Lefters in Amlied Microbioloav 1995, 21, 277-281

Large scale cultivation of Hydrogenobacter thermophilus on hydrogen

G.O. Hreggvidsson', T. G u s ~ ~ v s s o ~ ' ~ ~ , 0. Hoist3, K. Kronsbein', S. Hjorleifsdottir', T. Skjenstad' and J.K. Kristjansson'~* 'Department of Biotechnology, Technological Institute of Iceland and 21nstitute of Biology, University of Iceland, Keldnaholt, Reykjavik, Iceland, and 3Departrnent of Biotechnology, Chemical Center, Lund University, Lund, Sweden

JRN/129: received 24 April 1995 and accepted 29 April 1995

G.O. HREGGVIDSSON, T. GUSTAVSSON, 0. HOLST, K. KRONSBEIN, S. HJORLEIFSDOTTIR,

T. SKJENSTAD AND J .K. KRISTJANSSON. 1995.

T h e thermophilic hydrogen-oxidizing eubacterium, Hydrogenobacter thermophilus was grown with a semi-continuous gas feed in a 150 1 fermentor, on different ratios of H2, O2 (air) and C02 and o n media of variable mineral salts composition. T h e best gas ratio during the exponential phase was approximately 0.5 : 1.0 : 0.03 atm of H2 : air : C02. Cell densities were increased by increasing the concentration of ammonium and phosphate in the medium. The maximum cell density obtained under the most favourable conditions was 2.62 g wet wt I-', corresponding to an absorption of 1.6 at 600 nm. The maximum specific growth rate (p) was 0.44 h-I.

INTRODUCTION

Hydrogen-oxidizing eubacteria of the genus Hydrogenobacter are aerobic or microaerophilic Gram-negative rods, and obli- gately chemolithoautotrophic. They are extreme thermo- philes with most strains having an optimum growth temperature close to 72°C (Kawasumi et al. 1984; Kryukov et al. 1983; Kristjansson et al. 1985). C 0 2 is fixed by the reductive TCA pathway (Shiba et ul. 1985), which is very rare among eubacteria. As an alternative to hydrogen some strains can use elemental sulphur or thiosulphate as electron donors (Alfredsson et ul. 1986; Beffa et al. 1992). This bac- terium was first isolated from hot springs in Japan (Kawasumi et al. 1984) and Kamchatka (Kryukov et al. 1983) and described as Hydrogenobacter thermophilus and Culdero- bacterium hydrogenophilum, respectively. I t has also been iso- lated from hot springs in Iceland and Italy (Kristjansson et al. 1985 ; Aragno 1992).

Phylogenetic analysis based on 16s rRNA sequencing has revealed that the genera Aguifex and Hydrogenobacter, are of the same lineage, distinctly separate from all other known eubacteria. This cluster of hydrogen-oxidizing thermophilic bacteria apparently forms the lowest phylogenetic branch in the eubacterial tree (Burggraf et al. 1992 ; Shima et al. 1994).

Correspondence to :Dr Jaboh K . Kristjansson, Department of Biotechnology, Technological Institute of Iceland, University qf'lceland, Keldnaholt. IS-1 12, Relikjavik, Iceland.

0 1995 The Society for Applied Bacteriology

Studies on thermophilic hydrogen-oxidizing bacteria are few and mainly concern the ecology, energetics and taxonomy of these organisms (Aragno 1992). Little work has been done on their enzymes, probably reflecting difficulties in handling these organisms and growing them in sufficient yields for relatively easy purification of enzymes. The thermophilic hydrogen-oxidizing eubacteria will undoubtedly receive more attention in future due to their unique metabolic type and phylogenetic status.

There has been little work concerning the optimization of growth conditions for Hydrogenobacter. Bonjour and Aragno (1986) studied the growth of Hydrogenobacter in a 2 1 fer- mentor of thiosulphate and on a mixture of hydrogen and thiosulphate. They estimated the cell yield to be 10 g mol-' of thiosulphate but accurate measurements could not be done due to precipitation of solid sulphur that occurs in the thio- sulphate cultures (Bonjour and Aragno 1986). They also found the growth rate on thiosulphate to be twice as fast as on hydrogen.

In this paper the authors report a study on the cultivation of Hydrogenobacter on hydrogen in a 150 1 fermentor in order to obtain high cell densities. A fed-batch technique was devised for administering the gasses and by varying gas ratios and mineral composition of the medium cell densities were increased into the range comparable to those routinely obtained with other aerobic thermophilic eubacteria, such as Thermus.

