Biohydrogen production from beet molasses by sequential dark and photofermentation

international journal of hydrogen energy 35 (2010) 511–517

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Biohydrogen production from beet molasses by sequential dark and photofermentation Ebru O¨zgu¨r a,*, Astrid E. Mars b, Begu¨m Peksel c, Annemarie Louwerse b, Meral Yu¨cel c, _ Erog˘lu a Ufuk Gu¨ndu¨z c, Pieternel A.M. Claassen b, Inci a

Middle East Technical University, Department of Chemical Engineering, 06531, Ankara, Turkey Wageningen UR, Agrotechnology & Food Sciences Group, Wageningen UR, P.O. Box 17, 6700 AA Wageningen, The Netherlands c Middle East Technical University, Department of Biology, 06531, Ankara, Turkey b

article info

abstract

Article history:

Biological hydrogen production using renewable resources is a promising possibility to

Received 19 August 2009

generate hydrogen in a sustainable way. In this study, a sequential dark and photo-

Received in revised form

fermentation has been employed for biohydrogen production using sugar beet molasses as

27 October 2009

a feedstock. An extreme thermophile Caldicellulosiruptor saccharolyticus was used for the

Accepted 28 October 2009

dark fermentation, and several photosynthetic bacteria (Rhodobacter capsulatus wild type,

Available online 20 November 2009

R. capsulatus hup mutant, and Rhodopseudomonas palustris) were used for the photofermentation. C. saccharolyticus was grown in a pH-controlled bioreactor, in batch mode, on

Keywords:

molasses with an initial sucrose concentration of 15 g/L. The influence of additions of NHþ 4

Biohydrogen

and yeast extract on sucrose consumption and hydrogen production was determined. The

Dark fermentation

highest hydrogen yield (4.2 mol of H2/mol sucrose) and maximum volumetric productivity

Photofermentation

(7.1 mmol H2/Lc.h) were obtained in the absence of NHþ 4 . The effluent of the dark

Molasses

fermentation containing no NHþ 4 was fed to a photobioreactor, and hydrogen production was monitored under continuous illumination, in batch mode. Productivity and yield were improved by dilution of the dark fermentor effluent (DFE) and the additions of buffer, ironcitrate and sodium molybdate. The highest hydrogen yield (58% of the theoretical hydrogen yield of the consumed organic acids) and productivity (1.37 mmol H2/Lc.h) were attained using the hup mutant of R. capsulatus. The overall hydrogen yield from sucrose increased from the maximum of 4.2 mol H2/mol sucrose in dark fermentation to 13.7 mol H2/mol sucrose (corresponding to 57% of the theoretical yield of 24 mol of H2/mole of sucrose) by sequential dark and photofermentation. ª 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

Today, the energy supply of the world’s population mostly depends on limited resources of fossil fuels. The utilization of fossil fuels causes global climate change due to emissions of greenhouse gasses during their combustion. Hydrogen has the potential to be a clean energy carrier, provided that it is produced in a renewable way, since it only produces water

upon combustion. Among the different technologies employed, fermentative biohydrogen production offers an opportunity to utilize renewable resources, including agricultural waste products, which facilitates waste recycling. Sequential dark and photofermentation of organic compounds is a promising method of producing renewable hydrogen. During dark fermentation, sugars are converted to H2, CO2 and short-chain organic acids with a theoretical

* Corresponding author. Tel.: þ90 312 2102696; fax: þ90 312 2102600. ¨ zgu¨r). E-mail address: [email protected] (E. O 0360-3199/$ – see front matter ª 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2009.10.094

