Vol. 139, No. 1
JOURNAL OF BACTERIOLOGY, July 1979, p. 225-230 0021-9193/79/07-0225/06$02.00/0
Growth of Nocardia rhodochrous on Acetylene Gast D. KANNER AND R. BARTHA*
Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, New Jersey 08903 Received for publication 7 May 1979
Soil sediment enrichment cultures yielded a coryneform bacterium capable of growing in a mineral salts solution with acetylene gas as its only source of carbon and energy. Based on morphological and physiological traits as well as on cell wall analysis, the bacterium was characterized as a strain of Nocardia rhodochrous. Maximal growth rates (generation time 2.7 to 3.0 h) on acetylene were obtained at 5 to 20% acetylene, 25 to 40% oxygen, pH 7.0, and 26 to 28°C. Yields (grams of dry cells produced per gram of acetylene consumed) ranged between 90 and 110%. N. rhodochrous exhibits a growth factor requirement for the pyrimidine moiety of thiamine. Acetylene utilization is not an obligate trait, and a wide range of alternate carbon sources is utilized. Ethylene is neither produced nor consumed. The only previous report on acetylene utilization appeared in 1932. The Mycobacterium lacticola strain described in that report strongly resembles N. rhodochrous.
Acetylene gas (HC = CH) is not a natural product and is a highly unusual substrate for microbial growth. Our interest in the microbial utilization of acetylene gas initially arose from three separate considerations. A popular nitrogenase assay is based on acetylene reduction (10), and acetylene conversions by other enzyme systems could interfere with this assay. Acetylene can be derived from coal via calcium carbide, and acetylene utilization by microorganisms should be explored as one of several options for conversion of coal into single cell protein. Finally, the microbial metabolism of acetylene gas may shed some light on the biodegradation pathways of the more than 600 complex acetylenic compounds that were reported to occur as natural products (2). An extensive literature search has located only a single documented report on the microbial utilization of acetylene gas (1). In this 1932 report, a bacterium described as Mycobacterium lacticola was shown to grow in a mineral saltssoil extract medium incubated under mixtures of acetylene and air. Stationary culture conditions, long (2 to 4 weeks) incubation periods, and the lack of modem analytical tools limit the present usefulness of this report and prompted our reexamination of the problem. A more recent report on effects of acetylene gas on ethylene uptake by soil bacteria (5) contains a cursory statement about the isolation of an acetyleneutilizing bacterium, but the statement is not substantiated by any descriptive or experimental
data. We report here the isolation and growth characteristics of a Nocardia rhodochrous strain that is capable of using acetylene gas as its sole source of carbon and energy.
MATERIALS AND METHODS
A mixture of agricultural field soil and lake sediment from the grounds of the New Jersey Agricultural Experiment Station, New Brunswick, N.J. was moistened with a mineral salts solution (Na2HPO4, 0.4%, KH2PO4, 0.15%; NH4C1, 0.1%; MgSO4.7H20, 0.02%; FeNH4-citrate, 0.0005%; Hoagland's trace element solution, 1 ml/liter of medium; pH after steam sterilization, 7.0) and incubated under an atmosphere of 10% acetylene in air at 28°C. After 2 weeks, small amounts of this soil were inoculated into 25-ml Erlenmeyer flasks each containing 5 ml of the same mineral solution. The flasks were mounted on a hardboard inset that fitted in a vacuum desiccator and allowed agitation of the arrangement on a reciprocating shaker. The contents of flasks showing growth after several passages in the acetylene-air atmosphere were streaked on mineral salts agar plates (2% agar) and incubated under acetylene-air. Cultures were purified by restreaking on both mineral salts agar and Trypticase soy agar. Stock cultures of the strain were maintained on Trypticase soy and mineral salts agars (C2H2 as carbon source) and were transferred monthly. Taxonomy. Preparation of cell walls and their analysis for sugars, amino acids, and mycolic acids were all performed according to the methods of Lechevalier and Lechevalier (15). Physiological tests employed, except for growth on 5% NaCl, were those of Gordon (7, 8) and Gordon and Mihm (9). Growth on 5% NaCl was performed according to the method of Goodfellow (6). Gas purification. Cylinder acetylene was purified t Paper of the Journal Series, New Jersey Agricultural according to Rutledge (16) by passage of the gas Experiment Station, Cook College, Rutgers University. 225
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shaking at 240 rpm and, except for the temperature studies, at 28°C. Inocula for all experiments in this study were pregrown on acetylene and originated from mineral salts agar (C2H2-grown) stock cultures. Growth was routinely followed by monitoring both optical density (OD; at 660 nm) and protein concentration in the medium. Analyses. OD was measured using a Gilford Stasar II spectrophotometer. PrQtein was measured by a modified Lowry procedure using bovine serum albumin as a standard (13). Acetylene in headspace gas was measured gas chromatographically by using a Hewlett-Packard model 5700A instrument with dual-flame ionization detectors and on-column injection of samples with a gas-tight syringe. The stainless-steel columns (1.8 m by 3 mm) were packed with Porapak N, 80-100 mesh. Operating conditions were as follows: nitrogen carrier, 30 ml/ min; hydrogen, 30 ml/min; air, 250 ml/min. Detector temperature was 250°C; column oven was 75°C. Under these conditions, acetylene retention time was 105 s, and ethylene retention time was 65 s. Acetylene concentration was determined by quantitating peak areas and comparing them to a standard curve. Prior to sample withdrawal, internal pressure of the flasks was equalized to ambient by opening the stopcock of the long needle. Optimization experiments. In all experiments, flasks were run in triplicate, and each experiment was performed at least twice. Growth curves were plotted and used for the determination of growth rate constants (u). These were plotted versus the parameter under investigation to determine the optimal condition. Optimal acetylene concentration was determined by holding 02 steady at 20% and varying C2H2, with N2 making up the balance. Optimal 02 concentration was then determined by holding C2H2 constant at 10% fc and varying 02. The effect of pH on growth was tested between pH 4 and 10. Buffers for these experiments were chosen to