Gary M. King

Ecological Aspects of Methane Oxidation, a Key Determinant of Global Methane Dynamics

Methane oxidation became a subject of scientific inquiry when Alessandro Volta observed in 1776 that gas bubbles collected from a pond were combustible. Methane was subsequently exploited as a source of heat and light. However, in spite of its commercial significance, the biological and ecological aspects of methane oxidation were largely ignored until the pioneering work of Sohngen (1906), who first isolated methane-oxidizing bacteria (MOB). [Quayle (1987) notes that Lowe probably isolated the first MOB in 1892 without recognizing their ability to oxidize methane.] Little additional progress was made until the 1960s, at which time the systematic efforts of several groups provided methodological tools and details on the taxonomy, physiology, and biochemistry of C1 metabolism. Aside from purely academic motivations, this work was stimulated by: (1) the potential use of methanotrophic bacteria as sources of “single cell protein”; (2) the role of methylotrophic bacteria in food spoilage; (3) the possible use of methanotrophs in the bioremediation of certain halogenated organic pollutants or as agents for commercial biotransformations (Higgins et al., 1980). Ecological studies were slower in development, but a number of important observations established the ubiquity of methanotrophs, the impact of methane oxidation in freshwater and some marine systems, and the potential for anaerobic as well as aerobic methane oxidation (see Hanson, 1980, and Rudd and Taylor, 1980, for earlier reviews).

Distribution, diversity and ecology of aerobic CO-oxidizing bacteria.

Numerous studies indicate that carbon monoxide (CO) participates in a broader range of processes than any other single molecule, ranging from subcellular to planetary scales. Despite its toxicity to many organisms, a diverse group of bacteria that span multiple phylogenetic lineages metabolize CO. These bacteria are globally distributed and include pathogens, plant symbionts and biogeochemically important lineages in soils and the oceans. New molecular and isolation techniques, as well as genome sequencing, have greatly expanded our knowledge of the diversity of CO oxidizers. Here, we present a newly emerging picture of the distribution, diversity and ecology of aerobic CO-oxidizing bacteria.

Methane release from soils of a Georgia salt marsh

Abstract A seasonal study of methane release from marsh soils to the atmosphere indicates that ebullition is a significant process varying both seasonally and spatially. Release rates are higher during summer months than winter months and ebullition is greatest in the short Spartina alterniflora marshes and least in the tall S. alterniflora marshes. The annual amounts of methane released in the short and tall marshes are 53.1 and 0.4 gm −3 which represents a loss of 8.8 and 0.002% of the net carbon fixation in the two respective marsh types. In vitro experimentation shows that methane production is sensitive to changes in temperature and addition of H 2 and CO 2 .

PANGEA: pipeline for analysis of next generation amplicons

High-throughput DNA sequencing can identify organisms and describe population structures in many environmental and clinical samples. Current technologies generate millions of reads in a single run, requiring extensive computational strategies to organize, analyze and interpret those sequences. A series of bioinformatics tools for high-throughput sequencing analysis, including pre-processing, clustering, database matching and classification, have been compiled into a pipeline called PANGEA. The PANGEA pipeline was written in Perl and can be run on Mac OSX, Windows or Linux. With PANGEA, sequences obtained directly from the sequencer can be processed quickly to provide the files needed for sequence identification by BLAST and for comparison of microbial communities. Two different sets of bacterial 16S rRNA sequences were used to show the efficiency of this workflow. The first set of 16S rRNA sequences is derived from various soils from Hawaii Volcanoes National Park. The second set is derived from stool samples collected from diabetes-resistant and diabetes-prone rats. The workflow described here allows the investigator to quickly assess libraries of sequences on personal computers with customized databases. PANGEA is provided for users as individual scripts for each step in the process or as a single script where all processes, except the χ2 step, are joined into one program called the ‘backbone’.

Molecular and Culture-Based Analyses of Aerobic Carbon Monoxide Oxidizer Diversity†

ABSTRACT Isolates belonging to six genera not previously known to oxidize CO were obtained from enrichments with aquatic and terrestrial plants. DNA from these and other isolates was used in PCR assays of the gene for the large subunit of carbon monoxide dehydrogenase (coxL). CoxL and putative coxL fragments were amplified from known CO oxidizers (e.g., Oligotropha carboxidovorans and Bradyrhizobium japonicum), from novel CO-oxidizing isolates (e.g., Aminobacter sp. strain COX, Burkholderia sp. strain LUP, Mesorhizobium sp. strain NMB1, Stappia strains M4 and M8, Stenotrophomonas sp. strain LUP, and Xanthobacter sp. strain COX), and from several well-known isolates for which the capacity to oxidize CO is reported here for the first time (e.g., Burkholderia fungorum LB400, Mesorhizobium loti, Stappia stellulata, and Stappia aggregata). PCR products from several taxa, e.g., O. carboxidovorans, B. japonicum, and B. fungorum, yielded sequences with a high degree (>99.6%) of identity to those in GenBank or genome databases. Aligned sequences formed two phylogenetically distinct groups. Group OMP contained sequences from previously known CO oxidizers, including O. carboxidovorans and Pseudomonas thermocarboxydovorans, plus a number of closely related sequences. Group BMS was dominated by putative coxL sequences from genera in the Rhizobiaceae and otherα -Proteobacteria. PCR analyses revealed that many CO oxidizers contained two coxL sequences, one from each group. CO oxidation by M. loti, for which whole-genome sequencing has revealed a single BMS-group putative coxL gene, strongly supports the notion that BMS sequences represent functional CO dehydrogenase proteins that are related to but distinct from previously characterized aerobic CO dehydrogenases.

