Oxygen Sensitivity of Methanogenic Bacteria

System. Appl, Microbiol. 4, 305-312 (1983)

Mikrobiologisches Institut ETH Zurich, ETH-Zentrum, 8092 Zurich, Switzerland

Oxygen Sensitivity of Methanogenic Bacteria ANDREAS KIENER and THOMAS LEISINGER

Received December 27, 1982

Summary Exponentially growing cells of five methanogenic bacteria were plated on solid media with efficiencies greater than 80%. This permitted the determination of the oxygen sensitivity of these strains under standardized conditions involving the exposure of suspensions of starved cells in non-reduced buffer to air. The death curves of Methanobacterium thermoautotropbicum, Methanobrevibacter arboriphilus and Methanosarcina barkeri were biphasic. Exposures to air for up to 30 h were without effect on the number of colony forming units whereas longer periods of contact with oxygen led to a rapid decrease in viability. In Methanococcus uoltae and Methanococcus uannielii the number of surviving cells upon exposure to air dropped exponentially without lag leading to 99% kill within 10 h.

Key words: Archaebacteria - Methanogenic bacteria - Plating efficiency - Oxygen sensitivity

Introduction

Growth of methanogenic bacteria as well as methane formation by this group of archae bacteria are observed only under strictly anaerobic conditions in media with a redox potential below - 330 mV (Smith and Hungate, 1958; Zehnder, 1978). This well established fact has led to the view that methanogens are extremely intolerant to oxygen. While scattered observations of qualitative nature indicate that this does not need to be the case for all methanogens (e.g. Zehnder and Wuhrmann, 1977), quantitative information on the oxygen sensitivity of these organisms are not available. We therefore have conducted a study on the oxygen tolerance of five species of methanogenic bacteria. The data obtained may help to understand how methanogens are able to colonize newly formed anaerobic ecosystems originating in aerobic environments. They may also be useful for judging the necessity of strictly anaerobic conditions in manipulating methanogens.