fulfill the following criteria: (i) the organism should not utilize them as carbon or nitrogen sources; (ii) their presence in the mineral salts medium at pH 7.0 should not affect growth of the strain on acetylene. The buffers used, all at 0.025 M concentration, were: tartrate-hydrochloric acid, pH 4.0, phthalate-NaOH, pH 5.5; phosphate, pH 7.0; bicine (N,N-bis[2-hydroxyethyl]glycine)-NaOH, pH 8.5; carbonate-bicarbonate, pH 10.0. A control experiment comparing growth on acetylene in mineral salts at pH 7.0 with and without the buffering compounds was run before each pH variance experiment, and no inhibition or enhancement of growth was noted. In these experiments, NH4Cl was added to all media after sterilization to avoid losses at high pH during autoclaving. Alternatively, the problem was avoided by performing an additional set of experiments using KNO3 instead of NH4C1 as the nitrogen source. B Gases and chemicals. Acetylene (purified), nitroA gen (high purity), and oxygen (extra dry) were purFIG. 1. Flasks used in growing N. rhodochrous on chased from Matheson Gas, East Rutherford, N.J. 4acetylene gas. (A) Flask for routine cell cultivation; Methyl-5-,-hydroxyethylthiazole and 2-methyl-5(B) flask allowing quantitative sampling ofthe head- ethoxymethyl-6-aminopyrimidine were a gift of Merck, Sharp and Dohme Laboratories. All other space gas. cf, Cotton filter; sc, stopcock; fc, filter cap. chemicals were of reagent grade or better. For detailed description see text.
through dry-ice traps, concentrated H2S04, solid KOH, and activated alumina. Culture flasks. The bacterium was cultured on acetylene gas in 1-liter screw-cap Erlenmeyer flasks (Beilco Glass, Vineland, N.J.) containing 50 to 150 ml of mineral salts solution (Fig. 1A). Two stainless-steel syringe needles (16 gauge) were inserted through the Teflon-lined screw cap; one needle extended into the culture medium and the other one extended into the headspace of the flask. The needles were permanently secured with epoxy cement. Periodically, the flasks were gassed with an atmosphere of the desIred composition by bubbling excess volumes of gas through the culture solution. The incoming gas was sterilized by passage through an attached cotton filter. Culture broth was sampled by a sterile syringe attached to the long needle. During incubation the cotton filters were capped, thus making the flask airtight. The described culture flask was modified slightly (Fig. 1B) for gas chromatographic experiments when sampling of the flask atmosphere was desired. Here, both needle openings were sealed to the atmosphere with Luer-lock stainless-steel stopcocks. In this case, the on-line cotton filter on the short needle was placed inside the headspace, allowing injection or withdrawal of gas while preventing contamination. Both culture broth and flask atmosphere were sampled by syringe through the appropriate stopcock. Culture conditions. The mineral salts medium used for culture on acetylene and, in some experiments, on acetate and fructose, was identical to the enrichment medium except that thiamine hydrochloride (500 ,tg/liter) was added. The organism was grown on acetylene in the above-mentioned flasks with rotary
NOCARDIA RHODOCHROUS GROWTH ON ACETYLENE GAS
VOL. 139, 1979
RESULTS
Description and taxonomy. The bacterium isolated by our enrichment procedure is a grampositive non-acid-fast coryneform rod. It is catalase positive and an obligate aerobe. Analysis of cell wall sugars and amino acids reVealed the presence of a type IV (15) cell wall, with galactose, arabinose, and meso-diaminopimelic acid as major constituents. The isolate contains mycolic acids of the nocardomycolic (approximate chain length C5o) type. These chemical features are characteristic of the genus Nocardia (15). Its physiological characteristics (Table 1) place it in the Nocardia rhodochrous taxon (14). Like other members of this taxon, our N. rhodochrous strain utilizes a variety of amnino acids and sugars for growth, but fructose is preferred to glucose. The isolate also grows on hexadecane and paraffin oil. Young cultures are cream colored; older ones exhibit pink pigmentation. Thiamine requirements. When grown in defined media, the organism exhibits a growth factor requirement for thiamine, with either filter-sterilized or autoclaved thiamine hydrochloride being equally effective in promoting growth. Since thiamine hydrochloride is unstable to autoclaving at pH 7.0 (12), it was suspected that our N. rhodochrous strain requires only one constituent of the thiamine molecule or can re-
227
synthesize the molecule from its constituent halves. Experiments in which 4-methyl-5-,B-hydroxyethylthiazole and 2-methyl-5-ethoxymethyl-6-aminopyrimidine were added separately and in combination to miniimal media proved that the pyrimidine moiety alone can satisfy the apparent thiamine requirement (data not shown). Acetylene purification. Purified cylinder acetylene (99.6% in acetone) contains, in addition to some entrained acetone, trace amounts of phosphine, sulfur compounds, chlorine, hydrogen, and methane (3). We were initially interested in whether those impurities inhibited or stimulated growth of the bacterium on acetylene. The results of preliminary experiments showed that growth rates and final yields were identical in cultures fed acetylene after purification or directly from the cylinder. Consequently, acetylene was used in all subsequent experiments directly from the cylinder. Optimization experiments. Figure 2 shows the relationship between growth rate on C2H2 and temperature. Optimal growth temperature was approximately 26 to 280C, and the organism was therefore routinely cultured at 280C. Slow growth occurred at 50C, and was particularly noticeable when Trypticase soy broth or other complex media served as growth substrates. Acetylene concentration versus growth rate is
TABLE 1. Physiological characteristics of acetylene-utilizing isolate Determination
Hydrolysis of:Casein Xanthine Tyrosine Adenine Starch Gelatin Esculin Urea Production of Nitrate reductase
+ +
+ +
-
Growth at/in/on:
50C
370C Lysozyme broth 5% NaCl
Survival after 50°C for 8 h
+
_ + +
Result
Utilization of: Pyruvate Succinate Tartrate Ethanol
+
Acid from: Arabinose Cellobiose Dulcitol Erythritol Fructose Glucose Galactose Glycerol Inositol Lactose Maltose
+ +
+
+
Mannose
+ +
-
Mannitol Melibiose Raffinose Rhamnose Salicin Sorbitol Sucrose Trehalose
+
Xylose
-
Utilization of: Acetate Benzoate Citrate Lactate Malate Oxalate Propionate
Determination
Result
+
+ + +
+ + +
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J. BACTERIOL.