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international journal of hydrogen energy 35 (2010) 511–517

maximum hydrogen yield of 4 mol of H2/mole of hexose sugar, when all sugars are fermented to acetate, CO2 and H2. In practice, the hydrogen yields are lower due to the formation of biomass, and more reduced alternative fermentation products like lactate, ethanol, and butyrate. The hydrogen yields are generally higher when thermophiles are applied due to more favorable thermodynamics for hydrogen production at elevated temperatures [1–3]. The effluent of the dark fermentation is used as the substrate for photosynthetic bacteria during the second photofermentative step, in which short-chain organic acids are assimilated to H2 when light is present, producing maximally 4 and 6 mol of H2 per mole of acetate and lactate, respectively. Because of this, the combined two-step process has theoretical maximum hydrogen yield of 12 mol of H2/mole of hexose sugar. This process is being developed within the EU 6th framework project ‘‘HYVOLUTION’’ [4]. Studies with pure sugars showed that the overall yield of H2 can be improved by combining the dark fermentation with the photofermentation [5–7]. The yields achieved by two-step processes on pure sugars range from 2.4 to 6.3 mol H2/mol glucose [8]. However, studies from real feedstocks are rather limited [9,10]. In the present study, the overall hydrogen yield was improved to 13.7 mol of H2/mol of sucrose by the sequential operation of dark fermentation and photofermentation using sugar beet molasses as feedstock. Sugar beet molasses is a by-product of the sugar industry that is obtained as a thick syrup during the crystallization of sucrose. It contains a high amount of sucrose (ca. 50%), and is rich in organic nitrogen, vitamins and salt that may support bacterial growth. It has been described as a suitable feedstock for dark fermentations under mesophilic conditions [11–15]. In this study, an efficient H2 production from sugar beet molasses by sequential operation of dark and photofermentation was aimed. The dark fermentation was operated under extreme thermophilic conditions. Caldicellulosiruptor saccharolyticus was chosen as the hydrogen producing bacterium since the hydrogen yields that are obtained with this species are usually very high [16,17]. Several photosynthetic bacteria (Rhodobacter capsulatus wild type, R. capsulatus hup mutant, and Rhodopseudomonas palustris) were applied during the photofermentative step. The effects of additions of buffer and nutrients on the hydrogen productivity and yield were investigated.

2.

Materials and methods

2.1.

Microorganisms and culture conditions

C. saccharolyticus DSM 8903 was obtained from the Deutsche Sammlung von Microorganismen und Zellkulturen (DSMZ). It was grown overnight in small serum flasks on sucrose at 70  C. The growth medium contained per liter: 0.3 g KH2PO4, 0.3 g K2HPO4, 2.5 mg FeCl3$6H2O, 1 ml SL-10 trace element solution (www.dsmz.de), 0.5 mg resazurin, 0.4 g MgCl2$6H2O, 0.75 g cysteine-HCl, 4 g sucrose, 0.45 g NH4Cl, 0.5 g YE (Duchefa, The Netherlands), and 50 mM of 3-(N-morpholino)propanesulfonic acid (pH 7.0). The fermentation was started by inoculating with 10% of a pre-culture into molasses media.

R. capsulatus (DSM1710) and R. palustris (DSM127) were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany. The mutant strain of R. capsulatus (YO3) lacking uptake hydrogenase gene (hup) was obtained previously in our laboratory [18]. Bacteria were activated in modified SB medium [19] containing acetate (20 mM) and glutamate (10 mM) as carbon and nitrogen sources, and 20 mM of potassium phosphate buffer, pH 6.4. Ten percent inoculation of grown culture was carried out into H2 production media of molasses DFE.

2.2.

Dark fermentation

Dark fermentation was performed in a pH-controlled jacketed 2-L bioreactor (Applikon, The Netherlands) with a working volume of 1 L at 72  1  C. The medium contained per Litre: 0.3 g KH2PO4, 0.3 g K2HPO4, 2.5 mg FeCl3$6H2O, 1 ml SL-10 trace element solution (www.dsmz.de), 0.5 mg resazurin, 0.4 g MgCl2$6H2O, 0.75 g cysteine-HCl, and 27.5 g of molasses, which contained 15 g of sucrose. When applicable, 0.9 g NH4Cl and 1 g of YE (Duchefa, The Netherlands) were added. The pH was maintained at 6.9  0.1 (measured at room temperature) by the automatic addition of 2 M of NaOH. The fermentation was started by inoculating with 10% of a pre-culture that was grown overnight in small serum flasks on sucrose at 70  C. Immediately after inoculation, the cultures were stirred at 100 rpm and sparged with nitrogen at 1 l/h. When the hydrogen concentration in the off-gas became >0.1–1%, the stirring speed was increased to 350 rpm, and sparging with nitrogen was increased to 7 l/h.

