Photoproduction of Molecular Hydrogen by Rhodobacter sulfidophilus

System. Appl. Microbiol. 8, 19-23 (1986)

Photoproduction of Molecular Hydrogen by Rhodobacter

sulfidophilus P. STEVENS, N. PLOVIE, P. DE VOS, and

J.

DE LEY

Laboratorium voor Microbiologie en microbiele Genetica, Faculteit der Wetenschappen, Rijksuniversiteit, B-9000 Gent, Belgium

Received September 7, 1985

Summary Light-dependent anaerobic hydrogen production by the marine photosynthetic organism Rhodobacter sulfidophilus was investigated and demonstrated with DL-malate, DL-Iactate, acetate or butyrate as Hdonor. The H2 production was optimal when the pH of the medium was stabilized at 6.5-6.9 either by increasing the phosphate buffer concentration up to 80 mM or by continuous pH monitoring and correction.

Key words: Rhodobacter sulfidophilus- Hydrogen production - Waste treatment - Hydrogen recycling Introduction Hydrogen gas is one of the possible alternative energy vectors of the future to replace or complement fossil hydrocarbons. It is also a pollution-free fuel. Therefore, several microbial hydrogen-evolving systems have become of interest. Indeed, biological hydrogen production has been observed by a great number of microbial species (De Vos et al., 1983; Karube et al., 1982; Zhang et al., 1983; Zurrer, 1982; Feijtel et aI., 1985). Among these, the photosynthetic bacteria are particularly important because they use solar energy to convert the reducing equivalents from organic compounds into H 2. Light-dependent production of hydrogen gas was first observed in cultures of Rhodospirillum rubrum (Gest and Kamen, 1949 a, b), growing photoheterotrophically on media containing dicarboxylic acids of the TCA cycle and glutamate as nitrogen source. H2 is not evolved in large quantities when the cells are growing on ammonium salts or on molecular nitrogen as nitrogen source (Gest and Kamen, 1949 a, b). The inhibitory effect of NH4-ions and N2 on H2 production provides evidence for a correlation between light-dependent H2 production and nitrogen fixation (Wall et al., 1975). The hydrogenase activity of nitrogenase is responsible for the hydrogen production from NADH + H+ (Gottschalk, 1979): 5 ATP

+ NADH + H+ ~ H2 + 5 ADP + 5 Pi + NAD+ nitrogenase/hydrogenase

Light-dependent evolution of hydrogen has been observed with other members of the Rhodospirillaceae as

well. Indeed, our own findings (Stevens et aI., 1983) and these of others (Segers et al., 1983; Willison et al., 1982; Hillmer and Gest, 1977) show that Rhodobacter capsulatus (Van Niel, 1944) Imhoff et al., 1984 (Rhodopseudomonas capsulata AL ) (Imhoff et aI., 1984) is able of evolving large amounts of hydrogen gas from organic compounds. Matsunaga and Mitsui (1982) demonstrated hydrogen production from an unidentified marine photosynthetic bacterium. We examined the H2 production of four strains of the marine Rhodobacter sulfidophilus (Hansen and Veldkamp , 1973) Imhof{ et al., 1984 (Rhodopseudomonas sulfidophila AL) (Imhoff et al., 1984). We found that these organisms are able to produce appreciable amounts of H2 from different H-donors in defined conditions. These organisms may be of particular interest whenever H2 production is envisaged in marine or highsalt conditions. The present paper deals with these observations.

Materials and Methods Organisms used We used four strains of Rhodobacter sulfidophilus: LMG 5202 (= W4), LMG 5205 (= W12) (Hansen and Veldkamp, 1973), LMG 5203 (= 981) and LMG 5204 (= 989) (De Bont et al.,1981).

20

P. Stevens, N. Plovie, P. de Vos, and J. de Ley

Medium and incubation conditions for gas production The RSM medium used for growth and possible gas production contained per liter 10 mM phosphate buffer (KzHP0 4, KH zP0 4 ) pH 6.9,1 g L-glutamic acid, 120 mg MgS04' 7H zO, 75 mg CaCl z . 2H zO, 1 ml each of trace elements solution and vitamin solution (Imhoff and Truper, 1977),5.2 mg di-Na-EDTA, 11.8 mg FeS04 . 7H zO, 0.5 g ascorbic acid, 0.7 g Na ZS04, 30 g NaCl, 30 mM organic substrate. This medium was used to test growth and gas production wjth different organic substrates (DLlactate, DL-malate, acetate and butyrate) as H-donors, and different phosphate buffer concentrations.