shown in Fig. 3. A broad optimum of acetylene concentration was observed in the 5 to 20% range, and thereafter routine growth experiments were conducted at 10% C2H2. The effect of 02 concentration on growth rate is shown in Fig. 4. Again, the optimum was found to be broad, and experiments thereafter were conducted at 30% 02. With either NH4' or N03 as nitrogen source, optimal pH for growth on acetylene was almost exactly 7.0 (Fig. 5), obviating a need to alter the initial pH of our mineral salts medium. Growth on acetylene. A typical growth experiment on acetylene, with gas uptake plotted as well as OD and protein, is shown in Fig. 6. A NaN3 (0.2%) poisoned flask served as a control, and showed no increase in OD or protein and no uptake of C2H2. In numerous similar growth experiments, OD reached a value of 2 to 4 and protein reached a value of 400 to 600 ,g/ml after roughly 25 h of incubation. Generation times of 2.7 to 3.0 h on acetylene were identical to generation times on acetate and fructose, but slightly slower than the 2.2-h doubling time on Trypticase soy broth. Growth on C2H2 is therefore comparable to growth on other defined carbon sources in a mineral salts solution. The time course of acetylene uptake closely corresponded to increases of OD and protein in the culture solution (Fig. 6). The data indicated a high C2H2 utilization efficiency by N. rhodochrous. In two such experiments, as average of 55.9% (standard deviation, ±15.3) of the acetylene removed from the headspace was converted to protein. Assuming that 50% of the cell dry weight is protein, this corresponds to a utilization efficiency (grams of cell dry weight produced per gram of acetylene utilized) of about 110%. In one experiment where dry weight yield was measured directly, utilization efficiency was 95.7%. Using gas chromatography on Porapak N, we
Q2L
21
0.1
10
15
I
A
I
5
25 TEMPERATURE (C) 20
I
I
30
35
40
FIG. 2. Effect of incubation temperature on growth rate of N. rhodochrous
on
acetylene gas.
I6 AC
5
10 40 20 30 ACETYLENE CONCENTRATION (%W
FIG. 3. Effect ofacetylene gas concentration in the headspace on growth rate of N. rhodochrous.
have not detected the accumulation of ethylene or other detectable volatiles in the headspace of the culture flasks during growth on acetylene. Attempts to grow N. rhodochrous on ethylene or to show ethylene utilization by this organism were unsuccessful. The metabolic pathway and enzymology of acetylene utilization are currently under investigation and will be the subject of a separate publication.
DISCUSSION The central conclusion of the 1932 report by Birch-Hirschfeld (1), i.e., the microbial utilization of acetylene gas, is clearly confirmed by our findings. When comparing finer details of the two studies, the changes during the last 50 years in taxonomic, cultural, and analytical techniques should be borne in mind. Birch-Hirschfeld used stationary mineral solution with soil extract (0.1 g/liter) and incubated the cultures for 2 to 4 weeks before analysis. We used submerged cultures with agitation and recorded growth curves instead of endpoint analyses. Such differences in methodology undoubtedly influence the obtained results, but a comparison of the two studies is nevertheless intriguing. Taxonomy. The question may be asked, whether the Mycobacterium lacticola isolated by Birch-Hirschfeld was similar to or identical with the N. rhodochrous isolated by us. Since the original culture was not preserved, an absolutely conclusive answer to this question is not possible. Nevertheless, nothing in the described morphological and physiological characteristics of M. lacticola differs greatly from the characteristics of our N. rhodochrous strain. The two isolates share somewhat unusual metabolic features, such as the preference for fructose over
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229
I1
O2
CONCENTRATION (%)
FIG. 4. Effect of oxygen concentration in the headspace on the growth rate of N. rhodochrous.
pH
FIG. 5. Effect of pH on the growth rate of N. rhodochrous on acetylene gas with either ammonium or nitrate nitrogen.
