A sealed-tube method for offline δ13 C analysis of CO2 via a Gas Bench II continuous-flow isotope ratio mass spectrometer.

RATIONALE: The isotopic measurement of environmental sample CO2 via isotope ratio mass spectrometry (IRMS) can present many analytical challenges. In many offline applications, exceedingly few samples can be prepared per day. In such applications, long-term storage (months) of sample CO2 is desirable, in order to accumulate enough samples to warrant a day of isotopic measurements. Conversely, traditional sample tube cracker systems for dual-inlet IRMS offer a capacity for only 6-8 tubes and thus limit throughput. Here we present a simple method to alleviate these concerns using a Gas Bench II gas handling device coupled with continuous-flow IRMS. METHODS: Sample preparation entails the cryogenic purification and quantification of CO2 on a vacuum line. Sample CO2 splits are expanded from a known volume to several sample ports and allowed to isotopically equilibrate (homogenize). Equilibrated CO2 splits are frozen into 3 mm outer diameter Pyrex break-seals and sealed under vacuum with a torch to a length of 5.5 cm. Sample break-seals are scored, placed into 12 mL Labco Exetainer® vials, purged with ultrahigh-purity helium, cracked inside the capped helium-flushed vials and subsequently measured via a Gas Bench equipped IRMS instrument using a CTC Analytics PAL autosampler. RESULTS: Our δ13 C results from NIST and internal isotopic standards, measured over a time period of several years, indicate that the sealed-tube method produces accurate δ13 C values to a precision of ±0.1‰ for samples containing 10-35 µgC. The tube cracking technique within Exetainer vials has been optimized over a period of 10 years, resulting in decreased sample failure rates from 5-10% to <1%. CONCLUSIONS: This technique offers an alternative method for δ13 C analyses of CO2 where offline isolation and long-term storage are desired. The method features a much higher sample throughput than traditional dual-inlet IRMS cracker setups at similar precision (±0.1‰).

Illuminating microbial species-specific effects on organic matter remineralization in marine sediments.

Marine microorganisms play a fundamental role in the global carbon cycle by mediating the sequestration of organic matter in ocean waters and sediments. A better understanding of how biological factors, such as microbial community composition, influence the lability and fate of organic matter is needed. Here, we explored the extent to which organic matter remineralization is influenced by species-specific metabolic capabilities. We carried out aerobic time-series incubations of Guaymas Basin sediments to quantify the dynamics of carbon utilization by two different heterotrophic marine isolates (Vibrio splendidus 1A01; Pseudoalteromonas sp. 3D05). Continuous measurement of respiratory CO2 production and its carbon isotopic compositions (13 C and 14 C) shows species-specific differences in the rate, quantity and type of organic matter remineralized. Each species was incubated with hydrothermally-influenced versus unimpacted sediments, resulting in a ~2-fold difference in respiratory CO2 yield across the experiments. Genomic analysis indicated that the observed carbon utilization patterns may be attributed in part to the number of gene copies encoding for extracellular hydrolytic enzymes. Our results demonstrate that the lability and remineralization of organic matter in marine environments is not only a function of chemical composition and/or environmental conditions, but also a function of the microorganisms that are present and active.

Oceanic efflux of ancient marine dissolved organic carbon in primary marine aerosol.

Breaking waves produce bubble plumes that burst at the sea surface, injecting primary marine aerosol (PMA) highly enriched with marine organic carbon (OC) into the atmosphere. It is widely assumed that this OC is modern, produced by present-day biological activity, even though nearly all marine OC is thousands of years old, produced by biological activity long ago. We used natural abundance radiocarbon (14C) measurements to show that 19 to 40% of the OC associated with freshly produced PMA was refractory dissolved OC (RDOC). Globally, this process removes 2 to 20 Tg of RDOC from the oceans annually, comparable to other RDOC losses. This process represents a major removal pathway for old OC from the sea, with important implications for oceanic and atmospheric biogeochemistry, the global carbon cycle, and climate.

Properties of Seawater Surfactants Associated with Primary Marine Aerosol Particles Produced by Bursting Bubbles at a Model Air-Sea Interface.

