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The Pennsylvania State University The Graduate School Department of Crop and Soil Sciences DISTRIBUTION AND DIVERSITY OF SULFUR-REDUCING PROKARYOTES IN SULFUR-RICH PEAT SOILS A Thesis in Soil Science by Carolina Elvira María Yáñez Prieto © 2006 Carolina Elvira María Yáñez Prieto Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2006 The thesis of Carolina Elvira María Yáñez Prieto was reviewed and approved* by the following: Carmen Enid Martínez Assistant Professor of Soil Environmental Chemistry Thesis Advisor Chair of Committee Mary Ann Bruns Associate Professor of Soil Science and Microbial Ecology Peter J. Landschoot Professor of Turfgrass Science Katherine H. Freeman Professor of Geosciences and Associate Head of Graduate Programs and Research, Department of Geosciences John M. Regan Assistant Professor of Environmental Engineering David M. Sylvia Professor of Soil Microbiology Head of the Department of Crop and Soil Sciences *Signatures are on file in the Graduate School iii ABSTRACT Geochemical transfers between the Lockport Dolomite Formation and overlying wetlands near the town of Manning in Western New York have resulted in peat soils that contain high levels of sulfur, zinc, and cadmium. These peat soils are artificially drained for agricultural purposes and they have undergone drastic seasonal changes in redox for the last 60 years. Sulfur cycle reactions driven by prokaryotes potentially influence the availability, mobility and the biogeochemical control of Zn and Cd retention in the peats. The objectives of this research were to determine the presence of sulfate-reducing prokaryotes (SRP), to detect enzymatic pathways used for sulfur respiration, to evaluate the influence of temporal and spatial changes in sulfate-reducing populations and to determine the oxidation states of S present in peat soils from the Manning region. Surface peat samples were collected from the Manning peatland region of Western New York in July 2002 (dry season). The surface soils were collected from the edge of a field, along a Zn phytotoxicity gradient starting in an area where there was no plant growth and moving to a zone of typical plant growth. Additionally, intact soil vertical profiles (soil cores) were collected during dry (July 2002) and wet (March 2004) seasons. The soil cores were collected along a lateral transect where the center of the field had high enough levels of Zn (phytotoxic levels) to prevent plant growth and moving to areas of stunted, and then typical plant growth. The diversity of prokaryotes involved in sulfur reduction was studied in peat soils containing high natural levels of sulfur (4,200 to 40,500 mg kg-1), zinc (137.5 to 71175 mg kg-1) and cadmium (<1 to 310.7 mg kg-1). Diversity in microbial populations was iv analyzed using classical and molecular approaches and the microbiological data were coupled to the spectroscopic characterization (S-XANES) of the oxidation states of sulfur in the peats under investigation. Sulfur-XANES analyses reveal that sulfide/thiol groups (reduced forms of S) and sulfonate groups (oxidized forms of S) are the two dominant S species in surface peats. While the percentage of the total S present in oxidized forms is relatively constant (~10%) in vertical profiles, about 40% and 60% of the total S exist as sulfide/thiol groups (reduced forms of S) in surface and deep soils, respectively. The use of ribosomal intergenic spacer analyses (RISA) for the characterization of bacterial communities revealed the abundance and diversity of microorganisms. Dominant bacterial populations inhabiting surface soils were very similar, while those located in deep soils were dynamic, responding to changes induced by depth and season. Enumerations of sulfate-reducing prokaryotes (SRPs) in surface soils indicated that SRPs using acetate or lactate as electron donors represent only a small fraction (~103 g-1 dry soil) of total cell densities determined by microscopy (~109 g-1 dry soil). Enrichment for SRPs showed that typical sulfate reducing bacteria (delta subgroup of Proteobacteria, among others) do not seem to be present in surface soils although 40% of the total S is in reduced (thiol/sulfide) forms. Instead, sulfur cycling in these metalliferous surface soils is driven by Gram-positive bacteria related to Clostridium spp. and may involve the use of sulfonates as electron acceptors. Results of PCR-amplification of enzymes involved in the sulfur respiration pathway [adenosine-5’-phosphosulfate reductase (apsA) and dissimilatory sulfite reductase (dsrAB)] in DNA from surface soils supported these observations. No dsrAB genes were detected and phylogenetic association of cloned sequences showed that apsA sequences recovered from these soils had diverged from v apsA of known SRPs. Alternative pathways for sulfur dissimilation are proposed for these surface soils. The enzyme anaerobic sulfite reductase (asrA) was detected in the peats and phylogenetic analysis of asrA sequences revealed a wide distribution and diversity of this gene. By analysis of dsrAB genes in deep peat soils, we identified sulfate-reducing prokaryotes in soil profile samples deeper than 45 cm. Furthermore, the sequences of dsrAB genes present in peat soil samples from both dry and wet seasons have not been previously described in the literature. Thus, our results suggest that novel sulfur-reducing prokaryotes are present in these peat soils and that they might be specialized colonists in these metalliferous soil environments. vi TABLE OF CONTENTS LIST OF FIGURES .....................................................................................................viii LIST OF TABLES.......................................................................................................xi ACKNOWLEDGEMENTS.........................................................................................xii Chapter 1 Introduction ................................................................................................1 SULFUR CYCLE.................................................................................................2 METALLIFEROUS SOILS OF THE MANNING REGION..............................6 OBJECTIVES.......................................................................................................8 REFERENCES .....................................................................................................10 Chapter 2 Sulfur-Reducing Prokaryotes in Surface Peats Subjected to Variable Redox Conditions .................................................................................................12 ABSTRACT .........................................................................................................13 INTRODUCTION ................................................................................................15 MATERIALS AND METHODS .........................................................................17 RESULTS.............................................................................................................23 DISCUSSION.......................................................................................................40 CONCLUSIONS ..................................................................................................44 REFERENCE .......................................................................................................46 Chapter 3 Novel Functional Genes and Enzymatic Pathways Involved in Dissimilatory Reduction of Sulfur Compounds in Agriculturally Drained Peats......................................................................................................................57 ABSTRACT .........................................................................................................58 INTRODUCTION ................................................................................................60 MATERIALS AND METHODS .........................................................................63 RESULTS AND DISCUSSION...........................................................................66 CONCLUSIONS ..................................................................................................78 REFERENCE .......................................................................................................79 Chapter 4 Sulfate-Reducing Bacteria Populations in Sulfur-Rich Peat Soils as a Function of Soil Depth and Moisture Content......................................................87 ABSTRACT .........................................................................................................88 INTRODUCTION ................................................................................................90 MATERIALS AND METHODS .........................................................................92 RESULTS.............................................................................................................97 DISCUSSION.......................................................................................................109 vii CONCLUSIONS ..................................................................................................113 REFERENCES .....................................................................................................114 Chapter 5 General Conclusions ..................................................................................121 viii LIST OF FIGURES Figure 1-1: Biological sulfur cycle. .............................................................................4