glucose and the utilization of paraffin. The soil extract requirement of M. lacticola that was not scrutinized further may correspond to the thiamine requirement of N. rhodochrous. In all of our acetylene enrichment cultures, we encountered only a single morphological type capable of acetylene utilization. The admittedly limited information presently available indicates that the ability of acetylene utilization is a unique trait, and that the two organisms, isolated on different continents and 50 years apart, are closely related if not identical. Optimal growth conditions. The broad optima of acetylene (5 to 20%) and of oxygen (25 to 40%) were measured in the headspace of the culture flasks, where the gases act as reservoirs only. Presumably, sharper optima would be obtained for dissolved gas concentrations that interact with enzyme systems directly, but because
'a
z
Ji
g
0
44
c4.0
I
44
44
HOURS
FIG. 6. Typical growth curve ofN. rhodochrous on acetylene gas, showing the consumption of acetylene and the increases in optical density and in protein. Data were plotted cumulatively by subtracting initial OD (0.120) and protein (5 pg/ml) values.
of technical difficulties we had to settle for the former approach. The solubility of acetylene gas in water is high (1.03 g/liters) when compared
230
KANNER AND BARTHA
with the solubility of some other substrate gases, e.g., hydrogen (cw2 mg/liter). Consequently, the lowest atmospheric concentration of acetylene (5%) already allowed a maximal growth rate. The acetylene gas optima (10 to 80%) reported by Birch-Hirschfeld (1) are meaningless, since calculations show that oxygen was limiting in every case. Oxygen, being less soluble in water (8 mg/liter), was required by N. rhodochrous in higher headspace concentrations for optimal growth. M. lacticola, cultivated as a surface film, showed a slightly lower optimum. Temperature and pH optima determined by us and by BirchHirschfeld (1) are in good agreement. Efficiency of acetylene conversion. The high utilization efficiency for acetylene by N. rhodochrous is not unique. Similar values (around 100%) were reported for yeasts growing on petroleum hydrocarbons (4, 11). Acetylene is also a highly reduced and energy-rich substrate (310 kcal [ca. 1,298 kJ] per mol). The substantially lower efficiencies reported by BirchHirschfeld (1) are artifacts of endpoint determinations after long incubation periods. Some acetylene carbon converted to cell material was bound to be lost in her experiments due to long periods of endogenous cell respiration. The high conversion efficiency of N. rhodochrous is a favorable trait in relation to single cell protein production. Relation to nitrogenase assay. Ethylene is neither produced nor consumed by N. rhodochrous. Consequently, a direct interference by this or similar organisms with the nitrogenase assay is unlikely. The abundance of N. rhodochrous in soils and sediments, without prior enrichment, appears to be low. Therefore, a significant competition for acetylene substrate by N. rhodochrous and similar organisms in nitrogenase assays is not expected. ACKNOWLEDGMENTS The technical asistance of John Sherwood in the initial enrichment and isolation work is gratefully acknowledged. We thank Mary Lechevalier for her guidance in our taxonomic studies, and Merck, Sharpe and Dohme Laboratories for the gift of thiamine constituents.
J. BACTERIOL. D.K. was supported by a Rutgers Graduate Fellowship during the course of this work. The work was also supported by the New Jersey Agricultural Experiment Station.
LITERATURE CITED 1. Birch-Hhrschfeld, L 1932. Die Umsetzung von Acetylen durch Mycobacterium lacticola. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 2 86:113-130. 2. Bohimann, F., T. Burkhardt, and C. Zdero. 1973. Naturally occurring acetylenes, p. 2. Academic Press, London. 3. Braker, W., and A. L Mossman (ed.). 1971. Matheson gas data book, 5th ed., p. 1-2. Matheson Gas Products, East Rutherford, N.J. 4. Coty, V. F., and R. I. Leavitt. 1971. Microbial protein from hydrocarbons. Dev. Ind. Microbiol. 12:61-71. 5. de Bont, J. A. M 1976. Bacterial degradation of ethylene and the acetylene reduction test. Can. J. Microbiol. 22: 1060-1062. 6. Goodfellow, M. 1971. Numerical taxonomy of some nocardioform bacteria. J. Gen. Microbiol. 69:33-80. 7. Gordon, R. E. 1966. Some criteria for the recognition of Nocardia madurae. J. Gen. Microbiol. 45:355-364. 8. Gordon, R. E. 1967. The taxonomy of soil bacteria, p. 293-321. In T. Gray, R. G. Parkinson, and B. Parkinson (ed.), The ecology of soil bacteria. Liverpool University Press. 9. Gordon, R. E., and J. M. Mihm. 1957. A comparative study of some strains received as nocardiae. J. Bacteriol. 73:15-27. 10. Hardy, R. W. F., R. P. Holsten, E. K. Jackson, and R. C. Burns. 1968. The acetylene-ethylene assay for Nr fixation: laboratory and field evaluation. Plant Physiol. 43:1185-1207. 11. Kanazawa, M. 1975. The production of yeast from nparaffins, p. 438-453. In S. R. Tannenbaum and D. I. C. Wang (ed.), Single cell protein II. MIT Press, Cambridge, Mass. 12. Koser, S. 1968. Vitamin requirements of yeasts and bacteria, p. 173-189. Charles C Thomas Publisher, Springfield, Ill. 13. Layne, E. 1975. Spectrophotometric and turbidometric methods for measuring proteins. Methods Enzymol. 3: 447-454. 14. Lechevalier, M. P., and H. A. Lechevalier. 1974. Nocardia amarae sp. nov., an actinomycete common in foaming activated sludge. Int. J. Syst. Bacteriol. 24: 278-288. 15. Lechevalier, M. P., and H. A. Lechevalier. 1978. The chemotaxonomy of actinomycetes. Actinomycete Taxonomy Workshop. Society for Industrial Microbiology, Rice University, Houston, Texas, 13 August 1978. 16. Rutledge, T. F. 1968. Acetylenic compounds, preparation and substitution reactions, p. 14. Reinhold Book Corp., New York.
