ASSIMILATION OF CARBON DIOXIDE BY HYDROGEN BACTERIA*

BY FRED H. BERGMANN,t JACK C. TOWNE,$ AND R. H. BURRIS

(From the Department of Biochemistry, College of Agriculture, University of Wisconsin, Madison, Wisconsin)

(Received for publication, August 12, 1957)

Autotrophic organisms can use carbon dioxide as their sole source of carbon. Within this group, the chemosynthetic bacteria secure energy for carbon dioxide assimilation by the oxidation of simple inorganic sub- stances, although the photosynthetic organisms obtain this energy from light. The phylogenetic relationships of the chemosynthetic bacteria to the photosynthetic and to the heterotrophic organisms have been a matter for much speculation (1,2). The demonstration of biochemical similarities or differences among the organisms of these three groups could offer clues to their interrelationships.

Recently, the main pathway of carbon dioxide fixation by photosynthetic tissue has been clarified. It has been demonstrated that PGAl is the first stable photosynthetic product (3, 4) and that ribulose diphosphate is the primary carbon dioxide acceptor (5). The carboxylating enzyme involved has been termed carboxydismutase.

Similar reactions have been demonstrated in several chemoautotrophic bacteria. Santer and Vishniac (6) showed that ribulose diphosphate greatly stimulated the uptake of Cl402 by cell-free extracts of Thiobacillus thioparus and that radioactive PGA was formed. Trudinger (7) demon- strated a similar system in extracts of Thiobacillus denitri$cans. He was able to show that this organism potentially was capable of synthesizing hexose phosphates from carbon dioxide by a cyclic mechanism similar to that described for photosynthetic tissue by Calvin (8). However, the quantitative significance of the carboxydismutase system for the steady state fixation of carbon dioxide during the growth of these organisms remained uncertain. More recently, Suzuki and Werkman (9) reported

* Published with the approval of the Director of the Wisconsin Agricultural Ex- periment Station. Supported in part by a grant from the Office of Naval Research.

t Present address, Department of Microbiology, Medical School, Washington University, St. Louis, Missouri.

$ Present address, Institute for Psychosomatic and Psychiatric Research and Training, Michael Reese Hospital, Chicago, Illinois.

1 The following abbreviations are used: PGA for phosphoglyceric acid, R-5-P for ribose 5.phosphate, ATP for adenosine triphosphate, Tris for tris(hydroxymethyl)- aminomethane.

13

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14 CO2 FIXATION BY I-IYDROGEN BACTERIA

that PGA is the first stable product of carbon dioxide fixation by whole cells of Thiobacillus thiooxidans.

In the present communication are similar results obtained with Hydro- genomonas facilis, a representative of another group of chemoautotrophic organisms, the hydrogen bacteria. These organisms, when grown under autotrophic conditions, obtain energy for the reduction of carbon dioxide by the oxidation of molecular hydrogen to water (the oxyhydrogen or Knallgas reaction). Some experiments with cell-free preparations from H. facilis also are presented.

Materials and Methods

Culture of Organism and Preparation of Cell-Free Extracts-A culture of H. jacilis, obtained from Dr. A. Schatz in 1951, was maintained by con- tinuous subculture under autotrophic conditions. The bacteria were grown in a modified Ruhland’s salt medium which contained the following macronutrients per liter of distilled water: NaHC03 1 .O gm., NH&l 1.0 gm., KHzPOb 0.5 gm., MgS04.7H20 0.1 gm., NaClO.1 gm., CaCL 0.1 gm., and Fe(NH4)2(S0&.6H20 8 mg. A solution of trace elements, described by Cohen and Burris (lo), was added to this medium. The cells were aerated with a gas mixture consisting of 6 parts of hydrogen, 2 parts of oxygen, and 1 part of carbon dioxide by volume; species of the Hydro- genomonas assimilate these gases in approximately the proportions sup- plied (11, 12). The incubation temperature was 30”.

