Banker GEOL394. Jonathan Banker GEOL394. Advisor: Dr. Farquhar

Transcription

1 Banker GEOL Jonathan Banker GEOL Advisor: Dr. Farquhar

2 Banker GEOL Table of Contents Abstract.... Introduction.... Methods.... Culturing of Bacteria.... Sample Preparation.... Gas Source Mass Spectrometry.... Data.... Discussion of Uncertainty.... Discussion...0. Conclusion.... Acknowledgements.... References.... Appendices.... Appendix.... Appendix...

3 Banker GEOL Abstract Desulfobacterium autotrophicum is a sulfate reducer commonly found in marine-shelf sediments. During the metabolic process, D. autotrophicum reduces sulfate to sulfide. The nature of its metabolism is primitive, and understanding how environmental factors influence sulfur isotope fractionation in this model organism can provide insight into sulfur isotope signatures in Earth s rock record. In addition, studies of anaerobic bacteria and how they influence their environment can potentially be used to find evidence of life on other planetary bodies. The genome sequence of D. autotrophicum has been completely mapped, yielding a large quantity of information about the organism. Among sulfate reducers, D. autotrophicum is particularly versatile in the number of electron donors and carbon sources that it can utilize. From 00-00, Dr. James Farquhar and collaborators conducted a variety of experiments in which D. autotrophicum were cultured. During the experiments, growth conditions such as temperature and electron donor concentration were manipulated to explore the effect that these variables would have on the magnitude of fractionation between sulfate in the growth medium and excreted sulfide. For this study, the isotopic concentration of the sulfur in the initial sulfate and the sulfide that was excreted, as a result of the bacterium s metabolism, was analyzed. This study has found that there are varying degrees of fractionation that relate to the use of different substrates. In addition, higher levels of fractionation were observed in experiment that utilized a smaller concentration of the electron donor. Fractionation between the initial sulfate and the excreted sulfide also changed as a function of time.

4 Banker GEOL.Introduction D. autotrophicum is a species of anaerobic bacteria that belongs to the genus Desulfobacteriaceae. Members of this genus and other sulfate reducers are dominantly found in anoxic marine sediments (Strittmatter et. al 00). D. autotrophicum are single cell organisms that lack a membrane enclosed nucleus and organelles. According to Subhraveti et al. (0) D. autotrophicum has a total of protein genes, RNA genes, and psuedogenes. It is. million base pairs in size and it has 0 total pathways. At. million base pairs, it is million base pairs larger than other common sulfate reducing bacteria that have been completely sequenced (Strittmatter et. al 00). Between 00 and 00, Dr. Farquhar spent a year on sabbatical in Denmark. At the University of Southern Denmark, Dr. Farquhar cultured D. autotrophicum under a variety of conditions with the intention of controlling the fractionations between sulfate and sulfide by varying the substrate, temperature, and concentration of electron donors. These samples were then brought back to the United States to be analyzed by using gas source mass spectrometry in University of Maryland s Stable Isotope Laboratory. Throughout this paper, variations in the isotope abundance rations will be expressed using delta and cap-delta notation. The exponents. and 0. are used to approximate low temperature equilibrium reactions. S = 000*(( S/ S)sample/( S/ S)reference ), S = S 00*((+ S/000) 0. ), S = S 000*( + S/000. ). (Farquhar et. al, 00). Capital delta notation is used as a proxy for mixing between sulfur pools in the cell. Because the metabolic pathway is not strictly a one way sequence of chemical reactions. S values become more positive due to back reactions that take place in the cell (Brunner and Bernasconi, 00; Farquhar et. al, 00) Fractionation observed between the initial sulfate and the excreted sulfide is caused by the multi-step metabolism of D. autotrophicum. As seen in Figure, D. autotrophicum initially uptakes external sulfate ions into the cell. This process is actively mediated by transport enzymes embedded in the cell wall (Brunner and Bernasconi, 00). Sulfate is then reacted with adenosine triphosphate [ATP] to form adenosine phosphosulfate Figure. Metabolic Pathway of D. autotrophicum.

