The use of disposable gaas generating kits for the growth of hydrogen-oxidizing bacteria and the determination of hydrogen autotrophy

Journal of Mtcrobzological Methods 3 (1984) 7-14 Elsevier

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JMM 00076

The use of disposable gas generating kits for the growth of hydrogen-oxidizing bacteria and the determination of hydrogen autotrophy L.I. Sly Department of Microbiology, University of Queensland, Brisbane, Queensland 4067 (Australia) (Received 2 March 1984) (Revised version received 14 May 1984) (Accepted 15 May 1984)

Summary A simplified procedure for the determination of autotrophic growth of hydrogen-oxidizing bacteria has been developed. The method uses commercially available disposable hydrogen and carbon dioxide kits, commonly used in anaerobic bacteriology, to produce a gaseous atmosphere containing by volume approximately 41% hydrogen, 6% carbon dioxide, 11% oxygen and 42% nitrogen. The atmosphere was suitable for the growth of strains assigned to the species Alcaligenes eutrophus, Alcaligenes paradoxus, Paracoccusdenitrificans, Pseudomonasfacilis, Pseudomonasflava, Pseudomonaspalleronii, Pseudomonas saccharophila and Rhodococcu~ sp. ('Nocardia opaca'). The method can also be used for the screening of hydrogen-oxidizing ability in bacterial isolates, thus eliminating the need for complex gas mixing devices or expensive gas mixtures. Key words: Autotrophic growth - Hydrogen autotrophy - Hydrogen oxidizing bacteria

Introduction The ability to oxidize hydrogen is an important physiological property of some bacteria which has been used for their classification and their identification. Although these bacteria f o r m a physiological group, they do not form a single taxonomic group. H y d r o g e n oxidizing bacteria have been assigned to various taxa including species in the currently recognized genera of Alcaligenes, Aquaspirillum, Arthrobacter, Bacillus, Flavobacterium, Mycobacterium, Nocardia, Paracoccus, Pseudomonas, Rhodococcus and Xanthobacter [1, 2]. These aerobic facultatively chemolithotrophic bacteria are capable of oxidizing molecular hydrogen when growing in a simple mineral m e d i u m [3, 4] with an a t m o s p h e r e containing hydrogen, oxygen and carbon dioxide gases. A variety of 0167-7012/84/$03.00 © 1984 Elsevier Science Publishers B.V.

gaseous formulations have been used. A critical component is the concentration of oxygen in the atmosphere. Oxygen concentrations between 2 and 20% by volume have been used [1, 3-9], with levels between 2 and 5% being required for oxygen sensitive strains [1, 9]. Carbon dioxide and hydrogen levels are less critical and concentrations in the ranges of 5-15% and 10--85%, respectively, have been used [1, 3-9]. Hydrogen levels may be reduced by the presence of nitrogen when air is used as the source of oxygen. Routinely, gas mixtures have been prepared by gas mixing devices [1] or by specially formulated commercial cylinders. In some laboratories the complexity of the former and the cost of the latter have inhibited the widespread application of the hydrogen autotrophy test in bacterial characterization and identification. Hydrogen and carbon dioxide gas generating kits [10-12] are commercially available and are used routinely in anaerobic microbiology, These kits offer a simple and convenient method for the generation of a standardized mixture of hydrogen and carbon dioxide. This paper describes the development of a procedure for the growth of hydrogen autotrophic bacteria in an atmosphere produced by a commercially available hydrogen and carbon dioxide gas generating kit. Methods

Microorganisms The strains of microorganisms used in this study were obtained from culture collections and are listed in Table 1 together with their origins and their strain designations. Growth conditions The medium of Doudoroff [3] was used. The liquid medium was dispensed in 10 ml quantities in 50 ml Erlenmeyer flasks with loosely fitting glass caps to allow TABLE 1 HYDROGEN OXIDIZING STRAINS USED IN THIS STUDY CAPABLE OF GROWING AUTOTROPHICALLY IN ATMOSPHERES PREPARED USING DISPOSABLE GAS GENERATING KITS Name

Strain No.

