B12B-01 INVITED 10:20h
Carbon-Isotope Fractionations of Autotrophic Bacteria: Relevance to Primary Production and Microbial Evolution in Hot Springs and Hydrothermal Vents
Terrestrial hot springs and marine hydrothermal vents are often dominated by autotrophic microorganisms. Species of the Bacteria Domain in these environments are known to use different pathways for CO$_{2}$ fixation. These may include the Calvin cycle, the Acetyl CoA pathway, the reverse TCA cycle, and the 3-HP pathway. Each cycle or pathway may be characterized by distinct patterns of carbon isotope fractionation. This presentation will summarize isotope fractionation patterns associated with known autotrophic bacteria and to use these patterns for interpreting natural isotopic variations. Examples will include hot springs from the Yellowstone National Park and Nevada desert, USA and Kamchatka, Russia, and hydrothermal vents from the East Pacific Rise. An attempt will be made to discuss isotopic variations within a particular pathway in the context of species evolution through horizontal gene transfer.
B12B-02 INVITED 10:35h
Compound Specific Isotope Analysis of Individual Biochemicals: Insights into Biogeochemistry
Stable isotopic determinations made on bulk organic materials are the weighted averages of the compositions of hundreds to thousands of chemical compounds, each of which has its own isotopic abundance. The stable isotope analysis of individual molecular components holds great potential as a method of tracing the source, biochemistry, diagenesis or indigeneity of a material. Through the advancements of continuous flow technologies, compound specific isotope analysis (CSIA) is unique in allowing for the resolution of a material's history or for partitioning the sources of organic inputs to a product or sink. Inscribed in the isotopic signature are indicators of the source material and pathways used in the formation of the compound. Perspectives on the use of this powerful tool will be presented. Isotope compositions of carbon and nitrogen on individual amino acids or of the isotopic compositions carbon on individual fatty acids or carbohydrates, hold the potential for delineation of production by an organism in an environment. The modification of the original signature can be used to suggest the contribution of new production of that same compound by microbial action or alteration of another substrate. Additionally, the partitioning of the signatures of individual amino acids between the D- and L- steroeoisomers can be utilized to further resolve new production within a sedimentary environment from the original primary photosynthetic source of production. Though standard mass balance approaches we suggest that perhaps a third of the organic matter preserved in an environment can be attributed to modified or new production from microbial processes. The questions as to the origins of individual molecular components, whether existing as environmental problems, or in potentially pristine materials, or serving as a proxy for bigger questions on depositional history or climate, can now be addressed. The tool of CSIA now uniquely offers an incredibly powerful probe into the origin of compound, and its history, and perhaps its eventual fate.
B12B-03 INVITED 10:50h
Application of compound-specific hydrogen isotope analyses to study anaerobic processes
Compound specific isotope analysis provides a potentially-powerful tool to study Earth system processes. In order to realize this goal we must first understand the many factors that impact isotopic distributions in nature. In pursuit of this goal we have initiated a series of investigations to assess the applicability of compound specific hydrogen isotope analyses for studying microbial processes in nature. Laboratory investigations thus far have focused on anaerobic microbial processes involving H$_{2}$, including methanogenesis and acetogenesis. Our experimental approach has been to grow pure and enrichment cultures of autotrophic, anaerobic bacteria and archaea under isotopically-defined conditions ($\delta$D of water and H$_{2}$ are known), and to relate these conditions to the $\delta$D of major metabolic products (such as methane and lipids). Investigations of methanogenic archaea indicate variable fractionation patterns between H$_{2}$, H$_{2}$O and CH$_{4}$. Fractionation is dependent on the microbial community structure and on the growth stage of the organism. Alternate biochemical routes of methanogenesis yield methane with $\delta$D variations as large as 150 per mil. These results help to explain much of the hydrogen isotope variability in methane from deep subsurface versus surficial environments. Studies with homoacetogenic bacteria show fractionations between H$_{2}$O and cellular lipids which are larger than has been observed in other culture studies. Results may be due to greater fractionation in the biochemical pathway, or may be due to the indirect incorporation of hydrogen atoms from H$_{2}$ into the lipids. Either way, these results indicate that the hydrogen isotopic composition of lipid biomarkers in nature may provide an indication of anoxia and/or H$_{2}$ cycling. All studies performed to date also indicate rapid isotopic equilibration of H$_{2}$ and H$_{2}$O, and indicate the likelihood of achieving isotopic equilibrium in nature. Ongoing investigations focus on the variability of hydrogen isotope fractionation in other microbial processes and on the hydrogen isotopic composition of organic metabolites in sediments and soils.
