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The ability to measure the radiocarbon content of compounds isolated from complex mixtures has begun to revolutionize our understanding of carbon transformations on earth. Because samples are often small, each new compound isolation method must be tested for background carbon contamination (C ex). Here, we present a new method for compound-specific radiocarbon analysis (CSRA) of higher plant-derived lignin phenols. To test for C ex, we compared the Δ14C values of unprocessed lignin phenol containing standard materials (woods, leaves, natural vanillin, and synthetic vanillin) with those of lignin phenols liberated by CuO oxidation and purified by two-dimensional high-pressure liquid chromatography (HPLC) coupled to mass spectrometry (MS) and UV detection. We assessed Cex associated with (1) microwave assisted CuO oxidation of bulk samples to lignin phenol monomers, (2) HPLC purification, and (3) accelerator mass spectrometry (AMS) sample preparation. The Δ14C of purified compounds (corrected for Cex) agreed, within error, with those of bulk materials for samples that were >10 μg C. This method will allow routine analysis of the Δ14C of lignin phenols isolated from terrestrial, aquatic, and marine settings, revealing the time scale for the processing of one of the single largest components of active organic carbon reservoirs on earth. © 2010 American Chemical Society.

This study demonstrates that intracrystalline organic matter in coral skeletons is well preserved over century timescales. The extent of preservation of organic matter in coral skeletons was investigated by measuring total organic carbon (TOC), total hydrolyzable amino acid (THAA), chloropigment, and lipid concentrations in 0-300 year old annual growth bands from Montastraea annularis (Florida Keys) and Porites lutea (Red Sea). Organic matter intrinsic to the calcium carbonate mineral (intracrystalline) was analyzed separately from total skeletal organic matter. The Red Sea coral had less TOC (0.02-0.04 wt%) than the Florida Keys coral (0.04-0.11 wt%), but a higher percent of intracrystalline organic matter in all annual bands measured. Carbon in the form of THAA, most likely from mineral-precipitating proteins, contributed 30-45% of the TOC in both corals. Carbon in lipids represented about 3% of the TOC in the coral skeletons. Chlorophyll-a and b were present in annual bands where endolithic algae were present, but these compounds were minor contributors to TOC. The distribution of specific organic compounds showed that organic matter was well preserved throughout the time period sampled in both the total and intracrystalline pools. Variations in THAA were not correlated with TOC over time, suggesting that organic matter that is involved in biomineralization, like amino acids, may be deposited in response to different environmental factors than are other components of skeletal organic matter. Differences in the quantity and composition of organic matter between the two corals investigated here were assessed using principal components analysis and suggest that location, species and skeletal structure may all influence organic matter content and possibly the degree of physical protection of organic matter by the coral skeleton. Further, our study suggests that intracrystalline organic matter may be better protected from diagenesis than non-intracrystalline organic matter and may therefore be a more reliable source of organic matter for paleoceanographic studies than total skeletal organic matter. © 2003 Elsevier Science Ltd.

Ammonia-oxidizing archaea (AOA) are among the most ubiquitous and abundant archaea on Earth, widely distributed in marine, terrestrial, and geothermal ecosystems. However, the genomic diversity, biogeography, and evolutionary process of AOA populations in subsurface environments are vastly understudied compared to those in marine and soil systems. Here, we report a novel AOA order Candidatus (Ca.) Nitrosomirales which forms a sister lineage to the thermophilic Ca. Nitrosocaldales. Metagenomic and 16S rRNA gene-read mapping demonstrates the abundant presence of Nitrosomirales AOA in various groundwater environments and their widespread distribution across a range of geothermal, terrestrial, and marine habitats. Terrestrial Nitrosomirales AOA show the genetic capacity of using formate as a source of reductant and using nitrate as an alternative electron acceptor. Nitrosomirales AOA appear to have acquired key metabolic genes and operons from other mesophilic populations via horizontal gene transfer, including genes encoding urease, nitrite reductase, and V-type ATPase. The additional metabolic versatility conferred by acquired functions may have facilitated their radiation into a variety of subsurface, marine, and soil environments. We also provide evidence that each of the four AOA orders spans both marine and terrestrial habitats, which suggests a more complex evolutionary history for major AOA lineages than previously proposed. Together, these findings establish a robust phylogenomic framework of AOA and provide new insights into the ecology and adaptation of this globally abundant functional guild., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Seasonal cycles within the marginal ice zones in polar regions include large shifts in temperature and salinity that strongly influence microbial abundance and physiology. However, the combined effects of concurrent temperature and salinity change on microbial community structure and biochemical composition during transitions between seawater and sea ice are not well understood. Coastal marine communities along the western Antarctic Peninsula were sampled and surface seawater was incubated at combinations of temperature and salinity mimicking the formation (cold, salty) and melting (warm, fresh) of sea ice to evaluate how these factors may shape community composition and particulate metabolite pools during seasonal transitions. Bacterial and algal community structures were tightly coupled to each other and distinct across sea-ice, seawater, and sea-ice-meltwater field samples, with unique metabolite profiles in each habitat. During short-term (approximately 10-day) incubations of seawater microbial communities under different temperature and salinity conditions, community compositions changed minimally while metabolite pools shifted greatly, strongly accumulating compatible solutes like proline and glycine betaine under cold and salty conditions. Lower salinities reduced total metabolite concentrations in particulate matter, which may indicate a release of metabolites into the labile dissolved organic matter pool. Low salinity also increased acylcarnitine concentrations in particulate matter, suggesting a potential for fatty acid degradation and reduced nutritional value at the base of the food web during freshening. Our findings have consequences for food web dynamics, microbial interactions, and carbon cycling as polar regions undergo rapid climate change., (© 2023. The Author(s).)

Background: Chromatographic peakpicking continues to represent a significant bottleneck in automated LC-MS workflows. Uncontrolled false discovery rates and the lack of manually-calibrated quality metrics require researchers to visually evaluate individual peaks, requiring large amounts of time and breaking replicability. This problem is exacerbated in noisy environmental datasets and for novel separation methods such as hydrophilic interaction columns in metabolomics, creating a demand for a simple, intuitive, and robust metric of peak quality., Results: Here, we manually labeled four HILIC oceanographic particulate metabolite datasets to assess the performance of individual peak quality metrics. We used these datasets to construct a predictive model calibrated to the likelihood that visual inspection by an MS expert would include a given mass feature in the downstream analysis. We implemented two novel peak quality metrics, a custom signal-to-noise metric and a test of similarity to a bell curve, both calculated from the raw data in the extracted ion chromatogram, and found that these outperformed existing measurements of peak quality. A simple logistic regression model built on two metrics reduced the fraction of false positives in the analysis from 70-80% down to 1-5% and showed minimal overfitting when applied to novel datasets. We then explored the implications of this quality thresholding on the conclusions obtained by the downstream analysis and found that while only 10% of the variance in the dataset could be explained by depth in the default output from the peakpicker, approximately 40% of the variance was explained when restricted to high-quality peaks alone., Conclusions: We conclude that the poor performance of peakpicking algorithms significantly reduces the power of both univariate and multivariate statistical analyses to detect environmental differences. We demonstrate that simple models built on intuitive metrics and derived from the raw data are more robust and can outperform more complex models when applied to new data. Finally, we show that in properly curated datasets, depth is a major driver of variability in the marine microbial metabolome and identify several interesting metabolite trends for future investigation., (© 2023. The Author(s).)