2.3.

Photofermentation

Glass bottles with 55 ml liquid volume were used for photobiological hydrogen production. The hydrogen gas produced by the bacteria was collected from the top by a thin hollow tube into a graded glass cylinder initially filled with water which was replaced by the hydrogen produced during the process. The photobioreactors were maintained at 30–33  C in an incubator. The illumination was provided by 100 W tungsten lamp, adjusted to provide a uniform light intensity of 150–200 W/m2 at the surface of the reactor. The initial pH of the medium was 6.7. Dark fermentor effluent (DFE) was centrifuged and sterilized by autoclaving prior to use, to remove contaminants and any colloidal materials that may interfere with light penetration. Several adjustments were carried out to optimize the conditions for photofermentative hydrogen production from DFE: the organic acid concentration of the DFE was decreased 3, 5 and 10 times by diluting the DFE with sterile distilled water, 20 mM of potassium phosphate buffer (pH 6.4) was added to the DFE to maintain the pH in a proper range for photofermentation, and iron (Fe-Citrate, 0.1 mM) and molybdenum (Na2MoO4.2H2O, 0.16 mM) were added.

2.4.

Analytical methods

The concentrations of H2 and CO2 in the headspace of the dark fermentor culture, and the concentrations of sucrose, ethanol, and organic acids in the culture supernatant were determined

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international journal of hydrogen energy 35 (2010) 511–517

C2 H4 O2 þ 2H2 O/4H2 þ 2CO2

(1)

Lactate :

C3 H6 O3 þ 3H2 O/6H2 þ 3CO2

(2)

The light conversion efficiency was determined as the ratio of the total energy value of the hydrogen that has been obtained to the total energy input to the photobioreactor by light radiation. It is calculated by the following equation:

100 0.3

50

mmol/L

0.6

100 0.3

50

0

1

2

time (days)

3.1.

Dark fermentation

C. saccharolyticus was grown in pH-controlled bioreactors on molasses with an initial sucrose concentration of 15 g/L. The influence of additions of NHþ 4 and YE on sucrose consumption and hydrogen production was determined. When NHþ 4 was present in the growth medium, the specific hydrogen production rate was 20 mmol of H2/g protein.h at the start of exponential production of hydrogen. The consumption of sucrose and production of hydrogen were largely completed within 1–1.5 day (Fig. 1A, B). Only a limited amount of NHþ 4 was consumed when YE was added to the culture medium, but this increased to 33% when YE was omitted (Table 1), suggesting that C. saccharolyticus mainly uses YE as nitrogen source when available. The addition of YE is necessary to obtain good growth of C. saccharolyticus on pure sugars. However, the fermentation patterns on molasses were similar in the presence and absence of YE (Fig. 1A,B), indicating that molasses can eliminate the need for expensive YE in the fermentation medium of C. saccharolyticus. When NHþ 4 was omitted from the medium, over 2 days were required to complete the fermentations (Fig. 1C, D). When YE was present, the specific hydrogen production rate at the start of exponential production of hydrogen was equal to the rates observed during the fermentations with NHþ 4 (Table 1). C. saccharolyticus was also able to grow without NHþ 4 and without YE, Apparently, molasses contains sufficient nutrients to support growth in the absence of NHþ 4 and YE, although hardly any hydrogen was produced and growth was very slow (Fig. 1D; Table 1). 200

3

0.0

−

0.9

B

YE + NH4+

150

0.6

100 0.3

50 0

0.0

C

150

0

Results and discussion

mmol/L

0.6

+ YE − NH4+

3.

