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Methane-oxidizing bacteria are culturable on 10 to 50% methane in air, but such cultured methanotrophs have not been found to sustain oxidation of atmospheric methane (1.8 parts per million per volume [ppmv]). Sustained oxidation of atmospheric methane in aerobic soils has been explained by the presence of uncultured methanotrophs with high affinities for methane (3, 6), cooxidation by nitrifiers (32), and mixotrophy (16, 22).

The metabolism of methane oxidation is initiated by the methane monooxygenase (MMO) (1). Methane is oxidized by the MMO to methanol with the consumption of NADH + H⁺ or reduced quinoles (20, 37). From methanol, a set of dehydrogenases supply reductive power by the oxidation of methanol, formaldehyde, and formate, producing carbon dioxide in a concerted pathway which offers formaldehyde to be assimilated for biosynthesis. During fortuitous oxidation this pathway is not fully operative beyond the MMO, and reductive power must be supplied endogenously from storage material as PHA (polyhydroxyalkanoates) or exogenously as methane metabolites for the sustained oxidation of various hydrocarbons such as n-alkanes, propylene, or trichloroethylene (5, 8, 10-12, 18, 21, 26, 31, 33). We hypothesized that a low availability of methane would result in a starvation effect comparable to fortuitous oxidation, with a restricted flow of reductive power back to the MMO which at a critical threshold value would bring the MMO to a halt.

Mixotrophy with parallel oxidation of methanol, formaldehyde, or formate liberated during the degradation of organic matter in soil (14, 25, 29) has the potential to feed the MMO with reductive power and sustain its methane oxidation. In this study, we tested whether addition of methanol or formate would sustain the oxidation of atmospheric methane by starved methanotrophs.

Soil collection. The soil used in experiments with indigenous methane-oxidizing bacteria was collected at different dates in 1995 and 1996 from 5 to 10 cm below the litter layer in a stand of 33- to 34-year-old Picea abies trees 35 km north of Copenhagen, Denmark. The soil was sieved (2-mm mesh) and stored at 5°C in the dark. Enrichment cultures of methanotrophic bacteria were obtained from five different agricultural soils collected from 0- to 10-cm depths at different dates in 1995. Two soils were taken from near the Agricultural University of Norway (K2 and H1), one from about 130 km north of Oslo (Ap), and two from Stend in western Norway, near Bergen (Kr and St) (Table 1).

Cultivation of methane oxidizers. Enrichment cultures of methane oxidizers were made by adding 5 g of soil to 10 ml of NMS (a modified version of the mineral medium used by Whittenbury et al. [34]: MgSO₄ · 7H₂O [1.0 g liter¹], CaCl₂ · 2H₂O [134.0 mg liter¹], EDTA ferric monosodium salt [4.0 mg liter¹], KNO₃ [1.0 g liter¹] [stock solution adjusted to pH 6.8], trace element solution [0.5 ml], KH₂PO₄ [272.0 mg liter¹], and Na₂HPO₄ · 2H₂O [356.0 mg liter¹] [this P buffer was adjusted separately to pH 6.8 before addition] [trace element solution contained EDTA disodium salt {500.0 mg liter¹}, FeSO₄ · 7H₂O {200.0 mg liter¹}, ZnSO₄ · 7H₂O {10.0 mg liter¹}, MnCl₂ · 4H₂O {3.0 mg liter¹}, H₃BO₃ {30.0 mg liter¹}, CoCl₂ · 6H₂O {20.0 mg liter¹}, CaCl₂ · 2H₂O {1.0 mg liter¹}, NiCl₂ · 6H₂O {2.0 mg liter¹}, and Na₂MoO₄ · 2H₂O {3.0 mg liter¹}]) in 120-ml serum flasks, which were capped with butyl rubber stoppers and supplied with a mixture of 17% CH₄ and 1.7% CO₂. The flasks were incubated at 30°C with shaking at 170 rpm in a Maxi-Shake (Heto Holten AS, Allerød, Denmark). Subculturing was done regularly as the cultures became visibly turbid. These cultures were, early in their turbidity, used for methane oxidation experiments.

Extraction of indigenous soil bacteria. Indigenous methane oxidizers were extracted and separated from the soil particles by the density gradient centrifugation method described by Priemé et al. (24). To prevent growth of protozoa, we added cycloheximide to a final concentration of 100 mg liter¹. The effect of cycloheximide on methane oxidation was found to be tolerable (up to 13,000 mg of cycloheximide liter¹ added to soil slurries produced <15% inhibition of methane oxidation activity [data not shown]).

Gas chromatography. Methane oxidation rates were determined by flame ionization gas chromatography of headspace samples collected from serum flasks closed with butyl rubber septa. Methanotrophic enrichment cultures in flasks were sampled for analysis of 1 ml of gas (13). Slurries or extracts of indigenous bacteria in 120-ml flasks were sampled likewise for analysis of 3 ml of gas (23).

Atmospheric methane oxidation in the presence of methanol or formate. In experiments with diluted methane enrichments, 10-ml cultures were incubated with laboratory air in 120-ml capped serum flasks incubated in the Maxi-Shake at 170 rpm and 30°C. Pulses (0.1 ml) of methanol (high-performance liquid chromatography grade; Rathburn, Walkerburn, United Kingdom) were added from a 1-ml syringe fixed by the flask septum. In a prolonged starvation experiment in which 60 ml of St culture (1.2 × 10⁸ cells ml¹) was incubated at room temperature with magnetic stirring (Variomag, Munich, Germany), intermittent flask closures for periods of 10 to 20 h allowed monitoring of the effect of methanol on atmospheric methane oxidation. In a follow-up experiment with 30 ml of St culture (6.0 × 10⁷ cells ml¹) in 60-ml serum flasks, methanol or formate was added after the atmospheric methane oxidation had declined to very low levels.