The experiments with whole cells were performed with 100 ml. volumes of culture grown in 500 ml. shaken flasks. 5 ml. of a 2 to 3 day-old culture served as the inoculum. The gas mixture was replaced frequently during growth. Under these conditions, the lag phase of growth is about 2 hours; growth (determined turbidimetrically) remains in its exponential phase for approximately 35 hours, and the generation time is about 5 hours. Exposures of whole cells to CY402 were performed at a tempera- ture of 24” with 26 to 29 hour-old cultures; at this time the culture con- tained about 0.1 mg. of cellular nitrogen per ml. Before exposure to NaHC403, the gas mixture was changed to one containing less CO2 (Hz- 02-CO2 = 39: 10: 1) to avoid excessive dilution of NaHC403. The cul- tures were equilibrated with this new gas mixture for half an hour before addition of NaHC40 3. Cells were exposed to Cl4 by rapidly injecting NaHCY403 solution into the actively metabolizing cultures, and the cul- tures were inactivated after preselected intervals with 5 ml. of 60 per cent perchloric acid per 100 ml. of culture or with sufficient hot 95 per cent ethanol to bring the final ethanol concentration to 70 per cent.

8 liter mass cultures used for the preparation of cell-free extracts were grown in a closed gas-circulating system described by Cohen and Burris

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F. H. BERGMANN, J. C. TOWNE, AND R. II. BURRIS 15

(10). 9fter 15 to 18 hours of growth, the organisms had assimilated 35 to 40 liters of the gas mixture, and a yield of 10 to 15 gm. of packed wet cells generally was obtained. The cell mass was washed twice with 0.1 M

Tris or glycylglycine buffer, pH 7.5, and then resuspended in 25 ml. of buffer containing 10 mg. of cysteine.

Cell-free preparations were made by subjecting suspensions of cells to the sonic vibrations of a 10 kc. magnetostrictive oscillator (Raytheon Company, Waltham, Massachusetts) at full power (250 watts). The apparatus was cooled by circulating ethanol-water or ethylene glycol at -10” through the cooling jacket. To prevent excessive heating, sonic treatment was interrupted every 5 minutes. Under these conditions, the temperature of the treated suspensions never exceeded 5”.

The suspensions, after treatment in the sonic oscillator, were centrifuged for 20 minutes at full speed in the high speed head of the International refrigerated centrifuge (about 20,000 X g). The opalescent supernatant solutions contained 5 to 9 mg. of protein N per ml. and had a red color. Spectrophotometric examination revealed cytochrome bands. Prepara- tions of this type had strong hydrogenase activity as measured with methylene blue or oxygen as the hydrogen acceptor.

Exposures of cell-free preparations to NaHCL403 in the presence of various substrates and cofactors were performed in standard Warburg respirometers.

Materials-Radioactive sodium bicarbonate solutions were prepared by converting BaC1403 (15 to 20 per cent carbon as C14, obtained from the Oak Ridge National Laboratory) to Cl402 gas and reabsorbing this in NaOH. The reaction was performed in standard Warburg respirometers with 100 ml. reaction vessels.

The disodium salt of ATP was a product of the Pabst Laboratories. The barium salts of various sugar phosphates were obtained from the Nutritional Biochemicals Corporation; these were converted to their sodium salts by decomposition with sodium sulfate or by allowing them to react with the hydrogen form of Dowex 50 and subsequently neutralizing them with NaOH. 5-Phosphoribonic acid was prepared from ribose 5-phosphate by the method of Robison and King (13). Other materials used in reaction mixtures or for cochromatography were reagent grade chemicals.

Estimation of Enzymes and Metabolites-Nitrogen was determined by a Nessler method (14) or by semimicro-Kjeldahl distillation (15). Inor- ganic phosphorus, total phosphorus, and pentoses were estimated by methods described by LePage (16). The anthrone method was used for carbohydrates (17). Glyceric acid and PGA were determined by the method of Rapoport (18). Amino acids were detected qualitatively with

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16 CO2 FIXATION BY HYDROGEN BACTERIA

ninhydrin, and organic acids eluted from columns were estimated by titrations. Phosphoriboisomerase was assayed by the method of Axelrod and Jang (19).

Assays for Radioactivity-In most of the work reported here, 1 ml. samples in an alkaline gelatin solution were plated out on 1 inch diameter flat copper planchets? Better reproducibility was obtained with t.his method than with “infinitely thin” samples. 1 volume of 10 per cent gelatin in 0.1 N NaOH was added to 9 volumes of the sample (highly acidic samples were first neutralized). 1 ml. aliquots of this mixture then were plated out on clean copper planchets and dried overnight in air. If the final solution had a pH below 10, the gelatin film tended to peel away from the planchet after drying. Corrections for coincidence, background, and self-absorption were made in the usual manner.

