Alex L. Sessions

Identification of Novel Methane-, Ethane-, and Propane-Oxidizing Bacteria at Marine Hydrocarbon Seeps by Stable Isotope Probing

ABSTRACT Marine hydrocarbon seeps supply oil and gas to microorganisms in sediments and overlying water. We used stable isotope probing (SIP) to identify aerobic bacteria oxidizing gaseous hydrocarbons in surface sediment from the Coal Oil Point seep field located offshore of Santa Barbara, California. After incubating sediment with 13C-labeled methane, ethane, or propane, we confirmed the incorporation of 13C into fatty acids and DNA. Terminal restriction fragment length polymorphism (T-RFLP) analysis and sequencing of the 16S rRNA and particulate methane monooxygenase (pmoA) genes in 13C-DNA revealed groups of microbes not previously thought to contribute to methane, ethane, or propane oxidation. First, 13C methane was primarily assimilated by Gammaproteobacteria species from the family Methylococcaceae, Gammaproteobacteria related to Methylophaga, and Betaproteobacteria from the family Methylophilaceae. Species of the latter two genera have not been previously shown to oxidize methane and may have been cross-feeding on methanol, but species of both genera were heavily labeled after just 3 days. pmoA sequences were affiliated with species of Methylococcaceae, but most were not closely related to cultured methanotrophs. Second, 13C ethane was consumed by members of a novel group of Methylococcaceae. Growth with ethane as the major carbon source has not previously been observed in members of the Methylococcaceae; a highly divergent pmoA-like gene detected in the 13C-labeled DNA may encode an ethane monooxygenase. Third, 13C propane was consumed by members of a group of unclassified Gammaproteobacteria species not previously linked to propane oxidation. This study identifies several bacterial lineages as participants in the oxidation of gaseous hydrocarbons in marine seeps and supports the idea of an alternate function for some pmoA-like genes.

Compound-Specific δ34S Analysis of Volatile Organics by Coupled GC/Multicollector-ICPMS

We have developed a highly sensitive and robust method for the analysis of delta(34)S in individual organic compounds by coupled gas chromatography (GC) and multicollector inductively coupled plasma mass spectrometry (MC-ICPMS). The system requires minimal alteration of commercial hardware and is amenable to virtually all sample introduction methods. Isobaric interference from O(2)(+) is minimized by employing dry plasma conditions and is cleanly resolved at all masses using medium resolution on the Thermo Neptune MC-ICPMS. Correction for mass bias is accomplished using standard-sample bracketing with peaks of SF(6) reference gas. The precision of measured delta(34)S values approaches 0.1 per thousand for analytes containing >40 pmol S and is better than 0.5 per thousand for those containing as little as 6 pmol S. This is within a factor of 2 of theoretical shot-noise limits. External accuracy is better than 0.3 per thousand. Integrating only the center of chromatographic peaks, rather than the entire peak, offers significant gain in precision and chromatographic resolution with minimal effect on accuracy but requires further study for verification as a routine method. Coelution of organic compounds that do not contain S can cause degraded analytical precision. Analyses of crude oil samples show wide variability in delta(34)S and demonstrate the robustness and precision of the method in complex environmental samples.

Sulfur isotopes of organic matter preserved in 3.45-billion-year-old stromatolites reveal microbial metabolism

The 3.45-billion-year-old Strelley Pool Formation of Western Australia preserves stromatolites that are considered among the oldest evidence for life on Earth. In places of exceptional preservation, these stromatolites contain laminae rich in organic carbon, interpreted as the fossil remains of ancient microbial mats. To better understand the biogeochemistry of these rocks, we performed microscale in situ sulfur isotope measurements of the preserved organic sulfur, including both Δ33S and . This approach allows us to tie physiological inference from isotope ratios directly to fossil biomass, providing a means to understand sulfur metabolism that is complimentary to, and independent from, inorganic proxies (e.g., pyrite). Δ33S values of the kerogen reveal mass-anomalous fractionations expected of the Archean sulfur cycle, whereas values show large fractionations at very small spatial scales, including values below -15‰. We interpret these isotopic patterns as recording the process of sulfurization of organic matter by H2S in heterogeneous mat pore-waters influenced by respiratory S metabolism. Positive Δ33S anomalies suggest that disproportionation of elemental sulfur would have been a prominent microbial process in these communities.