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Materials and Methods Organisms: Methanobacterium thermoautotrophicum Marburg (DSM 2133) was obtained from G.Fuchs, Marburg University, Marburg, Germany. Methanobrevibacter arboriphilus strain AZ (DSM 744) and Methanosarcina barkeri strain MS (DSM 800) were obtained from A. Zehnder, EAWAG, Diibendorf, Switzerland. Methanococcus vannielii (DSM 1224) and Methanococcus voltae (DSM 1537) were from the German collection of Micro-organisms (Gotringen, German y). Anaerobe facilities and gas processing: Experiments were performed in a Freter-type anaerobe chamber (Coy Laboratory Products, Inc., Ann Arbor, Michigan, USA). It was equipped with copper tubing leading to gas mixtures or to a vacuum pump. The oxygen concentration inside the chamber was continously monitored with an oxygen analyser (K.Gerhard, Blankenbach , Germany). It remained between 2 and 4 ppm. The cycles of gas exchange in the entry lock of the anaerobe chamber and in the gassing manifold were electronically programmed. Gas mixtures used for gassing of sterile stoppered serum flasks were passed through autoclavable teflon filters (pore size 0.45 pm). The H . /CO. gas mixture (80% /20%) and the N 2 /C0 2 gas mixture (80% /20%) were obtained from the manufacturer with less than 2 ppm O 2, The N 2 /H 2 gas mixture (92%/8%) used for the anaerobe chamber was of technical grade. Before use the hydrogen-containing gas mixtures were passed through a column filled with palladium catalyst RO-20 (BASF, Ludwigshafen, Germany) . The N. /C0 2 gas mixture was passed through an Oxisorb Cartridge (MesserGriessheim, Diisseldorf, Germany ). Media: The methanogens used in this study were cultured as described by Balch et a!. (1979). The growth medium was medium 1 of Balch et a!. (1979) prepared with a trace minerals solution containing 0.1 gIl of NiCl 2 (Schonheit et a!., 1979) and 0.1 gIl of Na2Se03 (Jones and Stadtman, 1981) in addition to the components listed. For the cultivation of Methanococcus vannielii and Methanococcus voltae 18 gIl of NaCl were added to the medium. The reducing agents and solid NaHC0 3 were added inside the anaerobe chamber after the medium had been boiled under the N 2 /C0 2 gas mixture . Growth in liquid media: The bacteria were grown in 120 ml serum flasks containing 25 ml of medium. The bottles were incubated at 37 °C (55 °C for Metbanobacterium thermoautotrophicum) on a rotary shaker at 60 rpm. Growth was followed by measuring the absorbance at 546 nm (1 ern light path) in an Eppendorf phorometer. Total cell counts were determined by use of a Petroff-Hausser counting chamber. For each determination the chamber was filled twice. Cells occurring in pairs, chains or packets were counted as a single cel!. The number of viable cells was determined by plating appropriate dilutions of the cultures in soft agar as described below. The dilutions were prepared in growth medium whose pH had been adjusted to 7.0 with 4 M HC!. Growth on solid media: 16 gIl of standard bacteriological agar were added to solidify the media described above. Plastic petri dishes were filled inside the anaerobe chamber with hot agar-medium. To maintain the correct pH the solidified agar plates were stored in pressure cylinders containing the H. /C0 2 gas mixture at 2-2.5 bar. The soft agar-overlay method proved advantageous for obtaining discrete single colonies of methanogenic bacteria. Soft agar was prepared by the addition of 7 g/I of agar to the growth medium. After autoclaving, 50 ml portions of melted agar were transferred inside the anaerobe chamber to screw cap bottles . These were stored at 45 °C in a block heater inside the chamber. Plating was done by pipetting 2.2 ml of soft agar and 0.1 ml of appropriately diluted cell suspensions into a prewarmed test tube and immediately pouring this mixture on an agar plate. After inoculation the plates were filled into pressure cylinders (up to 16 plates per cylinder). The cylinders were pressurized to 2.5 bar with the H 2 /CO. gas mixture and incubated at 37 °C in the anaerobe chamber except for the cylinders containing plates with Mb. thermoautotrophicum which were incubated outside the chamber at 55°C. During incubation each cylinder contained a few pellets of palladium catalyst and a small amount of anhydrous CaCI 2. Incubation lasted 5-7 days in the case of Mb. thermo-

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autotrophieum, Me. vannielii and Me. voltae, and 10-14 days for Mbr. arboriphilus and Ms. barkeri. Determination of methane: The methane content of culture headspaces was determined by gas-chromatography as described by Daughton et ai. (1979). Buffers: Nonreduced oxygen-free buffer contained in g/I: NaHCO., 5; K2HPO" 0.3; KH 2PO" 0.3; (NH')2 S0" 0.3; NaCI, 4.5; MgSO,' 7H 20, 0.13; CaCI2· 2H 20, 0.008. The bufferfor methanococci was supplemented with 18gil of NaCI. For preparing the buffer, the mineral solution without NaHCO, was boiled for 30 min while being continuously gassed by the N 2/C02 gas mixture. After boiling the flask was stoppered, cooled and transferred to the anaerobe chamber where solid NaHCO, was added. 9 ml portions of buffer were then distributed into 60 ml serum flasks. After sealing, the gas phase of the flasks was exchanged for the N 2/C0 2 atmosphere at 3 bar by use of a gassing manifold. The serum flasks were autoclaved, brought back into the chamber and stored after reducing the pressure to 1 bar. Aerobic buffer contained the same components as the nonreduced oxygen-free buffer. To saturate it with air it was stirred vigorously for 30 min in room atmosphere. Serum flasks (60 ml) were filled with 9 ml of buffer, sealed, autoclaved and stored outside the anaerobe chamber. Determination of oxygen sensitivity: All manipulations relating to the the determination of oxygen sensitivity were carried out in the anaerobe chamber. Bacterial cells, harvested in the late exponential growth phase, were washed twice with nonreduced oxygen-free buffer by use of a table centrifuge. 10 to 15 ml of washed cell suspension were transferred with a syringe to a stoppered serum flask whose atmosphere was subsequently exchanged for the N 2/C0 2 gas mixture at 1 bar. The bacterial cells were then allowed to equilibrate with the nonreduced environment during one hour of incubation at 37°C or at 55 °C for Mb. thermoautotrophieum. After this period portions of the cell suspension were transferred with a syringe to a series of control flasks containing nonreduced oxygen-free buffer under the N 2/C0 2 atmosphere and to a series of test flasks containing aerobic buffer and air in the gas phase. The A546 of the cell suspension in the test and control flasks was 0.1 except in the case of Ms. barkeri. After incubation for various periods at 3rC or 55 DC flasks were cooled to room temperature. Before determining the number of viable cells in the suspensions exposed to oxygen and the respective controls, the headspace volumes of test and control flasks were exchanged for the N 2/C0 2 gas mixture by three cycles of 15 s of evacuation and 3 s of gassing each.