Analysis of Facultative Lithotroph Distribution and Diversity on Volcanic Deposits by Use of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

ABSTRACT A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass.

Short-term endproducts of sulfate reduction in a salt marsh: Formation of acid volatile sulfides, elemental sulfur, and pyrite☆

Rates of sulfate reduction, oxygen uptake and carbon dioxide production in sediments from a short Spartina alterniflora zone of Great Sippewissett Marsh were measured simultaneously during late summer. Surface sediments (0–2 cm) were dominated by aerobic metabolism which accounted for about 45% of the total carbon dioxide production over 0–15 cm. Rates of sulfate reduction agreed well with rates of total carbon dioxide production below 2 cm depth indicating that sulfate reduction was the primary pathway for sub-surface carbon metabolism. Sulfate reduction rates were determined using a radiotracer technique coupled with a chromous chloride digestion and carbon disulfide extraction of the sediment to determine the extent of formation of radiolabelled elemental sulfur and pyrite during shortterm (48 hr) incubations. In the surface 10 cm of the marsh sediments investigated, about 50% of the reduced radiosulfur was recovered as dissolved or acid volatile sulfides, 37% as carbon disulfide extractable sulfur, and only about 13% was recovered in a fraction operationally defined as pyrite. Correlations between the extent of sulfate depletion in the marsh sediments and the concentrations of dissolved and acid volatile sulfides supported the results of the radiotracer work. Our data suggest that sulfides and elemental sulfur may be major short-term end-products of sulfate reduction in salt marshes.

Molecular Analysis of Carbon Monoxide-Oxidizing Bacteria Associated with Recent Hawaiian Volcanic Deposits

ABSTRACT Genomic DNA extracts from four sites at Kilauea Volcano were used as templates for PCR amplification of the large subunit (coxL) of aerobic carbon monoxide dehydrogenase. The sites included a 42-year-old tephra deposit, a 108-year-old lava flow, a 212-year-old partially vegetated ash-and-tephra deposit, and an approximately 300-year-old forest. PCR primers amplified coxL sequences from the OMP clade of CO oxidizers, which includes isolates such as Oligotropha carboxidovorans, Mycobacterium tuberculosis, and Pseudomonas thermocarboxydovorans. PCR products were used to create clone libraries that provide the first insights into the diversity and phylogenetic affiliations of CO oxidizers in situ. On the basis of phylogenetic and statistical analyses, clone libraries for each site were distinct. Although some clone sequences were similar to coxL sequences from known organisms, many sequences appeared to represent phylogenetic lineages not previously known to harbor CO oxidizers. On the basis of average nucleotide diversity and average pairwise difference, a forested site supported the most diverse CO-oxidizing populations, while an 1894 lava flow supported the least diverse populations. Neither parameter correlated with previous estimates of atmospheric CO uptake rates, but both parameters correlated positively with estimates of microbial biomass and respiration. Collectively, the results indicate that the CO oxidizer functional group associated with recent volcanic deposits of the remote Hawaiian Islands contains substantial and previously unsuspected diversity.

Biodiversity links above and below the marine sediment–water interface that may influence community stability

Linkages across the sediment–water interface (SWI) between biodiversity and community stability appear to exist but are very poorly studied. Processes by which changes in biodiversity could affect stability on the other side of the SWI include carbon transfer during feeding, decomposition of organic matter, nutrient recycling, organism recruitment and structural stabilisation of sediments. The importance of these processes will clearly vary among habitats. Direct disturbance to communities on one side of the SWI, such as created by overfishing, habitat destruction, and species invasions, has the potential to impact communities on the other side of the SWI through the many functional links. Hypotheses are proposed to suggest further areas of research to fill the large gaps in our knowledge concerning the nature and intensity of such linkages. The linkage between benthic and pelagic diversity is likely to be tighter where there is a close energetic connection between the domains, such as polar and shallow coastal waters, and where communities are dominated by selective detritivores. The quantity of carbon reserves in the sediment and the predominant mode of larval development of sediment communities probably influence the stability of below SWI communities in the face of changes in above SWI diversity. The organisms, including hyperbenthos, that are found at the SWI may be of crucial importance to the linkage and stability of above and below SWI communities.

Characteristics and significance of atmospheric carbon monoxide consumption by soils

Abstract CO consumption by soils is an important but uncertain component of atmospheric CO budgets. Global estimates range from 15 to 640 Tg CO yr −1 . A number of such estimates have been used in models of CO dynamics in the atmosphere. Uncertainties in these models and in CO sources readily accommodate a wide range of consumption estimates. A review of empirical evidence suggests that a range of 190–580 Tg yr −1 derived from Seiler (1978) encompasses the likely sink strength, although the lower limit is most probable since CO production in temperate soils and the effects of illumination have not yet been adequately incorporated. Various evidence also indicates that soil water content and organic matter concentrations are major parameters affecting CO exchange, with temperature exerting more modest effects except in arid systems. The microbes active in CO consumption remain unknown, although ammonia- and methane-oxidizing bacteria seem to contribute little.