−

YE − NH4+

0.0

D

150

mmol/L

mmol/L

150

0

where VH2 is the volume of produced H2 in l, rH2 is the density of the produced hydrogen gas in g/L, I is the light intensity in W/m2, A is the irradiated area in m2 and t is the duration of hydrogen production in hours [21].

0.9

A

cell density (g protein/L)

+ YE + NH4+

cell density (g protein/L)

200

(3)

0.6

100 0.3

50 0

0

2

1

cell density (g protein/L)

Acetate :

hð%Þ ¼ ð33:6  rH2  VH2 Þ  100=ðI  A  tÞ

3

cell density (g protein/L)

as previously described [15]. The cell density in the dark fermentor was determined by the micro-biuret assay with bovine serum albumin as a standard [20]. For this, cells were harvested by centrifugation and washed prior to protein analysis. The molecular weight of C. saccharolyticus was previously determined to be 24.6 g (mol C)1 [15]. The cell dry weight of C. saccharolyticus is similar to the protein content as determined with the micro-biuret assay (unpublished data). The total carbon and total nitrogen contents of the DFE of molasses were analyzed spectrophotometrically (DR/2400, Hach-Lange, Germany). A detailed elemental composition of the effluent was determined by atomic absorption spectroscopy (Philips, PU9200X). The evolved hydrogen during photofermentation was measured by gas chromatography (Agilent Technologies 6890N) equipped with a thermal conductivity detector and a Supelco Carboxen 1000 column (60/80 mesh). The oven, injector and detector temperatures were 140, 160 and 170  C, respectively. Argon was used as a carrier gas at a flow rate of 25 ml/min. The organic acid concentrations in the culture medium of the photofermentation were analyzed by HPLC on a MetaCarb 87H column (300  7.8 mm, by Varian ProStar). The biomass concentration in the photofermentor was determined by measuring the optical density at 660 nm with a visible spectrophotometer (Shimadzu UV-1201V). An optical density of 1 at 660 nm corresponds to a cell density of 0.55 g dry weight/L. The molar yield of the photofermentation was calculated as the percent of the ratio of moles of hydrogen that was produced to the theoretical moles of hydrogen that can be produced from utilized organic acids (acetate and lactate). The stoichiometric equations for hydrogen production from acetate and lactate are given below:

0.0

time (days)

Fig. 1 – Fermentation profiles of batch cultivations of C. saccharolyticus in pH-controlled bioreactors on molasses (15 g/L sucrose) in the presence and absence of NHD 4 and YE. The production of H2 (B), acetate (C), lactate (-), and cell biomass (A), and the consumption of sucrose (>) are indicated.

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international journal of hydrogen energy 35 (2010) 511–517

Table 1 – Fermentation data of batch cultivations of C. saccharolyticus in pH-controlled bioreactors on molasses (15 g/L sucrose) in the presence and absence of NHD 4 and YE. a qH b mmol H2/ Molar yield (mol/mol sucrose) C-balance Sucrose NHþ Fermentation NHþ 4 YE Max QH 4 2 2 mmol H2/ (g protein.h) consumption consumption H2 CO2 Acetate Lactate Ethanol (l.h) % %

A B C D

þ þ – –

þ – þ –

6.8 4.0 7.1 0.9

20 20 20 3

2.0 2.0 4.2 0.9

1.5 1.4 2.1 0.8

1.1 0.9 1.9 0.7

2.1 2.0 1.5 2.5

0.3 0.3 0.0 0.1

0.93 0.86 0.91 0.88

97 97 94 79

5 33 n.a.c n.a.c

a Max QH2: maximum volumetric hydrogen productivity. b qH2: specific hydrogen productivity after 6 h of fermentation. c n.a.: not applicable.

