B52A-01 INVITED
Sulfur and oxygen isotope studies of sulfate reduction
I will discuss insights into sulfur and oxygen isotope fractionations of dissimilatory sulfate reduction and specifically insight provided by experiments with natural populations of sulfate-reducing bacteria from Faellestrand, Denmark. The experiments yielded relatively large magnitude sulfur isotope fractionations for dissimilatory sulfate reduction (up to approximately 45 ‰ for 34S/32S), with higher δ18O accompanying higher δ34S, similar to that observed in previous studies. The seawater used in the experiments was spiked by addition of 17O-labelled water and the 17O content of residual sulfate was found to depend on the fraction of sulfate reduced in the experiments. The 17O data provides evidence for recycling of sulfur from metabolic intermediates and for an 18O/16O fractionation of ~25-30 ‰ for dissimilatory sulfate reduction, a magnitude that is consistent with isotopic exchange between a sulfite species and cell water. The molar ratio of oxygen exchange to sulfate reduction was found to be about 2.5. Using recent models of sulfur isotope fractionations we find that our combined sulfur and oxygen isotopic data places constraints on the proportion of sulfate recycled to the medium (78-96 %), the proportion of sulfur intermediate sulfite that was recycled by way of APS to sulfate and released back to the external sulfate pool (~70%) and also that a fraction of the sulfur intermediates between sulfite and sulfide were recycled to sulfate. These parameters can be constrained because of the independent information provided by δ18O, δ34S, 17O labels, and Δ33S.
B52A-02
Micron-scale resolution of sulfur cycling in a microbial mat
Microbial mats, finely laminated layers of metabolically diverse microbial communities, likely dominated biogeochemical cycling throughout most of Earth history. To further our understanding of biogeochemical processes within microbial mats, we have investigated sulfur cycling (as recorded by sulfate reduction) in mats from Guerrero Negro, Baja California Sur. We have pursued a combined microbiological (CARD-FISH) and microgeochemical (nanoSIMS) approach to understanding sulfur cycling in the hopes to establish the link between microbial metabolic activity and the establishment of geochemical gradients at the micron-scale. CARD-FISH analysis indicates that viable sulfate reducers form coherent bands (on the order of 100um in thickness) throughout at least the upper ~5mm of microbial mat. There appears to be no degradation in banding with depth below the uppermost few millimeters. We present a high-resolution spatial profile of sulfide abundance and isotopic (d34S) composition on the micron-scale using a Cameca NanoSIMS 50L ion microprobe. We find fine-scale (0.1-1mm) banding of sulfide throughout the mats. In addition, there are micron- scale (~4um) laminations observed using the NanoSIMS both in optical CCD and element scanning mode. 2D maps of sulfide abundance and d34S profile from the mat surface down to a depth of ~1cm were obtained at ~50um resolution with a typical analytical error in d34S of ±1 permil (1sigma). Horizontal banding (parallel to mat laminations) of d34S is observed at the scale of 50 – 200 um. These profiles trend toward depleted d34S with depth by up to 25 permil within the upper 1 cm of the mat. Control experiments in standard solutions did not reveal any banding or the same scale of isotopic variability as observed in the microbial mat. We therefore believe that the banding and isotopic variability observed in the microbial mat are not an analytical artefact, but rather reflect very-fine-scale lamination in microbial activity preserved at depth within the mat, as is supported by CARD-FISH observations. As such, the laminations at depth within these mats are not merely relict architecture but reflect ongoing metabolic activity that is subject to the same sharp spatial gradients as are found in the upper few mm of the mat surface. The environmental conditions that maintain such rigid laminations at depth (~1 cm), far from the sharp redox gradients of the mat surface, remain poorly constrained. Thus, our results suggest that the full scope of sulfur cycling with microbial mats is far from completely understood.