Glycine betaine (GBT) is a compatible solute in high concentrations in marine microorganisms. As a component of labile organic matter, GBT has complex biochemical potential as a substrate for microbial use that is unconstrained in the environment. Here we determine the uptake kinetics and metabolic fate of GBT in two natural microbial communities in the North Pacific characterized by different nitrate concentrations. Dissolved GBT had maximum uptake rates of 0.36 and 0.56 nM h -1 with half-saturation constants of 79 and 11 nM in the high nitrate and low nitrate stations respectively. During multiday incubations, most GBT taken into cells was retained as a compatible solute. Stable isotopes derived from the added GBT were also observed in other metabolites, including choline, carnitine and sarcosine, suggesting that GBT was used for biosynthesis and for catabolism to pyruvate and ammonium. Where nitrate was scarce, GBT was primarily metabolized via demethylation to glycine. Gene transcript data were consistent with SAR11 using GBT as a source of methyl groups to fuel the methionine cycle. Where nitrate concentrations were higher, more GBT was partitioned for lipid biosynthesis by both bacteria and eukaryotic phytoplankton. Our data highlight unexpected metabolic pathways and potential routes of microbial metabolite exchange., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Complex assemblages of microbes in the surface ocean are responsible for approximately half of global carbon fixation. The persistence of high taxonomic diversity despite competition for a small suite of relatively homogeneously distributed nutrients, that is, 'the paradox of the plankton', represents a long-standing challenge for ecological theory. Here we find evidence consistent with temporal niche partitioning of nitrogen assimilation processes over a diel cycle in the North Pacific Subtropical Gyre. We jointly analysed transcript abundances, lipids and metabolites and discovered that a small number of diel archetypes can explain pervasive periodic dynamics. Metabolic pathway analysis of identified diel signals revealed asynchronous timing in the transcription of nitrogen uptake and assimilation genes among different microbial groups-cyanobacteria, heterotrophic bacteria and eukaryotes. This temporal niche partitioning of nitrogen uptake emerged despite synchronous transcription of photosynthesis and central carbon metabolism genes and associated macromolecular abundances. Temporal niche partitioning may be a mechanism by which microorganisms in the open ocean mitigate competition for scarce resources, supporting community coexistence., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Extracellular vesicles are small (~50-200 nm diameter) membrane-bound structures released by cells from all domains of life. While vesicles are abundant in the oceans, their functions, both for cells themselves and the emergent ecosystem, remain a mystery. To better characterize these particles - a prerequisite for determining function - we analysed the lipid, protein, and metabolite content of vesicles produced by the marine cyanobacterium Prochlorococcus. We show that Prochlorococcus exports a diverse array of cellular compounds into the surrounding seawater enclosed within discrete vesicles. Vesicles produced by two different strains contain some materials in common, but also display numerous strain-specific differences, reflecting functional complexity within vesicle populations. The vesicles contain active enzymes, indicating that they can mediate extracellular biogeochemical reactions in the ocean. We further demonstrate that vesicles from Prochlorococcus and other bacteria associate with diverse microbes including the most abundant marine bacterium, Pelagibacter. Together, our data point toward hypotheses concerning the functional roles of vesicles in marine ecosystems including, but not limited to, possibly mediating energy and nutrient transfers, catalysing extracellular biochemical reactions, and mitigating toxicity of reactive oxygen species., (© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Thaumarchaeota and Thermoplasmatota are the most abundant planktonic archaea in the sea. Thaumarchaeota contain tetraether lipids as their major membrane lipids, but the lipid composition of uncultured planktonic Thermoplasmatota representatives remains unknown. To address this knowledge gap, we quantified archaeal cells and ether lipids in open ocean depth profiles (0-200 m) of the North Pacific Subtropical Gyre. Planktonic archaeal community structure and ether lipid composition in the water column partitioned into two separate clusters: one above the deep chlorophyll maximum, the other within and below it. In surface waters, Thermoplasmatota densities ranged from 2.11 × 10 6 to 6.02 × 10 6 cells/L, while Thaumarchaeota were undetectable. As previously reported for Thaumarchaeota, potential homologs of archaeal tetraether ring synthases were present in planktonic Thermoplasmatota metagenomes. Despite the absence of Thaumarchaeota in surface waters, measurable amounts of intact polar ether lipids were found there. Based on cell abundance estimates, these surface water archaeal ether lipids contributed only 1.21 × 10 -9 ng lipid/Thermoplasmatota cell, about three orders of magnitude less than that reported for Thaumarchaeota cells. While these data indicate that even if some tetraether and diether lipids may be derived from Thermoplasmatota, they would only comprise a small fraction of Thermoplasmatota total biomass. Therefore, while both MGI Thaumarchaeota and MGII/III Thermoplasmatota are potential biological sources of archaeal GDGTs, the Thaumarchaeota appear to be the major contributors of archaeal tetraether lipids in planktonic marine habitats. These results extend and confirm previous reports of planktonic archaeal lipid sources, and further emphasize the need for Thermoplasmatota cultivation, to better characterize the membrane lipid constituents of marine planktonic Thermoplasmatota, and more precisely define the sources and patterns of archaeal tetraether lipid distributions in marine plankton., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Leu, Poff, Carlson, Ingalls and DeLong.)

Chemoautotrophic bacteria from the SUP05 clade often dominate anoxic waters within marine oxygen minimum zones (OMZs) where they use energy gained from the oxidation of reduced sulfur to fuel carbon fixation. Some of these SUP05 bacteria are facultative aerobes that can use either nitrate or oxygen as a terminal electron acceptor making them ideally suited to thrive at the boundaries of OMZs where they experience fluctuations in dissolved oxygen (DO). SUP05 metabolism in these regions, and therefore the biogeochemical function of SUP05, depends largely on their sensitivity to oxygen. We evaluated growth and quantified differences in gene expression in Ca. T. autotrophicus strain EF1 from the SUP05 clade under high DO (22 μM), anoxic, and low DO (3.8 μM) concentrations. We show that strain EF1 cells respire oxygen and nitrate and that cells have higher growth rates, express more genes, and fix more carbon when oxygen becomes available for aerobic respiration. Evidence that facultatively aerobic SUP05 are more active and respire nitrate when oxygen becomes available at low concentrations suggests that they are an important source of nitrite across marine OMZ boundary layers., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Light fuels photosynthesis and organic matter production by primary producers in the sunlit ocean. The quantity and quality of the organic matter produced influence community function, yet in situ measurements of metabolites, the products of cellular metabolism, over the diel cycle are lacking. We evaluated community-level biochemical consequences of oscillations of light in the North Pacific Subtropical Gyre by quantifying 79 metabolites in particulate organic matter from 15 m every 4 h over 8 days. Total particulate metabolite concentration peaked at dusk and represented up to 2% of total particulate organic carbon (POC). The concentrations of 55/79 (70%) individual metabolites exhibited significant 24-h periodicity, with daily fold changes from 1.6 to 12.8, often greater than those of POC and flow cytometry-resolvable biomass, which ranged from 1.2 to 2.8. Paired metatranscriptome analysis revealed the taxa involved in production and consumption of a subset of metabolites. Primary metabolites involved in anabolism and redox maintenance had significant 24-h periodicity and diverse organisms exhibited diel periodicity in transcript abundance associated with these metabolites. Compounds with osmotic properties displayed the largest oscillations in concentration, implying rapid turnover and supporting prior evidence of functions beyond cell turgor maintenance. The large daily oscillation of trehalose paired with metatranscriptome and culture data showed that trehalose is produced by the nitrogen-fixing cyanobacterium Crocosphaera , likely to store energy for nighttime metabolism. Together, paired measurements of particulate metabolites and transcripts resolve strategies that microbes use to manage daily energy and redox oscillations and highlight dynamic metabolites with cryptic roles in marine microbial ecosystems. IMPORTANCE Fueled by light, phytoplankton produce the organic matter that supports ocean ecosystems and carbon sequestration. Ocean change impacts microbial metabolism with repercussions for biogeochemical cycling. As the small molecule products of cellular metabolism, metabolites often change rapidly in response to environmental conditions and form the basis of energy and nutrient management and storage within cells. By pairing measurements of metabolites and gene expression in the stratified surface ocean, we reveal strategies of microbial energy management over the day-night cycle and hypothesize that oscillating metabolites are important substrates for dark respiration by phytoplankton. These high-resolution diel measurements of in situ metabolite concentrations form the basis for future work into the specific roles these compounds play in marine microbial communities., (Copyright © 2021 Boysen et al.)