Large amounts of lactate were produced besides acetate during all batch fermentations, which reduced the molar hydrogen yields to 10–55% of the theoretical maximum of 8 mol of H2 per mol of sucrose. Furthermore, low amounts of ethanol were observed in some of the cultures (Table 1), but other organic acids, like butyrate or succinate were not detected. The highest hydrogen yield (4.2 mol of H2/mol sucrose) and maximum hydrogen productivity (7.1 mmol of H2/Lc.h) were obtained with the fermentation on molasses with YE without NHþ 4 (Table 1). Previously, a very high hydrogen yield of 3.3 mol of H2/mol hexose was obtained with batch cultivations of C. saccharolyticus on hydrolysate of the lignocellulosic energy crop Miscanthus at a similar initial sugar concentration of 14 g/L [16]. The hydrogen yield and productivity of the dark fermentation with Miscanthus hydrolysate were considerably higher than those with molasses since the formation of large amounts of lactate was not observed with Miscanthus hydrolysate.

3.2.

Photofermentation

3.2.1. Suitability of the DFE of molasses for photofermentation The ammonium content of the DFE is especially important, because NHþ 4 is the inhibitor of the nitrogenase enzyme of purple non-sulphur bacteria [22–25]. No hydrogen production could be observed from dark fermentor effluents containing more than 2 mM ammonium (unpublished data). The best performing dark fermentation on molasses was done without NHþ 4 and the DFE of this fermentation is therefore very suitable for photofermentative hydrogen production. The composition of the DFE of molasses was determined, and compared with the composition of the defined SB medium used in our previous studies for hydrogen production (given in Table 2) [26]. The DFE contained almost ten times more total carbon and nitrogen than the defined SB medium, which might require dilution of the effluent since high organic acid concentrations decreases the yield [27]. The total nitrogen content was high, despite the absence of NHþ 4 , due to nitrogenous compounds (like amino acids) that are present in the molasses itself. The carbon to nitrogen ratio, which is important for efficient hydrogen production, was almost the same as in SB medium. Some of the elements (like Mn, Zn, Ni, and Cu) were higher in the DFE of molasses. However, the amount of iron, which is crucial for nitrogenase activity, was

low in the effluent. Molybdenum, another important cofactor of the nitrogenases of PNS bacteria, was even undetectable within the precision of the instrument.

3.2.2. Effect of dilution on hydrogen production by R. capsulatus on molasses effluent Hydrogen production was not observed from DFE without buffering, as the pH increased up to 10.0. Since negatively charged organic acids are taken into the cell during photofermentation, cells restore their membrane potential by the efflux of negatively charged OH ions, causing increase in pH. Uyar et al. [28] have compared the photofermentative hydrogen production from volatile fatty acids present in dark fermentation effluents. The organic acids such as malate, propionate, lactate and butyrate required low buffering (3.5 mM potassium phosphate), however, acetate containing medium required 6 times higher amounts of buffer in order to keep pH at a favorable range for hydrogen production. The low buffering capacity of DFE of molasses is not sufficient for hydrogen production. Therefore, 20 mM of potassium

Table 2 – The composition of the DFE of molasses compared with synthetic SB medium.

Total N (M) Total C (M) NHþ 4  N C/N molar ratio Fe (mM) Mn (mM) Zn (mM) Ni (mM) Mg (mM) Ca (mM) Co (mM) Cu (mM) Mo (mM) Acetate (mM) Lactate (mM) Sucrose (mM) Glutamate(mM) Biotin (ug/L) Thiamin (ug/L) Niacin(ug/L) a n.a.: not available.