~

Equipment For qualitative experiments on growth and Hz production, 100 ml all-glass vials were used with an inlet for flushing and an outlet for Hz production. The gas produced was captured under inverted test tubes under water. Illumination was provided by a bank of tubular incandescent lamps ( 6 x 125 W); the temperature was about 30°C. For quantitative experiments, Hz production from the different H-donors was carried out in our fermentation vessels, as described earlier (Stevens et aI., 1983), illuminated with a 60 W lamp at 30°C. Hz production at constant pH was carried out in a 2.5 liter all-glass fermentor of our own design (Fig. 1). The temperature was controlled by means of a double-walled system. Illumination was provided by 6 tubular tungsten lamps of 60 W. A number of in- and outlets are shown in Fig. 1.

~ ~

4

.~ ~ ~

~ ~

~• f~

13

~~

..,E C>

III

I I

I I I I

I

10

t

I

I I I

I I

I

I I

I

Hydrogen detection The gas produced was analyzed by means of a gas chromatograph (Intersmat IGC 112M; TC detector) as reported previously (De Vos et aI., 1983).

~I I I I I

I

U

11

---",.;----~~

14

Determination of lactate, malate, acetate, butyrate In general, for lactate, acetate and butyrate we followed the methods for sample preparation, detection and quantitative determination as described in the anaerobe laboratory manual (The Staff of the Anaerobe Laboratory Manual,1977). For malate we followed the same methods as for lactate. The quantitative determination of acetate and butyrate was performed by using valeric acid as internal standard, for lactate and malate the peak height was used. Samples were taken at the beginning and the end of each experiment. For the gas chromatographic analyses we used a Tracor 550 gas chromatograph (FID) with a 6 it long Chromosorb W-AW 80/100 mesh (liquid phase 10% 20M terephtalic acid) column.

Cell mass determination (dry weight determination) Dry weight was determined by collecting 3 ml of the cell suspension on a membrane filter (diameter 47 mm, pore size 0.45 ~m). The filter was dried before weighing.

Hz yield The experimental Hz yields for the different H-donors were calculated in %, using the following theoretical maximal conversions as 100%: lactic acid + 3 HzO ~ 3 COz + 6 Hz; malic acid + 3 HzO ~ 4 COz + 6 Hz; acetic acid + 2 HzO ~ 2 CO 2 + 4 Hz; butyric acid + 6 HzO ~ 4 CO 2 + 10 H2 When based on the amount of substrate supplied, we call it "virtual yield", on the amount of substrate used, we call it "real yield".

Fig. 1. Fermentor (2.5 1) for testing quantitative hydrogen production with photosynthetic bacteria. 1 Flush tube; 2 opening for thermometer; 3 pH-electrode; 4 opening for sampling; 5 opening for acid supply; 6 opening for base supply; 7 opening for gasoutlet; 8, 9 openings for medium in and out; 10 double wall with cooling water; 11 inlet cooling water; 12 outlet cooling water; 13 tubular tungsten lamps (60 W); 14 thermostatic bath; 15 pH controller.

Results and Discussion

Rhodobacter sulfidophilus is able to grow on a basal medium with a whole range of organic compounds (Hansen and Veldkamp, 1973). This medium cannot be used for Hz production because of the excess of NHt present. Therefore, we used a glutamate containing medium (RSM) on which growth and gas production were tested qualitatively with DL-Iactate, DL-malate, acetate an butyrate. Growth and weak gas production occurred with each of the four H-donors. An important observation was that the pH increased up to values between 8 and 9.5 during fermentation. It is interesting to note that the hydrogenase uptake enzyme of Rhodobacter capsulatus, responsible for Hz recyling, has an optimal activity between pH 8 and 9 (Colbeau et ai., 1978). An important hydrogen recycling

Hydrogen Rhodobacter sulfidophilus

could thus be an explanation for the weak hydrogen gas production; furthermore, in any chemical or biological system H2 production decreases with increasing pH because the proton-concentration, the substrate for H 2, decreases.

around seven, an increasing consumption of DL-Iactate and a slight increase of the biomass. The highest yield (33.6%) of DL-Iactate conversion to H2 was reached with strain LMG 5202 at a buffer concentration of 90 mM. This is still low compared to the DLlactate conversion by Rhodobacter capsulatus (Stevens et al., 1983).