B52A-03
Visualizing Single Cell Biology: Nanosims Studies of Carbon and Nitrogen Metabolism in Diazotrophic Cyanobacteria
Filamentous nitrogen fixing (diazotrophic) cyanobacteria are key players in global nutrient cycling, but the relationship between CO2- and N2-fixation and intercellular exchange of these elements remains poorly understood in many genera. These bacteria are faced with the challenge of isolating regions of N-fixation (O2 inhibited) and photosynthetic (O2 producing) activity. We used isotope labeling in conjunction with a high-resolution isotope and elemental mapping technique (NanoSIMS) to quantitatively describe 13C and 15N uptake and transport in two aquatic cyanobacteria grown on NaH13CO3 and 15N2. The technical challenges of tracing isotopes within individual bacteria can be overcome with high resolution Secondary Ion Mass Spectrometry (NanoSIMS). In NanoSIMS analysis, samples are sputtered with an energetic primary beam (Cs+, O-) liberating secondary ions that are separated by the mass spectrometer and detected in a suite of electron multipliers. Five isotopic species may be analyzed concurrently with spatial resolution as fine as 50nm. A high sensitivity isotope ratio ‘map' can then be generated for the analyzed area. Using sequentially harvested cyanobacteria in conjunction with enriched H13CO3 and 15N2 incubations, we measured temporal enrichment patterns that evolve over the course of a day's growth and suggest tightly regulated changes in fixation kinetics. With a combination of TEM, SEM and NanoSIMS analyses, we also mapped the distribution of C, N and Mo (a critical nitrogenase co-factor) isotopes in intact cells. Our results suggest that NanoSIMS mapping of metal enzyme co-factors may be a powerful method of identifying physiological and morphological characteristics within individual bacterial cells, and could be used to provide a 3-dimensional context for more traditional analyses such as immunogold labeling. Finally, we resolved patterns of isotope enrichment at multiple spatial scales: sub-cellular variation, cell-cell differences along filaments, inter-species transfers (with Rhizobium epibionts), and within-cell depth profiles. Spatial enrichment patterns were correlated with morphological features evidenced in TEM images of microtomed filaments. These features indicate how 15N and 13C "hotspots" are dispersed throughout individual cells in different species, and may indicate isolated locations of increased N2 fixation, sites of amino acid/protein synthesis, or cyanophycin storage granules. This combination of Nano-Secondary Ion Mass Spectrometry (NanoSIMS) analysis and high resolution microscopy allows isotopic analysis to be linked to morphological features and holds great promise for fine-scale studies of bacteria metabolism.
B52A-04 INVITED
Culture Studies of Nitrogen and Oxygen Isotope Effects Associated with Nitrate Assimilation and Denitrification
The isotope effects of nitrate-consuming reactions such as nitrate assimilation and denitrification are potential indicators of the physiological state of the organisms carrying out these reactions. Moreover, an understanding of these isotope effects is needed to use the stable isotopes to investigate the fluxes associated with these reactions in modern and ancient environments. We have used batch cultures to investigate the nitrogen (N) and oxygen (O) isotope effects of (1) nitrate assimilation by eukaryotic and prokaryotic algae and by heterotrophic bacteria, and (2) nitrate reduction by denitrifying bacteria. We observe intra- and inter-specific variation in isotope effect amplitudes and, in the case of denitrifiers, indications of isotope effect decreases during individual nitrate drawdown experiments. However, the measured N and O isotope effect ratio is close to 1 for all studied organisms, with the exception of an unusual denitrifier (Rhodobacter sphaeroides) that possesses only periplasmic (non-respiratory) nitrate reductase. This observation and other findings are consistent with nitrate reductase being the predominant source of isotope fractionation and with most isotope effect amplitude variability arising from variable degrees to which nitrate imported into the cell is reduced versus effluxed back into the environment; the more efflux, the more complete the expression of the fractionation imparted by nitrate reduction. If this is the case, then isotope effect amplitudes in the field should be related to physiological conditions in the environment, a prediction that, we argue, is supported by recent studies of (1) nitrate assimilation in the polar ocean and (2) denitrification in sediment porewaters.
B52A-05
Anaerobic Biodegradation of Pristane by Nitrate Reducing Bacteria
In recent sediments, microbial biodegradation provides a control on the long-term preservation of organic matter, through the preferential loss of certain biomolecules and the alteration and concentration of other more recalcitrant molecules. Biodegradation of hydrocarbons derived from membrane lipids, has been demonstrated by both aerobic and strictly anaerobic culturing experiments. The isoprenoid pristane, once considered stable under anaerobic conditions, is in fact degraded by a denitrifying microcosm (BREGNARD et al., 1997) and a methanogenic, sulphate-reducing enrichment culture (GROSSI, 2000). We recently demonstrated pristane biodegradation and accompanying loss of nitrate by an activated sludge isolate. The measured nitrate consumption accounts for a 7.1 +/- 0.4 mg loss of pristane, 4.74% of the initial substrate, in 181 days, assuming pristane conversion to CO2. We have characterized the microorganisms active in the biodegradation process, through the creation of a 16S rDNA clone library, as well as fluorescence in situ hybridization (FISH). Experiments are in progress to enrich cultures of sulfate reducing bacteria that utilize pristane as a sole carbon source and to characterize reaction mechanisms in pristane-oxidizing pathways.