5 Banker GEOL [APS]. Next, electrons are donated to form sulfite. Subsequently, additional electrons are donated to form hydrogen sulfide, which is the end product that is released from the cell. This process is a sequence of forward chemical reactions and back reactions. If the process was only a forward process with 00% yield, the composition of the sulfur isotopes between the initial sulfate and the final sulfide would be the same, and there would not be any fractionation. External sulfate, internal sulfate (sulfate within the cell), APS, sulfite and HS each constitute an individual sulfur pool. A sulfur pool is a discrete reservoir of sulfur with its own isotopic composition. Each step in the reduction process has a fractionation factor associated with it. There is a fractionation of approximately associated with the initial uptake of external sulfate into the cell. There is a very small fractionation associated with the transfer of sulfur from internal sulfate to APS and it has been deemed negligible when accounting for the overall fractionation associated with the entire metabolic pathway. There is a fractionation factor of approximately associated with the transfer of sulfur from APS to sulfite and a fractionation factor of approximately 0 associated with the transfer of sulfur from sulfite to sulfide (Brunner and Bernasconi, 00). Each of these steps contributes to the overall fractionation of sulfur isotopes as sulfur flows through the cell. For example, when sulfur in the internal sulfate is reacted with ATP and forms APS, there is a measurable change in the isotope ratios between the sulfur in the sulfate and the sulfur in the APS. Flow through the cell coupled with mixing of the sulfur pools are the key contributors to the fractionation associated with sulfate reducers. Mixing between the various sulfur pools (i.e. reversals in the pathway) leads to changes in the S of the sulfur (Farquhar et. al, 00). The overall magnitude of fractionation between external sulfate and secreted sulfide is controlled by environmental factors such as temperature, electron donor type, and the availability, or concentration, of the electron donor (Brunner & Bernasconi, 00). These environmental factors impact the magnitude of fractionation by manipulating the regular flow of sulfur through the cell. However, it has recently been suggested that environmental factors may Previous culture with D. autotrophicum also influence the manner 0 in which sulfite reductase fractionates sulfur (Bradley 0 et al., 0). Sulfite reductase is a transport 0 enzyme within the cell that is responsible for enabling and catalyzing the 0 reduction of sulfite to Temperature (Celcius) H S. Fractionation ( ) Figure. Fractionation, represented in as an epsilon value, changes as a function of temperature. Experiment with D. autotrophicum from Johnston et. al (00). This potential complicating factor is beyond the scope of this paper, because testing it would require additional methods not used during the culturing of the bacteria.

6 Banker GEOL Studies have shown that fractionation between sulfate in the medium and excreted sulfide depends in part on temperature. Figure illustrates fractionation expressed in epsilon notation ( S sulfate - S sulfide ). Fractionation changes as a function of temperature. Figure also shows that there is a significant impact on fractionation for organisms cultured with different electron donors (butyrate and hydrogen plus carbon dioxide). The white data points represent fractionation between sulfate and sulfide using butyrate as an electron donor). The gray datum represents fractionation between sulfate and sulfide when using bars of hydrogen and carbon dioxide as an electron donor S (normalized to sulfate) S vs. S (Previous work) S (normalized to sulfate) Figure. The umbrella-shaped field represents constraints on fractionation associated with microbial metabolism. Figure displays data from the same experiments in a different context. The umbrella shaped field represents constraints on fractionation due to the chemical and physical processes during metabolism (Brunner and Bernasconi, 00; Farquhar et. al, 00). These constraints are based on the current model of dissimilatory sulfate reduction. Older models were more simplistic but were unable to account for fractionation greater than - (Rees, ). This was because the Rees model assumed that the reaction between sulfite and sulfide was too fast to be irreversible. In this context, fractionation is examined between a pair of samples that were extracted at the same time within the experiment. Examining fractionation in this fashion requires a term that describes the observed relationship between the pair. Equation one was utilized to calculate the values in figures and. () The lines on the parameter of the field are referred to as mixing lines and represent the maximum and minimum amount of fractionation, in S and S, associated with the metabolic pathway. For these purposes, a mixing line is line that describes possible sulfur isotopic compositions. The possible compositions are a mix between different sulfur pools in the metabolic pathway. The top line represents the maximum amount of fractionation (more positive S). The individual curves along the bottom represent individual processes within the pathway. The transfer of sulfur from sulfite to sulfide is an example of an individual process