Alcahgenes eutrophus Alcaligenesparadoxus Paracoccus denttrificans Pseudomonasfacilis Pseudomonasflava Pseudomonaspalleronii Pseudomonassaccharophila Rhodococcus sp ('Nocardta opaca')

UQM1296 (= UQM1905 (= UQM 1274 (= UQM1918 (= UQM1920 (= UQM1917 (= UQMI53 (= UQM1301 (=

Source ~ DSM428) DSM30034) ATCC 17741 ) DSM649) DSM619) DSM63) ATCC15946) DSM427)

DSM DSM ATCC DSM DSM DSM ATCC DSM

Abbreviations: ATCC, American Type Culture Collection, Rockville, Md, USA; DSM, Deutsche Sammlung von Mikroorganismen, G6ttingen, FRG; UQM, Culture Collection, Department of Microbiology, University of Queensland, Brisbane, Australia.

W m a C

b

Fig. 1. Culture apparatus consisting of: a, anaerobic jar with the catalyst removed; b, hydrogen and carbon &oxide gas generating kit; c, rubber football bladder to collect excess gas generated; and d, gas collecting apparatus to collect or discard excess gas from bladder after mixing. adequate exchange of gases. Solid medium was prepared by adding 1.5 g agar (Difco Laboratories, Detroit, USA) per 100 ml of liquid medium. All media were sterilized by autoclaving at 121°C for 15 min. The culture apparatus is shown in Fig. 1. The apparatus consisted of a standard 3200 ml metal anaerobic jar (Baird and Tatlock Ltd., Romford, England) with the cold catalyst removed. The culture vessels, either Erlenmeyer flasks, agar plates, or a combination of these, were placed in the anaerobic jar. The atmosphere for the determination of hydrogen autotrophic growth was produced in the anaerobic jar without evacuation by a disposable hydrogen and carbon dioxide gas generating kit (Code No. BR38, Oxoid Ltd., Basingstoke, England). The manufacturer's specifications indicated that approximately 1800 ml hydrogen and 350 ml carbon dioxide were produced. The gas generating kit was placed in the anaerobic jar, and 10 ml of tap water was added according to the manufacturer's instructions. The lid of the anaerobic jar was placed quickly in position and clamped. The excess gas produced was vented through an open stopcock to a rubber football bladder to maintain atmospheric pressure in the growth apparatus for safety and to facilitate gas mixing. After gas evolution ceased in approximately 2 h the second stopcock was connected to a gas collection apparatus, and the excess gas was expelled by gently squeezing the bladder. The gas volume was measured as a check on the efficacy of the gas generating kit, and the gas was then safely discarded directly into the open atmosphere through a fumehood. R e m e m b e r , the gas atmosphere contains hydrogen and it should not be vented into the laboratory or anywhere near naked flames.

I0 Both stopcocks were then closed, the bladder was removed and the jar was incubated at 28°C. Cultures were passaged at least three times before positive growth was recorded. Control atmospheres without hydrogen were prepared with a disposable carbon dioxide generating kit (Code No. BR39, Oxoid Ltd., Basingstoke, England). The 350 ml carbon dioxide produced was supplemented with 1800 ml nitrogen gas in place of the hydrogen.

Gas analysis The composition of the gaseous atmosphere was determined with a Hewlett Packard gas chromatograph (Model 5840 A) fitted with dual columns packed with Porapak N (Waters Associates Inc., Milford, USA) and molecular sieve 5A (Union Carbide, New York, USA). The carrier gas was argon at a flow rate of 30 ml/min. Split gas samples were analyzed isothermally at 90°C. Hydrogen, oxygen and nitrogen were separated on the molecular sieve 5A column and detected by a thermal conductivity detector at 250°C. Carbon dioxide was analyzed after catalytic hydrogenation to methane using a nickel catalyst at 350°C. The methane was detected by a flame ionization detector at 150°C after passage through the Porapak N column. The response from both detectors was automatically recorded on a single chromatogram. Results and Discussion