Chromatographic Methods-Radioactive reaction mixtures were analyzed by the following three chromatographic techniques: (a) Ion exchange chromatography on Dowex 1 formate resin with formic acid and ammonium formate as eluents (20, 21). The samples, before analysis, were dried in a stream of air at 45” (21) or in vacua over NaOH at 37”. Ammonium formate was removed by lyophilization. (b) Partition chromatography on silica gel columns by the method of Liberman (22) ; and (c) two-direc- tional paper chromatography and subsequent radioautography as described by Benson et al. (3). Various other paper chromatographic systems occa- sionally were used as adjuncts to these three basic techniques. Compounds were considered as identified when shown to cochromatograph well with authentic samples in two, and preferably three, different systems.

Results

Results with Whole Cells--Whole cells of H. facilis fixed NaH(Y403 at a linear rate for several minutes. The percentage of the total Cl4 fixed in the perchloric acid-insoluble or ethanol-insoluble fractions (mostly protein) increased markedly with time (Fig. l), showing that synthesis of polymeric cell constituents was rapid.

A kinetic analysis was conducted on the radioactive perchloric acid-soluble components formed during 5, 15, 45, 135, and 600 seconds of exposure to NaHC1403 (1 me. per 100 ml. of culture). After a 5 second exposure, 96 per cent of the assimilated Cl4 was found in the perchloric acid-soluble fraction. After 600 seconds, this fraction contained only 44 per cent of the Cl4 fYixed. After 5 seconds, peaks 3 and 4 (Fig. 2) contained the bulk of this fixed U4. The soluble fractions were chromatographically separated

2 We are grateful to Dr. M. J. Johnson and to several members of his laboratory for acquainting us with the method for counting radioactive samples in gelatin films.

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F. H. BERGMANN, J. C. TOWNE, AND R. H. BURRIS 17

on Dowex 1 formate resin. The distribution of Cl4 among the chromato- graphic fractions changed markedly with time (Fig. 2).

Peak 3 appeared to be a mixture of hexose monophosphates; glucose 6-phosphate exhibited chromatographic behavior similar to this peak in several solvent systems. Both in the Dowex 1 formate system and on

Cl4 FIXED

SECONDS

FIG. 1. Gross distribution of radioactivity in whole cells of H. fucilis after ex- posure to NaHWOs. 100 ml. cultures of H. jucilis were exposed to 1 mc. of NaHC140a and were inactivated by the addition of 5 ml. of 60 per cent perchloric acid and rapid chilling. The residue was reextracted with 3 per cent perchloric acid, perchloric acid was removed from the combined supernatant solutions as the potassium salt, and the protein fraction was recovered from the residue. Similar results were ob- tained by exposing cells to COZ gas rather than to NaHWOz, and by killing with hot ethanol, rather than with perchloric acid.

two-directional paper chromatograms, the radioactivity in peak 3 showed a wider “spread” than authentic glucose 6-phosphate, indicating that other phosphorylated sugars were present. Treatment of the compounds of peak 3 with phosphatase produced two labeled free sugars; these were separated by two-directional paper chromatography and were located on the papers by radioautography and by spraying with naphthoresorcinol. The movement of one sugar was identical with fructose, but the other was slightly displaced from glucose.

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18 CO2 FIXATION BY HYDROGEN BACTERIA

I,000 I I I I I

750 - 5 second exposure 4 Recovery: 66.4 % 3

500 -

250 -

0 I,

750 - 15 second exposure Recovery: 78.8 %

500 -

250 - a

z 45 second exposure

Recovery: 76.4 %

750 - 135 second exposure = Recovery: 71%

500 -

750 750 - - 600 second exposure 600 second exposure Recovery: 62.5 % Recovery: 62.5 %

500 500 - -

250 250 - -

0’ 0’

TUBE NUMBER FIG. 2

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F. H. BERGMANN, J. C. TOWNE, AND R. H. BURRIS 19

The elution pa,ttern from Dowex 1 of added phosphoglyceric acid, estimated by a calorimetric method for glyceric acid (18) and by total phosphorus, coincided exactly with the pattern of radioactivity of peak 4. When peak 4 was dephosphorylated with alkaline phosphatase, the resulting glyceric acid again coincided with an authentic sample both on Dowex 1 columns and on silica gel partition columns.

The compound of peak 2 (Fig. 2), containing about 7 per cent of the total Cl4 fixed during a 5 second exposure, was identified as malic acid by cochromatography with an authentic sample on Dowex 1 formate and on silica gel columns.