JOURNAL OF BACTERIOLOGY, July 1979, p. 225-230 0021-9193/79/07-0225/06$02.00/0
Growth of Nocardia rhodochrous on Acetylene Gast D. KANNER AND R. BARTHA*
Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, New Jersey 08903 Received for publication 7 May 1979
Soil sediment enrichment cultures yielded a coryneform bacterium capable of growing in a mineral salts solution with acetylene gas as its only source of carbon and energy. Based on morphological and physiological traits as well as on cell wall analysis, the bacterium was characterized as a strain of Nocardia rhodochrous. Maximal growth rates (generation time 2.7 to 3.0 h) on acetylene were obtained at 5 to 20% acetylene, 25 to 40% oxygen, pH 7.0, and 26 to 28°C. Yields (grams of dry cells produced per gram of acetylene consumed) ranged between 90 and 110%. N. rhodochrous exhibits a growth factor requirement for the pyrimidine moiety of thiamine. Acetylene utilization is not an obligate trait, and a wide range of alternate carbon sources is utilized. Ethylene is neither produced nor consumed. The only previous report on acetylene utilization appeared in 1932. The Mycobacterium lacticola strain described in that report strongly resembles N. rhodochrous.
Acetylene gas (HC = CH) is not a natural product and is a highly unusual substrate for microbial growth. Our interest in the microbial utilization of acetylene gas initially arose from three separate considerations. A popular nitrogenase assay is based on acetylene reduction (10), and acetylene conversions by other enzyme systems could interfere with this assay. Acetylene can be derived from coal via calcium carbide, and acetylene utilization by microorganisms should be explored as one of several options for conversion of coal into single cell protein. Finally, the microbial metabolism of acetylene gas may shed some light on the biodegradation pathways of the more than 600 complex acetylenic compounds that were reported to occur as natural products (2). An extensive literature search has located only a single documented report on the microbial utilization of acetylene gas (1). In this 1932 report, a bacterium described as Mycobacterium lacticola was shown to grow in a mineral saltssoil extract medium incubated under mixtures of acetylene and air. Stationary culture conditions, long (2 to 4 weeks) incubation periods, and the lack of modem analytical tools limit the present usefulness of this report and prompted our reexamination of the problem. A more recent report on effects of acetylene gas on ethylene uptake by soil bacteria (5) contains a cursory statement about the isolation of an acetyleneutilizing bacterium, but the statement is not substantiated by any descriptive or experimental
data. We report here the isolation and growth characteristics of a Nocardia rhodochrous strain that is capable of using acetylene gas as its sole source of carbon and energy.
MATERIALS AND METHODS
A mixture of agricultural field soil and lake sediment from the grounds of the New Jersey Agricultural Experiment Station, New Brunswick, N.J. was moistened with a mineral salts solution (Na2HPO4, 0.4%, KH2PO4, 0.15%; NH4C1, 0.1%; MgSO4.7H20, 0.02%; FeNH4-citrate, 0.0005%; Hoagland's trace element solution, 1 ml/liter of medium; pH after steam sterilization, 7.0) and incubated under an atmosphere of 10% acetylene in air at 28°C. After 2 weeks, small amounts of this soil were inoculated into 25-ml Erlenmeyer flasks each containing 5 ml of the same mineral solution. The flasks were mounted on a hardboard inset that fitted in a vacuum desiccator and allowed agitation of the arrangement on a reciprocating shaker. The contents of flasks showing growth after several passages in the acetylene-air atmosphere were streaked on mineral salts agar plates (2% agar) and incubated under acetylene-air. Cultures were purified by restreaking on both mineral salts agar and Trypticase soy agar. Stock cultures of the strain were maintained on Trypticase soy and mineral salts agars (C2H2 as carbon source) and were transferred monthly. Taxonomy. Preparation of cell walls and their analysis for sugars, amino acids, and mycolic acids were all performed according to the methods of Lechevalier and Lechevalier (15). Physiological tests employed, except for growth on 5% NaCl, were those of Gordon (7, 8) and Gordon and Mihm (9). Growth on 5% NaCl was performed according to the method of Goodfellow (6). Gas purification. Cylinder acetylene was purified t Paper of the Journal Series, New Jersey Agricultural according to Rutledge (16) by passage of the gas Experiment Station, Cook College, Rutgers University. 225
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J. BACTERIOL.
