Pathways of Carbon Assimilation and Ammonia Oxidation Suggested by Environmental Genomic Analyses of Marine Crenarchaeota

Marine Crenarchaeota represent an abundant component of oceanic microbiota with potential to significantly influence biogeochemical cycling in marine ecosystems. Prior studies using specific archaeal lipid biomarkers and isotopic analyses indicated that planktonic Crenarchaeota have the capacity for autotrophic growth, and more recent cultivation studies support an ammonia-based chemolithoautotrophic energy metabolism. We report here analysis of fosmid sequences derived from the uncultivated marine crenarchaeote, Cenarchaeum symbiosum, focused on the reconstruction of carbon and energy metabolism. Genes predicted to encode multiple components of a modified 3-hydroxypropionate cycle of autotrophic carbon assimilation were identified, consistent with utilization of carbon dioxide as a carbon source. Additionally, genes predicted to encode a near complete oxidative tricarboxylic acid cycle were also identified, consistent with the consumption of organic carbon and in the production of intermediates for amino acid and cofactor biosynthesis. Therefore, C. symbiosum has the potential to function either as a strict autotroph, or as a mixotroph utilizing both carbon dioxide and organic material as carbon sources. From the standpoint of energy metabolism, genes predicted to encode ammonia monooxygenase subunits, ammonia permease, urease, and urea transporters were identified, consistent with the use of reduced nitrogen compounds as energy sources fueling autotrophic metabolism. Homologues of these genes, recovered from ocean waters worldwide, demonstrate the conservation and ubiquity of crenarchaeal pathways for carbon assimilation and ammonia oxidation. These findings further substantiate the likely global metabolic importance of Crenarchaeota with respect to key steps in the biogeochemical transformation of carbon and nitrogen in marine ecosystems.     

Effect of Glucose on Carbon Dioxide Assimilation and Substrate Oxidation by Ferrobacillus ferrooxidans

Ferrobacillus ferrooxidans, grown on either elemental sulfur or ferrous sulfate, was able to use either substrate as an energy source for the assimilation of CO(2). In both cases, 0.01 mumole of carbon was incorporated per mumole of oxygen utilized. Glucose inhibited substrate oxidation and CO(2) fixation. Sulfur and iron oxidation were inhibited 5 to 15% and 40 to 50%, respectively, in the presence of 10% glucose. Under the same conditions, CO(2) assimilation was inhibited 50% with elemental sulfur as the energy source, and was almost totally inhibited when ferrous iron was used.     

The Assimilation of Ammonia by Nitrogen-starved Cells of Chlorella vulgaris: Part II. The Assimilation of Ammonia to other Compounds

Ammonia is rapidly assimilated by nitrogen-starved Chlorellacells and converted into soluble organic nitrogenous compounds.During the first 20 minutes of assimilation most of the ammoniawhich has been used can be accounted for as free or combined-amino-nitrogen and amide-nitrogen. Later a smaller proportionof the assimilated ammonia is found in these fractions whileappreciable quantities of basic amino-acids are formed. Theassimilation of ammonia is accompanied by an increase of therates of oxygen absorption and carbon dioxide production; thevalue of the respiratory quotient decreases. Non-reducing sugarand acid-hydrolysable polysaccharide are metabolized rapidly. Possible interpretations of these facts are discussed.     

Carbon Dioxide Assimilation and Methane Oxidation in Various Zones of the Rainbow Hydrothermal Field

Rates of carbon dioxide assimilation and methane oxidation were determined in various zones of the Rainbow Hydrothermal Field (36°N) of the Mid-Atlantic Ridge. In the plume above the hydrothermal field, anomalously high methane content was recorded, the microbial population density (up to 10⁵ cells/ml) was an order of magnitude higher than the background values, and the CO₂ assimilation rate varied from 0.01 to 1.1 g C/(l day). Based on the data on CO₂ assimilation, the production of organic carbon due to bacterial chemosynthesis in the plume was calculated to be 930 kg/day or 340 tons/year (about 29% of the organic carbon production in the photic zone). In the black smoke above active smokers, the microbial population density was as high as 10⁶ cells/ml, the rate of CO₂ assimilation made up 5–10 g C/(l day), the methane oxidation rate varied from 0.15 to 12.7 l/(l day), and the methane concentration ranged from 1.05 to 70.6 l/l. In bottom sediments enriched with sulfides, the rate of CO₂ assimilation was at least an order of magnitude higher than in oxidized metal-bearing sediments. At the base of an active construction, whitish sediment was found, which was characterized by a high methane content (92 l/dm³) and a high rate of methane oxidation (1.7 l/(dm³ day)).     