278 G.O. HREGGVIDSSON ETAL.

MATERIALS AND METHODS

The strain used in this study (IT1 553) was isolated from an alkaline hot spring in Iceland as described by Kristjansson et al. ( 1985). Phenotypic and physiological characteristics of the strain are similar or identical to those ascribed to Hydro- genobacter thermophilus (Kawasumi et al. 1984).

The standard medium used for growing Hydrogenobacter (Kristjansson et al. 1985) contained the following (g 1-I): (NHJ2S04, 1.3 ; K,HPO4. 3Hz0, 0.47 ; MgS0,. 7H20, 0.25 ; CaCl,. 2H20, 0.07 ; Trace element solution, 1 ml.

The trace element solution had the following composition : nitrilotriacetic acid, 12.8 g I - ' ; FeCIZ. 4H20, 1.0 g 1-I; MnCI,. 4H20, 0.5 g 1 - ' ; CoCl,. 6H20, 0.3 g 1-' ; ZnCl,, 0.2 g 1-' ; CuCl,. 2H,O, 50 mg 1-' ; Na2Mo0,; 2H20, 50 mg I-'. H3B03, 20 mg 1-' ; NiCl,. 6H20, 20 mg 1-' ; pH was adjusted to 6.0 with HCl.

The medium was prepared in a 10-fold concentration and kept at pH 4 to prevent precipitation. Before use it was adjusted to pH 7.8 with NaOH and diluted to appropriate concentrations in the cultivation bottles. The bottles were sterilized at 121°C for 30 min.

Additional ammonium and phosphate were supplied dur- ing large scale cultivations as 100 times concentrated solutions of (NH,),SO, and K,HPO,. 3 H 2 0 relative to the standard medium concentration.

Growth in static cultures and effects of mineral concentration

Static batch cultivations for inoculation were grown with finite amounts of gasses in 50 ml and 1 1 bottles. The medium took up one-fifth and one-half of the volume, respectively. The bottles were closed with a rubber stopper and sealed. Gasses were supplied sequentially into the bottles through the rubber stopper with a needle, and the quantity of the individual gasses monitored by following the corresponding partial pressures. The composition of the gas mixture was H2, 0, and CO, in the volumetric ratio 60 : 21 : 10. The O2 was supplied as air so that the partial pressures of H2 : air : COz were 0.6, 1.0 and 0.1 atm, respectively.

Studies on the effect of mineral concentration on static cell cultures were carried out in 50 ml bottles. The culture volumes were 10 ml and the inoculum was 1%. The bottles were placed open into a 21 anaerobic jar filled with the gas mixture, as described above. Growth was measured under these conditions in media with varying concentrations of ammonium and phosphate, from one to three times the con- centration of the standard medium. Cultures were incubated for 48 h at 72°C and the cell densities measured as O.D. at 600 nm at the end of the cultivation.

Cultures continuously supplied with gasses

Static batch cultures with continuous gassing were carried out in 20 1 flasks with 15 I of medium. The bottles were closed

with a rubber stopper, fitted with tubes for inlet and outlet of gasses. The inlet tube acting as sparger was punctured at intervals and led into the medium. The sterile filtered gas mixture was continuously supplied at the following flow rates : air, 0.5 1 min-' ; H2, 0.1 1 min-' ; CO,, 0.02 1 min-I. Inoculum was 1% and the flasks were incubated in a closed water bath at 72°C for 3-6 d. Cell densities were measured at 600 nm at the end of the cultivation.

Large scale cultivations

The large scale cultivations were carried out at 72°C in a 150 I fermentor (Bioengineering AG, Wald, Switzerland), with a working volume of 100 1. Inoculum was 1-3940, grown as described above for small static cultures, as 500 ml cultures in 1 1 bottles. Dissolved 02, pH and stirrer speed were moni- tored on line, whereas growth of the culture was monitored off line at 600 nm and 436 nm. Stirrer speed was 0-150 rev min-l during the lag phase, but was increased to 500 rev min- ' during the exponential phase. Cultures were repeat- edly fed with a gas mixture, containing H2, air and C 0 2 in variable ratios during the early cultivations. During the later cultivations a ratio close to the deduced optimum of 0.5 : 1.0 : 0.03 atm partial pressures for the respective gasses was used. The gasses were supplied to the culture through a sparger by manually-operated valves. Partial pressures were monitored with a barometer connected to the fermentor. Before each gas refilling the culture was first thoroughly flushed with pressurized air. The utilization of individual components in the gas mixture was followed, the level of 0, directly, but that of other gas components only indirectly by monitoring growth in conjunction with changes in oxygen consumption. Some of the cultures were supplemented dur- ing growth with additional ammonium and phosphate. The concentrated solution of ammonium and phosphate was pumped through a sterile hose connected to an inlet at the top of the fermentor.