H2 production from DL-lactate Based on the above observations, we screened a range of phosphate buffer concentrations (10 to 100 mM) to keep the pH at 7 during the tests for quantitative hydrogen gas production from DL-Iactate with growing cultures of four Rhodobacter sulfidophilus strains (Table 1). The amount of hydrogen gas produced and the difference in DL-Iactate concentration before and after fermentation allowed us to calculate the yield of DL-Iactate conversion to H2 in these batch cultures. The data from Table 1 indicate that hydrogen production increased rapidly with increasing phosphate concentrations and approached a maximum around 80 mM phosphate buffer. Higher phosphate buffer concentrations affected the hydrogen production only slightly, except for strain LMG 5205 where hydrogen production decreased rapidly at these high values. Increasing phosphate buffer concentration resulted (Table 1) in an increasing stabilizing effect of the final pH

21

H2 production from DL-malate, acetate and butyrate At 80 mM phosphate buffer concentration, the H2 production of Rhodobacter sulfidophilus LMG 5202, LMG 5203, LMG 5204, LMG 5205 was measured quantitatively for three other organic compounds: DL-malate, acetate and butyrate (Table 2). By comparison with the DL-Iactate conversion (Table 1), virtual yields from DL-malate, acetate and butyrate conversion to H2 were much lower, except for the H2 production on acetate with strain LMG 5203. From DLmalate the highest virtual yield of 18.6% was reached with strain LMG 5202; from butyrate, hydrogen production was very poor with very virtual low yields of 1.2% to 3.9%. For all strains, important amounts of unconverted DL-malate and butyrate were found after fermentation (Table 2). However, in some cases, the cell mass was twice as much as compared to growth on DL-Iactate (Table 1 and 2).

Table 1. H2 production from 10.5 mmol DL-lactate with growing cultures of four Rhodobacter sulfidophilus strains at different phosphate buffer concentrations. For the definition of yield see Materials and Methods Phosphate buffer concentration (mM)

10

20

30

40

50

60

70

80

90

100

Strain LMG 5202 H2 produced (mmol) DL-lactate (left) (mmol) Virtual yield (%) Real yield (%) Final pH Cell mass (mg/ml)

5.4 2.03 8.5 10.6 7.95 3.26

11.0 0.49 17.4 18.2 7.63 3.70

11.2 0.63 17.7 18.8 7.33 3.52

11.4 0.81 18.0 19.5 7.39 3.64

12.2 0.88 19.2 21.0 7.16 3.62

11.8 0.84 18.7 20.3 7.10 3.78

13.1 0.77 20.8 22.4 7.05 3.96

20.4 0 32.4 32.4 7.01 3.38

21.2 0 33.6 33.6 6.99 3.44

20.4 0 32.4 32.4 6.98 3.50

Strain LMG 5205 H2 produced (mmol) DL-lactate (left) (mmol) Virtual yield (%) Real yield (%) Final pH Cell mass (mg/ml)

5.8 2.73 9.2 12.4 7.65 2.90

8.5 1.37 13.4 15.5 7.47 3.33

7.5 2.63 11.9 15.8 7.35 3.47

9.4 2.59 14.9 19.8 7.21 3.63

9.7 3.50 15.4 23.1 7.14 4.43

10.5 0.88 16.7 18.2 7.07 3.73

13.1 0.95 20.8 22.9 7.12 4.30

13.0 2.70 20.7 27.8 6.98 4.17

7.2 2.08 11.4 14.2 6.98 4.63

3.3 5.92 5.2 11.9 6.87 3.03

Strain LMG 5203 H2 produced (mmol) DL-lactate (left) (mmol) Virtual yield (%) Real yield (%) Final pH Cell mass (mg/ml)