B52A-06
Rubisco and Atmospheric Oxygen: the Archean Record.
Carbon isotopic evidence, from c.2.9 Ga stromatolites from Steep Rock, Ontario, Canada, c.2.9 Ga stromatolites from Mushandike, Zimbabwe, and from 2.7 to 2.65 Ga stromatolites in the Belingwe belt, Zimbabwe, implies that in all three localities the reef-building autotrophs included organisms using Forms I and II Rubisco. This inference, strong though not conclusive, is supported by other geochemical evidence that these stromatolites formed in oxic conditions. Collectively, the implication is that oxygenic photosynthesisers were present c.2.9 Ga ago, and abundant 2.7-2.65 Ga ago. Rubisco specificity and compensation may explain the paradox that, despite the inferred evolution of oxygenesis c.2.9 Ga ago, the air was anoxic in much of the Late Archaean. The Earth System may be bistable under the warming Sun, with liquid oceans occurring in either anoxic greenhouse (H2O with abundant CH4 plus CO2, but no O2) or oxic (H2O with more abundant CO2, free O2 but little CH4) states. Build-up of a very high atmospheric inventory of CO2 in the c.2.3 Ga glaciation and subsequent oxygenesis to the Rubisco CO2 compensation line may have allowed the atmosphere to reach stability in an oxygen-rich system. Since then, Form I Rubisco specificity and consequent compensation limits may have maintained the long-term atmospheric disproportion between O2 and CO2, which is now close to both CO2 and O2 compensation barriers.
B52A-07
Supply and Demand in Subseafloor Basalt Aquifers
Subseafloor basalt aquifers contain 26 million cubic kilometers of water populated by unknown microbes that mediate the fluxes of elements between ridge flanks and seawater. The energy supporting this deep biosphere originates in the fundamental disequilibria between mid-ocean ridge basalts and seawater. Quantifying energy supplies depends in part on sampling and analysis of basalt aquifer fluids and in part on theoretical models of energy flow in complex natural systems. These approaches are joined through affinity diagrams that provide quantitative frameworks for testing models of the feedback between metabolism and weathering of oceanic crust. Fluid compositions can result from conductive cooling of hydrothermal fluids or mixing of hydrothermal fluids and seawater in regions proximal to the ridge, and conductive warming of seawater in basalt aquifers distal from the ridge. In all cases, temperature changes can be accompanied by diverse fluid-rock reactions. As a consequence, fluids can reach similar temperatures through multiple geochemical pathways, leading to diverse compositions. It is expected that these differences engender different habitats. Those influenced by deep hydrothermal fluids, such as post-eruptive fluids sampled at ridges and seamounts of the northeast Pacific, tend to be somewhat more acidic than habitats influenced by conductive heating of seawater. New results indicate that even at relatively low temperatures, these fluids provide ample energy for biosynthetic pathways including lipid and amino acid synthesis. For conductively heated habitats, preliminary results indicate that nitrate reduction must be coupled to oxidation of iron-bearing silicates for overall affinities to decrease in response to metabolism. In the extreme case of restricted fluids, in which water is the oxidant, hydrolytic oxidation of olivine provides sufficient hydrogen to reduce nicotineamide adenine dinucleotide at prevailing pH and silica activities. These are examples of how coupling biochemical demands with geochemical energy supplies defines habitats as alteration products and permits the integration of metabolism into assessments of elemental cycles.
B52A-08
Life in Subseafloor Sediments of the South Pacific Gyre
The middle of the South Pacific Gyre (SPG) is farther from continents and productive oceanic zones than any other site on Earth. To understand the nature of life in the most oxidized and food-limited subseafloor sediments, expedition Knox-02RR surveyed and cored the sediment at ten sites throughout the Gyre and one site at the southern margin of the Gyre. The sediment has accumulated extraordinarily slowly at all of the SPG sites (0.1 to 1 m/myr). These sediments contain a living community that is characterized by very low biomass and very low metabolic activity. At every depth in the cored SPG sediments, cell abundances are two to six orders of magnitude lower than at the same depths in all previously explored subseafloor communities. The net rate of respiration by the subseafloor sedimentary community at each SPG site is one to three orders of magnitude lower than the rates of subseafloor communities at previously explored sites. Due to the low rates of respiration and the thinness of the sediment, interstitial waters are oxic throughout the sediment column in most of this region. Consequently, the subseafloor sedimentary community of this region is predominantly aerobic, unlike previously explored subseafloor communities. Generation of hydrogen by radiolysis of water may be a significant food source for this subseafloor community.