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Photofermentative production of hydrogen from organic acidsby the purple sulfur bacterium Thiocapsa roseopersicina

Andrea Nyilasi a, Eva Molnos a,b,c, Szabolcs Lanyi c, Iosif Nagy b, Gabor Rakhely a,Kornel Lajos Kovacs a,d,*aDepartment of Biotechnology, University of Szeged, Kozep fasor 52, Szeged H-6726, Hungaryb Faculty of Applied Chemistry and Material Science, Politehnica University of Bucharest, Str. Polizu 1-7, Bucures‚ti RO-011061, Romaniac Faculty of Technical and Social Sciences, Sapientia e Hungarian University of Transylvania, P-ta Libert�atii 1, Miercurea Ciuc RO-530104,

Romaniad Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Temesvari krt. 52, Szeged H-6726, Hungary

a r t i c l e i n f o

Article history:

Received 8 November 2012

Received in revised form

11 February 2013

Accepted 18 February 2013

Available online 23 March 2013

Keywords:

Biohydrogen

Nitrogenase

Organic acid

Photofermentation

Thiocapsa roseopersicina

* Corresponding author. Department of Biotefax: þ36 62 544352.

E-mail addresses: kovacs.kornel@brc.mta0360-3199/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.ijhydene.2013.02.0

a b s t r a c t

A mutant strain of the anaerobic purple sulfur bacterium Thiocapsa roseopersicina, con-

taining only nitrogenase as a functionally active enzyme for H2 generation was utilized to

study the production of H2 from organic acids (acetate, pyruvate and succinate). Two types

of potential substrates for H2 production, thiosulfate and salts of various organic acids,

were compared under photoheterotrophic growth conditions. Thiosulfate proved to be the

preferred electron donor for T. roseopersicina; the consumption of organic acids became

pronounced only following depletion of the thiosulfate supply. The system is suitable for

the generation of H2 from effluents of heterotrophic dark fermentation processes or waste

streams rich in inorganic reduced sulfur compounds and/or simple organic acids.

Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights

reserved.

1. Introduction products [5,6]. Furtherutilizationof theorganicacids is feasible

Various strategies have been proposed for biological H2 pro-

duction, including the direct and indirect biophotolysis of

water, photofermentation, dark fermentation, or hybrid

fermentation technologies [1e3]. H2 generation in light-

dependent (photofermentation) and light-independent (dark

fermentation) processes is of practical importance by virtue of

the combination of H2 production with the elimination of

organicwastematerials in a single step [4]. The dark anaerobic

fermentation of a biomass, e.g. cellulose, starch, protein or

lipids, usually yields various organic acids as metabolic

chnology, University of Sz

.hu, kornel@brc.hu (K.L.2013, Hydrogen Energy P84

energetically only if there is an additional energy input, and

these metabolites therefore accumulate in the dark fermen-

tationeffluents [4]. The additional energymaybe introduced in

the form of light and a photofermentation step will allow

complete conversion of the biomass to biohydrogen.

A diverse group of photosynthetic bacteria can utilize

organic acids as carbon and energy sources [7]. A two-step

strategy H2 production, involving dark heterotrophic and

photofermentation processes, has been proposed to approach

the theoretical maximal H2 yield from a biomass [8,9]. The

large-scale industrial application of this technology is limited

eged, Kozep fasor 52, Szeged H-6726, Hungary. Tel.: þ36 62 546940;

Kovacs).ublications, LLC. Published by Elsevier Ltd. All rights reserved.

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 5 5 3 5e5 5 4 45536

by the relatively low efficiency of H2 production by photo-

synthetic bacteria, which can be attributed to their slow

growth and ineffective utilization of light energy [4].

From among the different bacteria capable of photo-

fermentative H2 production, the anaerobic, purple sulfur

phototrophic bacterium Thiocapsa roseopersicina was chosen

for this studybecauseof its versatileH2 production capabilities

and because it harbors a nitrogenase in addition to several

hydrogenases [10,11]. In previous work, the H2 productivity of

the soluble cytoplasmatic hydrogenase Hox1 and nitrogenase

in T. roseopersicina were compared by using mutant strains,

and the affinity for simple organic acids was tested. The

overall yield of H2 production via the nitrogenase was higher

than that obtained with Hox1 during the initial phase. The H2

evolution by the Hox1 enzyme, however, was more sustain-

able, and the cumulative total H2 generation capabilities of the

two enzymes were therefore comparable [11,12]. Hox1 in T.

roseopersicina is a constitutive enzyme, which is an advantage

of Hox1-driven H2 production. Nonetheless, the copy number

of the nitrogenase is much higher than that of functionally

active Hox1, and the nitrogenase route may therefore offer

additional benefits for large-scale exploitation.