9.4 2.07 14.9 18.6 7.64 3.48

14.9 0.63 23.6 25.1 7.20 3.38

13.7 1.23 21.7 24.6 7.22 3.40

14.5 0.95 23.1 25.3 7.11 3.52

16.00 0.84 25.4 27.6 7.10 3.56

15.2 0.74 24.2 26.0 7.09 3.94

16.8 0.84 26.6 28.9 7.05 4.70

16.6 0.35 26.4 27.3 6.98 3.66

17.8 0.39 28.3 29.4 6.98 3.76

18.8 0 29.9 29.9 6.98 3.50

Strain LMG 5204 H2 produced (mmo!) DL-lactate (left) (mmol) Virtual yield (%) Real yield (%) Final pH Cell mass (mg/ml)

8.0 2.21 12.7 16.1 7.53 3.90

8.7 1.75 13.8 16.6 7.26 3.23

9.3 1.33 14.7 16.8 7.25 4.63

11.1 0.70 17.6 18.9 7.12 4.27

13.9 1.96 22.1 27.2 6.99 4.80

14.2 1.79 22.5 27.1 6.99 5.30

20.1 2.54 31.9 42.1 6.88 4.03

18.5 0.91 29.3 32.1 6.91 4.20

18.5 1.02 29.4 32.6 6.88 4.37

19.6 1.09 31.1 34.7 6.84 3.77

22

P. Stevens, N. Plovie, P. de Vos, and]. de Ley H2 produced H-donor left (mmol) (mmol)

Virtual yield

Real yield

(%)

(%)

Strain LMG 5202 DL-malate Acetate Butyrate

11.7 5.1 3.8

4.15 0 4.10

18.6 12.2 3.7

Strain LMG 5205 DL-malate Acetate Butyrate

2.4 6.5 4.1

7.11 0 6.51

Strain LMG 5203 DL-malate Acetate Butyrate

4.0 11.1 3.05

Strain LMG 5204 DL-malate Acetate Butyrate

10.5 4.1 1.3

Final pH

Cell mass (mg/ml)

30.7 12.2 6.0

7.35 7.57 7.83

5.63 5.33 6.80

3.7 15.5 3.9

11.5 15.5 10.3

7.27 7.56 7.78

4.53 4.67 4.40

3.85 0 7.11

6.4 26.4 2.8

10.0 26.4 8.7

7.23 7.34 7.46

4.97 5.90 6.63

4.13 0 8.09

16.7 9.8 1.2

27.5 9.8 5.3

7.15 7.94 7.47

4.17 3.20 7.40

That hydrogen production from DL-malate, acetate and butyrate was less efficient than on lactate is not surprising because even at 80 mM phosphate buffer concentration the final pH was not stabilized at seven. H2 production from DL-lactate at a defined pH To examine the hypothesis that the increase in H2 production was correlated with the pH stabilizing effect of the buffer, we used a 2.5 I light-fermentor, equiped with an automatic pH controler. Hydrogen production from 2 I RSM medium (30 mM lactate, 10 mM phosphate buffer) was studied with Rhodobacter sulfidophilus LMG 5202. The experiments with floating pH (between 6.5 and 8.61) could be compared to former experiments at 10 mM phosphate buffer (Table 1), but now in slightly different conditions: (i) 2 liter volume, (ii) stirred, (iii) a different type of reactor. The three other experiments were carried out at three selected pH values : pH 6.5, which is the lowest pH to allow growth for Rhodobacter sulfidophilus LMG 5202; pH 6.9, which is the pH optimum for growth; and pH 8.5, which is the optimum pH of the hydrogenase uptake enzyme of Rhodobacter capsulatus (Colbeau et al., 1978). The results were compiled in Table 3. As postulated, H2 production reached its maximum at the lowest pH. The yields of Hz production at floating pH and at constant pH 6.9 corresponded quite well with those from Table 1, where Hz production was tested out with 10