The RND-family transporter, HpnN, is required for hopanoid localization to the outer membrane of Rhodopseudomonas palustris TIE-1

Rhodopseudomonas palustris TIE-1 is a Gram-negative bacterium that produces structurally diverse hopanoid lipids that are similar to eukaryotic steroids. Its genome encodes several homologues to proteins involved in eukaryotic steroid trafficking. In this study, we explored the possibility that two of these proteins are involved in intracellular hopanoid transport. R. palustris has a sophisticated membrane system comprising outer, cytoplasmic, and inner cytoplasmic membranes. It also divides asymmetrically, producing a mother and swarmer cell. We deleted genes encoding two putative hopanoid transporters that belong to the resistance–nodulation–cell division superfamily. Phenotypic analyses revealed that one of these putative transporters (HpnN) is essential for the movement of hopanoids from the cytoplasmic to the outer membrane, whereas the other (Rpal_4267) plays a minor role. C30 hopanoids, such as diploptene, are evenly distributed between mother and swarmer cells, whereas hpnN is required for the C35 hopanoid, bacteriohopanetetrol, to remain localized to the mother cell type. Mutant cells lacking HpnN grow like the WT at 30 °C but slower at 38 °C. Following cell division at 38 °C, the ΔhpnN cells remain connected by their cell wall, forming long filaments. This phenotype may be attributed to hopanoid mislocalization because a double mutant deficient in both hopanoid biosynthesis and transport does not form filaments. However, the lack of hopanoids severely compromises cell growth at higher temperatures more generally. Because hopanoid mutants only manifest a strong phenotype under certain conditions, R. palustris is an attractive model organism in which to study their transport and function.

Quantifying Microbial Utilization of Petroleum Hydrocarbons in Salt Marsh Sediments by Using the 13C Content of Bacterial rRNA

ABSTRACT Natural remediation of oil spills is catalyzed by complex microbial consortia. Here we took a whole-community approach to investigate bacterial incorporation of petroleum hydrocarbons from a simulated oil spill. We utilized the natural difference in carbon isotopic abundance between a salt marsh ecosystem supported by the 13C-enriched C4 grass Spartina alterniflora and 13C-depleted petroleum to monitor changes in the 13C content of biomass. Magnetic bead capture methods for selective recovery of bacterial RNA were used to monitor the 13C content of bacterial biomass during a 2-week experiment. The data show that by the end of the experiment, up to 26% of bacterial biomass was derived from consumption of the freshly spilled oil. The results contrast with the inertness of a nearby relict spill, which occurred in 1969 in West Falmouth, MA. Sequences of 16S rRNA genes from our experimental samples also were consistent with previous reports suggesting the importance of Gamma- and Deltaproteobacteria and Firmicutes in the remineralization of hydrocarbons. The magnetic bead capture approach makes it possible to quantify uptake of petroleum hydrocarbons by microbes in situ. Although employed here at the domain level, RNA capture procedures can be highly specific. The same strategy could be used with genus-level specificity, something which is not currently possible using the 13C content of biomarker lipids.