The aim of the present study was to evaluate the feasibility

of utilizing the organic acids generated from dark fermenta-

tion as substrates for H2 production by photofermentation

with T. roseopersicina. Reduced sulfur compounds such as Na-

thiosulfate or Na-sulfide are the primary electron donors for

photosynthesis in this bacterium [13,14], and the levels of

consumption of thiosulfate in the presence of the organic

acids and the concomitant H2 evolution were therefore

determined.

2. Materials and methods

2.1. Bacterial strains and growth conditions

AmutantofT. roseopersicinaBBS (wild-typestrain), strainM539,

in which the hypF gene had been knocked out, was used

throughout this study. A functional HypF accessory protein is

required for the appropriate assembly of the active center of

[NiFe]-hydrogenases [15]. All the hydrogenases in T. rose-

opersicina are [NiFe]-hydrogenases, and the mutation in hypF

therefore rendered the bacterium unable to produce active

[NiFe]-hydrogenases. Strain M539 is able to evolve H2 only via

the inducible nitrogenase under nitrogen-fixing conditions

[16]. T. roseopersicina strain M539 (DhypF ) was routinely culti-

vated anaerobically in liquid Pfennig’s mineral culture me-

dium (2%NaCl, 0.1% KH2PO4, 0.1%MgCl2, 0.1% KCl, 0.1%NH4Cl

as nitrogen source, 0.2% NaHCO3 as carbon source, 0.4%

Na2S2O3, 20mg/ml vitaminB12, 3.3mg/l Fe-EDTA, 2.975mg/lNa2-

EDTA, 0.3mg/l H3BO3, 0.2mg/l CaCl2, 0.1mg/l ZnSO4, 0.03mg/l

MnCl2, 0.03 mg/l Na2MoO4, 0.713 mg/l NiCl2, 0.01 mg/l CuCl2)

with continuous illumination (35 mmol photons/m2 s light in-

tensity) at 25 �C [10,11,16]. The pH was maintained at 7.2.

The liquid medium used in the H2 production experiments

was the same as that employed for bacterial growth, except

that it did not containNH4Cl andwas supplementedwith salts

of organic acids (acetate, pyruvate or succinate) in various

initial concentrations. The thiosulfate concentration was

varied between 0 and 4.0 g/l. The mineral reaction mix was

filter-sterilized, dispensed into serum bottles (total working

volume 125 ml) under aseptic conditions and inoculated with

3% (v/v) fresh T. roseopersicina culture, and the bottles were

sealed with a rubber septum and Al crimp. The headspace of

the bottles was purged with ultrapure N2 gas to maintain

anaerobic conditions and to provide N-source. The samples

were incubated at 25 �C under continuous illumination for 2

weeks. The standard deviation of at least 3 independent ex-

periments are indicated in the figures.

2.2. Analytical methods

The H2 gas that accumulated in the headspace was sampled

every third day with a gas-tight Hamilton syringe (200 ml) and

was measured by gas chromatography (Agilent Technologies

6890N equipped with a thermal conductivity detector and a

Molesieve 30 m � 0.53 mm � 25 mm column). The operational

temperature of both the oven and the detector was 160 �C. N2

was used as carrier gas.

The concentration of residual organic acid in the culture

medium was determined by using a high-performance liquid

chromatograph (Hitachi Elite) equipped with an ICSep ICE-

COREGEL 64H column and a refractive index detector. 0.1 N

H2SO4 served as the mobile phase at a flow rate of 0.8 ml/min.

The column temperature was 50 �C and the detector temper-

ature was 41 �C. The sample volume used for organic acid

determination was 50 ml, obtained after centrifugation of

0.5 ml of cell suspension at 13,000 rpm for 10 min. The cell

suspensions were harvested after each H2 gas determination,

and were stored at �20 �C until further analyses.

The thiosulfate content of the medium was determined

spectrophotometrically on the supernatant of the cell sus-

pension after centrifugation at 22,500 rpm for 15 min. After

dilution, the thiosulfate content was measured by UV ab-

sorption at 230 nm using quartz cells. The calibration plot for

thiosulfate was linear (A230nm ¼ 0.019 Cthiosulfate þ 0.0029;

R2 ¼ 0.9973) for standard Pfennig mineral medium supple-

mented with 0e4.0 g thiosulfate/l.