7 Banker GEOL within the metabolic pathway. The white data points in figure represent fractionation associated with butyrate (Johnston et. al, 00)..Methods. Culturing of Bacteria Dr. Farquhar began the experiment by culturing D. autotrophicum at 0 C and C. As previously mentioned, it has been well established that the degree of fractionation changes as a function of temperature and substrate. This experiment is attempting to confirm these statements as well as to determine if the magnitude of fractionation changes as a function of electron donor availability. Each individual culture was primarily artificial seawater with a concentration of 0.g/L of NaSO as well as the various electron donors mentioned in the subsequent paragraph. At the beginning of each experiment, a sample of the original sulfate and sulfide was extracted from the medium. Throughout the duration of each experiment, a sulfate sample and a sulfide sample were taken every few days until the experiment was over. By conducting the experiment in this fashion, fractionation can also be observed as a function of time. While the primary purpose is to determine how the isotope ratios change from the beginning sulfate to the final sulfide, observing how the ratios change over time could potentially yield far more information about the bacterium s metabolism than the S sulfate (initial) - S sulfide (final) could alone. Each experiment has a starting sulfate with a known isotopic composition and the excreted sulfide. Therefore, each fractionation data point expressed in epsilon represents a pair of two samples. The experiment was set up to determine the magnitude of fractionation associated with the reduction of the sulfate to sulfide during the bacteria s metabolism. The first two experiments were cultured with CO + H, offering concentrations as high as bars of pressure and as low as only 0cc of H. The second two experiments used a short chain fatty acid, butyrate, commonly found in butter. Sodium butyrate (NaC H COO) was the specific butyrate compound used in the experiments. The two butyrate experiments required concentrations of butyrate at 0mM and 0mM. The experiments used for this project were conducted at a constant C. This project focused on the experiments conducted with 0mM of butyrate as well as the bar and 0cc hydrogen experiments. Each procedure was repeated three times to account for reproducibility.. Sample Preparation Once the samples were transported to the United States, each pair was reduced to silver sulfide using well established methods in the Stable Isotope Laboratory at the University of Maryland. The sulfate and sulfides that were extracted from the cultures were precipitated as BaSO and ZnS, respectively. Then, the barium sulfate was reduced to Ag S (acanthite) by

8 Banker GEOL reacting the barium sulfate with heat and a Thode solution that is composed of hydrochloric acid, hypophosphorus acid, and hydriodic acid to form H S. The H S was then precipitated in a silver nitrate solution to form the Ag S. The sulfur in the ZnS samples was extracted in a similar procedure but with N HCl. The samples were then aged for one week and then rinsed with water and ammonium hydroxide before drying in an oven.. Gas Source Mass Spectrometry Depending on the amount of silver sulfide per sample available,.~.0mg of silver sulfide was weighed out and placed into a cm ethanol-cleansed aluminum foil packet. After that, the packet was loaded into a nickel reaction vessel. Once the vessel and the manifold were leak checked to ensure a closed system, the packet was reacted with F for a minimum of hours at ~00 C. Ag S + F = SF + AgF The impurities were extracted and the SF peak was detected using gas chromatography. It was then isolated between two liquid N cooled traps. After that, the SF was fed into the bellows that ultimately pass it to the source. Finally, isotope ratios ( and values) for each isotope are retrieved and archived in Microsoft Excel.. Data Sample ( ) ( ) ( ) ( ) ( ) 0 (SO) uncertainty SO (SO) uncertainty (SO) uncertainty (ZnS) uncertainty (ZnS) uncertainty (ZnS) uncertainty B (SO) uncertainty (SO) uncertainty

9 Banker GEOL B(SO) uncertainty B(SO) uncertainty Ddelta(ZnS) uncertainty (ZnS) uncertainty B (SO) uncertainty (SO) uncertainty Bdelta(ZnS) uncertainty Gdelta(ZnS) uncertainty A(SO) uncertainty C(SO) uncertainty E (SO) uncertainty endB(SO) uncertainty A(ZnS) uncertainty C(ZnS) uncertainty E(ZnS) uncertainty A(ZnS) uncertainty Discussion of Uncertainty In graphical figures, error bars are used to represent the uncertainties in the data. The error bars attached to the data points in this study are too small to be seen with the human eye. Uncertainties were calculated using different methods for analyzing and calculating. To analyze a sample, the mass spectrometer completes a sequence of eight cycles. Each cycle entails a measurement of the abundance of the four stable isotopes of sulfur of the sample and makes a

10 Banker GEOL comparison to the measurement of a standard gas. For this project, each sample was analyzed by completing four sequences per sample, totaling measurements per samples. As seen in the example in Appendix b, the average was calculated for each isotope ratio. Then, the standard deviation was calculated. Finally, the standard error (uncertainty) was calculated by dividing the standard deviation by the square root of the number of values. For reported values such as fractionation expressed in epsilon notation, the uncertainties were calculated using error propagation. To calculate the uncertainties, the standard deviation for each value was multiplied by the partial derivative, one, and then squared. Next, those values are added together and the square root of the sum yielded the uncertainty.. Discussion As mentioned previously, every sample analyzed in this study comes from an experiment that cultured D. autotrophicum at C. C is the established optimal growth temperature for D. autotrophicum (Johnston et. al, 00). As seen in figure, fractionation can be expressed in Epsilon notation. Figure graphically displays the same experiments, with the addition of data from this study. By averaging the fractionations resulting from the experiments that used a high concentration of hydrogen as an electron donor, there is a difference of.±. between the experiment using 0cc of hydrogen and the experiment using bars of hydrogen. Fractionation( ) Experimental results with D. auto (Johnston et al., 00) Experiment with hydrogen and CO Johnston et al., 00 Butyrate Hydrogen 0cc Temperature (Celcius) Runing average ( points Figure. All cultured at the same temperature, different environmental factors produce different levels of fractionation. Furthermore, differences between the experiments using hydrogen and the experiments using butyrate yielded a range of fractionation between.± 0.0 and.± 0.0. The differences observed in this study represent different magnitudes of fractionation as a result of different growth conditions. Changing substrate and the concentration of the electron donor 0