Phytoplankton transform inorganic carbon into thousands of biomolecules that represent an important pool of fixed carbon, nitrogen, and sulfur in the surface ocean. Metabolite production differs between phytoplankton, and the flux of these molecules through the microbial food web depends on compound-specific bioavailability to members of a wider microbial community. Yet relatively little is known about the diversity or concentration of metabolites within marine plankton. Here, we compare 313 polar metabolites in 21 cultured phytoplankton species and in natural planktonic communities across environmental gradients to show that bulk community metabolomes reflect the chemical composition of the phytoplankton community. We also show that groups of compounds have similar patterns across space and taxonomy, suggesting that the concentrations of these compounds in the environment are controlled by similar sources and sinks. We quantify several compounds in the surface ocean that represent substantial understudied pools of labile carbon. For example, the N-containing metabolite homarine was up to 3% of particulate carbon and is produced in high concentrations by cultured Synechococcus , and S-containing gonyol accumulated up to 2.5 nM in surface particles and likely originates from dinoflagellates or haptophytes. Our results show that phytoplankton composition directly shapes the carbon composition of the surface ocean. Our findings suggest that in order to access these pools of bioavailable carbon, the wider microbial community must be adapted to phytoplankton community composition. IMPORTANCE Microscopic phytoplankton transform 100 million tons of inorganic carbon into thousands of different organic compounds each day. The structure of each chemical is critical to its biological and ecosystem function, yet the diversity of biomolecules produced by marine microbial communities remained mainly unexplored, especially small polar molecules which are often considered the currency of the microbial loop. Here, we explore the abundance and diversity of small biomolecules in planktonic communities across ecological gradients in the North Pacific and within 21 cultured phytoplankton species. Our work demonstrates that phytoplankton diversity is an important determinant of the chemical composition of the highly bioavailable pool of organic carbon in the ocean, and we highlight understudied yet abundant compounds in both the environment and cultured organisms. These findings add to understanding of how the chemical makeup of phytoplankton shapes marine microbial communities where the ability to sense and use biomolecules depends on the chemical structure., (Copyright © 2021 Heal et al.)

Intense bottom-ice algal blooms, often dominated by diatoms, are an important source of food for grazers, organic matter for export during sea ice melt, and dissolved organic carbon. Sea-ice diatoms have a number of adaptations, including accumulation of compatible solutes, that allows them to inhabit this highly variable environment characterized by extremes in temperature, salinity, and light. In addition to protecting them from extreme conditions, these compounds present a labile, nutrient-rich source of organic matter, and include precursors to climate active compounds (e.g., dimethyl sulfide [DMS]), which are likely regulated with environmental change. Here, intracellular concentrations of 45 metabolites were quantified in three sea-ice diatom species and were compared to two temperate diatom species, with a focus on compatible solutes and free amino acid pools. There was a large diversity of metabolite concentrations between diatoms with no clear pattern identifiable for sea-ice species. Concentrations of some compatible solutes (isethionic acid, homarine) approached 1 M in the sea-ice diatoms, Fragilariopsis cylindrus and Navicula cf. perminuta, but not in the larger sea-ice diatom, Nitzschia lecointei or in the temperate diatom species. The differential use of compatible solutes in sea-ice diatoms suggests different adaptive strategies and highlights which small organic compounds may be important in polar biogeochemical cycles., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

Ammonia-oxidizing archaea (AOA) are among the most abundant and ubiquitous microorganisms in the ocean, exerting primary control on nitrification and nitrogen oxides emission. Although united by a common physiology of chemoautotrophic growth on ammonia, a corresponding high genomic and habitat variability suggests tremendous adaptive capacity. Here, we compared 44 diverse AOA genomes, 37 from species cultivated from samples collected across diverse geographic locations and seven assembled from metagenomic sequences from the mesopelagic to hadopelagic zones of the deep ocean. Comparative analysis identified seven major marine AOA genotypic groups having gene content correlated with their distinctive biogeographies. Phosphorus and ammonia availabilities as well as hydrostatic pressure were identified as selective forces driving marine AOA genotypic and gene content variability in different oceanic regions. Notably, AOA methylphosphonate biosynthetic genes span diverse oceanic provinces, reinforcing their importance for methane production in the ocean. Together, our combined comparative physiological, genomic, and metagenomic analyses provide a comprehensive view of the biogeography of globally abundant AOA and their adaptive radiation into a vast range of marine and terrestrial habitats.

Ammonia oxidation to nitrite and its subsequent oxidation to nitrate provides energy to the two populations of nitrifying chemoautotrophs in the energy-starved dark ocean, driving a coupling between reduced inorganic nitrogen (N) pools and production of new organic carbon (C) in the dark ocean. However, the relationship between the flux of new C production and the fluxes of N of the two steps of oxidation remains unclear. Here, we show that, despite orders-of-magnitude difference in cell abundances between ammonia oxidizers and nitrite oxidizers, the two populations sustain similar bulk N-oxidation rates throughout the deep waters with similarly high affinities for ammonia and nitrite under increasing substrate limitation, thus maintaining overall homeostasis in the oceanic nitrification pathway. Our observations confirm the theoretical predictions of a redox-informed ecosystem model. Using balances from this model, we suggest that consistently low ammonia and nitrite concentrations are maintained when the two populations have similarly high substrate affinities and their loss rates are proportional to their maximum growth rates. The stoichiometric relations between the fluxes of C and N indicate a threefold to fourfold higher C-fixation efficiency per mole of N oxidized by ammonia oxidizers compared to nitrite oxidizers due to nearly identical apparent energetic requirements for C fixation of the two populations. We estimate that the rate of chemoautotrophic C fixation amounts to ∼1 × 10 13 to ∼2 × 10 13 mol of C per year globally through the flux of ∼1 × 10 14 to ∼2 × 10 14 mol of N per year of the two steps of oxidation throughout the dark ocean., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Cobalamin (vitamin B 12 ) is an essential enzyme cofactor for most branches of life. Despite the potential importance of this cofactor for soil microbial communities, the producers and consumers of cobalamin in terrestrial environments are still unknown. Here we provide the first metagenome-based assessment of soil cobalamin-producing bacteria and archaea, quantifying and classifying genes encoding proteins for cobalamin biosynthesis, transport, remodeling, and dependency in 155 soil metagenomes with profile hidden Markov models. We also measured several forms of cobalamin (CN-, Me-, OH-, Ado-B 12 ) and the cobalamin lower ligand (5,6-dimethylbenzimidazole; DMB) in 40 diverse soil samples. Metagenomic analysis revealed that less than 10% of soil bacteria and archaea encode the genetic potential for de novo synthesis of this important enzyme cofactor. Predominant soil cobalamin producers were associated with the Proteobacteria, Actinobacteria, Firmicutes, Nitrospirae, and Thaumarchaeota. In contrast, a much larger proportion of abundant soil genera lacked cobalamin synthesis genes and instead were associated with gene sequences encoding cobalamin transport and cobalamin-dependent enzymes. The enrichment of DMB and corresponding DMB synthesis genes, relative to corrin ring synthesis genes, suggests an important role for cobalamin remodelers in terrestrial habitats. Together, our results indicate that microbial cobalamin production and repair serve as keystone functions that are significantly correlated with microbial community size, diversity, and biogeochemistry of terrestrial ecosystems.