The bacterial growth was monitored by determining the

total protein contents of the cell suspensions by the micro-

Lowry method [17], using bovine serum albumin as standard.

3. Results and discussion

Photoheterotrophic bacteria are capable of utilizing a wide

variety of substrates as carbon and nitrogen sources. The

metabolic pathways involved are wide-ranging and differ be-

tween species or even between different strains of the same

species. A variety of substrates can be used for growth, but

only a few are suitable for efficient H2 production [18].

The substrate concentration is one of the most important

parameters in the optimization of the H2 production. The

experiments with organic acids were therefore conducted

with different initial concentrations of the selected substrates.

The biomass accumulation of T. roseopersicina cannot be

monitored with the commonly used method of determining

the optical density of the culture at 660 nm, because sulfur

globules are stored inside the cell during growth on

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 5 5 3 5e5 5 4 4 5537

thiosulfate. These globules scatter light and therefore inter-

fere with optical density measurements. For this reason, the

rate of bacterial growth was followed by determining the total

protein content of the culture, which is proportional to the

quantity of biomass.

3.1. Growth and production of H2 from acetate andthiosulfate

A common main product of dark anaerobic fermentation is ac-

etate [6], which can be converted to H2 through photo-

fermentation. In the presence of both thiosulfate and acetate,

the metabolic pathways leading to H2 production interact with

each other. Fig. 1 demonstrates that T. roseopersicina grows best

at a thiosulfate concentration of 4 g/l. Decreasing the reduced

sulfur concentration seriously limits cell growth. At 4 g/l thio-

sulfate, acetate didnot support cell growth; thepropagation rate

decreased following consumption of the thiosulfate. The situa-

tion changed significantly when thiosulfate became growth-

limiting. The cultures were less dense, but the cells started to

utilize the available acetate for growth. Fig. 2 shows the changes

in time of cumulative H2 formation via nitrogenase in the pres-

ence of various amounts of acetate and thiosulfate.

Supplementation of the culture medium with acetate

resulted in increased H2 production relative to the control

(culture without acetate), irrespective of the thiosulfate con-

tent. The cumulative H2 production reported relative to the

culture volume was nearly the same (270e280 ml) when the

acetate concentration was increased up to 6 g/l (data not

shown), indicating that an initial thiosulfate concentration of

4 g/l is a saturating concentration for both growth and H2

production. It is noteworthy that, at all thiosulfate concen-

trations, the addition of acetate led to enhanced H2 evolution.

It may be noteworthy that at the saturating initial concen-

tration of thiosulfate (4 g/l) addition of acetate resulted in

partial growth inhibition when practically all thiosulfate was

consumed by the bacteria (Fig. 1). There is no obvious

Fig. 1 e Total protein contents of cultures, demonstrating bacte

concentrations (> e without acetate; D e 2 g/l acetate; , e 4 g

rationalization of this effect, which manifested itself with the

other organic acids as well (see Figs. 4 and 7). At lower initial

thiosulfate concentrations acetate can contribute to cell pro-

liferation to a limited degree, nevertheless it is primarily used

for H2 generation by T. roseopersicina cells. This tendency is

more pronounced at low thiosulfate concentrations: although

the cells need the electrons from thiosulfate to support

photosynthetic growth, acetate cannot replace thiosulfate in

this role; instead the low number of cells evolve H2 from

acetate.

A relatively low initial concentration of acetate was suffi-

cient to support H2 production with high efficacy. Elevation of

the initial acetate concentration from 2 to 6 g/l did not in-

crease the cumulated H2 evolution significantly. The decrease

in the absolute values in response to lower thiosulfate con-

tents is explained by the restricted growth under these

conditions.

The highest H2 concentration in the headspace was 73.8%

(v/v), obtained at 4 g/l thiosulfate and 6 g/l acetate. When 2 g/l

thiosulfate and 6 g/l acetate were added to the initial medium,

the H2 concentration decreased somewhat, to 68.1% (v/v).

These levels are particularly promising when compared with

the H2 concentrations in the headspace of control cultures

containing 4 or 2 g/l thiosulfate alone, which evolved 22.1 and

11.1% H2 (v/v), respectively. It should be noted that the H2-

evolving activity of nitrogenase, unlike that of [NiFe]-

hydrogenases, is not subject to product inhibition.