11 Banker GEOL produced different levels of fractionation due to the reduction of sulfate to sulfide within the bacterial cell. Figure is a recreation of figure, but with the addition of data from this study. This graph simultaneously adds evidence to the conclusions made by Brunner and Bernasconi (00) and it validates the experimental design of this study. The second claim is based on data from this study harmoniously coinciding with the Brunner and Bernasconi model for dissimilatory sulfate reduction, specifically the constraints on fractionation S (normalized to sulfate) S vs. S 0mM Butyrate Hydrogen 0cc Hydrogen bar 0 S (normalized to sulfate) Figure. A recreation of figure with the addition of data from this study.. Conclusions Desulfobacterium autotrophicum is a well studied single cell organism. It is a known sulfate reducer and due to its versatility, it is a useful strain for studying this primitive metabolic pathway. Fractionation occurs as sulfur moves through the pathway, changing from one form to another (i.e. sulfite to sulfide). This study has demonstrated that the magnitude of fractionation associated with microbial metabolism varies as a function of both substrate content and availability of the electron donor to the bacteria. Although this study has found new information about the way environmental factors manipulate the level of fractionation between sulfate and excreted sulfide, there is still a lot to learn. Biological sulfate reduction signatures can be found early in the Earth's rock record. Understanding how this fractionation occurs provides insight into the world's global sulfur cycle. This study is but a small piece of a much larger puzzle.

12 Banker GEOL. Acknowledgements I would like to thank Dr. James Farquhar, Brian Harms, and Dr. Joost Hoek for their discussion of the literature pertaining to this complex field. I would particularly like to thank Dr. Farquhar for his patience while answering numerous questions and for his assistance in calculating the values in Figure. The author would also like to thank Brian Harms and Dr. Joost Hoek for their constructive feedback and training in mass spectrometry. Lastly, the author would also like to thank Dr. Phil Candela and the Dept. of Geology for providing an experience that provides opportunities to utilize and improve many of the necessary professional skills that other undergraduate majors do not offer.. References Bradley, A., Leavitt, E., and Johnston, D. (0) Revisiting the dissimilatory sulfate reduction pathway. Geobiology., -. Brunner B. and Bernasconi S. (00) A revised isotope fractionation model for dissimilatory sulfate reduction in sulfate reducing bacteria. Geochemica et Cosmochemica Acta.0, 0, -. Farquhar J., Johnston, D., and Wing, B. (00) Implications of conservation of mass effects on mass-dependent isotope fractionations: Influence of network structure on sulfur isotope phase space of dissimilatory sulfate reduction. Geochemica et Cosmochemica Acta. () -. Johnston D., et al. (00) Multiple sulfur isotope fractionations in biological systems: a case study with sulfate reducers and sulfur disproportionators. American Journal of Science. 0,. Johnston D., Farquhar J., Canfield D. (00) Sulfur isotope insights into microbial sulfate reduction: When microbes meet models. Geochim. Cosmochim. Acta., -. Rees C. () Steady-state model for sulfur isotope fractionation in bacterial reduction processes. Geochim. Cosmochim. Acta.,, -. Spring S., et al. (00) Complete genome sequence of Desulfotomaculum acetoxidans type strain ( T ). Standards in Genomic Sciences. (). Strittmatter A., et al. (00) Genome sequence of Desulfobacterium autotrophicum HRM, a marine sulfate reducer oxidizing organic carbon completely to carbon dioxide. Environmental Microbiology. (), 0.

13 Banker GEOL Subhraveti, P., et al. (0) Summary of Desulfobacterium Autotrophicum, Strain HRM, version. Accessed //. < Appendices. University of Maryland Honor Code I pledge on my honor that I have neither given nor received any unauthorized assistance or plagiarized on this assignment. Jonathan Banker Date. Appendix - Data a) Epsilon Values Experiment Epsilon uncert. 0cc bar bar mm but b) Example of uncertainty calculation 0endB(SO) d d d dc orr D D

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15 Banker GEOL average stdev std error