B12B-04 INVITED 11:05h
Relating species and functional diversity using stable isotope probing
Microbial communities play an essential role in biogeochemical cycles and analysis of laboratory cultures has provided much information on biochemical processes and physiological characteristics of functional groups of microorganisms responsible for these processes. However, the majority of microorganisms cannot be grown readily in laboratory culture and cultivation-independent molecular techniques are required for analysis of community structure and diversity. These techniques have demonstrated considerable microbial diversity in natural communities and have revealed the existence of abundant microorganisms belonging to novel, previously unsuspected microbial groups. Molecular analysis of natural communities typically provides little information on links between specific microorganisms and the biogeochemical processes that they carry out. We are therefore ignorant of the significance of microbial diversity for ecosystem processes and of the ecosystem function of uncultivated, but abundant microbial groups. Stable isotope probing enables identification of which members of a community are involved in the utilisation of specific substrates, particularly carbon substrates. It involves amendment of environmental samples, or field application with 13C-labelled carbon substrates and, after a period of exposure, extraction of nucleic acids and separation of 13C-labelled (heavy) and 12C-labelled (light) nucleic acid pools by density gradient centrifugation. The heavy nucleic acid pool will be derived only from organisms assimilating the labelled substrate.Molecular analysis of this pool provides information on identity and relative abundance of active members of the community. The technique therefore enables in situ functional analysis of microbial groups without the requirement for laboratory cultivation. Stable isotope probing has been used to determine which organisms are involved in the degradation of specific organic substrates, including recalcitrant compounds, and has been used, in field studies, to determine microorganisms responsible for utilisation of plant root exudates under different environmental conditions. It has the potential to increase greatly our understanding of the links between diversity and ecosystem function and identification of the role of novel, previously uncharacterised microbial functional groups.
B12B-05 11:20h
Benthic Microbial Response to Varying Organic Matter Inputs: An In-Situ Labeling Study in the Deep Sea
Particulate organic carbon (POC) arriving at the sea floor has been altered during vertical transport from surface waters, lateral transport along the continental slope or resuspension in the benthic boundary layer. Both availability and quality (reactivity) of POC play key roles for microbially mediated organic carbon turnover, thereby being critical for carbon cycling and transfer through the ocean. The export rate of POC is not a continuous process but strongly pulsed. To investigate the influence of POC quality on the benthic turnover rates, a series of in situ-experiments was carried out in the Benguela Upwelling Region (SW-Africa). Using a benthic chamber lander, we investigated the response of the sediment community on pulsed supplies of fresh and degraded $^{13}$C-labeled phytodetritus. Sediment community oxygen consumption (SCOC) and bacterial secondary production (BSP) did not differ significantly between the experimental treatments, showing that the artificial input did not alter the biogeochemical station characteristics. However, clear differences in benthic response were observed when following the added $^{13}$C-label. The mineralisation of the added material was 1.8 to 12.8 times higher for fresh than for altered phytodetritus. Highest recovery of label occurred at the sediment surface, with single indication for non-local transport within the uppermost 4cm of sediment. Following evidence that macro- as well as microorganisms play a major role in the early diagenesis of POC, label incorporation into macrofauna and bacterial fatty acids (PLFAs) was investigated. PLFA data suggest the presence of considerable numbers of sulphate-reducing bacteria, which displayed lower incorporation rates of added label relative to aerobic bacteria in comparable studies. The pathways for carbon cycling as well as the involvement of different benthic compartments seem to depend on the quality of POC arriving at the seafloor, thereby closely coupling its sources and transport to its diagenetic degradation via a biological link.
http://www.rcom-bremen.de
B12B-06 11:35h
Lipid Biomarkers and Stable Isotope Signatures of Microbial Mats in Hot Springs of Kamchatka, Russia
Various hot springs of the Uzon Caldera, Kamchatka, were analyzed for their chemical and stable isotope composition to better understand the relationship(s) between thermophilic microorganisms and the environments in which they live. The springs had water temperatures ranging from 40-90\deg C and pH ranging from 5.6-5.9. Gases that emanated from the springs were composed predominantly of CO$_{2}$ (20 to 90%), with lesser amounts of CH$_{4}$, ($<$ 20%), H$_{2}$, NH$_{3}$ and SO$_{2}$. Because the springs were acidic, they contained little dissolved inorganic carbon (DIC: millimol L$^{-1}$) and sulfide ($<$ 200 ppb), yet in some cases where microbial activity was relatively high, these constituents reached the millimol L$^{-1}$ and ppm range, respectively. Total biomass displayed a relatively large range of carbon isotope compositions that ranged from -5.7 to -22.4 per mil, which may reflect the large range of carbon sources, varied CO$_{2}$ fixation pathways, or other unknown mechanisms. Microbial mats were freeze-dried and extracted for lipid biomarker analysis. The lipids were separated into hydrocarbon, sterol, ether lipid, free fatty acid, and phospholipid fatty acid (PLFA) fractions. Among these fractions, PLFA indicated the community structure and abundance for Bacteria while the ether lipid fraction provided analogous information for Archaea. Results of PLFA showed 16:0 as the most abundant fatty acid (33-44%), which is universal in all living organisms. Other significant biomarkers included 18:1$\omega$ (19 to 24%), 18:2$\omega$ (5 to 13%), 16:1$\omega$ (3 to 12%), and 18:0 (2 to 7%). These biomarkers are characteristic of cyanobacteria, green-sulfur bacteria, and green non-sulfur bacteria, respectively, which are common autotrophic organisms in terrestrial hot springs. On the other hand, biomarkers of heterotrophic bacteria, such as iso- and anteiso-15:0 were low (2-8%), indicating that the bacterial carbon cycle was dominated by autotrophic organisms. Analogous archaeal constituents were present in significant abundance in the ether lipids fraction.