Thaumarchaeota are responsible for a significant fraction of ammonia oxidation in the oceans and in soils that range from alkaline to acidic. However, the adaptive mechanisms underpinning their habitat expansion remain poorly understood. Here we show that expansion into acidic soils and the high pressures of the hadopelagic zone of the oceans is tightly linked to the acquisition of a variant of the energy-yielding ATPases via horizontal transfer. Whereas the ATPase genealogy of neutrophilic Thaumarchaeota is congruent with their organismal genealogy inferred from concatenated conserved proteins, a common clade of V-type ATPases unites phylogenetically distinct clades of acidophilic/acid-tolerant and piezophilic/piezotolerant species. A presumptive function of pumping cytoplasmic protons at low pH is consistent with the experimentally observed increased expression of the V-ATPase in an acid-tolerant thaumarchaeote at low pH. Consistently, heterologous expression of the thaumarchaeotal V-ATPase significantly increased the growth rate of E. coli at low pH. Its adaptive significance to growth in ocean trenches may relate to pressure-related changes in membrane structure in which this complex molecular machine must function. Together, our findings reveal that the habitat expansion of Thaumarchaeota is tightly correlated with extensive horizontal transfer of atp operons.

In the surface ocean, phytoplankton transform inorganic substrates into organic matter that fuels the activity of heterotrophic microorganisms, creating intricate metabolic networks that determine the extent of carbon recycling and storage in the ocean. Yet, the diversity of organic molecules and interacting organisms has hindered detection of specific relationships that mediate this large flux of energy and matter. Here, we show that a tightly coupled microbial network based on organic sulfur compounds (sulfonates) exists among key lineages of eukaryotic phytoplankton producers and heterotrophic bacterial consumers in the North Pacific Subtropical Gyre. We find that cultured eukaryotic phytoplankton taxa produce sulfonates, often at millimolar internal concentrations. These same phytoplankton-derived sulfonates support growth requirements of an open-ocean isolate of the SAR11 clade, the most abundant group of marine heterotrophic bacteria. Expression of putative sulfonate biosynthesis genes and sulfonate abundances in natural plankton communities over the diel cycle link sulfonate production to light availability. Contemporaneous expression of sulfonate catabolism genes in heterotrophic bacteria highlights active cycling of sulfonates in situ. Our study provides evidence that sulfonates serve as an ecologically important currency for nutrient and energy exchange between microbial autotrophs and heterotrophs, highlighting the importance of organic sulfur compounds in regulating ecosystem function.

From June to August 2018, the eruption of Kīlauea volcano on the island of Hawai'i injected millions of cubic meters of molten lava into the nutrient-poor waters of the North Pacific Subtropical Gyre. The lava-impacted seawater was characterized by high concentrations of metals and nutrients that stimulated phytoplankton growth, resulting in an extensive plume of chlorophyll a that was detectable by satellite. Chemical and molecular evidence revealed that this biological response hinged on unexpectedly high concentrations of nitrate, despite the negligible quantities of nitrogen in basaltic lava. We hypothesize that the high nitrate was caused by buoyant plumes of nutrient-rich deep waters created by the substantial input of lava into the ocean. This large-scale ocean fertilization was therefore a unique perturbation event that revealed how marine ecosystems respond to exogenous inputs of nutrients., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Diatoms perform an estimated 20% of global photosynthesis, form the base of the marine food web, and sequester carbon into the deep ocean through the biological pump. In some areas of the ocean, diatom growth is limited by the micronutrient cobalamin (vitamin B 12 ), yet the biochemical ramifications of cobalamin limitation are not well understood. In a laboratory setting, we grew the diatom Thalassiosira pseudonana under replete and low cobalamin conditions to elucidate changes in metabolite pools. Using metabolomics, we show that the diatom experienced a metabolic cascade under cobalamin limitation that affected the central methionine cycle, transsulfuration pathway, and composition of osmolyte pools. In T. pseudonana, 5'-methylthioadenosine decreased under low cobalamin conditions, suggesting a disruption in the diatom's polyamine biosynthesis. Furthermore, two acylcarnitines accumulated under low cobalamin, suggesting the limited use of an adenosylcobalamin-dependent enzyme, methylmalonyl CoA mutase. Overall, these changes in metabolite pools yield insight into the metabolic consequences of cobalamin limitation in diatoms and suggest that cobalamin availability may have consequences for microbial interactions that are based on metabolite production by phytoplankton., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

A hallmark of the SUP05 clade of marine Gammaproteobacteria is the ability to use energy obtained from reduced inorganic sulfur to fuel autotrophic fixation of carbon using RuBisCo. However, some SUP05 also have the genetic potential for heterotrophic growth, raising questions about the roles of SUP05 in the marine carbon cycle. We used genomic reconstructions, physiological growth experiments and proteomics to characterize central carbon and energy metabolism in Candidatus Thioglobus singularis strain PS1, a representative from the SUP05 clade that has the genetic potential for autotrophy and heterotrophy. Here, we show that the addition of individual organic compounds and 0.2 μm filtered diatom lysate significantly enhanced the growth of this bacterium. This positive growth response to organic substrates, combined with expression of a complete TCA cycle, heterotrophic pathways for carbon assimilation, and methylotrophic pathways for energy conversion demonstrate strain PS1's capacity for heterotrophic growth. Further, our inability to verify the expression of RuBisCO suggests that carbon fixation was not critical for growth. These results highlight the metabolic diversity of the SUP05 clade that harbours both primary producers and consumers of organic carbon in the oceans and expand our understanding of specific pathways of organic matter oxidation by the heterotrophic SUP05., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Wide salinity ranges experienced during the seasonal freeze and melt of sea ice likely constrain many biological processes. Microorganisms generally protect against fluctuating salinities through the uptake, production, and release of compatible solutes. Little is known, however, about the use or fate of glycine betaine (GBT hereafter), one of the most common compatible solutes, in sea-ice diatoms confronted with shifts in salinity. We quantified intracellular concentrations and used [ 14 C]-labeled compounds to track the uptake and fate of the nitrogen-containing osmolyte GBT and its precursor choline in three Antarctic sea-ice diatoms Nitzschia lecointei, Navicula cf. perminuta, and Fragilariopsis cylindrus at -1°C. Experiments show that these diatoms have effective transporters for GBT, but take up lesser amounts of choline. Neither compound was respired. Uptake of GBT protected cells against hyperosmotic shock and corresponded with reduced production of extracellular polysaccharides in N. lecointei cells, which released 85% of the retained GBT following hypoosmotic shock. The ability of sea-ice diatoms to rapidly scavenge and release compatible solutes is likely an important strategy for survival during steep fluctuations in salinity. The release and recycling of compatible solutes may play an important role in algal-bacterial interactions and nitrogen cycling within the semi-enclosed brines of sea ice., (© 2019 Phycological Society of America.)