Oxidation and Assimilation of Atmospheric Methane by Soil Methane Oxidizers

The metabolism of atmospheric methane in a forest soil was studied by radiotracer techniques. Maximum (sup14)CH(inf4) oxidation (163.5 pmol of C cm(sup-3) h(sup-1)) and (sup14)C assimilation (50.3 pmol of C cm(sup-3) h(sup-1)) occurred at the A(inf2) horizon located 15 to 18 cm below the soil surface. At this depth, 31 to 43% of the atmospheric methane oxidized was assimilated into microbial biomass; the remaining methane was recovered as (sup14)CO(inf2). Methane-derived carbon was incorporated into all major cell macromolecules by the soil microorganisms (50% as proteins, 19% as nucleic acids and polysaccharides, and 5% as lipids). The percentage of methane assimilated (carbon conversion efficiency) remained constant at temperatures between 5 and 20(deg)C, followed by a decrease at 30(deg)C. The carbon conversion efficiency did not increase at methane concentrations between 1.7 and 1,000 ppm. In contrast, the overall methane oxidation activity increased at elevated methane concentrations, with an apparent K(infm) of 21 ppm (31 nM CH(inf4)) and a V(infmax) of 188 pmol of CH(inf4) cm(sup-3) h(sup-1). Methane oxidizers from soil depths with maximum methanotrophic activity respired approximately 1 to 3% of the assimilated methane-derived carbon per day. This apparent endogenous respiration did not change significantly in the absence of methane. Similarly, the potential for oxidation of atmospheric methane was relatively insensitive to methane starvation. Soil samples from depths above and below the zone with maximum atmospheric methane oxidation activity showed a dramatic increase in the turnover of the methane assimilated (>20 times increase). Physical disturbance such as sieving or mixing of soil samples decreased methane oxidation and assimilation by 50 to 58% but did not alter the carbon conversion efficiency. Ammonia addition (0.1 or 1.0 (mu)mol g [fresh weight](sup-1)) decreased both methane oxidation and carbon conversion efficiency. This resulted in a dramatic decrease in methane assimilation (85 to 99%). In addition, ammonia-treated soil showed up to 10 times greater turnover of the assimilated methane-derived carbon (relative to untreated soil). The results suggest a potential for microbial growth on atmospheric methane. However, growth was regulated strongly by soil parameters other than the methane concentration. The pattern observed for metabolism of atmospheric methane in soils was not consistent with the physiology of known methanotrophic bacteria.     

Anaerobic Carbon Metabolism by the Tricarboxylic Acid Cycle : Evidence for Partial Oxidative and Reductive Pathways during Dark Ammonium Assimilation

Nitrogen-limited cells of Selenastrum minutum (Naeg.) Collins are able to assimilate NH₄⁺ in the dark under anaerobic conditions. Addition of NH₄⁺ to anaerobic cells results in a threefold increase in tricarboxylic acid cycle (TCAC) CO₂ efflux and an eightfold increase in the rate of anaplerotic carbon fixation via phosphoenolpyruvate carboxylase. Both of these observations are consistent with increased TCAC carbon flow to supply intermediates for amino acid biosynthesis. Addition of H¹⁴CO₃⁻ to anaerobic cells assimilating NH₄⁺ results in the incorporation of radiolabel into the α-carboxyl carbon of glutamic acid. Incorporation of radiolabel into glutamic acid is not simply a short-term phenomenon following NH₄⁺ addition as the specific activity of glutamic acid increases over time. This indicates that this alga is able to maintain partial oxidative TCAC carbon flow while under anoxia to supply α-ketoglutarate for glutamate production. During dark aerobic NH₄⁺ assimilation, no radiolabel appears in fumarate or succinate and only a small amount occurs in malate. During anaerobic NH₄⁺ assimilation, these metabolites contain a large proportion of the total radiolabel and radiolabel accumulates in succinate over time. Also, the ratio of dark carbon fixation to NH₄⁺ assimilation is much higher under anaerobic than aerobic conditions. These observations suggest the operation of a partial reductive TCAC from oxaloacetic acid to malate, fumarate, and succinate. Such a pathway might contribute to redox balance in an anaerobic cell maintaining partial oxidative TCAC activity.     

Oxidation of Ammonia by Spheroplasts of Nitrosomonas europaea

The ammonia-oxidizing activity of Nitrosomonas europaea spheroplasts was restored by addition of hydroxylamine or by preincubation with Mg(2+).     

Fractionation of Nitrogen Isotopes during the Uptake and Assimilation of Ammonia by Plants

Two varieties (Nihonbare and Koshihikari) of rice plants (Oryzasativa L.) were grown hydro-ponically with two levels (20 and100 mg N liter ^–1) of ammonia. Variations in levels ofnatural abundance of ¹⁵N (¹⁵N) were analyzed in the ammoniaand organic nitrogen of shoots and roots, as well as in theammonia in the culture solution. There was substantial fractionationof nitrogen isotopes during the uptake of ammonia. When plantsabsorbed a large proportion of ammonia from a solution witha low concentration, less negative ¹⁵N values in plants andhigh positive ¹⁵N values in the ammonia remaining in solutionwere observed. The reverse was found when a smaller fractionof ammonia was absorbed from a solution with a higher concentrationof ammonia. The ^l5N values of ammonia in shoots and roots werehigher than in the respective constituent organic nitrogen,suggesting the fractionation of nitrogen isotopes during theassimilation of ammonia. Wild-type and mutant cells of the cyanobacterium(blue-green alga) Synechococcus PCC 7942 were grown in nitrate-or ammonia-containing medium as the source of nitrogen. Fractionationof nitrogen isotopes during the uptake of nitrate was limited,whereas that during the uptake of ammonia was considerable. ¹ In this report, the term ammonia refers indiscriminately toboth NH₃ or NH₄⁺. (Received June 13, 1991; Accepted September 12, 1991)     

