Highaffinity Methane Oxidizers

Bender and Conrad (1992) noted that methane uptake in upland soils displayed a typical hyperbolic Michaelis-Menten response to methane concentration. This is expected for an enzymatic reaction. However, an intriguing feature was that the apparent affinity for methane was several orders of magnitude higher in upland soils (Ks ~10-100 nM) than in pure cultures of methanotrophs and wetland soils (Ks ~1-10 |M) (Fig. 10.2). This was interpreted to mean that the active methano-trophs in upland soils are oligotrophs and possess a high-affinity form of MMO that allows survival on the trace level of atmospheric methane. Energetic calculations support this proposal. Soil microorganisms can grow on atmospheric trace gases only if their half-saturation constants (Ks) are sufficiently low or their enzyme levels (cellular Vmax) are sufficiently high to supply their maintenance energy requirement, which is about 4.5 kJ/(C-mol microbial bio-mass)/h (Tijhuis et al., 1993). Given that the Vmax measured for methanotrophs is about 250 mmol CH4/(C-mol microbial biomass)/ h, a Ks of <110 nM CH4 would be required to allow maintenance on atmospheric methane alone (Conrad, 1999).

The findings of Bender and Conrad (1992) have been widely cited (more than 100 times to date) and the hypothesis that there are high-affinity methanotrophs has fuelled much research. However, there are difficulties with this hypothesis. It assumes that true affinity constants (Ks) are being measured in most experiments but, in fact, what is measured are kinetic constants (Km), which are not necessarily the same as affinity constants. In addition, because concentrations of the other two reactants for MMO (O2 and reductant) may vary, and because there may be diffusion limitation steps, only apparent kinetic coefficients are measured (Km(app)). It is impossible to apply simple Michaelis-Menten kinetics to an enzyme like MMO that has three reactants. The Km(app) for one reactant will change in response to concentrations of the other reactants. Although it seems counterintuitive, a limitation of one substrate (e.g. reductant) may actually cause the Km(app) for another substrate (e.g. methane) to decrease (Dunfield and Conrad, 2000). This is fully consistent with certain kinetic mechanisms for multireactant enzymes (Segel, 1975). Probably due to such an effect, the Km(app) of a Methylocystis strain (LR1) was observed to decrease with starvation (Dunfield et al., 1999; Dunfield and Conrad, 2000). This work did not conclude that a high-affinity enzyme was being expressed in this metha-notroph during starvation, but rather suggested that there may be no such thing as a high-affinity enzyme.

There are therefore two possibilities to explain high-affinity methane oxidation: either this is an inappropriate application of a simple Michaelis-Menten model to a complex system, or there truly is a high-affinity enzyme. A final verdict probably must await isolation of the unknown atmospheric methane oxidizers from soil.

The results of Bender and Conrad (1992) are also often extrapolated too far. 'High-affinity' methane oxidizer is frequently used interchangeably with 'atmospheric' methane oxidizer, a useful generalization in many instances but not necessarily true. A high-affinity kinetic curve will show a zero-order rate at high methane

Fig. 10.2. Summary of reported Km(app) values for methane oxidation measured in various natural environments and in methanotroph cultures. The upper and lower box lines represent the 25th and 75th percentile values (= 50% of all values). The horizontal line in the box represents the 50th percentile (median) and the square symbol represents the mean. Error bars denote the 5th and 95th percentiles and asterisks indicate the upper and lower limits. (Data from articles cited in Knief et al., 2005b. Figure from Knief et al., 2005b. With permission from Blackwell Publishing.)

Fig. 10.2. Summary of reported Km(app) values for methane oxidation measured in various natural environments and in methanotroph cultures. The upper and lower box lines represent the 25th and 75th percentile values (= 50% of all values). The horizontal line in the box represents the 50th percentile (median) and the square symbol represents the mean. Error bars denote the 5th and 95th percentiles and asterisks indicate the upper and lower limits. (Data from articles cited in Knief et al., 2005b. Figure from Knief et al., 2005b. With permission from Blackwell Publishing.)

concentrations, and a low-affinity curve will show a first-order rate at low methane concentrations. Most low-affinity methano-trophs can oxidize atmospheric methane (Knief and Dunfield, 2005), provided they have a supply of reductant, the key co-substrate for MMO. It is not always correct to assume that high-affinity activity is responsible for methane oxidation at 1.7 ppmv and low-affinity activity is responsible for methane oxidation at higher mixing ratios. This false assumption has sometimes been used even when the relevant experimental data show a single hyperbolic curve in a soil, without any low-affinity activity (e.g. Bull et al., 2000). Concurrent low-affinity and high-affinity activity has only been observed in soils after long preincubation under high (>10% v/v) methane mixing ratios (Bender and Conrad, 1992), and in many soils a low-affinity activity cannot be induced at all (Bradford et al., 2001a; Reay and Nedwell, 2004).

The difference between high-affinity and low-affinity oxidation is also not clearly delineated. Forest soils oxidizing atmospheric methane show the lowest measured ^m(app) values. Some other soils that oxidize atmospheric methane display an 'intermediate-affinity', with -Km(app) values higher than in forests, but lower than in wetlands (Fig. 10.2). It is difficult to draw a sharp dividing line between high-affinity and low-affinity oxidation because the measured values span such a broad range. There may be many different variants of MMO with different kinetic properties. We do know that the soluble form of MMO has a lower affinity than the particulate form (Hanson and Hanson, 1996). Alternatively, the broad range may only reflect the difficulties described above in measuring apparent kinetic constants.

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