The methanol dehydrogenase gene, mxaF, as a functional and phylogenetic marker for proteobacterial methanotrophs in natural environments.

The mxaF gene, coding for the large (α) subunit of methanol dehydrogenase, is highly conserved among distantly related methylotrophic species in the Alpha-, Beta- and Gammaproteobacteria. It is ubiquitous in methanotrophs, in contrast to other methanotroph-specific genes such as the pmoA and mmoX genes, which are absent in some methanotrophic proteobacterial genera. This study examined the potential for using the mxaF gene as a functional and phylogenetic marker for methanotrophs. mxaF and 16S rRNA gene phylogenies were constructed based on over 100 database sequences of known proteobacterial methanotrophs and other methylotrophs to assess their evolutionary histories. Topology tests revealed that mxaF and 16S rDNA genes of methanotrophs do not show congruent evolutionary histories, with incongruencies in methanotrophic taxa in the Methylococcaceae, Methylocystaceae, and Beijerinckiacea. However, known methanotrophs generally formed coherent clades based on mxaF gene sequences, allowing for phylogenetic discrimination of major taxa. This feature highlights the mxaF gene's usefulness as a biomarker in studying the molecular diversity of proteobacterial methanotrophs in nature. To verify this, PCR-directed assays targeting this gene were used to detect novel methanotrophs from diverse environments including soil, peatland, hydrothermal vent mussel tissues, and methanotroph isolates. The placement of the majority of environmental mxaF gene sequences in distinct methanotroph-specific clades (Methylocystaceae and Methylococcaceae) detected in this study supports the use of mxaF as a biomarker for methanotrophic proteobacteria.

Molecular characterization of methanotrophic communities in forest soils that consume atmospheric methane.

Methanotroph abundance was analyzed in control and long-term nitrogen-amended pine and hardwood soils using rRNA-targeted quantitative hybridization. Family-specific 16S rRNA and pmoA/amoA genes were analyzed via PCR-directed assays to elucidate methanotrophic bacteria inhabiting soils undergoing atmospheric methane consumption. Quantitative hybridizations suggested methanotrophs related to the family Methylocystaceae were one order of magnitude more abundant than Methyloccocaceae and more sensitive to nitrogen-addition in pine soils. 16S rRNA gene phylotypes related to known Methylocystaceae and acidophilic methanotrophs and pmoA/amoA gene sequences, including three related to the upland soil cluster Alphaproteobacteria (USCalpha) group, were detected across different treatments and soil depths. Our results suggest that methanotrophic members of the Methylocystaceae and Beijerinckiaceae may be the candidates for soil atmospheric methane consumption.

Carbon monoxide uptake kinetics in unamended and long-term nitrogen-amended temperate forest soils.

The effect of nitrogen (N) additions on the dynamics of carbon monoxide consumption in temperate forest soils is poorly understood. We measured soil CO profiles, potential rates of CO consumption and uptake kinetics in temperate hardwood and pine control plots and plots amended with 50 and 150 kg N ha-1 year-1 for more than 15 years. Soil profiles of CO concentrations were above atmospheric levels in the high-N plots of both stands, suggesting that in these forest soils the balance between consumption and production may be shifted so that either production is increased or consumption decreased. Highest rates of CO consumption were measured in the organic horizon and decreased with soil depth. In the N-amended plots, CO consumption increased in all but one soil depth of the hardwood stand, but decreased in all soil depths of the pine stand. CO enzyme affinities increased with soil depth in the control plots. However, enzyme affinities in the most active soil depths (organic and 0-5 cm mineral) decreased in response to low levels of N in both stands. In the high-N plots, affinities dramatically-increased in the hardwood stand, but decreased in the organic horizon and increased slightly in the 0-5 cm mineral soil in the pine stand. These findings indicate that long-term N addition either by fertilization or deposition may alter the size, composition and/or physiology of the community of CO consumers so that their ability to act as a sink for atmospheric CO has changed. This change could have a substantial effect on the lifetime of greenhouse gases such as CH4 and therefore the future of Earth's climate.

Effects of long-term nitrogen fertilization on the uptake kinetics of atmospheric methane in temperate forest soils.

To determine whether repeated, long-term NH(4) (+) fertilization alters the enzymatic function of the atmospheric CH(4) oxidizer community in soil, we examined CH(4) uptake kinetics in temperate pine and hardwood forest soils amended with 150 kg N ha(-1) y(-1) as NH(4)NO(3) for more than a decade. The highest rates of atmospheric CH(4) consumption occurred in the upper 5 cm mineral soil of the control plots. In contrast to the results of several previous studies, surface organic soils in the control plots also exhibited high consumption rates. Fertilization decreased in situ CH(4) consumption in the pine and hardwood sites relative to the control plots by 86% and 49%, respectively. Fertilization increased net N mineralization and relative nitrification rates and decreased CH(4) uptake most dramatically in the organic horizon, which contributed substantially to the overall decrease in field flux rates. In all cases, CH(4) oxidation followed Michaelis-Menten kinetics, with apparent K(m) (K(m(app))) values typical of high-affinity soil CH(4) oxidizers. Both K(m(app)) and V(max(app)) were significantly lower in fertilized soils than in unfertilized soils. The physiology of the methane consumer community in the fertilized soils was distinct from short-term responses to NH(4) (+) addition. Whereas the immediate response to NH(4) (+) was an increase in K(m(app)), resulting from apparent enzymatic substrate competition, the long-term response to fertilization was a community-level shift to a lower K(m(app)), a possible adaptation to diminish the competitiveness of NH(4) (+) for enzyme active sites.