The substrate degradation rate is a key factor in optimizing

H2 production, especially when coupled with wastewater

treatment. Fig. 3 illustrates the consumption of acetate and

thiosulfate in time.

It is noteworthy that the kinetics of thiosulfate consump-

tion was not influenced by the presence of acetate, regardless

of the initial thiosulfate content. At least 75% of the thiosulfate

was metabolized during the first 3 days of incubation, while

the acetate concentration did not decrease significantly. At a

very low thiosulfate concentration (0.5 g/l), the severely

rial growth at different thiosulfate and acetate

/l acetate; D e 6 g/l acetate).

Fig. 2 e Cumulative production of H2 by nitrogenase in the presence of various amounts of thiosulfate and acetate. (A.

Variation with time: > e without acetate; D e 2 g/l acetate; , e 4 g/l acetate; D e 6 g/l acetate; B. H2 produced by incubation

for 6 days: , e without acetate; e 2 g/l acetate; e 4 g/l acetate; - e 6 g/l acetate).

Fig. 3 e Variation of thiosulfate and acetate concentrations with time. Dotted lines depict thiosulfate concentrations.

(Without marker e without acetate, D e 2 g/l acetate, , e 4 g/l acetate, D e 6 g/l acetate. Continuous lines depict acetate

concentrations: B e 0.5 g/l thiosulfate, 3 e 2 g/l thiosulfate, * e 4 g/l thiosulfate).

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 5 5 3 5e5 5 4 45538

Fig. 4 e Total protein contents of cultures, reflecting bacterial growth at different thiosulfate and pyruvate concentrations.

(> e without pyruvate; D e 2 g/l pyruvate; , e 4 g/l pyruvate; D e 6 g/l pyruvate.)

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 5 5 3 5e5 5 4 4 5539

starving cells started to utilize acetate earlier, probably due to

the life-threatening stressful conditions. The results corrob-

orate that thiosulfate is consumed by the bacterium primarily

to support photosynthetic growth.

Overall, the data suggest that T. roseopersicina is able to

produce a considerable amount of H2 from acetate, but the cell

growth is limited by thiosulfate. Acetate has a very limited

capacity to replace thiosulfate in supporting bacterial growth.

The supplementation with acetate is primarily beneficial for

H2 production, and nitrogenase utilizes the chemical energy of

these two substrates in partially overlapping, but different

pathways.

3.2. Growth and production of H2 from pyruvate andthiosulfate

Pyruvate is a central metabolite of the carbohydrate meta-

bolism of most organisms, and should therefore be wide-

spread in anaerobic fermentation effluents. We reported

earlier that supplementation of the culture medium contain-

ing 4 g/l thiosulfate (basal medium) with pyruvate resulted in

higher H2 production rates [12], but the systematic co-

metabolism of thiosulfate and pyruvate was not studied.

The growth curves in the presence of various initial amounts

of thiosulfate and pyruvate are shown in Fig. 4. As in the case

of acetate, pyruvate does not appear to support bacterial

growth if a sufficient amount of thiosulfate is present in the

environment. Unexpectedly, pyruvate becomes inhibitory

when the thiosulfate has been consumed by the cells. At lower

thiosulfate concentrations, pyruvate is probably utilized as an

electron source for photoheterotrophic growth, but only a very

small fraction of this substrate can substitute thiosulfate in its

physiological function.

The cumulative production of H2 via nitrogenase at

different initial concentrations of thiosulfate and pyruvate is

depicted in Fig. 5.

A similar response to that of acetate, as regards growth and

H2 evolution, was observed when pyruvate was added to the

growthmediumtogetherwith thiosulfate.Thesupplementation

withpyruvate resulted ina substantial increase inH2production

relative to the control at all thiosulfate concentrations. In these

experiments, the maximum H2 production (w265 ml) was

attained with an initial 4 g/l thiosulfate and 2 g/l pyruvate.

At an initial 2 g/l thiosulfate, the H2 production increased

from 122.7 ml to 228.6 ml when the pyruvate concentration

was raised from 2 to 6 g/l. Similarly, the addition of pyruvate

increased the H2 production at an initial thiosulfate concen-

tration of 0.5 g/l. However, when there was no thiosulfate in

the culture medium, only a pyruvate concentration of 6 g/l

produced ameasurable amount of H2 in the headspace, due to

the inefficient contribution of pyruvate to cell growth. These

results resembled those obtained with acetate supplementa-

tion, indicating that thiosulfate was needed for bacterial

growth and that, when the primary electron donor was un-

available for T. roseopersicina, no growth and consequently no

H2 production took place. Pyruvate could serve as a better

electron source than acetate for photosynthetic growth but its

contribution was limited. A headspace H2 concentration of

68.4% (v/v) was attained at 4 g/l thiosulfate and 2 g/l pyruvate

as substrate. At 2 g/l thiosulfate and 6 g/l pyruvate, the H2

concentration was 58.8% (v/v).