RESULTS AND DISCUSSION

Effects of mineral concentration and other factors on growth : static small batch cultivations

In order to evaluate the effects of aeration and mineral salts on the growth, a series of small static cultivations was carried out. Table 1 gives optical cell densities reached with different cultivation methods. It is evident from Table 1 that the gas supply is limiting in the small static cultures grown under overpressure in 50 ml bottles. When identical 10 ml cultures were grown under the same conditions in a 2 1 anaerobic jar, almost double the cell densities were obtained. Interestingly, a significant difference in final cell densities, measured at O.D. a t 600 nm, was observed between the 10 ml cultures

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CULT I VAT1 0 N 0 F H YDROGENOBA C TER 279

Table 1 The effect of mineral salt composition and gas supply on growth of Hydrugenobacter thermophilus IT1 553 in small static cultures

Culture type, Optical gas ratio Culture volume/ density H, : air : CO, vessel volume (NH,),SO,* K2HP0,* (600 nm)

Closed static cultures grown under overpressure, 0.6 : 1.0 : 0.1 atm 10 m1/50 mi I x I x 0.08-0.17

Closed static cultures, surplus of gases grown under overpressure, 10 m1/2 1 I x l x 0.43 0.6 : 1.0 : 0.1 atm 10 m1/2 I 2 x 2 x 0.58

10 m1/2 I 3 x 3 x 0.34 10 m1/2 1 2 x I x 0.32 10 m1/2 1 3 x l x 0.28 10 m1/2 1 I x 2 x 0.49 10 m1/2 I l x 3X 0.83

Open cultures, continuous gas supply, 0.53 : 0.37 : 0.12 atm 15 1/20 1 l x l x 0.14.2

*The medium of cultivation varies in concentration of these salts from one times to three times the concentration of the standard medium.

supplied with a surplus of the gas mixture and grown under overpressure and the static 15 1 cultures continuously sup- plied with gasses. This indicates that gas transfer to the liquid phase was limited to the latter cultures due to the combined effect of ineffective sparging and the absence of stirring.

Cultures grown in a medium with twice the strength of both ammonium and phosphate gave approximately twice the cell density obtained in the standard medium, while threefold strength of ammonium and phosphate was inhibitory (Table 1). When ammonium alone was increased, a more pronounced growth inhibition was observed. T h u s the final cell density decreased from an O.D. of 0.43 to an O.D. of 0.280 when the concentration of ammonium was increased threefold (from 0.1 to 0.3%), and the phosphate kept constant at the standard strength. T h e opposite effect was observed when only the phosphate was increased. At three times the usual strength of phosphate the final O.D. reached was 0.8, almost twice the next highest value obtained on twofold strength of both ammonium and phosphate. In part these results might be explained by an increased buffering capacity of the medium with higher phosphate concentration, but the above results indicate that ammonium above a certain limit has an adverse effect on growth.

Large scale cultivations

First the feasibility and efficiency of supplying the gasses in discrete batches to large scale cultures were examined. Results of such an experiment are presented in Fig. 1. Air was used instead of O,, because when pure 0, was supplied through the sparger the dissolved 0, concentration in the medium

became too high and growth did not occur. During the lag phase the gas mixture used consisted of H2 : air : CO, in the partial pressure ratio of 0.7 : 1.0 : 0.1 atm corresponding to a H,, 0, and CO, ratio of 3.3 : 1 :0.48. According to eqns 1 and 2 for energy generation and reductive fixation of CO,, the

0.5 O T

r a

Time (h) Fig. 1 Growth of Hydrugenubacter thermophilus IT1 553 in a 150 1 fermentor. Cultivation on a standard medium. 0, Absorbance at 600 nm; 0, pH. Black arrows below the curve indicate the points of flushing and addition of H2, air, and C02 in the ratios 0.8 : 1.0 : 0.1 atm. Two separate supplements of H, are marked with open arrows. The numbers below the growth curve give the partial pressure of H, of each addition. Arrows above the curve indicate additional air supplements in atm

0 1995 The Society for Applied Bacteriology, Letters in Applied Microbiology 21, 277-281

280 G.O. HREGGVIDSSON ET A L .

hydrogen should be sufficient to ensure complete utilization of both 0, and CO,, if neither one is limiting.

