Susan Q. Lang

Dissolved organic carbon measurement using a modified high-temperature combustion analyzer

The performance of a dissolved organic carbon (DOC) analyzer operating on the principle of high-temperature combustion (HTC) is subject to numerous design and operation characteristics. Here we describe modifications and performance tests of a commercial HTC analyzer (MQ Scientific, model MQ-1001), many of which are applicable to other HTC instruments. Design improvements include a new combustion column, auto-sampler needle assembly, and a smaller sparger/water trap that automatically maintains constant water level and pH. Techniques for monitoring and compensating for carrier gas flow rate fluctuations, as well as electronic improvements to the auto-sampler advance control and the high-pressure injection gas pulse, are also described. A new model LICOR 7000, nondispersive IR (NDIR) detector is shown to provide a 50-fold increase in sensitivity over the previous LICOR 6252 model. The total blank for the modified instrument is initially f27 ng C and declines during use to f9 ng C as the combustion column ages. For an instrument with a well-conditioned combustion column, approximately half of this background is resolvable into a reagent component coming largely from the deionized, UV-irradiated (DUV) water used to rinse the sample onto the combustion column: the other half is intrinsic to the instrument and appears associated with the quartz bead packing. Injection of varying volumes of DUV water with and without an added constant C background, indicates that our DUV water contains between 2 and 9 AM DOC, depending on the variable performance of the water purifier. Similar experiments indicate that the intrinsic instrument blank is variable over time and depends complexly on both the wait time between individual water injections and the overall time that the combustion column has been conditioned. The modified instrument fitted with the new LICOR 7000 detector measures 46.1F1.3 AM DOC in Sargasso Sea water reference material (44–45 AM) against a total instrument blank equivalent to about a fourth of this value. Overall, the modified MQ-1001 analyzer is capable of dependable, automated analysis of relatively challenging deep seawater samples with an average accuracy of about F3.8% of the consensus value. D 2003 Elsevier Science B.V. All rights reserved.

Stable isotope analysis of organic carbon in small (µg C) samples and dissolved organic matter using a GasBench preparation device

The stable isotopes of organic matter can provide valuable information on carbon cycling dynamics, microbial metabolisms, and past climates. Since bulk measurements may mask dynamic changes to critical portions of the organic pool, researchers are increasingly isolating individual compounds for isotopic analysis. The amount of carbon isolated is frequently small, requiring specialized equipment for its analysis. We present a simple and accurate method to measure the δ(13)C values of µg-amounts of organic compounds and dissolved organic matter in freshwaters using wet oxidation and a GasBench II preparation device. Samples containing 3 µg C can be analyzed with a precision of <0.4‰. For samples containing 1.2 µg C, the precision is <0.8‰. The blank is estimated to be ~0.2 µg C. The accuracy of the method is demonstrated for a wide range of compounds including those that are difficult to oxidize such as humic acid and phthalic acid. The δ(13)C values of DOC from river and riparian ground water determined by this method are comparable with those determined with an elemental analyzer on freeze-dried samples of DOC. The low detection limit and the ease with which it can be combined with isolation techniques such as liquid chromatography make this technique attractive for the off-line analysis of organic compounds, and open new possibilities for the development of methodologies for compound-specific carbon isotope analysis of complex mixtures separated by HPLC.

Record of archaeal activity at the serpentinite-hosted Lost City Hydrothermal Field.

Samples of young, outer surfaces of brucite-carbonate deposits from the ultramafic-hosted Lost City hydrothermal field were analyzed for DNA and lipid biomarker distributions and for carbon and hydrogen stable isotope compositions of the lipids. Methane-cycling archaeal communities, notably the Lost City Methanosarcinales (LCMS) phylotype, are specifically addressed. Lost City is unlike all other hydrothermal systems known to date and is characterized by metal- and CO2 -poor, high pH fluids with high H2 and CH4 contents resulting from serpentinization processes at depth. The archaeal fraction of the microbial community varies widely within the Lost City chimneys, from 1-81% and covaries with concentrations of hydrogen within the fluids. Archaeal lipids include isoprenoid glycerol di- and tetraethers and C25 and C30 isoprenoid hydrocarbons (pentamethylicosane derivatives - PMIs - and squalenoids). In particular, unsaturated PMIs and squalenoids, attributed to the LCMS archaea, were identified for the first time in the carbonate deposits at Lost City and probably record processes exclusively occurring at the surface of the chimneys. The carbon isotope compositions of PMIs and squalenoids are remarkably heterogeneous across samples and show highly (13) C-enriched signatures reaching δ(13) C values of up to +24.6‰. Unlike other environments in which similar structural and isotopic lipid heterogeneity has been observed and attributed to diversity in the archaeal assemblage, the lipids here appear to be synthesized solely by the LCMS. Some of the variations in lipid isotope signatures may, in part, be due to unusual isotopic fractionation during biosynthesis under extreme conditions. However, we argue that the diversity in archaeal abundances, lipid structure and carbon isotope composition rather reflects the ability of the LCMS archaeal biofilms to adapt to chemical gradients in the hydrothermal chimneys and possibly to perform either methanotrophy or methanogenesis using dissolved inorganic carbon, methane or formate as a function of the prevailing environmental conditions.