Sulfur-oxidizing bacteria from the SUP05 clade are abundant in anoxic and oxygenated marine waters that appear to lack reduced sources of sulfur for cell growth. This raises questions about how these chemosynthetic bacteria survive across oxygen and sulfur gradients and how their mode of survival impacts the environment. Here, we use growth experiments, proteomics, and cryo-electron tomography to show that a SUP05 isolate, " Candidatus Thioglobus autotrophicus," is amorphous in shape and several times larger and stores considerably more intracellular sulfur when it respires oxygen. We also show that these cells can use diverse sources of reduced organic and inorganic sulfur at submicromolar concentrations. Enhanced cell size, carbon content, and metabolic activity of the aerobic phenotype are likely facilitated by a stabilizing surface-layer (S-layer) and an uncharacterized form of FtsZ-less cell division that supports morphological plasticity. The additional sulfur storage provides an energy source that allows cells to continue metabolic activity when exogenous sulfur sources are not available. This metabolic flexibility leads to the production of more organic carbon in the ocean than is estimated based solely on their anaerobic phenotype. IMPORTANCE Identifying shifts in microbial metabolism across redox gradients will improve efforts to model marine oxygen minimum zone (OMZ) ecosystems. Here, we show that aerobic morphology and metabolism increase cell size, sulfur storage capacity, and carbon fixation rates in " Ca Thioglobus autotrophicus," a chemosynthetic bacterium from the SUP05 clade that crosses oxic-anoxic boundaries., (Copyright © 2019 Shah et al.)

Cobalamin (vitamin B 12 ) is a precious resource in natural systems that is produced by select prokaryotes and required by a broad range of organisms. In this way, the production of cobalamin reinforces numerous microbial interdependencies. Here we report the accumulation of an unusual form of cobalamin, nitrocobalamin (NO 2 -cobalamin), in a marine oxygen deficient zone (ODZ), isolates of ammonia-oxidizing archaea (AOA), and an anaerobic ammonium-oxidizing (anammox) bacteria enriched bioreactor. Low oxygen waters were enriched in NO 2 -cobalamin, and AOA isolates experiencing ammonia or copper stress produced more NO 2 -cobalamin, though there is wide strain-to-strain and batch-to-batch variability. NO 2 -cobalamin has no known biochemical role. We hypothesize that AOA and anammox bacteria are a source of marine NO 2 -cobalamin in the environment via a reactive nitrogen intermediate. These findings suggest connections between cobalamin forms and nitrogen transformations, physiological stress and ocean deoxygenation., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

High representation by ammonia-oxidizing archaea (AOA) in marine systems is consistent with their high affinity for ammonia, efficient carbon fixation, and copper (Cu)-centric respiratory system. However, little is known about their response to nutrient stress. We therefore used global transcriptional and proteomic analyses to characterize the response of a model AOA, Nitrosopumilus maritimus SCM1, to ammonia starvation, Cu limitation and Cu excess. Most predicted protein-coding genes were transcribed in exponentially growing cells, and of ~74% detected in the proteome, ~6% were modified by N-terminal acetylation. The general response to ammonia starvation and Cu stress was downregulation of genes for energy generation and biosynthesis. Cells rapidly depleted transcripts for the A and B subunits of ammonia monooxygenase (AMO) in response to ammonia starvation, yet retained relatively high levels of transcripts for the C subunit. Thus, similar to ammonia-oxidizing bacteria, selective retention of amoC transcripts during starvation appears important for subsequent recovery, and also suggests that AMO subunit transcript ratios could be used to assess the physiological status of marine populations. Unexpectedly, cobalamin biosynthesis was upregulated in response to both ammonia starvation and Cu stress, indicating the importance of this cofactor in retaining functional integrity during times of stress.

The goal of metabolomics is to measure the entire range of small organic molecules in biological samples. In liquid chromatography-mass spectrometry-based metabolomics, formidable analytical challenges remain in removing the nonbiological factors that affect chromatographic peak areas. These factors include sample matrix-induced ion suppression, chromatographic quality, and analytical drift. The combination of these factors is referred to as obscuring variation. Some metabolomics samples can exhibit intense obscuring variation due to matrix-induced ion suppression, rendering large amounts of data unreliable and difficult to interpret. Existing normalization techniques have limited applicability to these sample types. Here we present a data normalization method to minimize the effects of obscuring variation. We normalize peak areas using a batch-specific normalization process, which matches measured metabolites with isotope-labeled internal standards that behave similarly during the analysis. This method, called best-matched internal standard (B-MIS) normalization, can be applied to targeted or untargeted metabolomics data sets and yields relative concentrations. We evaluate and demonstrate the utility of B-MIS normalization using marine environmental samples and laboratory grown cultures of phytoplankton. In untargeted analyses, B-MIS normalization allowed for inclusion of mass features in downstream analyses that would have been considered unreliable without normalization due to obscuring variation. B-MIS normalization for targeted or untargeted metabolomics is freely available at https://github.com/IngallsLabUW/B-MIS-normalization .

Four mesophilic, neutrophilic, and aerobic marine ammonia-oxidizing archaea, designated strains SCM1 T , HCA1 T , HCE1 T and PS0 T , were isolated from a tropical marine fish tank, dimly lit deep coastal waters, the lower euphotic zone of coastal waters, and near-surface sediment in the Puget Sound estuary, respectively. Cells are straight or slightly curved small rods, 0.15-0.26 µm in diameter and 0.50-1.59 µm in length. Motility was not observed, although strain PS0 T possesses genes associated with archaeal flagella and chemotaxis, suggesting it may be motile under some conditions. Cell membranes consist of glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, with crenarchaeol as the major component. Strain SCM1 T displays a single surface layer (S-layer) with p6 symmetry, distinct from the p3-S-layer reported for the soil ammonia-oxidizing archaeon Nitrososphaera viennensis EN76 T . Respiratory quinones consist of fully saturated and monounsaturated menaquinones with 6 isoprenoid units in the side chain. Cells obtain energy from ammonia oxidation and use carbon dioxide as carbon source; addition of an α-keto acid (α-ketoglutaric acid) was necessary to sustain growth of strains HCA1 T , HCE1 T , and PS0 T . Strain PS0 T uses urea as a source of ammonia for energy production and growth. All strains synthesize vitamin B1 (thiamine), B2 (riboflavin), B6 (pyridoxine), and B12 (cobalamin). Optimal growth occurs between 25 and 32 °C, between pH 6.8 and 7.3, and between 25 and 37 ‰ salinity. All strains have a low mol% G+C content of 33.0-34.2. Strains are related by 98 % or greater 16S rRNA gene sequence identity, sharing ~85 % 16S rRNA gene sequence identity with Nitrososphaera viennensis EN76 T . All four isolates are well separated by phenotypic and genotypic characteristics and are here assigned to distinct species within the genus Nitrosopumilus gen. nov. Isolates SCM1 T (=ATCC TSD-97 T =NCIMB 15022 T ), HCA1 T (=ATCC TSD-96 T ), HCE1 T (=ATCC TSD-98 T ), and PS0 T (=ATCC TSD-99 T ) are type strains of the species Nitrosopumilusmaritimus sp. nov., Nitrosopumilus cobalaminigenes sp. nov., Nitrosopumilus oxyclinae sp. nov., and Nitrosopumilus ureiphilus sp. nov., respectively. In addition, we propose the family Nitrosopumilaceae fam. nov. and the order Nitrosopumilales ord. nov. within the class Nitrososphaeria.