DFE of Molasses

SB Medium

0.025 0.85 0 34 0.035 10.5 23.5 3.4 3.65 1.34 3.05 4.7 0 86 69 2.7 n.a.a n.a n.a n.a

0.002 0.070 0 35 0.1 0.51 0.51 0.084 2 0.3 0.84 0.12 0.16 30 – – 2 15 250 250

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international journal of hydrogen energy 35 (2010) 511–517

A 90 70

7,5

60 50

pH

H2 (mmol/Lc)

8

B

80

40

7

30 6,5

20 10 0

0

1

2

3

4

5

6

6

7

0

1

2

3

Time (days) 50

D

30 20 10 0

5

6

7

30 25

40

Lactate (mM)

Acetate (mM)

C

4

Time (days)

20 15 10 5

0

1

2

3

4

5

6

7

0

0

1

2

3

4

5

6

7

Time (days)

Time (days)

Fig. 2 – Hydrogen production (A), pH change (B), acetate utilization (C) and lactate utilization (D) by R. capsulatus (DSM1710) during photofermentation on 10 (C), 5 (-) and 3 (:) times diluted DFE of molasses.

phosphate buffer (pH 6.4) was added to the DFE, which maintained the pH in a tolerable range (6.8–7.4) for hydrogen production by PNS bacteria (Fig. 2B). As the total amounts of carbon and nitrogen of the effluent were high compared to the defined SB medium (Table 2), the effluent was diluted 10, 5, 3 and 2 times with sterile distilled water to determine the optimum initial organic acid concentration for efficient hydrogen production. The hydrogen production on undiluted DFE was nil (data not shown). Hydrogen production and growth were observed with all dilutions during photofermentation with the wild type strain of R. capsulatus (DSM 1710), and acetate and lactate were completely consumed

(Fig. 2). The best hydrogen productivity and yield were obtained with 3 times diluted DFE, with 67 mmol/Lc of cumulative hydrogen produced at the end of 5 days of incubation (Table 3, Fig. 2A).

3.2.3. Effect of additions of Fe and Mo on hydrogen production by different PNS bacterial strains on molasses effluent Fe and Mo are two essential cofactors for the nitrogenase activity of photosynthetic purple non-sulphur bacteria. It was previously reported that additions of Mo and Fe stimulated the hydrogen production by Rhodobacter sphaeroides, with optimum concentrations of 0.1 mM of Fe-citrate and 0.16 mM

Table 3 – The effect of dilution and additions of iron (0.1 mM) and molybdenum (0.16 mM) on hydrogen production yield, productivity and substrate/light conversion efficiency by R. capsulatus wild type, R. capsulatus hupL mutant and R. palustris. 20 mM of potassium phosphate buffer (pH 6.4) was added to the DFE of molasses. pH was between 6.8 and 7.6 for all reactors throughout the experiment. Bacteria

Max Biomass (gdw/Lc)

Max QH a 2 (mmol/Lc.h)

qH b (mmol/g 2 biomass.h)

Molar Yield (% of Theoretical Max)

Light Conversion Efficiency (%)

     þ

0.58 0.85 1.21 2.29 1.66 1.48

0.08 0.38 0.73 0.80 1.10 0.75

0.14 0.45 0.60 0.35 0.66 0.50

30 18 26 30 39 20

0.09 0.19 0.42 0.55 0.66 0.59

þ þ

 þ

1.14 1.03

0.72 1.37

0.63 1.33

34 58

0.49 0.83

þ þ

 þ

1.44 1.59

0.89 1.16

0.62 0.73

37 46

0.53 0.65

Dilution

Fe

Mo

R. capsulatus (DSM1710)

10 5 3 2 3 3

   þ þ þ

R. capsulatus hup (YO3)

3 3

R. palustris (DSM127)

3 3

a Max QH : Maximum volumetric hydrogen productivity. 2 b qH : Specific hydrogen productivity. 2

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international journal of hydrogen energy 35 (2010) 511–517

160 140

H2 (mmol/Lc)

120 100 80 60 40 20 0

0

1

2

3

4

5

6

7

8

9

Time (days)