An examination of the distribution of Cl4 in the acid-soluble fraction indicates that the percentage of total Cl4 decreased with time in phos- phoglyceric acid, in the sugar phosphate fraction, and in malic acid (Figs. 2 and 3). These compounds therefore can be considered as early products of carbon dioxide fixation in H. facilis. On the other hand, the relative amount of radioactivity in peak 5 (a mixture of glutamic and aspartic acids) and in the forerun (peak 1, Fig. 2) of neutral and cationic substances, including neutral amino acids, increased with time. After exposures to Cl402 for 45 seconds or more, glutamic acid was by far the predominant radioactive amino acid in peak 5, although after a 5 or 15 second exposure the amounts of Cl4 found in glutamic and aspartic acids were approximately equal. The kinetic behavior of glutamic acid is thus that of a typical end product of carbon dioxide fixation, whereas aspartic acid, like malic acid, may be an early product of carbon dioxide fixation in this organism.

Recovery of Cl4 from Dowex 1 formate columns, compared to the radio- activity of the acid-soluble fractions added to the columns, was about

FIG. 2. Chromatography on Dowex 1 formate of the acid-soluble fractions of H. fucilis cells exposed to NaHC403. 100 ml. volumes of cultures of H. fucilis each were exposed to 1 mc. of NaHCr403. The cells were killed by the addition of 5 ml. of 60 per cent perchloric acid. The suspension was centrifuged, the residue reex- tracted with 3 per cent perchloric acid, and the combined supernatant solutions were adjusted to pH 7 with KOH; KC104 was removed by centrifugation before chromatography. A 50 ml. forerun obtained by washing with water has been plot,ted as a flat ten tube peak (peak 1) eluted before tube 1. 5 ml. fractions of increasing concentrations of formic acid (gradient elution as described by Busch et al. (20)) were collected up to tube 60. The column then was irrigated with 3.5 N formic acid- 0.5 N ammonium formate until forty more 5 ml. fractions had been collected; these fractions were lyophilized before assay. Recovery refers to the total Cl4 found in the eluted fractions compared to the amount of Cl4 added to the column. The or- dinates have been normalized so that each elution pattern corresponds to 10,000 c.p.m. added to a column. In practice, two to three times as much radioactivity usually was used. Peak I, forerun (neutral and cationic materials); peak 2, malic acid; peak 3, “sugar phosphates;” peak 4, phosphoglyceric acid; peak 5, glutamic and aspartic acids.

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20 CO2 FIXATION B‘J I HYDROGEN BACTERIA

u 2! : 30- FORE-RUN FORE-RUN

40

30

20

IO

40

30

20

IO

5 15 45 135 600 TIME, seconds

I I I I I

MALIC ACID

I

5 I5 45 135 600

? ? I I I I I I , , I F

\ PHOSPHOGLYCERIC \ PHOSPHOGLYCERIC . . - : - : ACID ACID

I t I I I I I I I 5 5 I5 45 I35 600 I5 45 I35 600

40 I I I I 1

GLUTAMIC and 3. _ ASPARTIC ACIDS

5 I5 45 135 600

I I I I I I

5 I5 45 135 600

FIG. 3

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F. H. BERGMANN, J. C. TOWNE, AND R. H. BURRIS 21

70 per cent. Orgel, Dewar, and Koffler (23) have reported that acetic and formic acids, compounds which generally are lost during chromatog- raphy on anion exchange columns, are early products of CO2 fixation by H. facilis. However, chromatographic separation of our reaction mixtures, or of the corresponding stea.m distillates, on silica gel revealed negligible incorporation of Cl4 into the volatile organic acids under the conditions of our experiments. Similarly, no significant amounts of radioactive keto acids, either in the free form or as their dinitrophenylhydrazones, could be detected. As no significant trend toward better recoveries of CL4 with increasing time of exposure of cells to NaHC403 was noted, it seems probable that no major early intermediates were missed, but rather that the low recoveries arose from systematic errors such as losses of radio- activity during lyophilization.

Results with Cell-Free Preparations-Freshly harvested cells of H. facilis yielded extracts whose ability to fix Cl402 was stimulated by the addition of sugar phosphates. Ribose 5-phosphate was the most active com- pound, being twice as active as glucose 6-phosphate or fructose B-phosphate. Pyruvic acid and tricarboxylic acid cycle intermediates were relatively ineffective in stimulating fixation of C402.