Recent studies point to the importance of oxygen (O 2 ) in controlling the distribution and activity of marine ammonia-oxidizing archaea (AOA), one of the most abundant prokaryotes in the ocean. The AOA are associated with regions of low O 2 tension in oceanic oxygen minimum zones (OMZs), and O 2 availability is suggested to influence their production of the ozone-depleting greenhouse gas nitrous oxide (N 2 O). We show that marine AOA available in pure culture sustain high ammonia oxidation activity at low μM O 2 concentrations, characteristic of suboxic regions of OMZs (<10 µM O 2 ), and that atmospheric concentrations of O 2 may inhibit the growth of some environmental populations. We quantify the increasing N 2 O production by marine AOA with decreasing O 2 tensions, consistent with the plausibility of an AOA contribution to the accumulation of N 2 O at the oxic-anoxic redox boundaries of OMZs. Variable sensitivity to peroxide also suggests that endogenous or exogenous reactive oxygen species are of importance in determining the environmental distribution of some populations., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Organisms within all domains of life require the cofactor cobalamin (vitamin B 12 ), which is produced only by a subset of bacteria and archaea. On the basis of genomic analyses, cobalamin biosynthesis in marine systems has been inferred in three main groups: select heterotrophic Proteobacteria, chemoautotrophic Thaumarchaeota, and photoautotrophic Cyanobacteria. Culture work demonstrates that many Cyanobacteria do not synthesize cobalamin but rather produce pseudocobalamin, challenging the connection between the occurrence of cobalamin biosynthesis genes and production of the compound in marine ecosystems. Here we show that cobalamin and pseudocobalamin coexist in the surface ocean, have distinct microbial sources, and support different enzymatic demands. Even in the presence of cobalamin, Cyanobacteria synthesize pseudocobalamin-likely reflecting their retention of an oxygen-independent pathway to produce pseudocobalamin, which is used as a cofactor in their specialized methionine synthase (MetH). This contrasts a model diatom, Thalassiosira pseudonana, which transported pseudocobalamin into the cell but was unable to use pseudocobalamin in its homolog of MetH. Our genomic and culture analyses showed that marine Thaumarchaeota and select heterotrophic bacteria produce cobalamin. This indicates that cobalamin in the surface ocean is a result of de novo synthesis by heterotrophic bacteria or via modification of closely related compounds like cyanobacterially produced pseudocobalamin. Deeper in the water column, our study implicates Thaumarchaeota as major producers of cobalamin based on genomic potential, cobalamin cell quotas, and abundance. Together, these findings establish the distinctive roles played by abundant prokaryotes in cobalamin-based microbial interdependencies that sustain community structure and function in the ocean., Competing Interests: The authors declare no conflict of interest.

Marine ammonia-oxidizing archaea (AOA) are among the most abundant of marine microorganisms, spanning nearly the entire water column of diverse oceanic provinces. Historical patterns of abundance are preserved in sediments in the form of their distinctive glycerol dibiphytanyl glycerol tetraether (GDGT) membrane lipids. The correlation between the composition of GDGTs in surface sediment and the overlying annual average sea surface temperature forms the basis for a paleotemperature proxy (TEX86) that is used to reconstruct surface ocean temperature as far back as the Middle Jurassic. However, mounting evidence suggests that factors other than temperature could also play an important role in determining GDGT distributions. We here use a study set of four marine AOA isolates to demonstrate that these closely related strains generate different TEX86-temperature relationships and that oxygen (O2) concentration is at least as important as temperature in controlling TEX86 values in culture. All of the four strains characterized showed a unique membrane compositional response to temperature, with TEX86-inferred temperatures varying as much as 12 °C from the incubation temperatures. In addition, both linear and nonlinear TEX86-temperature relationships were characteristic of individual strains. Increasing relative abundance of GDGT-2 and GDGT-3 with increasing O2 limitation, at the expense of GDGT-1, led to significant elevations in TEX86-derived temperature. Although the adaptive significance of GDGT compositional changes in response to both temperature and O2 is unclear, this observation necessitates a reassessment of archaeal lipid-based paleotemperature proxies, particularly in records that span low-oxygen events or underlie oxygen minimum zones.

Nitrification is a critical process for the balance of reduced and oxidized nitrogen pools in nature, linking mineralization to the nitrogen loss processes of denitrification and anammox. Recent studies indicate a significant contribution of ammonia-oxidizing archaea (AOA) to nitrification. However, quantification of the relative contributions of AOA and ammonia-oxidizing bacteria (AOB) to in situ ammonia oxidation remains challenging. We show here the production of nitric oxide (NO) by Nitrosopumilus maritimus SCM1. Activity of SCM1 was always associated with the release of NO with quasi-steady state concentrations between 0.05 and 0.08 μM. NO production and metabolic activity were inhibited by the nitrogen free radical scavenger 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). Comparison of marine and terrestrial AOB strains with SCM1 and the recently isolated marine AOA strain HCA1 demonstrated a differential sensitivity of AOB and AOA to PTIO and allylthiourea (ATU). Similar to the investigated AOA strains, bulk water column nitrification at coastal and open ocean sites with sub-micromolar ammonia/ammonium concentrations was inhibited by PTIO and insensitive to ATU. These experiments support predictions from kinetic, molecular and biogeochemical studies, indicating that marine nitrification at low ammonia/ammonium concentrations is largely driven by archaea and suggest an important role of NO in the archaeal metabolism., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Interactions between primary producers and bacteria impact the physiology of both partners, alter the chemistry of their environment, and shape ecosystem diversity. In marine ecosystems, these interactions are difficult to study partly because the major photosynthetic organisms are microscopic, unicellular phytoplankton. Coastal phytoplankton communities are dominated by diatoms, which generate approximately 40% of marine primary production and form the base of many marine food webs. Diatoms co-occur with specific bacterial taxa, but the mechanisms of potential interactions are mostly unknown. Here we tease apart a bacterial consortium associated with a globally distributed diatom and find that a Sulfitobacter species promotes diatom cell division via secretion of the hormone indole-3-acetic acid, synthesized by the bacterium using both diatom-secreted and endogenous tryptophan. Indole-3-acetic acid and tryptophan serve as signalling molecules that are part of a complex exchange of nutrients, including diatom-excreted organosulfur molecules and bacterial-excreted ammonia. The potential prevalence of this mode of signalling in the oceans is corroborated by metabolite and metatranscriptome analyses that show widespread indole-3-acetic acid production by Sulfitobacter-related bacteria, particularly in coastal environments. Our study expands on the emerging recognition that marine microbial communities are part of tightly connected networks by providing evidence that these interactions are mediated through production and exchange of infochemicals.

Rationale: Vitamin B(12) is an essential nutrient for more than half of surveyed marine algae species, but methods for directly measuring this important cofactor in seawater are limited. Current mass spectrometry methods do not quantify all forms of B(12), potentially missing a significant portion of the B(12) pool., Methods: We present a method to measure vitamins B(1), B(2), B(6), B(7) and four forms of B(12) dissolved in seawater. The method entails solid-phase extraction, separation by ultra-performance liquid chromatography, and detection by triple-quadrupole tandem mass spectrometry using stable-isotope-labeled internal standards. We demonstrated the use of this method in the environment by analyzing B(12) concentrations at different depths in the Hood Canal, part of the Puget Sound estuarine system in Washington State., Results: Recovery of vitamin B(12) forms during the preconcentration steps was >71% and the limits of detection were <0.275 pM in seawater. Standard addition calibration curves in three different seawater matrices were used to determine analytical response and to quantify samples from the environment. Hydroxocobalamin was the main form of B(12) in seawater at our field site., Conclusions: We developed a method for quantifying four forms of B(12) in seawater by liquid chromatography/mass spectrometry with the option of simultaneous analysis of vitamins B(1), B(2), B(6), and B(7). We validated the method and demonstrated its application in the field., (Copyright © 2014 John Wiley & Sons, Ltd.)