Ubiquitous Dissolved Inorganic Carbon Assimilation by Marine Bacteria in the Pacific Northwest Coastal Ocean as Determined by Stable Isotope Probing

In order to identify bacteria that assimilate dissolved inorganic carbon (DIC) in the northeast Pacific Ocean, stable isotope probing (SIP) experiments were conducted on water collected from 3 different sites off the Oregon and Washington coasts in May 2010, and one site off the Oregon Coast in September 2008 and March 2009. Samples were incubated in the dark with 2 mM ¹³C-NaHCO₃, doubling the average concentration of DIC typically found in the ocean. Our results revealed a surprising diversity of marine bacteria actively assimilating DIC in the dark within the Pacific Northwest coastal waters, indicating that DIC fixation is relevant for the metabolism of different marine bacterial lineages, including putatively heterotrophic taxa. Furthermore, dark DIC-assimilating assemblages were widespread among diverse bacterial classes. Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes dominated the active DIC-assimilating communities across the samples. Actinobacteria, Betaproteobacteria, Deltaproteobacteria, Planctomycetes, and Verrucomicrobia were also implicated in DIC assimilation. Alteromonadales and Oceanospirillales contributed significantly to the DIC-assimilating Gammaproteobacteria within May 2010 clone libraries. 16S rRNA gene sequences related to the sulfur-oxidizing symbionts Arctic96BD-19 were observed in all active DIC assimilating clone libraries. Among the Alphaproteobacteria, clones related to the ubiquitous SAR11 clade were found actively assimilating DIC in all samples. Although not a dominant contributor to our active clone libraries, Betaproteobacteria, when identified, were predominantly comprised of Burkholderia. DIC-assimilating bacteria among Deltaproteobacteria included members of the SAR324 cluster. Our research suggests that DIC assimilation is ubiquitous among many bacterial groups in the coastal waters of the Pacific Northwest marine environment and may represent a significant metabolic process.     

Betaine Fermentation and Oxidation by Marine Desulfuromonas Strains

Two bacterial strains were dominant in anaerobic enrichment cultures with betaine (N,N,N-trimethylglycine) as a substrate and intertidal mud as an inoculum. One was a coccoid bacterium which was a trimethylamine (TMA)-fermenting methanogen similar to Methanococcoides methylutens. The other strain, a rod-shaped, gram-negative, motile bacterium, fermented betaine. On the basis of its ability to oxidize acetate and ethanol to CO₂ with sulfur as an electron acceptor, its inability to reduce sulfate and sulfite, its morphology, the presence of c-type cytochromes, and other characteristics, the isolated strain PM1 was identified as Desulfuromonas acetoxidans. Although only malate and fumarate were known as substrates for fermentative growth of this species, the type strain (DSM 684) also fermented betaine. Strain PM1 grew with a doubling time of 9.5 h at 30°C on betaine and produced approximately 1 mol of TMA per mol of betaine, 0.75 mol of acetate, and presumably CO₂ as fermentation products but only in the presence of selenite (100 nM). In this fermentation, betaine is probably reductively cleaved to TMA and acetate, and part of the acetate is then oxidized to CO₂ to provide the reducing equivalents for the initial cleavage reaction. In the presence of sulfur, betaine was converted to TMA and presumably CO₂ with the formation of sulfide; then, only traces of acetate were produced.     

Assimilation of Inorganic Nitrogen by Marine Invertebrates and Their Chemoautotrophic and Methanotrophic Symbionts

Symbioses between marine invertebrates and their chemoautotrophic and methanotrophic symbionts are now known to exist in a variety of habitats where reduced chemical species are present. The utilization of chemical energy and reliance on C₁ compounds by these symbioses are well documented. Much less is known about their metabolism of nitrogen. Earlier work has shown that the tissues of organisms in these associations are depleted of ¹⁵N compared with those of other marine organisms, indicating that local sources of nitrogen are assimilated and that novel mechanisms of nitrogen metabolism may be involved. Although these symbioses have access to rich sources of ammonium (NH₄⁺ and NH₃) and/or nitrate, several investigators have proposed that N₂ fixation may account for some of these isotope values. Here we report that [¹⁵N]ammonium and, to a lesser degree, [¹⁵N]nitrate are assimilated into organic compounds by Solemya reidi, a gutless clam containing S-oxidizing bacteria, and seep mussel Ia, an undescribed mytilid containing methanotrophic bacteria. In contrast, Riftia pachyptila, the giant hydrothermal vent tube worm symbiotic with S-oxidizing bacteria, assimilated nitrate but not exogenous ammonium. The rates of assimilation of these sources are sufficient to at least partially support C₁ compound metabolism. N₂ assimilation was not exhibited by the symbionts tested.