The results verify that T. roseopersicina is able to produce a

significant amount of H2 from pyruvate, but the presence of

thiosulfate is indispensable for bacterial growth. The role of

pyruvate in H2 production becomes significant when the

thiosulfate content is low, as shown in Fig. 6.

In contrast with the contribution from acetate, the con-

sumption of the primary electron donor was influenced by the

presence of pyruvate. As the pyruvate concentration in the cul-

ture medium was increased, the thiosulfate consumption rate

decreased. Itwasnoteworthy that, inanenvironmentcompletely

devoid of thiosulfate, pyruvatewas consumed at a rate similar to

Fig. 5 e Cumulative production of H2 by nitrogenase in the presence of different amounts of thiosulfate and pyruvate. (A.

Variation with time: > e without pyruvate; D e 2 g/l pyruvate; , e 4 g/l pyruvate; D e 6 g/l pyruvate; B. H2 produced by

incubation for 6 days: , e without pyruvate; e 2 g/l pyruvate; e 4 g/l pyruvate; - e 6 g/l pyruvate).

Fig. 6 e Variation of thiosulfate and pyruvate concentrations with time. Dotted lines depict the thiosulfate concentrations.

(Without marker e without pyruvate, D e 2 g/l pyruvate, , e 4 g/l pyruvate, D e 6 g/l pyruvate. Continuous lines represent

the pyruvate concentrations: B e 0.5 g/l thiosulfate, 3 e 2 g/l thiosulfate, * e 4 g/l thiosulfate).

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i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 5 5 3 5e5 5 4 4 5541

that observed in the thiosulfate-containing medium. Pyruvate

could therefore effectively replace thiosulfate as an electron

donor to the photosynthetic apparatus, although its primary

contribution is toward photoheterotrophic H2 production.

3.3. Growth and production of H2 from succinate andthiosulfate

Succinate is another organic acid which has a beneficial effect

on H2 production by nitrogenase in T. roseopersicina [12]. Fig. 7

presents growth curves in the presence of succinate, while

Fig. 8 illustrates H2 production via nitrogenase at various

concentrations of thiosulfate and succinate.

Similarly aswith acetate and pyruvate, supplementation of

the culture medium with succinate resulted in an increased

H2 production relative to the control. The cumulative H2 pro-

duction expressed on the basis of culture volume was highest

when the initial concentrations of thiosulfate and succinate

were 4 g/l and 6 g/l, respectively. Up to 6 g/l an increase in

succinate concentration was accompanied by an elevated H2

production (from 214.6 ml to 275.4 ml in the presence of 4 g/l

thiosulfate).

At an initial thiosulfate concentration of 0.5 g/l, the succinate

concentration didnot affect theH2 production significantly. The

results are in line with the observation that succinate did not

influence the proliferation of the bacterium (Fig. 7).

The highest H2 concentration in the headspace was 70.8%

(v/v) at initial concentrations of 4 g/l thiosulfate and 6 g/l

succinate.

The results indicate that T. roseopersicina is able to produce

a significant amount of H2 from succinate provided that the

thiosulfate-dependent growth is maintained.

The kinetics of the substrate consumption was similar to

the results obtained with the previously tested organic acids.

Fig. 9 shows that succinate is not taken up as long as thio-

sulfate is available. The majority of the thiosulfate was uti-

lized during the logarithmic growth phase in the first 3 days.

Fig. 7 e Total protein contents of cultures, reflecting bacterial g

(> e without succinate; D e 2 g/l succinate; , e 4 g/l succinate

Succinate could not replace thiosulfate in its growth-

supporting and H2-production functions, except at an initial

thiosulfate concentration of 0.5 g/l. This interesting case

suggests that the distinction between the two electron sources

is not absolute.

3.4. Rate of H2 production and efficiency of substrateconsumption

Two indicators are frequently used to evaluate the H2 pro-

duction performance of a specific substrate, i.e., the H2 pro-

duction rate, expressed on the basis of the culture volume or

bacterial dry weight, and the substrate consumption, i.e., how

much of the substrate is utilized for H2 production [18].