Ammonia-oxidizing archaea (AOA) are now implicated in exerting significant control over the form and availability of reactive nitrogen species in marine environments. Detailed studies of specific metabolic traits and physicochemical factors controlling their activities and distribution have not been well constrained in part due to the scarcity of isolated AOA strains. Here, we report the isolation of two new coastal marine AOA, strains PS0 and HCA1. Comparison of the new strains to Nitrosopumilus maritimus strain SCM1, the only marine AOA in pure culture thus far, demonstrated distinct adaptations to pH, salinity, organic carbon, temperature, and light. Strain PS0 sustained nearly 80% of ammonia oxidation activity at a pH as low as 5.9, indicating that coastal strains may be less sensitive to the ongoing reduction in ocean pH. Notably, the two novel isolates are obligate mixotrophs that rely on uptake and assimilation of organic carbon compounds, suggesting a direct coupling between chemolithotrophy and organic matter assimilation in marine food webs. All three isolates showed only minor photoinhibition at 15 µE ⋅ m(-2) ⋅ s(-1) and rapid recovery of ammonia oxidation in the dark, consistent with an AOA contribution to the primary nitrite maximum and the plausibility of a diurnal cycle of archaeal ammonia oxidation activity in the euphotic zone. Together, these findings highlight an unexpected adaptive capacity within closely related marine group I Archaea and provide new understanding of the physiological basis of the remarkable ecological success reflected by their generally high abundance in marine environments.

Archaeal ammonia oxidizers (AOAs) are increasingly recognized as prominent members of natural microbial assemblages. Evidence that links the presence of AOA with in situ ammonia oxidation activity is limited, and the abiotic factors that regulate the distribution of AOA natural assemblages are not well defined. We used quantitative PCR to enumerate amoA (encodes α-subunit of ammonia monooxygenase) abundances; AOA amoA gene copies greatly outnumbered ammonia-oxidizing bacteria and amoA transcripts were derived primarily from AOA throughout the water column of Hood Canal, Puget Sound, WA, USA. We generated a Michaelis-Menten kinetics curve for ammonia oxidation by the natural community and found that the measured Km of 98±14 nmol l(-1) was close to that for cultivated AOA representative Nitrosopumilus maritimus SCM1. Temperature did not have a significant effect on ammonia oxidation rates for incubation temperatures ranging from 8 to 20 °C, which is within the temperature range for depths of measurable ammonia oxidation at the site. This study provides substantial evidence, through both amoA gene copies and transcript abundances and the kinetics response, that AOA are the dominant active ammonia oxidizers in this marine environment. We propose that future ammonia oxidation experiments use a Km for the natural community to better constrain ammonia oxidation rates determined with the commonly used (15)NH4(+) dilution technique.

Sixty percent of the world ocean by area is contained in oligotrophic gyres [Longhurst A (1995) Prog Oceanog 36:77-16], the biomass of which is dominated by picophytoplankton, including cyanobacteria and picoeukaryotic algae, as well as picoheterotrophs. Despite their recognized importance in carbon cycling in the surface ocean, the role of small cells and their detrital remains in the transfer of particulate organic matter (POM) to the deep ocean remains disputed. Because oligotrophic marine conditions are projected to expand under current climate trends, a better understanding of the role of small particles in the global carbon cycle is a timely goal. Here we use the lipid profiles, radiocarbon, and stable carbon isotopic signatures of lipids from the North Pacific Subtropical Gyre to show that in the surface ocean, lipids from submicron POM (here called extra-small POM) are distinct from larger classes of suspended POM. Remarkably, this distinct extra-small POM signature dominates the total lipids collected at mesopelagic depth, suggesting that the lipid component of mesopelagic POM primarily contains the exported remains of small particles. Transfer of submicron material to mesopelagic depths in this location is consistent with model results that claim the biological origin of exported carbon should be proportional to the distribution of cell types in the surface community, irrespective of cell size [Richardson TL, Jackson GA (2007) Science 315:838-840]. Our data suggest that the submicron component of exported POM is an important contributor to the global biological pump, especially in oligotrophic waters.

Organic molecules from known biological sources (biomarkers) that are preserved over geological time are critical tools in the study of past conditions and events on earth. Polar molecules are typically recycled rapidly in marine environments and do not survive burial within aquatic sediments in unambiguously recognizable form. As such, geological biomarkers are formed almost exclusively from precursor biomolecules that have been altered, limiting their utility as paleoproxies. Here, we report that nitrogen-rich aliphatic long-chain polyamines (LCPAs), biosynthesized by diatoms in species-specific assemblages for the precipitation of nanopatterned siliceous cell walls (frustules), are preserved unaltered in the oldest available diatom fossils dating to the Lower Cretaceous (early Albian, 115-110 Ma). We further show that the cumulative LCPA pool accounts for 60% of the total C and 80% of the total N preserved in the Cretaceous age sediments. We suggest that silica glass formation by diatoms constitutes an important preservation mechanism for source-specific, polar biomolecules, protecting them indefinitely by encapsulation within the silicified frustule. LCPAs are a unique, source-specific carbon and nitrogen archive of diatom biomass, offering a promising tool for reconstruction of global cycles of carbon and nitrogen over geological timescales., (© 2013 Blackwell Publishing Ltd.)

There is great interest in the membrane lipids of archaea (glycerol dialkyl glycerol tetraethers [GDGTs]) as tracers of archaeal biomass because of their utility as paleoproxies and because of the biogeochemical importance of archaea. While core GDGTs (formed by hydrolysis of polar head groups of intact GDGTs after cell death) are appropriate for paleostudies, they have also been used to trace archaeal populations. Also, despite the small size (0.2 by 0.7 μm) of cultivated marine archaea, 0.7-μm glass-fiber filters (GFFs) are typically used to collect GDGTs from natural waters. We quantified both core and intact GDGTs in free-living (0.2- to 0.7-μm), suspended (0.7- to 60-μm), and aggregate (>60-μm) particle size fractions in Puget Sound (Washington State). On average, the free-living fraction contained 36% of total GDGTs, 90% of which were intact. The intermediate-size fraction contained 62% of GDGTs, and 29% of these were intact. The aggregate fraction contained 2% of the total GDGT pool, and 29% of these were intact. Our results demonstrate that intact GDGTs are largely in the free-living fraction. Because only intact GDGTs are present in living cells, protocols that target this size fraction and analyze the intact GDGT pool are necessary to track living populations in marine waters. Core GDGT enrichment in larger-size fractions indicates that archaeal biomass may quickly become attached or entrained in particles once the archaea are dead or dying. While the concentrations of the two pools were generally not correlated, the similar sizes of the core and intact GDGT pools suggest that core GDGTs are removed from the water column on timescales similar to those of cell replication, on timescales of days to weeks.