Investigations of potential microbial methanogenic and carbon monoxide utilization pathways in ultra-basic reducing springs associated with present-day continental serpentinization: the Tablelands, NL, CAN.

Ultra-basic reducing springs at continental sites of serpentinization act as portals into the biogeochemistry of a subsurface environment with H2 and CH4 present. Very little, however, is known about the carbon substrate utilization, energy sources, and metabolic pathways of the microorganisms that live in this ultra-basic environment. The potential for microbial methanogenesis with bicarbonate, formate, acetate, and propionate precursors and carbon monoxide (CO) utilization pathways were tested in laboratory experiments by adding substrates to water and sediment from the Tablelands, NL, CAD, a site of present-day continental serpentinization. Microbial methanogenesis was not observed after bicarbonate, formate, acetate, or propionate addition. CO was consumed in the live experiments but not in the killed controls and the residual CO in the live experiments became enriched in 13C. The average isotopic enrichment factor resulting from this microbial utilization of CO was estimated to be 11.2 ± 0.2‰. Phospholipid fatty acid concentrations and δ13C values suggest limited incorporation of carbon from CO into microbial lipids. This indicates that in our experiments, CO was used primarily as an energy source, but not for biomass growth. Environmental DNA sequencing of spring fluids collected at the same time as the addition experiments yielded a large proportion of Hydrogenophaga-related sequences, which is consistent with previous metagenomic data indicating the potential for these taxa to utilize CO.

Rapid 14C analysis of dissolved organic carbon in non-saline waters

The radiocarbon content of dissolved organic carbon (DOC) in rivers, lakes, and other non-saline waters can provide valuable information on carbon cycling dynamics in the environment. DOC is typically prepared for 14 C analysis by accelerator mass spectrometry (AMS) either by ultraviolet (UV) oxidation or by freeze-drying and sealed tube combustion. We present here a new method for the rapid analysis of 14 C of DOC using wet chemical oxidation (WCO) and automated headspace sampling of CO 2 . The approach is an adaption of recently developed methods using aqueous persulfate oxidant to determine the δ 13 C of DOC in non-saline water samples and the 14 C content of volatile organic acids. One advantage of the current method over UV oxidation is higher throughput: 22 samples and 10 processing standards can be prepared in one day and analyzed in a second day, allowing a full suite of 14 C processing standards and blanks to be run in conjunction with samples. A second advantage is that there is less potential for cross-contamination between samples.

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids (BPCA)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as char and soot. PyC is ubiquitous in the environment due to its long persistence, and its abundance might even increase with the projected increase in global wildfire activity and the continued burning of fossil fuel. PyC is also increasingly produced from the industrial pyrolysis of organic wastes, which yields charred soil amendments (biochar). Moreover, the emergence of nanotechnology may also result in the release of PyC-like compounds to the environment. It is thus a high priority to reliably detect, characterize and quantify these charred materials in order to investigate their environmental properties and to understand their role in the carbon cycle. Here, we present the benzene polycarboxylic acid (BPCA) method, which allows the simultaneous assessment of PyCs characteristics, quantity and isotopic composition (13C and 14C) on a molecular level. The method is applicable to a very wide range of environmental sample materials and detects PyC over a broad range of the combustion continuum, i.e., it is sensitive to slightly charred biomass as well as high temperature chars and soot. The BPCA protocol presented here is simple to employ, highly reproducible, as well as easily extendable and modifiable to specific requirements. It thus provides a versatile tool for the investigation of PyC in various disciplines, ranging from archeology and environmental forensics to biochar and carbon cycling research.

Exploring the metabolic potential of microbial communities in ultra‐basic, reducing springs at The Cedars, CA, USA: Experimental evidence of microbial methanogenesis and heterotrophic acetogenesis

Present-day serpentinization generates groundwaters with conditions (pH > 11, Eh < −550 mV) favorable for the microbial and abiotic production of organic compounds from inorganic precursors. Elevated concentrations of methane, C2-C6 alkanes, acetate, and formate have been detected at these sites, but the microbial or abiotic origin of these compounds remains unclear. While geochemical data indicate that methane at most sites of present-day serpentinization is abiogenic, the stable carbon, hydrogen, and clumped isotope data as well as the hydrocarbon gas composition from The Cedars, CA, USA, are consistent with a microbial origin for methane. However, there is no direct evidence of methanogenesis at this site of serpentinization. We report on laboratory experiments in which the microbial communities in fluids and sediments from The Cedars were incubated with 13C labeled substrates. Increasing methane concentrations and the incorporation of 13C into methane in live experiments, but not in killed controls, demonstrated that methanogens converted methanol, formate, acetate (methyl group), and bicarbonate to methane. The apparent fractionation between methane and potential substrates (α13CCH4-CO2(g) = 1.059 to 1.105, α13CCH4-acetate = 1.042 to 1.119) indicated that methanogenesis was dominated by the carbonate reduction pathway. Increasing concentrations of volatile organic acid anions indicated microbial acetogenesis. α13CCO2(g)-acetate values (0.999 to 1.000), however, were inconsistent with autotrophic acetogenesis, thus suggesting that acetate was produced through fermentation. This is the first study to show direct evidence of microbial methanogenesis and acetogenesis by the native microbial community at a site of present-day serpentinization.