The suitability of the procedure described has been demonstrated by the growth of a number of hydrogen-oxidizing bacteria, including strains from the genera Alcaligenes, Paracoccus, Pseudomonas and Rhodococcus (Table 1). Excellent growth was obtained both on agar plates and in liquid culture in flasks. Examples of the growth responses of Alcaligenes eutrophus UQM1296, Paracoccus denitrificans UQM1274 and Pseudomonas saccharophila UQM153 are shown in Fig. 2. Gas chromatographic analysis of the autotrophic atmospheres produced by disposable hydrogen and carbon dioxide generating kits showed that they contained approximately 41% hydrogen, 6% carbon dioxide, 11% oxygen and 42% nitrogen (Table 2). The actual concentrations of hydrogen and nitrogen were not as closely consistent with the theoretical estimates as were the concentrations of oxygen and carbon dioxide. However, irrespective of this slight deviation, gas chromatographic analysis showed that the composition of the atmospheres produced were reproducible and that a variation of approximately + 1% to + 1.6% in the concentration of the component gases could be expected (Table 2). The composition of the component gases in the atmosphere produced falls within the ranges which have been frequently used for the growth of hydrogenoxidizing bacteria [1, 3-9]. The atmospheres, however, had an oxygen level more suited to the growth of oxygen tolerant strains. The gas chromatographic analysis also showed that the gaseous atmosphere was free of contaminant gases which might inhibit growth or confuse the interpretation of a test for hydrogen autotrophic growth (Fig. 3).

I1

Fig. 2. Examples of cultures grown autotrophically on agar plates (a) and m flasks (b) for 7 days at 28°C in atmospheres prepared using disposable gas generating kits producing hydrogen and carbon dioxide (hc) or carbon dioxide (c) only: A, Alcaligenes eutrophus UQM1296; B, Paracoccus denitrifzcans UQM1274; C, Pseudomonas saccharophila UQM153.

12 TABLE 2 COMPOSITION OF GASEOUS ATMOSPHERES PREPARED USING DISPOSABLE HYD R O G E N AND CARBON DIOXIDE GENERATING KITS Gaseous component

Hydrogen Carbon dioxide Oxygen Nitrogen

Composition (Vol %) Theorettcal ~

Analyzed h

33.6 65 11.9 47.8

41.1 5.5 11.1 42.4

± 1.60 ± 0.99 ± 0.92 ___1.12

Based on manufacturer's specifications of 1800 ml hydrogen and 350 ml carbon dioxide generated and assuming perfect mixing. Mean and standard deviation of gas chromatographic analyses of atmospheres produced by kits on three occasions.

w Z 0 o. Ix

0 n-

\ TIME

(mm)

Fig. 3. Gas chromatogram of a hydrogen autotrophic atmosphere prepared using a disposable hydrogen and carbon dioxide gas generating kit. Peak 1, hydrogen; peak 2, carbon dioxide; peak 3, oxygen; and peak 4, nitrogen.

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The fact that hydrogen autotrophic organisms have been assigned to a broad and increasing range of genera [1, 2] necessitates a simple procedure for the routine screening of hydrogen-oxidizing ability. The recent discovery [13] of naturally occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus, and the spontaneous loss of hydrogenase activity in one strain of A. eutrophus, reinforces the significance of an earlier report [6] of the spontaneous loss of hydrogen-oxidizing ability in Alcaligenes paradoxus after isolation. This discovery also emphasizes the need for those culturing or preserving these organisms to regularly check for the retention of hydrogen-oxidizing ability. The method described offers routine laboratories a simplified and effective procedure for the growth of hydrogen-oxidizing bacteria. The procedure makes use of equipment and facilities readily available in most microbiology laboratories, and eliminates the need for vacuum pumps, gas mixing devices or expensive gas mixtures which may be infrequently used. The use of this procedure should help to facilitate the more accurate assignment of strains to the species of hydrogen-oxidizing bacteria with almost the same level of convenience that gas generating kits have brought to anaerobic microbiology.

Acknowledgement I thank Christine Kastrissios and Elizabeth Marden for technical assistance, and Stewart Bell, Queensland Government Chemical Laboratory, for carrying out the gas chromatographic analyses.

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