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Cell counts. Total cell counts were determined in a Helber counting chamber (depth, 0.02 mm; 1/400 mm³ [Mellige]). Viable methanotrophs were estimated as most probable numbers on microtiter plates (35). Serial dilutions in NMS (1:10 per step) were incubated at 30°C in 2% CO₂- and 20% CH₄-enriched air. A well with visible turbidity not seen in the control plate incubated without added CH₄ after 3 weeks was considered a positive well.

Data analysis. Atmospheric methane oxidation rates were calculated according to first-order kinetics (dC/dt = Ck, where C is the methane concentration and k is the rate constant) by estimating k through linear regression of ln (C) against time (t). The regression coefficient (k) estimates the methane oxidation in parts per million per volume per hour at 1 ppmv in the gas phase. This was further transformed to nanograms of CH₄ per milliliter or per gram (dry weight) of soil, taking into account the molecular weight of methane, the headspace volume, the liquid volume, and the solubility of methane in water (36). The reported rates are thus estimates of the methane oxidation at 1 ppmv in the gas phase; comparison with ambient in situ flux measurements implies multiplication by 1.8.

Atmospheric methane oxidation by cultured methanotrophs. The mixotrophic oxidation of atmospheric methane was investigated in methanotrophic enrichment cultures from five agricultural soils. Diluted cultures exposed to air and supplemented with methanol grew and initiated oxidation of atmospheric methane after 40 to 70 h; maximum oxidation occurred in the presence of 0.5 to 5 mM methanol (data not shown). Undiluted cultures exposed to air maintained high levels of atmospheric methane oxidation during an initial starvation period of about 0 to 90 h, at which point PHA or a similar reserve material was visible by phase contrast microscopy as large refractive bodies in the cells. A decline in oxidation was quickly restored by addition of methanol, as seen for the St culture containing a minimum of 2.0 × 10⁶ methanotrophs ml¹ (Fig. 1). Prolonged exposure to air did not hinder the ability of methanol or formate to restore the oxidation of atmospheric methane. As shown in Fig. 2, a threshold value below which methane oxidation was halted was not observed when the starved methanotrophs were supplemented with methanol or formate.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Oxidation of atmospheric methane by the St culture. At 0 h, laboratory air replaced the 17% CH4 and 1.7% CO2 growth condition. Single pulses of methanol were added after 20.1 h (833 µM final concentration) and after 139.3 h (83.3 µM final concentration), as indicated by the arrows. Note the logarithmic vertical axis.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Oxidation of atmospheric methane in the St culture after 10 days of exposure to laboratory air. Equal volumes of CH3OH, NaCOOH (166.7 µM pulses), or water were added as indicated by the arrows. Uptake rates are denoted by k. Note the logarithmic vertical axis (k of 1 equals 30 nl of CH4 oxidized h1 at 1 ppmv of CH4).

Atmospheric methane oxidation by indigenous methane oxidizers. Methanol was also explored as a cosubstrate for the oxidation of atmospheric methane by indigenous forest soil bacteria. Addition of 0 to 5 mM methanol in daily to weekly doses for up to 7 months did not enhance the atmospheric methane oxidation by indigenous soil bacteria, either in soil slurries or in suspensions of extracted bacteria (data not shown). Also, we did not observe any immediate stimulation of oxidation following the addition of methanol to methane-starved slurries or suspensions (data not shown). On the contrary, the oxidation of atmospheric methane in slurries or suspensions was inhibited by the addition of methanol (Fig. 3). However, the inhibitory effect of methanol was transient. The recovery period depended on the initial concentration of added methanol; for example, at 8.8 mM CH₃OH, methane oxidation rates recovered completely within 8 days (data not shown). The CO₂ evolution was monitored during a similar experiment by which methanol depletion could be quantified as the difference in accumulated CO₂ between amended and unamended slurries. We found that the recovery of methane oxidation coincided with the exhaustion of the methanol. In soil slurries with no methanol addition, methane starvation caused a decrease in methane oxidation compared to slurries kept at 1.8 ppmv of methane (Fig. 4).

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Oxidation of atmospheric methane in soil slurries () and suspensions () of extracted soil bacteria. The curves represent fits to sigmoid functions. Error bars correspond to ±1 standard error (n = 3). Note the logarithmic horizontal axis.

View larger version (29K): [in this window] [in a new window]   FIG. 4.   Oxidation of atmospheric methane by soil slurries supplemented with daily doses of methanol for 17 days and supplied with atmospheric methane (solid bars) or deprived of methane (open bars). A recovery period of 4 days followed the last methanol dose before methane oxidation rates were measured. The addition of 32.1 mg of CH3OH g1 day1 corresponds to an initial CH3OH concentration of 0.44 mM. Error bars correspond to 1 standard error (n = 3).

Conclusions. Many characteristics of in situ oxidation of methane by soils have been reproduced by cultured methanotrophs (2, 9, 17). However, cultured methanotrophs do not sustain methane oxidation during prolonged exposure to atmospheric methane concentrations (28, 30). This has been an important contribution to the assumption that as-yet-uncultured methanotrophs with superior affinities for methane are the dominant oxidizers of atmospheric methane in aerobic soils (3, 6, 7, 9, 15, 17, 19, 27). However, the present investigation demonstrates that culturable methanotrophs sustain high rates of atmospheric methane oxidation if they are supplied with methanol or formate.