The results presented in the previous sections demon-

strated that the tested organic acids increased the H2 pro-

duction relative to the controls. For comparison, the average

H2 production rates and the substrate consumption are to be

seen in Fig. 10.

The highest H2 production rate was obtained on the use of

2 g/l acetate and 4 g/l thiosulfate, when a 3-fold increase was

achieved relative to the control without acetate. Under these

conditions, the organic substrate was completely consumed

by the bacterium. Elevation of the acetate concentration

resulted in a lower H2 production rate and the substrate con-

sumption dropped to below 70%. Lowering the thiosulfate

concentration in the culture medium facilitated acetate con-

sumption. Nevertheless, the H2 production rate decreased,

which may be of importance from a practical point of view.

The second best organic acid substrate for H2 production

was pyruvate, use of which also led to a high substrate con-

sumption, i.e. more than 77%. In this case, a decrease of the

thiosulfate concentration resulted in a sharp drop in the H2

production rate, but it still exceeded the corresponding con-

trol level. The pyruvate consumption did not increase with

decrease of the thiosulfate content in the culture medium as

in the acetate case. Remarkably, the pyruvate consumption

rowth at different thiosulfate and succinate concentrations

; D e 6 g/l succinate).

Fig. 8 e Cumulative production of H2 by nitrogenase in the presence of different amounts of thiosulfate and succinate. (A.

Variation with time: > e without succinate; D e 2 g/l succinate; , e 4 g/l succinate; D e 6 g/l succinate; B. H2 produced by

incubation for 6 days: , e without succinate; e 2 g/l succinate; e 4 g/l succinate; - e 6 g/l succinate).

Fig. 9 e Variation of thiosulfate and succinate concentrations with time. Dotted lines depict thiosulfate concentrations.

(Without marker e without succinate, D e 2 g/l succinate, , e 4 g/l succinate, D e 6 g/l succinate. Continuous lines

represent the succinate concentrations: B e 0.5 g/l thiosulfate, 3 e 2 g/l thiosulfate, * e 4 g/l thiosulfate).

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 5 5 3 5e5 5 4 45542

Fig. 10 e Average H2 production rates. A: and substrate conversion efficiencies, B: in the presence of different organic acids

at various concentrations (- e 4 g/l thiosulfate; e 2 g/l thiosulfate; e 0.5 g/l thiosulfate; , e without thiosulfate).

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 5 5 3 5e5 5 4 4 5543

was more than 70% in the culture initiated without thiosul-

fate, although H2 was not produced from the pyruvate.

The lowest average H2 production rates were obtainedwith

succinate as organic substrate for H2 production, but the

values were still higher than those corresponding to the con-

trols. Reduction of the thiosulfate concentration decreased the

H2 production rate; the succinate consumption increased.

4. Conclusions

The purple sulfur bacterium T. roseopersicina is able to produce

significant amounts of H2 from organic acid substrates, such as

acetate, pyruvate and succinate. The organic substrates were

taken up and/or consumed in distinct fashions. Decrease of the

thiosulfate concentration in the culture medium was not al-

ways accompanied by a higher organic substrate utilization,

suggesting that the inorganic reduced sulfur and organic acid

substrates were utilized in distinct metabolic pathways. Pho-

totrophic non-sulfur bacteria usually produce significantly

more H2 via their nitrogenase system [1,6,8,9,18] relative to T.

roseopersicina. H2 production from these organic materials by

purple sulfur bacteria can be an economically viable process if it

is associated with waste disposal. Further research is needed to

elucidate the metabolic pathways participating in H2 evolution

from the various electron sources.

Acknowledgments

This work was supported by EU projects HUSRB/1002/214/041

IPA, HURO/1001/193/2.2.2 CBC, HyVolution FP6-IP-SES6 019825

and FP7 Collaborative Project SOLAR-H2 FP7-Energy-212508

and IEE/10/235 SI2.591589 GreenGasGrids. Domestic funds

from TAMOP-4.2.1/B-09/1/KONV-2010-0005 and TAMOP-4.2.2/

B-10/1-2010-0012 are gratefully acknowledged. Financial

assistance from Sectoral Operational Program Human Re-

sources Development 2007e2013 of the Romanian Ministry of

Labor, Family and Social Protection through Financial Agree-

ment POSDRU/6/1.5/S/19, and from Domus Hungarica is

gratefully acknowledged by Eva Molnos.

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