Results

Plating efficiencies of methanogenic bacteria To assess the oxygen sensitivity of methanogenic bacteria quantitatively, it was essential to dispose of a method for the determination of the number of viable cells. The reliability of the method of plating described in "Materials and Methods" was evaluated by following the number of vaible cells and the total number of cells, i. e. the efficiency of plating, in growing cultures. In the same cultures methane formation and A546 were recorded. As shown by the data presented in Figs. 1 and 2, there was satisfactory agreement between these four parameters. Depending on the growth phase, the plating efficiencies varied between 65% and 95% for Mb. thermoautotrophicum, 50% and 130% for Mbr. arboriphilus and 80% and 110% for Me. vannielii. Plating efficiencies above 100% probably resulted from pairs of cells being considered as single cells in the total count, their members, however, being separated during plating for viable counts. This view is in accordance with the observation

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that the highest plating efficiencies were recorded during the exponential growth phase where the proportion of pairs of cells was highest. Plating efficiencies of Me. voltae and Ms. barkeri were above 80% as determined by plating single samples of exponentially growing cultures.

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With the overlay method of plating smearing of colonies by condensing water was avoided and the bacterial cells embedded in the agar-layer were protected against traces of oxygen during incubation. Since the agar-layer hindered the diffusion of gases, cells at the bottom of the soft agar overlay formed smaller colonies than cells growing just beneath the surface of the soft agar. Oxygen sensitivity

The toxicity of molecular oxygen to obligate anaerobic bacteria may be due to oxygen acting as an oxidant and/or to toxic products arising from the interaction of oxygen with components of the medium or the cells (Morris, 1976). Since it is difficult to discriminate experimentally between these principal possibilities, the molecular basis of oxygen toxicity is a matter of controversy (Fee, 1982; Halliwell, 1982). In the protocol for testing the oxygen sensitivity of methanogenic bacteria employed in the present study, washed cells were subjected to a one-hour starvation period in the N 2/C02 atmosphere before exposure to air. The starvation step led to a decrease of metabolic activity which may have reduced the cellular formation of toxic superoxide and hydroxyl radicals (Rowley and Halliwell, 1982) upon exposure to air. Since methanogens, in their natural environments, may contact air but not pure oxygen, the survival of five strains in air-saturated buffer was determined. As evident from Fig. 3 a the death curves of Mb. thermoautotrophicum, Mbr. arboriphilus and Ms. barkeri were biphasic. For these strains exposure to air

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Fig. 3. Oxygen sensitivity of methanogenic bacteria. Anaerobically harvested cells were washed in nonreduced oxygen-free buffer, starved for 1 h and either exposed to air (A) or, as a control, to 80% N 2 - 20% CO 2 (B). For details see Materials and Methods. At various intervals flasks were removed and used for the determination of the number of viable cells. The values presented are the arithmetic means of two platings with a mean deviation of less than ± 6%. (e) Ms. barkeri; (.) Mbr. arboriphilus; (0) Mb. thermoautotrophieum; (0) Me. vannielii; (.) Me. voltae.