Fig. 3 – Effect of iron (0.1 mM) and Mo (0.16 mM) addition on H2 production by PNS bacteria on modified molasses effluent. The effluent was diluted 3 times and all reactors contained 20 mM of KPi buffer at pH 6.4. H2 production by (D)wild type R. capsulatus with Fe addition, (:)wild type R. capsulatus with Fe and Mo addition, (B)hupL mutant of R. capsulatus with Fe addition, (C)hupL mutant of R. capsulatus with Fe and Mo addition,(,)R. palustris with Fe addition,(-)R. palustris with Fe and Mo addition.

of molybdenum. Besides, the genes regulating nitrogenase activity were repressed under Fe and Mo starvation [29,30]. Elemental analysis of DFE revealed low levels of Fe and Mo. Because of this, both cofactors were added to 3 times diluted DFE, and the effect on hydrogen production was monitored. For wild type R. capsulatus, the hydrogen production improved (80 mmol H2/Lc) after addition of Fe (0.1 mM) (Fig. 3). The productivity was increased from 0.73 mmol/Lc.h to 1.10 mmol/ Lc.h, and a yield of 39% (acetate and lactate) was achieved (Table 3). However, addition of Mo (0.16 mM) resulted in a significant decrease of the hydrogen production by wild type R. capsulatus (Fig. 3). In contrast, the addition of Mo largely improved the hydrogen production by the hup strain of R. capsulatus and by R. palustris. The highest hydrogen productivity (1.37 mmol/Lc.h) and yield (58%) were attained with the hup strain of R. capsulatus on DFE containing buffer, Fe and Mo (Table 3). These parameters were all higher than those obtained using defined SB medium containing acetate (40 mM) and lactate (7.5 mM) as carbon sources, and glutamate (2 mM) as nitrogen source, which resulted in a yield and productivity of 19% and 0.49 mmol/Lc.h, respectively (unpublished data). The light conversion efficiency is a significant measure of the energy utilized, and especially critical in indoor experiments. Light conversion efficiency decreased from 0.42 to 0.09% with dilution of the DFE from 3X to 10X (Table 3). The maximum light conversion efficiency was estimated as 0.83 that has been obtained for R. capsulatus hup- after 3X dilution and addition of Fe and Mo. This value is quite comparable to the results obtained previously on defined medium containing acetate and lactate mixtures [27].

4.

Conclusion

In this study, the feasibility was shown of the sequential operation of thermophilic dark fermentation and photofermentation with sugar beet molasses as the feedstock using

batch fermentation systems. The hydrogen yields that were obtained during the thermophilic dark fermentations on molasses were considerably lower than the theoretical maximum due to the formation of large amounts of lactate, with the highest yield being 4.2 mol of H2/mol sucrose. The effluent of this fermentation did not contain NHþ 4 and was therefore a very suitable hydrogen production medium for photosynthetic purple non-sulphur bacteria, provided that the effluent was diluted and buffered to keep the pH at a tolerable range. The DFE of molasses contained acetate and lactate as the major carbon sources for photofermentation. Since lactate was completely consumed by the PNS bacteria, the reducing equivalents that were consumed for the production of lactate during the dark fermentation were recovered as hydrogen during the photofermentation, restoring the overall hydrogen yield of the process. The additions of Fe and Mo significantly improved the hydrogen production by hup R. capsulatus and R. palustris. The best hydrogen production was attained with hup R. capsulatus on 3 times diluted molasses effluent supplemented with adequate amounts of buffer, Fe and Mo. When the hup strain of R. capsulatus was used during photofermentation, an overall hydrogen yield of the two-step fermentation process of 13.7 mol of H2/mol of sucrose was obtained, which corresponds to 57% of the theoretical yield of 24 mol of H2/mole of sucrose. Further investigation of sequential dark and photofermentation for hydrogen production on molasses should be based on the continuous operations.

Acknowledgements This study was financially supported by the Commission of the European Communities, Sixth Framework Programme, Priority 6, Sustainable Energy Systems (019825 HYVOLUTION).

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