B12B-07 11:50h
Phosphate Oxygen Isotopes as a Tracer for Sources and Cycling of Phosphate in San Francisco Bay
Phosphorous is an essential macro-nutrient for primary productivity, but tracing sources and cycling of P in marine systems has been difficult to assess because P has only one stable isotope and can not be used as an isotopic tracer. Recently a new technique (McLaughlin et al., 2004) has been developed to track sources and cycling of phosphate in aquatic systems. This approach takes advantage of the strong P-O bond in phosphate, which is resistant to inorganic hydrolysis. The exchange of oxygen isotopes therein only occurs due to intracellular biological cycling. Because the d18O of phosphate will largely be determined by the isotopic composition of the water in which it is being recycled and because the isotopic composition of rivers and oceans is significantly different, the d18O of phosphate may be used as a tracer for different sources of phosphate to an estuarine system which is not phosphate limited. Consequently, the d18O of phosphate may be useful for quantifying the mixing of different sources of phosphate in estuarine systems. We applied this method to enhance our understanding of P sources and cycling in the San Francisco Bay. To this end we conducted four sampling transects from Coyote Creek in the South Bay to the Sacramento and San Joaquin Rivers in the North between October 2002 and August 2004. Phosphate d18O ranged from 10.1 to 20.1 per mil, with highest values at the Golden Gate and lowest at the San Joaquin River. Most of the Bay samples showed strong positive correlations with salinity, water d18O, and the inverse of phosphate concentration, suggesting a simple two-component mixing of oceanic and riverine sources. These data suggest that phosphate d18O can be an effective tool for identifying P point sources and understanding phosphate dynamics in the ecosystem.
B12B-08 12:05h
Natural abundance 14C analysis of biomarkers: A further tool for understanding microbial metabolism in the environment
On April 27, 2004 the barge Bouchard 120 spilled ~400,000 liters of No. 6 fuel oil into Buzzards Bay, MA and contaminated ~200 km of rocky and sandy beaches. To investigate the short-term fate of the spilled oil on one of the rocky beaches (Nyes Neck), oil-covered rocks were collected and analyzed by comprehensive two-dimensional gas chromatography (GCxGC). Within several weeks, the major removal processes were evaporation and water-washing, but microbial degradation of n-alkanes became evident in mid June 2004 and continued through July and August. In order to confirm that the observed losses of n-alkanes were due to microbial activity, select rocks collected in mid July 2004 were analyzed for the stable carbon and radiocarbon content of phospholipid fatty acids (PLFA). These results indicate that the active community living on these oil-covered rocks was predominated by algae due to the presence of polyunsaturated C:20 PLFA that represented 48.4 mole percent of the PLFA at the site. The ubiquitous C16:0 and C18:0 PLFA made up 18.9 and 15.2 mole percent respectively, and PLFA commonly associated with bacteria, C16:1, C17:0, C18:unsaturated, and C20:0 made up a cumulative 14.4 mole percent. Compound specific 13C analysis yielded ambiguous results and gave no clear indication of petroleum metabolism by the microbial community. In contrast, 14C analysis of the individual PLFA clearly demonstrated that microbial uptake of the petroleum carbon was occurring. The 14C content of the algal PLFA (33.4 permil) closely matches that of Buzzards Bay dissolved inorganic carbon (51-62 permil), but the bacterial PLFA were strongly depleted in 14C (-230 permil). The ubiquitous C16 and C18 PLFA fell between these values (-8 and -39 permil respectively). These observed depletions could only be due to incorporation of the petroleum carbon, which has a 14C of -1000 permil. Thus compound specific radiocarbon analysis has proved to be an excellent tool for identification of microbial metabolism of petroleum carbon in the environment and provides a further tool to be added to that of 13C analysis. Additionally, the extent of 14C depletion of the bacterial PLFA gives an indication of the relative importance of petroleum carbon as a carbon source in this microbial community, the remainder of the carbon presumably coming from recycling of carbon from the bacterial and algal community and potential dissolved organic carbon in Buzzards Bay.