aromatic-acids by Rhodopseudomonas palustris. App Microbiol Biotechnol 1994;41(4):395–9.
[47] Ike A, Murakawa T, Kawaguchi H, Hirata K, Miyamoto K.
Photoproduction of hydrogen from raw starch using a
halophilic bacterial community. J Biosci Bioeng
1999;88(1):172–7.
[48] Hemschemeier A, Happe T. The exceptional photofermentative hydrogen metabolism of the green alga Chlamydomonas
reinhardtii. Biochem Soc Trans 2005;33:39–41.
[49] Eroğu E, Gündüz U, Yücel M, Türker L, Eroğu İ. Photobiological
hydrogen production by using olive mill wastewater as
a sole substrate source. Int J Hydrogen Energy 2004;29:
163–71.
[50] Fascetti E, Todini O. Rhodobacter sphaeroids RV cultivation and
hydrogen production in a one and two stage chemostat. Appl
Microbial Biotechnol 1995;44:300–5.
[51] Koku H, Eroglu I, Gunduz U, et al. Aspects of the metabolism
of hydrogen production by Rhodobacter sphaeroides. Int J
Hydrogen Energy 2002;27(11–12):1315–29.
[52] Maeda I, Miyasaka H, Umeda F, et al. Maximization
of hydrogen production ability in high-density suspension
of Rhodovulum sulfidophilum cells using intracellular
poly(3-hydroxybutyrate) as sole substrate. Biotechnol Bioeng
2003;81:474–81.
[53] Macler BA, Pelroy RA, Bassham JA. Hydrogen formation in
nearly stoichiometric amounts by a Rhodopseudomonas
sphaeroides mutant. J Bacteriol 1978;138(2):446–52.
[54] Hillmer P, Gest H. H2 metabolism in photosynthetic
bacterium Rhodopseudomonas capsulata: H2 production by
growing cultures. J Bacteriol 1977;129(2):724–31.