Surfactants account for minor fractions of total organic carbon in the ocean but can significantly influence the production of primary marine aerosol particles (PMA) at the sea surface via modulation of bubble surface tension. During September and October 2016, model PMA (mPMA) were produced from seawater by bursting bubbles at two biologically productive and two oligotrophic stations in the western North Atlantic Ocean. Total concentrations of surfactants extracted from mPMA and seawater were quantified and characterized via measurements of surface tension isotherms and critical micelle concentrations (CMCs). Surfactant CMCs in biologically productive seawater were lower than those in the oligotrophic seawater suggesting that surfactant mixtures in the two regions were chemically distinct. mPMA surfactants were enriched in all regions relative to those in the associated seawater. Surface tension isotherms indicate that mPMA surfactants were weaker than corresponding seawater surfactants. mPMA from biologically productive seawater contained higher concentrations of surfactants than those produced from oligotrophic seawater, supporting the hypothesis that seawater surfactant properties modulate mPMA surfactant concentrations. Diel variability in concentrations of seawater and mPMA surfactants in some regions is consistent with biological and/or photochemical processing. This work demonstrates direct links between surfactants in mPMA and those in the associated seawater.

Single-Cell Growth Rates in Photoautotrophic Populations Measured by Stable Isotope Probing and Resonance Raman Microspectrometry.

A new method to measure growth rates of individual photoautotrophic cells by combining stable isotope probing (SIP) and single-cell resonance Raman microspectrometry is introduced. This report explores optimal experimental design and the theoretical underpinnings for quantitative responses of Raman spectra to cellular isotopic composition. Resonance Raman spectra of isogenic cultures of the cyanobacterium, Synechococcus sp., grown in 13C-bicarbonate revealed linear covariance between wavenumber (cm-1) shifts in dominant carotenoid Raman peaks and a broad range of cellular 13C fractional isotopic abundance. Single-cell growth rates were calculated from spectra-derived isotopic content and empirical relationships. Growth rates among any 25 cells in a sample varied considerably; mean coefficient of variation, CV, was 29 ± 3% (σ/[Formula: see text]), of which only ~2% was propagated analytical error. Instantaneous population growth rates measured independently by in vivo fluorescence also varied daily (CV ≈ 53%) and were statistically indistinguishable from single-cell growth rates at all but the lowest levels of cell labeling. SCRR censuses of mixtures prepared from Synechococcus sp. and T. pseudonana (a diatom) populations with varying 13C-content and growth rates closely approximated predicted spectral responses and fractional labeling of cells added to the sample. This approach enables direct microspectrometric interrogation of isotopically- and phylogenetically-labeled cells and detects as little as 3% changes in cellular fractional labeling. This is the first description of a non-destructive technique to measure single-cell photoautotrophic growth rates based on Raman spectroscopy and well-constrained assumptions, while requiring few ancillary measurements.

Sequential bioavailability of sedimentary organic matter to heterotrophic bacteria.

Aquatic sediments harbour diverse microbial communities that mediate organic matter degradation and influence biogeochemical cycles. The pool of bioavailable carbon continuously changes as a result of abiotic processes and microbial activity. It remains unclear how microbial communities respond to heterogeneous organic matrices and how this ultimately affects heterotrophic respiration. To explore the relationships between the degradation of mixed carbon substrates and microbial activity, we incubated batches of organic-rich sediments in a novel bioreactor (IsoCaRB) that permitted continuous observations of CO2 production rates, as well as sequential sampling of isotopic signatures (δ13 C, Δ14 C), microbial community structure and diversity, and extracellular enzyme activity. Our results indicated that lower molecular weight (MW), labile, phytoplankton-derived compounds were degraded first, followed by petroleum-derived exogenous pollutants, and finally by higher MW polymeric plant material. This shift in utilization coincided with a community succession and increased extracellular enzyme activities. Thus, sequential utilization of different carbon pools induced changes at both the community and cellular level, shifting community composition, enzyme activity, respiration rates, and residual organic matter reactivity. Our results provide novel insight into the accessibility of sedimentary organic matter and demonstrate how bioavailability of natural organic substrates may affect the function and composition of heterotrophic bacterial populations.