Deeply-sourced formate fuels sulfate reducers but not methanogens at Lost City hydrothermal field

Hydrogen produced during water-rock serpentinization reactions can drive the synthesis of organic compounds both biotically and abiotically. We investigated abiotic carbon production and microbial metabolic pathways at the high energy but low diversity serpentinite-hosted Lost City hydrothermal field. Compound-specific 14C data demonstrates that formate is mantle-derived and abiotic in some locations and has an additional, seawater-derived component in others. Lipids produced by the dominant member of the archaeal community, the Lost City Methanosarcinales, largely lack 14C, but metagenomic evidence suggests they cannot use formate for methanogenesis. Instead, sulfate-reducing bacteria may be the primary consumers of formate in Lost City chimneys. Paradoxically, the archaeal phylotype that numerically dominates the chimney microbial communities appears ill suited to live in pure hydrothermal fluids without the co-occurrence of organisms that can liberate CO2. Considering the lack of dissolved inorganic carbon in such systems, the ability to utilize formate may be a key trait for survival in pristine serpentinite-hosted environments.

Metagenomic identification of active methanogens and methanotrophs in serpentinite springs of the Voltri Massif, Italy

The production of hydrogen and methane by geochemical reactions associated with the serpentinization of ultramafic rocks can potentially support subsurface microbial ecosystems independent of the photosynthetic biosphere. Methanogenic and methanotrophic microorganisms are abundant in marine hydrothermal systems heavily influenced by serpentinization, but evidence for methane-cycling archaea and bacteria in continental serpentinite springs has been limited. This report provides metagenomic and experimental evidence for active methanogenesis and methanotrophy by microbial communities in serpentinite springs of the Voltri Massif, Italy. Methanogens belonging to family Methanobacteriaceae and methanotrophic bacteria belonging to family Methylococcaceae were heavily enriched in three ultrabasic springs (pH 12). Metagenomic data also suggest the potential for hydrogen oxidation, hydrogen production, carbon fixation, fermentation, and organic acid metabolism in the ultrabasic springs. The predicted metabolic capabilities are consistent with an active subsurface ecosystem supported by energy and carbon liberated by geochemical reactions within the serpentinite rocks of the Voltri Massif.

Mineralizing Filamentous Bacteria from the Prony Bay Hydrothermal Field Give New Insights into the Functioning of Serpentinization-Based Subseafloor Ecosystems

Despite their potential importance as analogs of primitive microbial metabolisms, the knowledge of the structure and functioning of the deep ecosystems associated with serpentinizing environments is hampered by the lack of accessibility to relevant systems. These hyperalkaline environments are depleted in dissolved inorganic carbon (DIC), making the carbon sources and assimilation pathways in the associated ecosystems highly enigmatic. The Prony Bay Hydrothermal Field (PHF) is an active serpentinization site where, similar to Lost City (Mid-Atlantic Ridge), high-pH fluids rich in H2 and CH4 are discharged from carbonate chimneys at the seafloor, but in a shallower lagoonal environment. This study aimed to characterize the subsurface microbial ecology of this environment by focusing on the earliest stages of chimney construction, dominated by the discharge of hydrothermal fluids of subseafloor origin. By jointly examining the mineralogy and the microbial diversity of the conduits of juvenile edifices at the micrometric scale, we find a central role of uncultivated bacteria belonging to the Firmicutes in the ecology of the PHF. These bacteria, along with members of the phyla Acetothermia and Omnitrophica, are identified as the first chimneys inhabitants before archaeal Methanosarcinales. They are involved in the construction and early consolidation of the carbonate structures via organomineralization processes. Their predominance in the most juvenile and nascent hydrothermal chimneys, and their affiliation with environmental subsurface microorganisms, indicate that they are likely discharged with hydrothermal fluids from the subseafloor. They may thus be representative of endolithic serpentinization-based ecosystems, in an environment where DIC is limited. In contrast, heterotrophic and fermentative microorganisms may consume organic compounds from the abiotic by-products of serpentinization processes and/or from life in the deeper subsurface. We thus propose that the Firmicutes identified at PHF may have a versatile metabolism with the capability to use diverse organic compounds from biological or abiotic origin. From that perspective, this study sheds new light on the structure of deep microbial communities living at the energetic edge in serpentinites and may provide an alternative model of the earliest metabolisms.