shaking at 240 rpm and, except for the temperature studies, at 28°C. Inocula for all experiments in this study were pregrown on acetylene and originated from mineral salts agar (C2H2-grown) stock cultures. Growth was routinely followed by monitoring both optical density (OD; at 660 nm) and protein concentration in the medium. Analyses. OD was measured using a Gilford Stasar II spectrophotometer. PrQtein was measured by a modified Lowry procedure using bovine serum albumin as a standard (13). Acetylene in headspace gas was measured gas chromatographically by using a Hewlett-Packard model 5700A instrument with dual-flame ionization detectors and on-column injection of samples with a gas-tight syringe. The stainless-steel columns (1.8 m by 3 mm) were packed with Porapak N, 80-100 mesh. Operating conditions were as follows: nitrogen carrier, 30 ml/ min; hydrogen, 30 ml/min; air, 250 ml/min. Detector temperature was 250°C; column oven was 75°C. Under these conditions, acetylene retention time was 105 s, and ethylene retention time was 65 s. Acetylene concentration was determined by quantitating peak areas and comparing them to a standard curve. Prior to sample withdrawal, internal pressure of the flasks was equalized to ambient by opening the stopcock of the long needle. Optimization experiments. In all experiments, flasks were run in triplicate, and each experiment was performed at least twice. Growth curves were plotted and used for the determination of growth rate constants (u). These were plotted versus the parameter under investigation to determine the optimal condition. Optimal acetylene concentration was determined by holding 02 steady at 20% and varying C2H2, with N2 making up the balance. Optimal 02 concentration was then determined by holding C2H2 constant at 10% fc and varying 02. The effect of pH on growth was tested between pH 4 and 10. Buffers for these experiments were chosen to fulfill the following criteria: (i) the organism should not utilize them as carbon or nitrogen sources; (ii) their presence in the mineral salts medium at pH 7.0 should not affect growth of the strain on acetylene. The buffers used, all at 0.025 M concentration, were: tartrate-hydrochloric acid, pH 4.0, phthalate-NaOH, pH 5.5; phosphate, pH 7.0; bicine (N,N-bis[2-hydroxyethyl]glycine)-NaOH, pH 8.5; carbonate-bicarbonate, pH 10.0. A control experiment comparing growth on acetylene in mineral salts at pH 7.0 with and without the buffering compounds was run before each pH variance experiment, and no inhibition or enhancement of growth was noted. In these experiments, NH4Cl was added to all media after sterilization to avoid losses at high pH during autoclaving. Alternatively, the problem was avoided by performing an additional set of experiments using KNO3 instead of NH4C1 as the nitrogen source. B Gases and chemicals. Acetylene (purified), nitroA gen (high purity), and oxygen (extra dry) were purFIG. 1. Flasks used in growing N. rhodochrous on chased from Matheson Gas, East Rutherford, N.J. 4acetylene gas. (A) Flask for routine cell cultivation; Methyl-5-,-hydroxyethylthiazole and 2-methyl-5(B) flask allowing quantitative sampling ofthe head- ethoxymethyl-6-aminopyrimidine were a gift of Merck, Sharp and Dohme Laboratories. All other space gas. cf, Cotton filter; sc, stopcock; fc, filter cap. chemicals were of reagent grade or better. For detailed description see text.
through dry-ice traps, concentrated H2S04, solid KOH, and activated alumina. Culture flasks. The bacterium was cultured on acetylene gas in 1-liter screw-cap Erlenmeyer flasks (Beilco Glass, Vineland, N.J.) containing 50 to 150 ml of mineral salts solution (Fig. 1A). Two stainless-steel syringe needles (16 gauge) were inserted through the Teflon-lined screw cap; one needle extended into the culture medium and the other one extended into the headspace of the flask. The needles were permanently secured with epoxy cement. Periodically, the flasks were gassed with an atmosphere of the desIred composition by bubbling excess volumes of gas through the culture solution. The incoming gas was sterilized by passage through an attached cotton filter. Culture broth was sampled by a sterile syringe attached to the long needle. During incubation the cotton filters were capped, thus making the flask airtight. The described culture flask was modified slightly (Fig. 1B) for gas chromatographic experiments when sampling of the flask atmosphere was desired. Here, both needle openings were sealed to the atmosphere with Luer-lock stainless-steel stopcocks. In this case, the on-line cotton filter on the short needle was placed inside the headspace, allowing injection or withdrawal of gas while preventing contamination. Both culture broth and flask atmosphere were sampled by syringe through the appropriate stopcock. Culture conditions. The mineral salts medium used for culture on acetylene and, in some experiments, on acetate and fructose, was identical to the enrichment medium except that thiamine hydrochloride (500 ,tg/liter) was added. The organism was grown on acetylene in the above-mentioned flasks with rotary
NOCARDIA RHODOCHROUS GROWTH ON ACETYLENE GAS
VOL. 139, 1979
RESULTS
Description and taxonomy. The bacterium isolated by our enrichment procedure is a grampositive non-acid-fast coryneform rod. It is catalase positive and an obligate aerobe. Analysis of cell wall sugars and amino acids reVealed the presence of a type IV (15) cell wall, with galactose, arabinose, and meso-diaminopimelic acid as major constituents. The isolate contains mycolic acids of the nocardomycolic (approximate chain length C5o) type. These chemical features are characteristic of the genus Nocardia (15). Its physiological characteristics (Table 1) place it in the Nocardia rhodochrous taxon (14). Like other members of this taxon, our N. rhodochrous strain utilizes a variety of amnino acids and sugars for growth, but fructose is preferred to glucose. The isolate also grows on hexadecane and paraffin oil. Young cultures are cream colored; older ones exhibit pink pigmentation. Thiamine requirements. When grown in defined media, the organism exhibits a growth factor requirement for thiamine, with either filter-sterilized or autoclaved thiamine hydrochloride being equally effective in promoting growth. Since thiamine hydrochloride is unstable to autoclaving at pH 7.0 (12), it was suspected that our N. rhodochrous strain requires only one constituent of the thiamine molecule or can re-
227
synthesize the molecule from its constituent halves. Experiments in which 4-methyl-5-,B-hydroxyethylthiazole and 2-methyl-5-ethoxymethyl-6-aminopyrimidine were added separately and in combination to miniimal media proved that the pyrimidine moiety alone can satisfy the apparent thiamine requirement (data not shown). Acetylene purification. Purified cylinder acetylene (99.6% in acetone) contains, in addition to some entrained acetone, trace amounts of phosphine, sulfur compounds, chlorine, hydrogen, and methane (3). We were initially interested in whether those impurities inhibited or stimulated growth of the bacterium on acetylene. The results of preliminary experiments showed that growth rates and final yields were identical in cultures fed acetylene after purification or directly from the cylinder. Consequently, acetylene was used in all subsequent experiments directly from the cylinder. Optimization experiments. Figure 2 shows the relationship between growth rate on C2H2 and temperature. Optimal growth temperature was approximately 26 to 280C, and the organism was therefore routinely cultured at 280C. Slow growth occurred at 50C, and was particularly noticeable when Trypticase soy broth or other complex media served as growth substrates. Acetylene concentration versus growth rate is
TABLE 1. Physiological characteristics of acetylene-utilizing isolate Determination
Hydrolysis of:Casein Xanthine Tyrosine Adenine Starch Gelatin Esculin Urea Production of Nitrate reductase
+ +
+ +
-
Growth at/in/on:
50C
370C Lysozyme broth 5% NaCl
Survival after 50°C for 8 h
+
_ + +
Result
Utilization of: Pyruvate Succinate Tartrate Ethanol
+
Acid from: Arabinose Cellobiose Dulcitol Erythritol Fructose Glucose Galactose Glycerol Inositol Lactose Maltose