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for 10 to 30 h was without effect on the number of colony forming units. Up to a critical time of contact, oxygen apparently caused no or only reversible damage in these organisms. For the two methanococci tested the number of surviving cells upon exposure to air dropped exponentially without lag (Fig. 3 a). Fig. 3 b shows the results of control experiments in which the bacterial cells were subjected to all manipulations of the oxygen sensitivity test except that the flasks contained nonreduced oxygen-free buffer and the N 2/C02 atmosphere. While the number of viable cells of Me. voltae decreased exponentially during incubation in nonreduced oxygen-free buffer, the viability of the other strains was apparently not affected by incubation under the conditions of the control experiment. Since Me. voltae survived well in reduced (0.5 gil Na2S . 9H 20 and 0.5 gil cysteine' Hel) oxygen-free buffer, the decrease in its viability observed in the experiment described in Fig. 3 may be due to traces of oxygen present in the nonreduced oxygen-free buffer. Since Ms. barkeri grows in cell packets, the number of colonies of this organism on plates does not correlate with the number of viable cells in suspension, and the survival curves of Methanosareina presented in Figs. 3 a and 3 b have to be interpreted with caution. The colony size of Ms. barkeri depended on the time these bacteria were incubated under control conditions in nonreduced oxygen-free buffer or under test conditions with exposure to air before plating. As shown in Fig. 4, a cell suspension which was exposed neither to control - nor to test-conditions yielded large colonies upon plating. The same suspension, after preincubation with exposure to air (or after pre-incubation under control conditions), led to approxi-

Fig. 4. Effect of exposure to oxygen on the colony size of Ms. barkeri. Anaerobically harvested cells were washed in nonreduced oxygen-free buffer, starved for 1 h and either plated immediately (left) or plated after 29 h of exposure to air (right). Both agar plates were prepared in the same batch and were incubated under identical conditions for a period of ten days.

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mately the same number of colonies which, however, were of smaller size. A proportion of the Methanosarcina cells in the packets is thus killed by oxygen or by incubation in nonreduced buffer. We suspect that the adverse effect of nonreduced buffer, as in the case of Me. voltae, is caused by traces of oxygen. The killing may proceed from the outer region of the aggregates sparing those cells which are located in the interior of the packets until last. As the duration of the toxic actions increases, the number of surviving cells per packet decreases leading to colonies originating from a progressively smaller number of cells and therefore of smaller size after a given incubation time. To determine the effect of the test conditions on the observed survival of the strains, a similar experiment as described in Fig. 3 was performed using a different protocol. The starvation step was omitted and the washed cells were transferred into flasks containing pure oxygen at 1 bar. While the relative sensitivities of the five strains tested were the same as in the standard tests, the death rates increased about lO-fold. Mb. thermoautotrophieum and Mbr. arboripbilus exhibited 1 hand 3 h of lag, respectively, before the onset of the killing effect of oxygen. Discussion Five different strains of methanogenic bacteria which included representatives of the three orders in the taxonomic scheme of Balch et al. (1979) exhibited marked differences in oxygen tolerance (Fig. 3). Most striking was the ability of Mb. thermoautotrophicum, Mbr. arboripbilus and Ms. barkeri to survive for hours in the presence of air without decrease in the number of colony forming units. These strains were originally isolated from sludge digesters, i.e. ecosystems which are periodically subjected to oxygen stress and therefore may favour the development of air-tolerant methanogenic bacteria. In contrast to these comparatively robust strains, Me. uannielii and probably also Me. voltae were highly sensitive to oxygen, being killed without lag upon contact with air. Sea and lake sediments, the natural habitats of methanococci , are not exposed to oxygen thereby providing a safe environment for highly oxygen sensitive methanogens. Protection against oxygen in the three methanogens exhibiting some tolerance to oxygen may be afforded by different mechanisms. For Mb. thermoautotrophicum and Mbr. arboriphilus protection must occur at the level of the individual cell. Whether superoxide dismutase activity plays a role in the limited oxygen tolerance of these strains remains to be investigated. Superoxide dismutase has been detected in several methanogenic bacteria belonging to the orders Methanomicrobiales and Methanobaeteriales (Kirby, 1981; Kirby ct aI., 1981), whereas there are no reports available on the presence of this enzyme in the highly oxygen sensitive mcthanococci which belong to the order Methanoeoceales. In the case of Ms. barheri protection against oxygen occurs at the level of cell aggregates. The experiment illustrated by Fig. 4 suggests that individual cells of this organisms are also highly sensitive to oxygen. The arrangement of the cells in packets probably leads to the protection of the cells in the interior and thereby secures the survival of a cell packet as a colony forming unit during extended periods of oxygen stress. Attempts to extrapolate from the oxygen sensitivity of methanogens determined in this study to events in natural environments will have to consider that exposure to air in nature occurs under conditions which are less stringent than in the labor a21 Systematic and Applied Microbiology, Vol. 4