Natural polyamines are found in all three domains of life and long-chain polyamines (LCPAs) play a special role in silicifying organisms such as diatoms and sponges where they are actively involved in the complex formation and nanopatterning of siliceous structures. With chain lengths extending up to 20 N-methylated propylamine repeat units, diatom LCPAs constitute the longest natural polyamines. Mixtures of natural LCPAs are typically purified in bulk using ion-exchange, size-exclusion and dialysis and then analyzed either by direct infusion mass spectrometry or by MALDI-TOF. Here, we describe a novel ion-pairing liquid chromatographic method that allows baseline separation, detection and structural elucidation of underivatized aliphatic methylated and non-methylated LCPAs with a wide range of chain lengths. Complete separation of synthetic mixtures of LCPA species differing by either a propylamine or an N-methylpropylamine unit is achievable using this method and chromatographic separation of natural, diatom frustule bound LCPAs extracted from sediment core samples is greatly improved. Using electrospray ionization mass spectrometry (ESI-MS), we detected singly [M+H](+) and multiply [M+nH](n+) charged protonated ions. The abundance of multiply charged LCPA species increased linearly as a function of LCPA chain length (N) and multiprotonated ions [M+nH](n+) were more abundant for longer chain polyamines. The abundance of multiply charged LCPAs along with the concomitant disappearance of the singly charged protonated molecular ion significantly increases the complexity of the MS spectra, justifying the need for good chromatographic separation of complex LCPA mixtures. This analytical procedure will likely constitute a powerful tool for the characterization, quantification, as well as the purification of individual LCPAs in natural and synthetic samples for studies of silica precipitation as well as nitrogen and carbon isotopic analysis used in paleoceanographic studies., (Copyright © 2011 John Wiley & Sons, Ltd.)

The disintegration of ice shelves, reduced sea-ice and glacier extent, and shifting ecological zones observed around Antarctica highlight the impact of recent atmospheric and oceanic warming on the cryosphere. Observations and models suggest that oceanic and atmospheric temperature variations at Antarctica's margins affect global cryosphere stability, ocean circulation, sea levels and carbon cycling. In particular, recent climate changes on the Antarctic Peninsula have been dramatic, yet the Holocene climate variability of this region is largely unknown, limiting our ability to evaluate ongoing changes within the context of historical variability and underlying forcing mechanisms. Here we show that surface ocean temperatures at the continental margin of the western Antarctic Peninsula cooled by 3-4 °C over the past 12,000 years, tracking the Holocene decline of local (65° S) spring insolation. Our results, based on TEX(86) sea surface temperature (SST) proxy evidence from a marine sediment core, indicate the importance of regional summer duration as a driver of Antarctic seasonal sea-ice fluctuations. On millennial timescales, abrupt SST fluctuations of 2-4 °C coincide with globally recognized climate variability. Similarities between our SSTs, Southern Hemisphere westerly wind reconstructions and El Niño/Southern Oscillation variability indicate that present climate teleconnections between the tropical Pacific Ocean and the western Antarctic Peninsula strengthened late in the Holocene epoch. We conclude that during the Holocene, Southern Ocean temperatures at the western Antarctic Peninsula margin were tied to changes in the position of the westerlies, which have a critical role in global carbon cycling.

Several lines of evidence indicate that microorganisms in the meso- and bathypelagic ocean are metabolically active and respiring carbon. In addition, growing evidence suggests that archaea are fixing inorganic carbon in this environment. However, direct quantification of the contribution from deep ocean carbon sources to community production in the dark ocean remains a challenge. In this study, carbon flow through the microbial community at 2 depths in the mesopelagic zone of the North Pacific Subtropical Gyre was examined by exploiting the unique radiocarbon signatures (Delta(14)C) of the 3 major carbon sources in this environment. The radiocarbon content of nucleic acids, a biomarker for viable cells, isolated from size-fractionated particles (0.2-0.5 microm and >0.5 microm) showed the direct incorporation of carbon delivered by rapidly sinking particles. Most significantly, at the 2 mesopelagic depths examined (670 m and 915 m), carbon derived from in situ autotrophic fixation supported a significant fraction of the free-living microbial community (0.2-0.5 microm size fraction), but the contribution of chemoautotrophy varied markedly between the 2 depths. Results further showed that utilization of the ocean's largest reduced carbon reservoir, (14)C-depleted, dissolved organic carbon, was negligible in this environment. This isotopic portrait of carbon assimilation by the in situ, free-living microbial community, integrated over >50,000 L of seawater, implies that recent, photosynthetic carbon is not always the major carbon source supporting microbial community production in the mesopelagic realm.

The widespread occurrence and diversity of ammonia oxidizing Archaea suggests their contribution to the nitrogen cycle is of global significance. Their distribution appeared limited to low- and moderate-temperature environments until the recent finding of a diagnostic membrane lipid, crenarchaeol, in terrestrial hot springs. We report here the cultivation of a thermophilic nitrifier ('Candidatus Nitrosocaldus yellowstonii'), an autotrophic crenarchaeote growing up to 74 degrees C by aerobic ammonia oxidation. The major core lipid of this archaeon growing at 72 degrees C is crenarchaeol, providing the first direct evidence for its synthesis by a thermophile. These findings greatly extend the upper temperature limit of nitrification and document that the capacity for ammonia oxidation is broadly distributed among the Crenarchaeota.

An ammonia-oxidizing, carbon-fixing archaeon, Candidatus "Nitrosopumilus maritimus," recently was isolated from a salt-water aquarium, definitively confirming that chemoautotrophy exists among the marine archaea. However, in other incubation studies, pelagic archaea also were capable of using organic carbon. It has remained unknown what fraction of the total marine archaeal community is autotrophic in situ. If archaea live primarily as autotrophs in the natural environment, a large ammonia-oxidizing population would play a significant role in marine nitrification. Here we use the natural distribution of radiocarbon in archaeal membrane lipids to quantify the bulk carbon metabolism of archaea at two depths in the subtropical North Pacific gyre. Our compound-specific radiocarbon data show that the archaea in surface waters incorporate modern carbon into their membrane lipids, and archaea at 670 m incorporate carbon that is slightly more isotopically enriched than inorganic carbon at the same depth. An isotopic mass balance model shows that the dominant metabolism at depth indeed is autotrophy (83%), whereas heterotrophic consumption of modern organic carbon accounts for the remainder of archaeal biomass. These results reflect the in situ production of the total community that produces tetraether lipids and are not subject to biases associated with incubation and/or culture experiments. The data suggest either that the marine archaeal community includes both autotrophs and heterotrophs or is a single population with a uniformly mixotrophic metabolism. The metabolic and phylogenetic diversity of the marine archaea warrants further exploration; these organisms may play a major role in the marine cycles of nitrogen and carbon.

Glycerol dialkyl glycerol tetraethers (GDGTs) are core membrane lipids of the Crenarchaeota. The structurally unusual GDGT crenarchaeol has been proposed as a taxonomically specific biomarker for the marine planktonic group I archaea. It is found ubiquitously in the marine water column and in sediments. In this work, samples of microbial community biomass were obtained from several alkaline and neutral-pH hot springs in Nevada, United States. Lipid extracts of these samples were analyzed by high-performance liquid chromatography-mass spectrometry and by gas chromatography-mass spectrometry. Each sample contained GDGTs, and among these compounds was crenarchaeol. The distribution of archaeal lipids in Nevada hot springs did not appear to correlate with temperature, as has been observed in the marine environment. Instead, a significant correlation with the concentration of bicarbonate was observed. Archaeal DNA was analyzed by denaturing gradient gel electrophoresis. All samples contained 16S rRNA gene sequences which were more strongly related to thermophilic crenarchaeota than to Cenarchaeum symbiosum, a marine nonthermophilic crenarchaeon. The occurrence of crenarchaeol in environments containing sequences affiliated with thermophilic crenarchaeota suggests a wide phenotypic distribution of this compound. The results also indicate that crenarchaeol can no longer be considered an exclusive biomarker for marine species.