Fixation of carbon dioxide in the presence of R-5-P was linear for at least 25 minutes; the 50 minute point indicated that the rate of fixation had decreased by that time (Fig. 4). Fixation with added R-5-P usually was about ten times as high as the endogenous fixation, but in occasional preparations R-5-P stimulated only 3-fold. The reaction required oxygen, for in evacuated Thunberg tubes the rate of C140~ fixation was less than 20 per cent of the aerobic rate.

Attempts to increase the rate of the R-5-P-dependent fixation by the addition of an energy source indicated that ATP in high concentrations (0.01 M) inhibited the reaction strongly, but that lower levels of ATP produced 20 to 100 per cent stimulation of CY402 fixation.

Since the extracts had vigorous hydrogenase activity, the possibility of coupling the oxyhydrogen reaction to Cl402 fixation was also investigated. However, a gas mixture of 20 per cent oxygen and 80 per cent hydrogen depressed fixation compared to controls run in air.

FIG. 3. Change in distribution of Cl4 with time in various fractions eluted from Dowex 1 columns. The z axis is on a logarithmic scale beyond the 5 second point. The data from Fig. 2 have been recalculated here to give the per cent of total re- covered radioactivity found in each peak. The dashed lines in the graphs of “sugar phosphates” and phosphoglyceric acid are hypothetical. They refer to the expected kinetic behavior of these two groups if phosphoglyceric acid is a precursor of “sugar phosphates.”

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22 CO2 FIXATION BY HYDROGEN BACTERIA

IO 20 30 40 50

TIME IN MINUTES

FIG. 4. Influence of ribose &phosphate on the time course of COZ fixation by cell- free preparations from H. jucilis. The system contained the following: 1.0 ml. of cell-free preparation in Tris HCl buffer, 0.1 M, pH 7.0; 0.2 ml. of NaHCi40a (652,000 c.p.m.); 0.1 ml. of R-5-P (10 PM) or 0.1 ml. of water. The temperature was 30”. The reaction was stopped by the addition of 2 N HCl in acetone. O,no R-5-P was added, l , R-5-P was added.

DISCUSSION

The results with whole cells of H. fucilis show that PGA (or a compound producing PGA during the inactivation procedure) and hexose phosphates are the main early compounds formed during carbon dioxide flation by this organism. The more rapid relative decrease of the percentage of total Cl4 in PGA with time, as well as the analogy to similar systems found in other organisms, suggests that PGA is the precursor of the sugar phos- phates. PGA probably is formed by the carboxylation of ribulose diphos- phate .

Suzuki and Werkman (9) have found a similar pattern of labeling for one of the thiobacilli, another group of chemoautotrophic bacteria. The carboxylation of ribulose diphosphate thus may be the main mode of carbon dioxide fixation by many, if not all, of the chemoautotrophic microorganisms. The phylogenetic significance of such a biochemical relationship between photosynthetic and chemoautotrophic organisms remains to be assessed, for under certain conditions heterotrophic bacteria such as Escherichia coli also can carboxylate ribulose diphosphate (24).

Our data suggest that the carboxylation of pyruvate or a related com- pound to yield a C-4 dicarboxylic acid constitutes a second, quantitatively

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17. H. BERGMANN, J. C. TOWNE, AND R. II. BURRIS 23

less significant, route for fixation of carbon dioxide by the hydrogen bacteria. Reactions of this type previously have been demonstrated in extracts of H. fucilis by Judis et al. (25). The percentage of the total Cl* (calculated in terms of Cl* fixed in compounds soluble in perchloric acid) in malic and in aspartic acids decreased with time of exposure of the cells to NaHC1*03, indicating that these compounds also are early intermediates in fixation of carbon dioxide. The data do not establish the mechanism of this fixation, for an intermediate present in small concentration and subject to rapid turnover (such as oxalacetic acid) could have been missed in these experiments.