floating pH 6.5 to 8.61

35.6 Hz produced (mmol) 9.9 Virtual yield (%) Lag-phase of growth (days) 3 Lag-phase of H2 production 4 (days) 3.78 Cell mass (mg/ml)

mM phosphate buffer (floating pH) and with 80 or 90 mM phosphate buffer (pH stabilized at 7.0). At pH 8.5 there was no Hz production at all, although good growth occurred with a somewhat longer lag-phase than in the other experiments. We have thus demonstrated that H2 production with Rhodobacter sulfidophilus is pH-dependent. Hydrogen recycling may be the cause of a decreased . Hz evolution at these higher pH values for the following reasons: (i) the optimum activity of the hydrogen uptake enzyme of Rhodobacter capsulatus is between pH 8 and 9. A similar hydrogen recycling system in Rhodobacter sulfidophilus could be expected, because both species are genotypically very closely related (Gillis et al., 1982), (ii) the cell mass increases with increasing pH (Table 3), which may indicate that an additional incorporation of carbon occurred vIa the reduction of COz through the Calvin cycle.

Conclusion According to Kelley et al. (1979) hydrogen recycling in Rhodobacter sulfidophilus LMG 5202 would be responsible for the total uptake of the hydrogen produced during growth on malate. Using the RSM medium we found a net hydrogen gas production from DL-lactate,DL-malate, acetate or butyrate as H-donor. It was pointed out that H2

constant pH 6.5

constant pH 6.9

constant pH 8.5

132.8 36.9 3 4

117.7 32.7 2 3

0 0 6

3.06

5.36

Table 2. Hz production from 10 mmol DL-malate, acetate or butyate by four Rhodobacter sulfidophilus strains at a phosphate buffer concentration of 80 mM

5.80

Table 3. The effect of pH on Hz production from 60 mmol DL-lactate with Rhodobacter sulfidophilus LMG 5202 on RSM medium

Hydrogen Rhodobacter sulfidophilus

evolution is correlated with pH stabilization of the medium between 7 and 6.5. That Rhodobacter sulfidophilus is able to produce H2 is not surprising in view of its close genotypic relationship with Rhodobacter capsulatus (Gillis et aI., 1982). Still, the H2 yields of Rhodobacter sulfidophilus are lower than those of Rhodobacter capsulatus (Stevens et aI., 1983). However, the observation that a marine photosynthetic organism is able to produce hydrogen gas is valuable in view of its eventual application in waste treatment, in marine or high salt conditions. Acknowledgements. The senior author J. De Ley is indebted to the Belgian Ministry for Science Policy Programming for a personnel and research grant Geconcerteerde Onderzoeksacties on prokaryote H2 production. The authors wish to thank Dr. T. A. Hansen (Rijksuniversiteit, Groningen, the Netherlands) and Dr. H. G. Truper (Rheinische Friedrich-Wilhelms-Universitat, Bonn, FRG.) for kindly providing the Rhodobacter sulfidophilus strains W4, W12, 981 and 989.

References Colbeau, A., Chabert, j., Vigna is, P. M.: Hydrogenase activity in Rhodopseudomonas capsulata. Stability and stabilization of the solubilized enzyme. In: Hydrogenases: their catalytic activity, structure and function (H. G. Schlegel, K. Schneider, eds.) . Gottingen, Erich Goltze 1978 De Bont, j. A. M., Scholten, A., Hansen, T. A.: DNA:DNA hybridization of Rhodopseudomonas capsulata, Rhodopseudomonas sphaeroides and Rhodopseudomonas sulfidophila strains. Arch. Microbio!. 128, 271-274 (1981) De Vos, P. , Stevens, P., De Ley, j.: Hydrogen gas production from formate and glucose by different members of the Enterobacteriaceae. Biotechno!' Lett. 5, 69-74 (1983) Feijtel, T. c., Segers, L., Verstraete, W.: Hydrogen accumulation by Hruptake negative strains of Rhizobium. Plant and Soil 85, 77-84 (1985 ) Gest, H., Kamen, M. D. : Studies on the metabolism of photosynthetic bacteria. IV. Photochemical production of molecular hydrogen by growing cultures of photosynthetic bacteria. ]. Bact. 58, 239-244 (1949 a) Gest, H., Kamen, M. D.: Photoproduction of molecular hydrogen by Rhodospirillum rubrum. Science 109, 558-559 (1949 b) Gillis, M., Dejonghe, J., Smet, A., Onghenae, G., De Ley, j.: Intra- and intergeneric similarities of the ribosomal ribonucleic acid cistrons in the Rhodospirillaceae. In: IV International Symposium on Photosynthetic Prokaryotes, Bombannes France (1982) Gottschalk, G.: Fixation of molecular nitrogen. In: Bacterial