+ +
+
+
Mannose
+ +
-
Mannitol Melibiose Raffinose Rhamnose Salicin Sorbitol Sucrose Trehalose
+
Xylose
-
Utilization of: Acetate Benzoate Citrate Lactate Malate Oxalate Propionate
Determination
Result
+
+ + +
+ + +
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KANNER AND BARTHA
J. BACTERIOL.
shown in Fig. 3. A broad optimum of acetylene concentration was observed in the 5 to 20% range, and thereafter routine growth experiments were conducted at 10% C2H2. The effect of 02 concentration on growth rate is shown in Fig. 4. Again, the optimum was found to be broad, and experiments thereafter were conducted at 30% 02. With either NH4' or N03 as nitrogen source, optimal pH for growth on acetylene was almost exactly 7.0 (Fig. 5), obviating a need to alter the initial pH of our mineral salts medium. Growth on acetylene. A typical growth experiment on acetylene, with gas uptake plotted as well as OD and protein, is shown in Fig. 6. A NaN3 (0.2%) poisoned flask served as a control, and showed no increase in OD or protein and no uptake of C2H2. In numerous similar growth experiments, OD reached a value of 2 to 4 and protein reached a value of 400 to 600 ,g/ml after roughly 25 h of incubation. Generation times of 2.7 to 3.0 h on acetylene were identical to generation times on acetate and fructose, but slightly slower than the 2.2-h doubling time on Trypticase soy broth. Growth on C2H2 is therefore comparable to growth on other defined carbon sources in a mineral salts solution. The time course of acetylene uptake closely corresponded to increases of OD and protein in the culture solution (Fig. 6). The data indicated a high C2H2 utilization efficiency by N. rhodochrous. In two such experiments, as average of 55.9% (standard deviation, ±15.3) of the acetylene removed from the headspace was converted to protein. Assuming that 50% of the cell dry weight is protein, this corresponds to a utilization efficiency (grams of cell dry weight produced per gram of acetylene utilized) of about 110%. In one experiment where dry weight yield was measured directly, utilization efficiency was 95.7%. Using gas chromatography on Porapak N, we
Q2L
21
0.1
10
15
I
A
I
5
25 TEMPERATURE (C) 20
I
I
30
35
40
FIG. 2. Effect of incubation temperature on growth rate of N. rhodochrous
on
acetylene gas.
I6 AC
5
10 40 20 30 ACETYLENE CONCENTRATION (%W
FIG. 3. Effect ofacetylene gas concentration in the headspace on growth rate of N. rhodochrous.
have not detected the accumulation of ethylene or other detectable volatiles in the headspace of the culture flasks during growth on acetylene. Attempts to grow N. rhodochrous on ethylene or to show ethylene utilization by this organism were unsuccessful. The metabolic pathway and enzymology of acetylene utilization are currently under investigation and will be the subject of a separate publication.
DISCUSSION The central conclusion of the 1932 report by Birch-Hirschfeld (1), i.e., the microbial utilization of acetylene gas, is clearly confirmed by our findings. When comparing finer details of the two studies, the changes during the last 50 years in taxonomic, cultural, and analytical techniques should be borne in mind. Birch-Hirschfeld used stationary mineral solution with soil extract (0.1 g/liter) and incubated the cultures for 2 to 4 weeks before analysis. We used submerged cultures with agitation and recorded growth curves instead of endpoint analyses. Such differences in methodology undoubtedly influence the obtained results, but a comparison of the two studies is nevertheless intriguing. Taxonomy. The question may be asked, whether the Mycobacterium lacticola isolated by Birch-Hirschfeld was similar to or identical with the N. rhodochrous isolated by us. Since the original culture was not preserved, an absolutely conclusive answer to this question is not possible. Nevertheless, nothing in the described morphological and physiological characteristics of M. lacticola differs greatly from the characteristics of our N. rhodochrous strain. The two isolates share somewhat unusual metabolic features, such as the preference for fructose over
NOCARDIA RHODOCHROUS GROWTH ON ACETYLENE GAS
VOL. 139, 1979
229
I1
O2
CONCENTRATION (%)
FIG. 4. Effect of oxygen concentration in the headspace on the growth rate of N. rhodochrous.
pH
FIG. 5. Effect of pH on the growth rate of N. rhodochrous on acetylene gas with either ammonium or nitrate nitrogen.
