MICROBIOLOGICAL PREPARATION OF 3H-LABELLED METHANE

Small quantities of tritiated methane were prepared by fermenting [methyl-3H] methylamine hydrochloride using exponential phase, transiently starved, co-cultures ofmethanogenic bacteria. This method gave the expected 75% yield of C3H4 from the substrate and produced H2-free, low 3H20 background C3H4 for tracer studies.


INTRODUCTION
An increase in atmospheric methane concentration has been reported recently (1); methane is a radiatively and chemically active gas and has the potential to modify climate through greenhouse wanning as well as by modifying concentrations of other radiatively active gases through reactions with the OH radical. A search for biological sinks capable of modulating methane emissions has led to increased interest in the microbial ecology, physiology and biochemistry of aerobic methane oxidation as well as continued interest in the controversial and poorly understood phenomenon of anaerobic methane oxidation (2,3). This has produced a need for labelled methane to study methane turnover in a variety of environments.
It is important that the pool size of reactan ts under study not be affected appreciably by added tracer in measurements of in situ rates. Radiocarbon labelled methane (14CH4) can be synthesized with specific activities up to 7.99 x 1012 Bq/mmol (60 mCi/mmol) (4 ) and used to obtain reliable rates of methane oxidation in environments where methane concentration is in the mM range (2). However, in environments such as water columns where the methane is present in µM to nM concentrations, the use of 14CH4 could alter the methane pool size and require additional concentration/rate information . Tritiated methane (C3H4) can be obtained at much higher specific activities (up to 1.15 x 1016 Bq/mmol, 86 Ci/mmol) and is more suitable for experiments in environments with low methane concentration. Geochemical studies on stable C and H isotopes in naturally occurring methane suggest thatH isotope exchange between methane and water does not occur (5). Unfortunately, C3H4 is expensive and is oflimited commercial availability.
A commonly used procedure for the synthesis of 14CH 4 has been previously presented by Daniels and Zeikus (4 ). They suggested that tritiated methane could be produced with this procedure by using tritiated water in the medium. Previous work showed that the hydrogen in methane originates from the aqueous medium, and not from headspace H2 (6,7). This method has been attempted in this laboratory and was successful (unpublished results). Difficulties inherent in this method include: 1) The tritiated methane produced by this method would be contaminated by high levels of 3H20 vapour and 2) large amounts of radioactivity would have to be added to the aqueous medium to obtain C3H4 of high specific activity.
Another method for producing C3H4 similar to the method reported here would employ methanogens that have the ability to produce methane from tritiated acetate.
However, this method could present problems because methanogens that use acetate grow very slowly (Lacey Daniels, personal communication).
A few species of methanogenic bacteria use methanol and methylated amines for gTowth and methanogenesis (8). We describe the preparation of small quantities of high specific activity tritiated methane from [methyl-3H) methylamine hydrochloride using co-cultures of strains of methanogenic ba~te~ ~imilar to Methan-0coccoides methylutens and Methanosarcina bar~ri. .'