2H2 + 0, + 2H20

2Hz + C0.7 + (CH2O) + H2O (2)

During exponential growth, the proportion of hydrogen in the mixture was increased, so the ratio of H2 : air : CO, became 0.8 : 1.0 : 0.1 atm. Immediately after the oxygen in the mixture was depleted the culture was supplied with additional air (Fig. 1). Further growth was then achieved, thus establishing that oxygen was the limiting component. After successive reintroduction of air, H2 was used up and the growth stopped. Growth continued if either additional H, was supplied or the culture was replenished with a new batch of the gas mixture. This experiment indicated that the concentration of CO, is superabundant for optimum growth and accordingly the proportion of air (0,) in the gas mixture could be increased for achieving the most efficient utilization of H,. By repeated experiments the optimum ratio of H,, air and CO, for util- ization during the exponential phase was determined to be approximately 0.5 : 1.0 : 0.03 atm partial pressures, cor- responding to the ratio for H,, 0, and CO, of 2.4 : 1.0 : 0.14.

Maximum growth rate during exponential phase was 0.17 h-I. The culture apparently reached the stationary phase at an O.D. (600 nm) of 0.56, but it was still actively consuming 0,. Despite frequent recharging with the gas mixture, the culture did not resume growth. The final cell density was 0.85 g wet wt 1-I. The pH had remained fairly constant at around 7 during the lag phase, but at the onset of the exponential phase it started to decrease, indicating an in- adequate buffering capacity of the medium. At the end of the exponential phase the pH was 4.5, which may be the lower limit for growth.

In an attempt to increase the buffering capacity of the medium another 100 1 cultivation was carried out with double the strength of both phosphate and ammonium (Fig. 2). This time the stationary phase was reached at an O.D. of0.8. When the culture at this stage was supplemented with additional ammonium and phosphate, making the total concentration three times that of the standard medium, growth resumed, but the O.D. only increased to 1.1. The maximum growth rate was found to be 0.27 h-' in the early exponential phase and 0.08 h-' in the late exponential phase after the addition of ammonium and phosphate. The final cell density was 1.81 g wet wt 1 - I . Similar results were obtained on a medium containing the standard concentration of ammonium and three times the standard concentration of the phosphate. In this cultivation pH was adjusted with NaOH. Stationary phase was reached at an O.D. of 1.30 and no further growth was obtained by adding ammonium (results not shown).

Attempts were then made to increase cell densities with a more incremental batch feeding of ammonium and phosphate

1.00 r I

5 7 9 11 13 15 17 19 21 23 25 Time (h)

Fig. 2 Growth of HydroRenobacter thrrmophilus IT1 553 in a 150 1 fermentor. 0, Cultivation in the standard medium; 0, cultivation in double concentration of ammonium and phosphate. Arrows indicate additions of ammonium and phosphate. The numbers give the amounts added in proportion to that of the standard medium

during growth. Feeding was started when the culture had reached an O.D. of approximately 0.35 and a total of 4 I of 100 times concentrated solution of ammonium and phosphate were added in six portions of 0.3-1 I (Fig. 2). The pH remained relatively constant at around 7.0 throughout growth and the maximum growth rate during the exponential phase was 0.44 h-I. The final cell density obtained was 2.62 g wet wt l-', corresponding to an optical density of 1.60 at 600 nm. The growth rate obtained in this study is close to that reported for other Hydrogenobacter strains grown on H2 (Kawasumi et ul. 1984; Bonjour and Aragno 1986), while Bonjour and Aragno (1986) reported a maximum growth rate of 0.67 h-' when Hydrogenobacter was grown mixo- lithotrophically on H2 and thiosulphate. The cell density obtained is at least twofold higher than previously reported for small scale cultivation of Hydrogenobucter sp., grown on thiosulphate (Bonjour and Aragno 1986). Under the latter conditions the culture reached an O.D. of 1.5 at 436 nm which corresponds to about 0.80 a t 600 nm.

If the above results are summarized one can conclude that semi-continuous batch feeding of gasses is a feasible method for growing Hydrogenobacter to relatively high cell densities. T o ensure high cell densities, gas supply has to be adequate and the gasses have to be thoroughly mixed into the culture liquid by efficient sparging and/or stirring. The optimum gas ratio of H2, air and CO, for utilization during the exponential phase was found to be approximately 0.5 : 1.0 : 0.03 atm, respectively. Cell densities could be

0 1995 The Society for Applied Bacteriology, Letters in Applied Microbiology 21, 277-281

CULTIVATION OF HYDROGENOBACTER 281

increased substantially by increasing the concentration of ammonium and phosphate, preferably by incremental or con- tinuous feeding that counteracted the fall in pH.