Net nitrogen mineralization and net nitrification rates in soils following deforestation for pasture across the southwestern Brazilian Amazon Basin landscape.

Previous studies of the effect of tropical forest conversion to cattle pasture on soil N dynamics showed that rates of net N mineralization and net nitrification were lower in pastures compared with the original forest. In this study, we sought to determine the generality of these patterns by examining soil inorganic N concentrations, net mineralization and nitrification rates in 6 forests and 11 pastures 3 years old or older on ultisols and oxisols that encompassed a wide variety of soil textures and spanned a 700-km geographical range in the southwestern Brazilian Amazon Basin state of Rondônia. We sampled each site during October-November and April-May. Forest soils had higher extractable NO3--N and total inorganic N concentrations than pasture soils, but substantial NO3--N occurred in both forest and pasture soils. Rates of net N mineralization and net nitrification were higher in forest soils. Greater concentrations of soil organic matter in finer textured soils were associated with greater rates of net N mineralization and net nitrification, but this relationship was true only under native forest vegetation; rates were uniformly low in pastures, regardless of soil type or texture. Net N mineralization and net nitrification rates per unit of total soil organic matter showed no pattern across the different forest sites, suggesting that controls of net N mineralization may be broadly similar across a wide range of soil types. Similar reductions in rates of net N transformations in pastures 3 years old or older across a range of textures on these soils suggest that changes to soil N cycling caused by deforestation for pasture may be Basin-wide in extent. Lower net N mineralization and net nitrification rates in established pastures suggest that annual N losses from largely deforested landscapes may be lower than losses from the original forest. Total ecosystem N losses since deforestation are likely to depend on the balance between lower N loss rates from established pastures and the magnitude and duration of N losses that occur in the years immediately following forest clearing.

Forest- and pasture-derived carbon contributions to carbon stocks and microbial respiration of tropical pasture soils.

The clearing of tropical forest for pasture leads to important changes in soil organic carbon (C) stocks and cycling patterns. We used the naturally occurring distribution of13C in soil organic matter (SOM) to examine the roles of forest- and pasture-derived organic matter in the carbon balance in the soils of 3- to 81-year-old pastures created following deforestation in the western Brazilian Amazon Basin state of Rondônia. Different δ13C values of C3 forest-derived C (-28‰) and C4 pasture-derived C (-13‰) allowed determination of the origin of total soil C and soil respiration. The δ13C of total soil increased steadily across ecosystems from -27.8‰ in the forest to -15.8‰ in the 81-year-old pasture and indicated a replacement of forest-derived C with pasture-derived C. The δ13C of respired CO2 increased more rapidly from -26.5‰ in the forest to -17‰ in the 3- to 13-year-old pastures and indicated a faster shift in the origin of more labile SOM. In 3-year-old pasture, soil C derived from pasture grasses made up 69% of respired C but only 17% of total soil C in the top 10 cm. Soils of pastures 5 years old and older had higher total C stocks to 30 cm than the original forest. This occurred because pasture-derived C in soil organic matter increased more rapidly than forest-derived C was lost. The increase of pasture-derived C in soils of young pastures suggests that C inputs derived from pasture grasses play a critical role in development of soil C stocks in addition to fueling microbial respiration. Management practices that promote high grass production will likely result in greater inputs of grass-derived C to pasture soils and will be important for maintaining tropical pasture soil C stocks.

Soil warming and trace gas fluxes: experimental design and preliminary flux results.

We conducted several experiments to determine a procedure for uniformly warming soil 5° C above ambient using a buried heating cable. These experiments produced a successful design that could: 1) maintain a temperature difference of 5° C over a wide range of environmental conditions; 2) reduce inter-cable temperture variability to ca. 1.5° C; 3) maintain a temperature difference of 5° C near the edges of the plot; and 4) respond rapidly to changes in the environment. In addition, this design required electrical power only 42% of the time. Preliminary measurements indicate that heating increased CO2 emission by a factor of ca. 1.6 and decreased the C concentration in the O soil horizon by as much as 36%. In addition, warming the soil accelerated the emergence and early growth of the wild lily of the valley (Maianthemum canadense Desf.). The relationship between CO2 flux and soil temperature derived from our soil warming experiment was consistent with data from other hardwood forests around the world. Since the other hardwood forests were warmed naturally, it appears that for soil respiration, warming the soil with buried heating cables differs little from natural, aboveground warming. By warming soil beyond the range of natural variability, a multi-site, long-term soil warming experiment may be valuable in helping us understand how ecosystems will respond to global warming.