Neoarchean carbonate–associated sulfate records positive Δ33S anomalies

Dissecting ancient microbial sulfur cycling Before the rise of oxygen, life on Earth depended on the marine sulfur cycle. The fractionation of different sulfur isotopes provides clues to which biogeochemical cycles were active long ago (see the Perspective by Ueno). Zhelezinskaia et al. found negative isotope anomalies in Archean rocks from Brazil and posit that metabolic fluxes from sulfate-reducing microorganisms influenced the global sulfur cycle, including sulfur in the atmosphere. In contrast, Paris et al. found positive isotope anomalies in Archean sediments from South Africa, implying that the marine sulfate pool was more disconnected from atmospheric sulfur. As an analog for the Archean ocean, Crowe et al. measured sulfur isotope signatures in modern Lake Matano, Indonesia, and suggest that low seawater sulfate concentrations restricted early microbial activity. Science, this issue p. 703, p. 742, p. 739; see also p. 735 Positive sulfur isotope anomalies hint at unexpected photochemical processes in Earth’s early atmosphere. [Also see Perspective by Ueno] Mass-independent fractionation of sulfur isotopes (reported as Δ33S) recorded in Archean sedimentary rocks helps to constrain the composition of Earth’s early atmosphere and the timing of the rise of oxygen ~2.4 billion years ago. Although current hypotheses predict uniformly negative Δ33S for Archean seawater sulfate, this remains untested through the vast majority of Archean time. We applied x-ray absorption spectroscopy to investigate the low sulfate content of particularly well-preserved Neoarchean carbonates and mass spectrometry to measure their Δ33S signatures. We report unexpected, large, widespread positive Δ33S values from stratigraphic sections capturing over 70 million years and diverse depositional environments. Combined with the pyrite record, these results show that sulfate does not carry the expected negative Δ33S from sulfur mass-independent fractionation in the Neoarchean atmosphere.

Organic Reference Materials for Hydrogen, Carbon, and Nitrogen Stable Isotope-Ratio Measurements: Caffeines, n-Alkanes, Fatty Acid Methyl Esters, Glycines, l-Valines, Polyethylenes, and Oils

An international project developed, quality-tested, and determined isotope-δ values of 19 new organic reference materials (RMs) for hydrogen, carbon, and nitrogen stable isotope-ratio measurements, in addition to analyzing pre-existing RMs NBS 22 (oil), IAEA-CH-7 (polyethylene foil), and IAEA-600 (caffeine). These new RMs enable users to normalize measurements of samples to isotope-δ scales. The RMs span a range of δ(2)H(VSMOW-SLAP) values from -210.8 to +397.0 mUr or ‰, for δ(13)C(VPDB-LSVEC) from -40.81 to +0.49 mUr and for δ(15)N(Air) from -5.21 to +61.53 mUr. Many of the new RMs are amenable to gas and liquid chromatography. The RMs include triads of isotopically contrasting caffeines, C16 n-alkanes, n-C20-fatty acid methyl esters (FAMEs), glycines, and l-valines, together with polyethylene powder and string, one n-C17-FAME, a vacuum oil (NBS 22a) to replace NBS 22 oil, and a (2)H-enriched vacuum oil. A total of 11 laboratories from 7 countries used multiple analytical approaches and instrumentation for 2-point isotopic normalization against international primary measurement standards. The use of reference waters in silver tubes allowed direct normalization of δ(2)H values of organic materials against isotopic reference waters following the principle of identical treatment. Bayesian statistical analysis yielded the mean values reported here. New RMs are numbered from USGS61 through USGS78, in addition to NBS 22a. Because of exchangeable hydrogen, amino acid RMs currently are recommended only for carbon- and nitrogen-isotope measurements. Some amino acids contain (13)C and carbon-bound organic (2)H-enrichments at different molecular sites to provide RMs for potential site-specific isotopic analysis in future studies.

Experimental determination of carbonate‐associated sulfate δ34S in planktonic foraminifera shells

Understanding the coupling of oxygen, carbon, and sulfur cycles in the past is critical for reconstructing the history of biogeochemical cycles, paleoclimatic variations, and oceanic chemistry. The abundance of sulfur isotopes (δ^(34)S) in sulfate from ancient marine carbonates, or carbonate-associated sulfate (CAS), is commonly used, along with other archives (mainly evaporites and barite), to estimate the δ^(34)S of seawater throughout Earth history. Analyses of CAS from hand-picked foraminifera are potentially valuable because this group of organisms is used in numerous paleoceanographic studies. They could provide coupled, high-resolution records of δ^(13)C, δ^(18)O, and δ^(34)S isotopic changes directly linked to orbitally tuned records of climate change through the Cenozoic. Such measurements have not previously been possible due to limitations of sensitivity in conventional IRMS-based techniques. However, the recent development of CAS analysis by multicollector inductively coupled plasma mass spectrometry (MC-ICP-MS) now allows us to work on samples containing just a few nmol of sulfur with accuracy for δ^(34)S values approaching 0.1‰ and, consequently, to analyze hand-picked samples of foraminifera shells. Here we report the results of culture experiments with the planktonic species Orbulina universa, that establish a shell:seawater δ^(34)S calibration for future applications to the fossil record. Our new method uses <650 μg of carbonate (∼15 shells) per analysis. The results show that S isotopes are fractionated consistently by −1‰ between seawater and O. universa tests. We also demonstrate that O. universa faithfully records the [SO^(2−)_(4)]/[Ca^(2+)] ratio of the seawater in which it grew.