The method developed in this study is simple and requires minimum equipment and controls. Optimum composition of the gas mixture is easily controlled and efficient utilization of the Hz can be achieved throughout cultivation. Batch feeding of gasses decreases the hazard of handling the potentially explosive Hz, as compared with continuous gas flow. The main drawback of manual feeding is that during exponential growth, gas uptake is so rapid that the culture needs constant attention. On the other hand the culture can be cooled down to 15°C and continued after some time (overnight) without any obvious effect on growth (results not shown). The use of air instead of pure 0, necessitates regular flushing of the culture to remove NZ. It would be advantageous to use pure oxygen, but the inhibitory effect of oxygen must then be circumvented, either by fine tuning the 0, supply through the sparger or by supplying the oxygen in the head space.

It is to be expected that the physiological uniqueness (Iga- rashi and Kodama 1990) of Hydrogenobacter, its phylogenetic position (Burggraf ct al. 1992) and genotypic divergence within the group will be manifested at the enzymatic level. The thermophilic hydrogen-oxidizing bacteria are therefore still an unexploited source of novel enzymes. These enzymes are also likely to have variable properties across the phylo- genetic range of the group. The cell densities obtained in this study are sufficient to make the purification of interesting enzymes and proteins from thermophilic hydrogen-oxidizing bacteria a feasible task.

ACKNOWLEDGEMENTS

The authors acknowledge the support from EC Generic pro- ject ‘Biotechnology of Extremophiles’ Contract BIO-CT93- 02734, the Icelandic Ministry of Education, the Icelandic National Research Council (Grants 93235 and 93239), the Icelandic Science Foundation (Grant 92-N-050) and the Swedish National Board for Industrial and Technical Devel- opment (NUTEK).

REFERENCES

Alfredsson, G.A., Ingason, A. and Kristjansson, J.K. (1986) Growth of thermophilic, obligately autotrophic hydrogen-oxidizing bac- teria on thiosulphate. Letters in -4pplied Microbiology 2, 21-23.

Aragno, M. (1992) Aerobic, chemolithoautotrophic, thermophilic bacteria. In Thermophilic Bacteria ed. Kristjansson, J.K. pp. 77- 103. London : CRC Press.

Beffa, T., Berczy, M . and Aragno, M. (1992) Metabolism of inor- ganic sulphur compounds in highly thermophilic hydrogen-oxi- dizing bacteria. In Thermnphiles : Science and Technology. pp. 135. Conference Programme and Abstract Book. IceTec, Reykjavik, Iceland.

Bonjour, F. and Aragno, M. (1986) Growth of thermophilic, ohli- gatorily chemolithoautotrophic hydrogen-oxidizing bacteria related to Hydropnobacter thermophilus with thiosulphate and elemental sulfur as electron and energy source. FEMS Micrn- biological Letters 35, 11-15.

Burggraf, S., Olsen, G.J., Stetter, K.O. and Woese, C.R. (1992) A phylogenetic analysis of Aquijer jyrophilus. Systematic and -4pplied Microbiology 15, 352-356.

Igarashi, Y. and Kodama, T. (1990) Hydrogenobacter thermnphilus : its unusual physiological properties and phylogenetic position in the microbial world. F E M S Microbiological Reviems 87, 403406.

Kawasumi, T., Igarashi, Y., Kodama, T. and Minoda, Y. (1984) Hydrogenobacter thermophilus gen. nov., sp. nov., an extremely thermophilic aerobic hydrogen-oxidising bacterium. International Journal ofSystematic Bacteriology 34, 5-10,

Kristjansson, J.K., Ingason, A. and Alfredsson, G.A. (1985) Iso- lation of thermophilic autotrophic hydrogen-oxidizing bacteria similar to Hydrogenobactcr thermophilus, from Icelandic hot springs. Archives ojMicrobiology 140, 321-325.

Kryukov, V.R., Savel’eva, N.D. and Pusheva, M.A. (1983) Caldero- bacterium hydrngennphilum n. gen. n. spec., an extremely thermo- philic hydrogen bacterium and its hydrogenase activity. Microbiologyia 52, 781-788.

Shiba, H., Kawasumi, T., Igarashi, Y., Kodama, T. and Minoda, Y. (1985) The COz assimilation via the reductive tricarboxylic acid cycle in an obligately, aerobic hydrogenqxidizing bacterium Hydrogenobacter thermophilus. Archives of Microbiology 141, 198- 203.

Shima, S., Yanagi, M. and Saiki, H. (1994) The phylogenetic position of Hydrogenobacter ncidophilus based on 16s rRNA sequencing analysis. F E M S Microbiological Letters 119, 119-122.

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