23

metabolism (M. P. Starr, ed.). New York-Heidelberg-Berlin, Springer-Verlag 1979

Hansen, T. A., Veldkamp, H.: Rhodopseudomonas sulfidophila nov. spec.; a new species of the purple non-sulfur bacteria. Arch. Microbio!. 92, 45-58 (1973) Hillmer, P., Gest, H.: H2 metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: H2 production by growing cultures. J. Bact. 129, 724-731 (1977) Imhoff, J. F., Truper, H. G.: Ectothiorhodospira halochloris, sp. nov., a new extremely halophilic phototrophic bacterium containing bacteriochlorophyll b. Arch. Microbiol. 114, 115-121 (1977) Imhoff, J. F., Truper, H. G., Pfennig, N.: Rearrangement of the species and genera of the phototrophic "Purple nonsulfur Bacteria". Int. J. System. Bact. 34, 340--343 (1984) Karube, 1., Urano, N., Matsunaga, T., Suzuki, S.: Hydrogen production from glucose by immobilized growing cells of Clostridium butyricum. Europ. J. App!. Microbiol. Biotechnol. 16, 5-9 (1982) Kelley, B. c., Jouanneau, Y., Vignais, P. M.: Nitrogenase activity in Rhodopseudomonas sulfidophila W4. Arch. Microbiol. 122, 145-152 (1979) Matsunaga, T., Mitsui, A.: Seawater-based hydrogen production by immobilized marine photosynthetic bacteria. Biotechnol. Bioeng. Symp. 12,441-450 (1982) Segers, L., Verstraete, W. : Conversion of organic acids to H2 by Rhodospirillaceae grown with glutamate or dinitrogen as nitrogen source. Biotechnol. Bioeng. 25, 2843-2853 (1983) Stevens, P., Van der Sypt, H., De Vos, P., De Ley, J.: Comparative study on H2 evolution from DL-lactate, acetate and butyrate by different strains of Rhodopseudomonas capsulata in a new type of reactor. Biotechnol. Lett. 5, 369-374 (1983) The Staff of the Anaerobe Laboratory, Virginia Polytechnic Institute and State University: Chromatographic procedures for analysis of acid and alcohol products. In: Anaerobe Laboratory Manual (L. V. Holdeman, E. P. Cato, W. E. C. Moore, eds.). Virginia, Southern Printing Co., 1977 Van Niel, C. B.: The culture, general physiology, morphology and classification of the non-sulphur purple and brown bacteria. Bact. Rev. 8-10, 1-117 (1944) Wall, J. D., Weaver, P. F., Gest, H. : Genetic transfer of nitrogenase-hydrogenase activity in Rhodopseudomonas capsulata. Nature (Lond.) 258, 630--631 (1975) Willison, J. c., Jouanneau, Y., Michalski, W. P., Colbeau, A., Vignais, P. M.: Nitrogen fixation and H2 metabolism in the photosynthetic bacterium, Rhodopseudomonas capsulata. In: Special FEBS Meeting on Cell Differentiation and Function, Athens/Greece, 25-29 april 1982 Zhang, X. , Haskell, J. B., Tabita, F. R., Van Baalen, c.: Aerobic hydrogen production by the heterocystous cyanobacteria Anabaena spp. strains CA and IF. ]. Bact. 156, 1118-1122 (1983) ZUrrer, H. : Hydrogen production by photosynthetic bacteria. Experientia 38, 64-66 (1982)

Professor Dr. j. De Ley, Lab. Microbiologie, RUG, K.L.Ledeganckstraat 35, B-9000 Gent, Belgium