Fractionation of Hydrogen Isotopes by Sulfate- and Nitrate-Reducing Bacteria

Hydrogen atoms from water and food are incorporated into biomass during cellular metabolism and biosynthesis, fractionating the isotopes of hydrogen—protium and deuterium—that are recorded in biomolecules. While these fractionations are often relatively constant in plants, large variations in the magnitude of fractionation are observed for many heterotrophic microbes utilizing different central metabolic pathways. The correlation between metabolism and lipid δ2H provides a potential basis for reconstructing environmental and ecological parameters, but the calibration dataset has thus far been limited mainly to aerobes. Here we report on the hydrogen isotopic fractionations of lipids produced by nitrate-respiring and sulfate-reducing bacteria. We observe only small differences in fractionation between oxygen- and nitrate-respiring growth conditions, with a typical pattern of variation between substrates that is broadly consistent with previously described trends. In contrast, fractionation by sulfate-reducing bacteria does not vary significantly between different substrates, even when autotrophic and heterotrophic growth conditions are compared. This result is in marked contrast to previously published observations and has significant implications for the interpretation of environmental hydrogen isotope data. We evaluate these trends in light of metabolic gene content of each strain, growth rate, and potential flux and reservoir-size effects of cellular hydrogen, but find no single variable that can account for the differences between nitrate- and sulfate-respiring bacteria. The emerging picture of bacterial hydrogen isotope fractionation is therefore more complex than the simple correspondence between δ2H and metabolic pathway previously understood from aerobes. Despite the complexity, the large signals and rich variability of observed lipid δ2H suggest much potential as an environmental recorder of metabolism.

Comparison of three methods for the methylation of aliphatic and aromatic compounds

RATIONALE Methylation protocols commonly call for acidic, hot conditions that are known to promote organic 1 H/2 H exchange in aromatic and aliphatic C-H bonds. Here we tested two such commonly used methods and compared a third that avoids these acidic conditions, to quantify isotope effects with each method and to directly determine acidic-exchange rates relevant to experimental conditions. METHODS We compared acidic and non-acidic methylation approaches catalyzed by hydrochloric acid, acetyl chloride and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)/DMAP (4-dimethylaminopyridine), respectively. These were applied to two analytes: phthalic acid (an aromatic) and octacosanoic acid (an aliphatic). We analyzed yield by gas chromatography/flame ionization (GC/FID) and hydrogen and carbon isotopic compositions by isotope ratio mass spectrometry (GC/IRMS). We quantified the 1 H/2 H exchange rate on dimethyl phthalate under acidic conditions with proton nuclear magnetic resonance (1 H-NMR) measurements. RESULTS The δ2 H and δ13 C values and yield were equivalent among the three methods for methyl octacosanoate. The two acidic methods resulted in comparable yield and isotopic composition of dimethyl phthalate; however, the non-acidic method resulted in lower δ2 H and δ13 C values perhaps due to low yields. Concerns over acid-catalyzed 1 H/2 H exchange are unwarranted as the effect was trivial over a 12-h reaction time. CONCLUSIONS We find product isolation yield and evaporation to be the main concerns in the accurate determination of isotopic composition. 1 H/2 H exchange reactions are too slow to cause measurable isotope fractionation over the typical duration and reaction conditions used in methylation. Thus, we are able to recommend continued use of acidic catalysts in such methylation reactions for both aliphatic and aromatic compounds.