glucose and the utilization of paraffin. The soil extract requirement of M. lacticola that was not scrutinized further may correspond to the thiamine requirement of N. rhodochrous. In all of our acetylene enrichment cultures, we encountered only a single morphological type capable of acetylene utilization. The admittedly limited information presently available indicates that the ability of acetylene utilization is a unique trait, and that the two organisms, isolated on different continents and 50 years apart, are closely related if not identical. Optimal growth conditions. The broad optima of acetylene (5 to 20%) and of oxygen (25 to 40%) were measured in the headspace of the culture flasks, where the gases act as reservoirs only. Presumably, sharper optima would be obtained for dissolved gas concentrations that interact with enzyme systems directly, but because
'a
z
Ji
g
0
44
c4.0
I
44
44
HOURS
FIG. 6. Typical growth curve ofN. rhodochrous on acetylene gas, showing the consumption of acetylene and the increases in optical density and in protein. Data were plotted cumulatively by subtracting initial OD (0.120) and protein (5 pg/ml) values.
of technical difficulties we had to settle for the former approach. The solubility of acetylene gas in water is high (1.03 g/liters) when compared
230
KANNER AND BARTHA
with the solubility of some other substrate gases, e.g., hydrogen (cw2 mg/liter). Consequently, the lowest atmospheric concentration of acetylene (5%) already allowed a maximal growth rate. The acetylene gas optima (10 to 80%) reported by Birch-Hirschfeld (1) are meaningless, since calculations show that oxygen was limiting in every case. Oxygen, being less soluble in water (8 mg/liter), was required by N. rhodochrous in higher headspace concentrations for optimal growth. M. lacticola, cultivated as a surface film, showed a slightly lower optimum. Temperature and pH optima determined by us and by BirchHirschfeld (1) are in good agreement. Efficiency of acetylene conversion. The high utilization efficiency for acetylene by N. rhodochrous is not unique. Similar values (around 100%) were reported for yeasts growing on petroleum hydrocarbons (4, 11). Acetylene is also a highly reduced and energy-rich substrate (310 kcal [ca. 1,298 kJ] per mol). The substantially lower efficiencies reported by BirchHirschfeld (1) are artifacts of endpoint determinations after long incubation periods. Some acetylene carbon converted to cell material was bound to be lost in her experiments due to long periods of endogenous cell respiration. The high conversion efficiency of N. rhodochrous is a favorable trait in relation to single cell protein production. Relation to nitrogenase assay. Ethylene is neither produced nor consumed by N. rhodochrous. Consequently, a direct interference by this or similar organisms with the nitrogenase assay is unlikely. The abundance of N. rhodochrous in soils and sediments, without prior enrichment, appears to be low. Therefore, a significant competition for acetylene substrate by N. rhodochrous and similar organisms in nitrogenase assays is not expected. ACKNOWLEDGMENTS The technical asistance of John Sherwood in the initial enrichment and isolation work is gratefully acknowledged. We thank Mary Lechevalier for her guidance in our taxonomic studies, and Merck, Sharpe and Dohme Laboratories for the gift of thiamine constituents.
J. BACTERIOL. D.K. was supported by a Rutgers Graduate Fellowship during the course of this work. The work was also supported by the New Jersey Agricultural Experiment Station.
LITERATURE CITED 1. Birch-Hhrschfeld, L 1932. Die Umsetzung von Acetylen durch Mycobacterium lacticola. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 2 86:113-130. 2. Bohimann, F., T. Burkhardt, and C. Zdero. 1973. Naturally occurring acetylenes, p. 2. Academic Press, London. 3. Braker, W., and A. L Mossman (ed.). 1971. Matheson gas data book, 5th ed., p. 1-2. Matheson Gas Products, East Rutherford, N.J. 4. Coty, V. F., and R. I. Leavitt. 1971. Microbial protein from hydrocarbons. Dev. Ind. Microbiol. 12:61-71. 5. de Bont, J. A. M 1976. Bacterial degradation of ethylene and the acetylene reduction test. Can. J. Microbiol. 22: 1060-1062. 6. Goodfellow, M. 1971. Numerical taxonomy of some nocardioform bacteria. J. Gen. Microbiol. 69:33-80. 7. Gordon, R. E. 1966. Some criteria for the recognition of Nocardia madurae. J. Gen. Microbiol. 45:355-364. 8. Gordon, R. E. 1967. The taxonomy of soil bacteria, p. 293-321. In T. Gray, R. G. Parkinson, and B. Parkinson (ed.), The ecology of soil bacteria. Liverpool University Press. 9. Gordon, R. E., and J. M. Mihm. 1957. A comparative study of some strains received as nocardiae. J. Bacteriol. 73:15-27. 10. Hardy, R. W. F., R. P. Holsten, E. K. Jackson, and R. C. Burns. 1968. The acetylene-ethylene assay for Nr fixation: laboratory and field evaluation. Plant Physiol. 43:1185-1207. 11. Kanazawa, M. 1975. The production of yeast from nparaffins, p. 438-453. In S. R. Tannenbaum and D. I. C. Wang (ed.), Single cell protein II. MIT Press, Cambridge, Mass. 12. Koser, S. 1968. Vitamin requirements of yeasts and bacteria, p. 173-189. Charles C Thomas Publisher, Springfield, Ill. 13. Layne, E. 1975. Spectrophotometric and turbidometric methods for measuring proteins. Methods Enzymol. 3: 447-454. 14. Lechevalier, M. P., and H. A. Lechevalier. 1974. Nocardia amarae sp. nov., an actinomycete common in foaming activated sludge. Int. J. Syst. Bacteriol. 24: 278-288. 15. Lechevalier, M. P., and H. A. Lechevalier. 1978. The chemotaxonomy of actinomycetes. Actinomycete Taxonomy Workshop. Society for Industrial Microbiology, Rice University, Houston, Texas, 13 August 1978. 16. Rutledge, T. F. 1968. Acetylenic compounds, preparation and substitution reactions, p. 14. Reinhold Book Corp., New York.