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tory. In their natural environments, methanogenic bacteria are associated with particles of organic material and with facultatively anaerobic bacteria providing protection against oxygen. The hydrogen partial pressure and the concentration of the reducing agent sulfide in natural environments have been determined at 10- 6 to 10-3 bar (Zeikus, 1977; Thauer et al., 1977) and 10- 4 M (Hungate, 1966), respectively. They are thus much lower than in the laboratory media routinely employed. Since hydrogen and reducing agents facilitate the formation of free radicals upon contact with oxygen, the conditions in nature are probably more favorable than those in the laboratory for the survival of methanogens when they are accidentally exposed to air. Acknowledgements. We wish to thank Dr. H.Hippe and Dr. A.Zehnder for advice and introductions into anaerobe techniques. This work was supported by the Swiss National Foundation for Scientific Research (project No. 3.691.80).

References Balch, W.E., Fox, G.E., Magrum, L.J., Woese, C.R., Wolfe, R.S.: Methanogens: Reevaluation of a unique biological group. Microbiol. Rev. 43, 260-296 (1979) Daughton, C. G., Cook, A. M., Alexander, M.: Bacterial conversion of alkylphosphonates to natural products via carbon-phosphorus bond cleavage. J. Agricult. Food Chern. 27, 1375-1382 (1979) Fee, J. A.: Is superoxide important in oxygen poisoning? Trends Biochem. Sci. 7, 84-86 (1982) Halliwell, B.: Superoxide and superoxide-dependent formation of hydroxyl radicals are important in oxygen toxicity. Trends Biochem. Sci. 7, 270-272 (1982) Hungate, R. E.: The rumen and its microbes. New York, Academic Press 1966 Jones, ]. B., Stadtman, T. C.: Selenium-dependent and selenium-independent formate dehydrogenase of Methanococcus vannielii. J. BioI. Chern. 256, 656-663 (1981) Kirby, T. W.: Superoxide dismutase in methanogens. Fed. Proc. 40, 1666-1666 (1981) Kirby, T. W., Lancaster, ].R., Fridovich, I.: Isolation and characterization of the ironcontaining superoxide dismutase of Methanobacterium bryantii. Arch. Biochem. Biophys. 210, 140-148 (1981) Morris, J. G.: Oxygen and the obligate anaerobe. J. appl. Bact. 40, 229-244 (1976) Rowley, D. A., Halliwell, B.: Superoxide-dcpendent formation of hydroxyl radicals from NADH and NADPH in presence of iron salts.FEBS Lett. 142,39-41 (1982) Schonheit, P., Moll, J., Thauer, R.K.: Nickel, cobalt, and molybdenum requirement for growth of Methanobacterium thermoautotrophicum. Arch. Microbiol. 123, 105-107 (1979) Smith, P.H., Hungate, R.E.: Isolation and characterization of Methanobacterium ruminantium n. sp. J. Bact. 75, 713-718 (1958) Thauer, R. K., Jungermann, K., Decker, K.: Energy conversion in chemotrophic anaerobic bacteria. Bact. Rev. 41, 100-180 (1977) Zehnder, A.]. B.: Ecology of methane formation. In: Water Pollution Microbiology (R. Mitchell, ed.), vol. 2. New York, John Wiley and Sons 1978 Zehnder, A.J.B., Wuhrmann, K.: Physiology of a Methanobacterium strain AZ. Arch. Microbiol. 111, 199-205 (1977) Zeikus, J. G.: The biology of methanogenic bacteria. Bact. Rev. 41, 514-541 (1977) Professor Dr. Thomas Leisinger, Mikrobiologisches Institut ETH, ETH-Zentrum, Weinbergstr. 38, CH-8092 Zurich