To explain the stimulation of carbon dioxide fixation by R-5-P added to cell-free extracts of H. fucilis, it is logical to suggest that R-5-P was isomerixed to ribulose 5-phosphate and then phosphorylated to ribulose diphosphate, the acceptor of carbon dioxide in the carboxydismutase system. An active phosphoriboisomerase, the first enzyme required for this transformation, could be demonstrated in our extracts. Both the enzymatic activity and the R-5-P-dependent, fixation of carbon dioxide could be inhibited by 5-phosphoribonic acid, an inhibitor of phosphori- boisomerase (19). This suggests that PGA could have been formed during these experiments, and that the PGA then was rapidly metabolized via normal glycolytic pathways to pyruvate or phosphoenolpyruvate. Second- ary carboxylations could yield 4-carbon dicarboxylic acids as end products. However, the data do not rule out other interpretations, such as the conversion of R-5-P to phosphoenolpyruvate, which then could be car- boxylated.

The radioactive end products of the R-5-P-dependent cell-free fixation of Cl402 always were malic and fumaric acids, contrary to expectations. The identification of these compounds was based on cochromatography of authentic samples and the reaction mixture with several solvent systems on paper and with partition chromatography on silica gel. As fixation of NaHC1403 is rather weak in the cell-free as compared to the whole cell system, exposures to NaHC1*03 of less than 1 minute were not performed. Very early products of fixation having a small pool size thus could have been missed.

SUMMARY

1. Whole cells of Hydrogenomonas facilis, a chemoautotrophic bacterium, fix carbon dioxide rapidly into phosphoglyceric acid and hexose phosphates. After 5 seconds exposure to NaHC1*03, the bulk of the Cl* fixed is found in these compounds. A quantitatively less important pathway for fixa- tion of carbon dioxide involves direct formation of C-4 dicarboxylic acids, presumably by the carboxylation of a C-3 compound.

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24 CO2 FIXATION BY HYDROGEN BACTERIA

2. The rate of carbon dioxide fixation by cell-free extracts of H. fadis is markedly increased by the addition of ribose 5-phosphate. The pro- ducts of this fixation are malic and fumaric acids.

BIBLIOGRAPHY

1. Elsden, S. R., J. Gen. Microbial., 12, 332 (1955). 2. Rabinowitch, E. I., Photosynthesis and related processes, New York, 1, 123

(1945) . 3. Benson, A. A., Bassham, J. A., Calvin, M., Goodale, T. C., Haas, V. A., and

Stepka, W., J. Am. Chem. Sot., 72, 1710 (1950). 4. Fager, E. W., Rosenberg, J. L., and Gaffron, H., Federation Proc., 9,536 (1950). 5. Calvin, M., and Massini, P., Experientia, 8, 445 (1952). 6. Santer, M., and Vishniac, W., Biochim. et biophys. acta, 18, 157 (1955). 7. Trudinger, P. A., Biochem. J., 64, 274 (1956). 8. Calvin, M., Proceedings of the 3rd International Congress of Biochemistry,

Brussels, 211 (1956). 9. Suzuki, I., and Werkman, C. H., Bact. PTOC., 120 (1957).

10. Cohen, J. S., and Burris, R. H., J. Bact., 69, 316 (1955). 11. Marino, R. J., and Clifton, C. E., J. Bact., 69, 188 (1955). 12. Packer, L., and Vishniac, W., J. Bact., 70, 216 (1955). 13. Robison, R., and King, E. J., Biochem. J., 26, 323 (1931). 14. Johnson, M. J., J. Biol. Chem., 137,575 (1941). 15. Hiller, A., Plazin, J., and Van Slyke, D. D., J. Biol. Chem., 176, 1401 (1948). 16. LePage, G. A., in Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric

techniques and tissue metabolism, Minneapolis, 185 (1949). 17. Morris, D. L., Science, 107, 254 (1948). 18. Rapoport, S., Biochem. Z., 289, 406 (1937). 19. Axelrod, B., and Jang, R., J. Biol. Chem., 209, 847 (1954). 20. Busch, H., Hurlbert, R. B., and Potter, V. R., J. BioZ. Chem., 196, 717 (1952). 21. Palmer, J. K., Connecticut Agr. Exp. Sta., Bull. 689 (1955). 22. Liberman, S., Thesis, University of Wisconsin (1956). 23. Orgel, G., Dewar, N. E., and Koffler, H., Biochim. et biophys. acta., 21,409 (1956). 24. Fuller, R. C., Bact. Proc., 112 (1956). 25. Judis, J., Koffler, H., and Powelson, D. M., Proc. Sot. Exp. BioZ. and Med., 89,

665 (1955).

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BurrisFred H. Bergmann, Jack C. Towne and R. H.

BY HYDROGEN BACTERIAASSIMILATION OF CARBON DIOXIDE

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