MATERIALS AND METHODS .
The two species of methanogenic bacteria used to synthesize tritiated methane have been previously isolated in pure culture (9). Using variations of the Hungate technique (10,11), the same bacteria were obtained in co-culture for this work by serial dilution using anoxic marine sediment as a source of inoculum. Pure mono-cultures were not needed as both strains utilized methylamine to produce methane. Enrichment, isolation in co-cultu re and maintenance of the methanogenic bacteria was carried out in a slightly modified version of medium number 3 of Balch et al. (12). The medium used for this work omitted the trace vitamins and contained only 0.25 g/L yeast extract and, in lieu of trypticase, contained 0.125 g/L of Bacto-Peptone (Difeo). It also contained 0.78 g/L HEPES as a buffering agent. The medium was pre-reduced by boiling under a stream of Oz-free nitrogen and 16 ml was dispensed anaerobically (13) into "Balch" type test tubes (18 x 150 mm) which were sealed with butyl rubber stoppers secured by aluminum crimp seals (Belk o Glass, Vineland, NJ). After autoclaving, tubes were a.mended from sterile, anoxic stock solutions to reach the following final concentrations: a.mpicillin-sodiumsalt, 200 µg/ml; vancomycin, 100 µg/ml; streptomycin-sulfate, 100 µg/ml; sodium bicarbonate, 10 mM; methylamine hydrochloride 0.2% v/v; and sodium sulphide, 0 .03% v/v. All reagents used were analytical grade (Sigma Chemical Co.). The methanogens used to produce CSH4 were grown on a rota~y shaker at room temperature.
Growth of the co-culture was monitored by gas chromatographic analysis of methane in subsamples of the culture heads~~e over time. Successive subculturing revealed that the late exponential growth phase was reached after three days, so co-cultures used later for the production of tritiated methane were harvested after three days of growth and transiently starved. The transient starvation was accomplished by centrifuging the cells and resuspending the pellet in fresh medium containing no methylamine. This medium replacement was done by first flushing the headspace of tubes with a sterile flow of oxygen-free nitrogen using 21 ga gassing and venting needles. The venting needle was positioned near the inside edge of the tube and stopper so that when the tube was inverted the used medium was expelled. Careful handling of the tube ensured that the pellet remained intact. A tube of sterile, fresh, substrate-free medium was then gassed and vented (as above) and connected in series to the tube containing the pellet. By inverting the tube containing the fresh medium, new medium was added to the pellet containing tube.
Two separate co-cultures were starved by the method outlined above and monitored for further methane production. When the rate of methane production approached zero ( Fig. 1), an aliquot of tritiated methyla.mine hydrochloride solution was added by syringe.
The [methyl-3) methylamine hydrochloride, 1.33 x 1012 Bq, lOmCi, (6.39 x 1015 Bq/mmol; 48 Ci/mmol) was obtained commercially (Amersham Corporation, Arlington Heights, IL) and two separate syntheses were performed. The labelled substrate was transferred to a serum vial and made anoxic before addition by repeatedly gassing the headspace with oxygen-free nitrogen. After the· labelled methylamine hydrochloride was added to initiate the second synthesis, the vial that had contained the labelled substrate was rinsed with sterile anoxic distilled water; this rinse was added to the starved culture.
The final liquid volume of both cultures wa. s about 19 ml; the headspace volume was 8 ml.
The cultures were incubated for one day and were checked for the prod'!-ction of C3H4. A sample of the headspace of both cultures was diluted 100-fold and was assayed by the method of Zehnder et al. (14). Biological activity was halted by the addition of 0.2 11. of The headspace of the culture tubes was displaced by addition of anoxic distilled water into a 10-ml Glaspak syringe (Becton-Dickinson) fitted with a Mini-nert valve (Supelco).
The syringe contents were then transferred to evacuated 8-ml serum vials fitted with black butyl rubber and aluir.inum crimp seals. These two vials each contained about 0.5 g of granular P20s C J . T. Baker Chemical Co.) to absorb residual water vapour.
Labelled methane was removed from these vials for field experiments; equal volumes of mercury were added following removal to maintain constant pressure.

RESU LTS AND DISCUSSION
Production of methane from methylamine with a methanogen co-culture should proceed according to the following overall fermentation equation: (1) so that complete reaction should convert 75% of the labelled me thy lamine into C3H4. The remaining label should be present as 3H20 resu lting from oxidation during the fermentation. Later assays of killed controls used in the estimation of both aerobic and anaerobic methane oxidation in the water column of the Black Sea revealed some contamination of the tritiated methane with tritiated water (about 1.22 x 106 Bq/100 µl, 9.18 nCi/100 µI). Table I summarizes the results o_f the syntheses. in fact , the two cultures received di.ITerent starting activities. 2 NA, not appl icable u the tritiated methane from the two s yntheses was not pooled.
The results in Table I show that the synthesis went to completion since the overall expected 75% yield, as demonstrated by the activity of the C3H4 product, was obtained.
The C3H4 specific activity, determined after a series of oxidation rate experiments, was lower than the 4.75 x 1015 BqlnunoH 36 Ci/nunol) expected. Production of unlabelled methane during the synthesis is the only process that can simultaneously account for the correct overall yield and lower specific activity, indicating that methane production from unlabelled substrates remaining after transient starvation must have occurred. Possibly, this can be minimized by longer periods of t ransient starvation. Also, there are indications (unpublished observations) that elimination of the yeast extract from the medium used for transient starvation will further minimize unlabelled methane production.
Losses ofC3H4 due to dissolution in the distilled water used to transfer the methane from the culture vessel to the syringe also occurred; these can be minimized by using a saturated salt solution as the displacing fluid. For most experimental applications of C3H4, it is important to minimize the activity of 3H20, as this is the product of oxidation.
The transfer should be conducted with agitation, allowing time for the 3H20 to equilibrate and be diluted by the medium, reducing its gas phase activity.
Future attempts to produce tritiated methane might also make use of starved, resuspended, frozen cells similar to a procedure previously employed to produce 14CH4 (4 ).
Finally, it should be noted that any methanogens capable of producing methane from methyl groups could also be used to produce 14CH4. C-14 methan e produced by this method would be free of excess hydrogen (4) and would be valuable in experimental situations where added hydrogen is of concern. Cultures used to produce C-14 methane by this procedure would have to be rendered sufficiently alkaline to absorb the 14C02 produced by this process from the headspace gas.