J17 204 Morrill Science Center IVN University of Massachusetts 639 North Pleasant Street Amherst, MA 01003 Tel: 413-545-1204 Fax: 413-545-1578 VISUALIZATION OF SULFATE-REDUCING BACTERIA IN MICROCOSMS OF AN ACID MINE DRAINAGE SITE Caryl Ann Becerra and Klaus Nüsslein Department of Microbiology, University of Massachusetts, Amherst, MA Indication of Sulfate Reduction Activity Introduction Experimental Flow Chart Acid Mine Drainage AMD Attenuating AMD Attenuating AMD Generating Extracted DNA from sediment used to create microcosms. Generation of AMD involves both geochemical and microbial processes of the oxidation of iron and sulfur in minerals. See reaction below. Microcosms amended with Glycerol+N+P Microcosms amended with Algae extract The smell of H2S, another indication of sulfate reduction, was detected in microcosms of the AMD attenuating zone 400 300 ORP (mV) Effect of Different Substrates Algae and G+N+P Amended Microcosms 200 The resultant product, sulfuric acid, acidifies the water, which dissolves the metals in the ore deposits, causing elevated concentrations of metal contaminants in the water system. 100 Amending microcosms of the attenuating zone with Algae or Glycerol with N and P resulted in higher pH and lower ORP measurements during the incubation as compared to unamended microcosms 0 -100 Typically, AMD is exacerbated because of mining activity, thus AMD has become a widespread problem occurring in every industrial nation. 2) Microcosms of Davis Mine Samples were incubated in the dark, under anaerobic conditions at 4 °C, 12°C, or 16°C. (incubation temperatures reflect measured average temperatures of the sediment during the winter and summer months). Overall Reaction for the Generation of AMD Geochemical: FeS2 + 3.5 O2 + H2O → Fe2+ + 2 SO42- + 2H+ Microcosms of AMD attenuating and generating zones were compared by measuring: Triplicates + Killed Controls Biological: (ex. Thiobacillus ferrooxidans) 7 Fe2+ + 1.75 O2 + 7H+ → 7 Fe3+ + 3.5 H2O 6 AMD Attenuating Zone: Unamended Algae AMD Generating Zone: Unamended Algae Glycerol + N+P K illed Control Glycerol + N+P K illed Control Amending with Algae from the site raised the pH and lowered the ORP faster than amending with Glycerol with N and P 5 pH Microcosms are batch-type experiments of environmental samples treated under controlled laboratory conditions to allow easier determination of microbial activity. Acid mine drainage is naturally attenuated at Davis Mine Amending the microcosms of the AMD generating zone did not cause the pH or ORP to change considerably as compared to the killed controls 4 3 0 20 40 60 Days of Incubation pH – detect changes in acidity 80 100 Iron (II) concentration suggest reduction in (Left): both AMD attenuating and generating zones unamended microcosms and amended microcosms is higher in attenuating zone ORP (oxidation-reduction potential) – indicate whether reduction reactions are occurring. Reduction reactions like sulfate and iron reduction are known reactions that attenuate AMD Sulfate, reduced iron, total sulfur and iron concentrations Geochemical: FeS2 + 14 Fe3+ + 8 H2O → 15 Fe2+ + 2 SO42- + 16 H+ Three sets of controls: unamended, standard media, and killed controls Sulfate concentration suggest no significant difference between (Above): microcosms and killed controls unamended and amended microcosms attenuation and generating zone Day 33 Day 0 Natural Attenuation at Davis Mine 3) Visualizing Bacteria in Microcosms In some instances, AMD is naturally attenuated by the system. Used fluorescent in situ hybridization to detect sulfate-reducing bacteria (SRB385), eubacteria (EUB338), and all cells (DAPI). Davis Mine in Rowe, Massachusetts is an AMD site where natural attenuation is occurring. See picture above. Changes in microbial composition over time were observed and correlated to physico-chemical changes in the microcosms Within a 100 meters from the mine effluent, the acidic leachate is neutralized and the heavy metal content decreases. AMD Attenuating Zone Day 33 3) Visualizing Bacteria in Microcosms Unamended Microcosm on Day 0 Sulfate-reducing bacteria are present at both the AMD attenuating and generating site, however acidic conditions may limit SRB growth. AMD Generating Zone Day 33 This self-remediating phenomenon involves both geochemical and biological processes such as EUB - Hydrological: by the dilution of AMD SRB Results - Geochemical: buffering capacity of minerals - Biological: iron and sulfate reduction EUB SRB 1) Bacterial Community Composition Samples were taken from differing geochemical zones at Davis Mine Microcosms of the AMD attenuating zone have larger bacterial populations than microcosms of the AMD generating zone The 16S rDNA of extracted DNA taken from the sediment used as microcosm inoculum was amplified AMD Attenuating Zone (W14) •pHavg = 4.91 Larger number of sulfate-reducing bacteria is found in microcosms of the AMD attenuating zone Community analysis was conducted to compare differences in the microbial communities of the two zones in order to possibly reveal potential groups responsible for the geochemical characteristics •Lower concentration of dissolved metals AMD Generating Zone (W15) W14 •Higher concentration of dissolved metals Black precipitate, an indication of sulfate reduction, appeared in microcosms of the AMD attenuating zone only The appearance of black precipitate was enhanced when microcosms were amended with algae or with glycerol with or without N +P 1) Microbial Community Analysis Acid mine drainage (AMD) is an acidic, iron-rich leachate that is generated when sulfur-rich minerals are exposed to oxygen and moisture. See picture on left. •pHavg = 2.58 AMD Generating Both zones contain acidophiles W15 Iron oxidizers were detected in the AMD generating zone but not the AMD attenuating zone Although sulfate and iron reducers were not detected, a large proportion of unknown members exist in the AMD attenuating zone Davis Mine Average Concentration Jul 2004 - Mar 2005 All concentrations are in ug/L Partial Map of Davis Mine pH W14 W15 4.91 2.98 2&3 Fe 45016.1 30543.3 SO4 2- 2) Microcosms of Davis Mine 579514.46 601257.38 AMD Attenuating Unamaneded: AMD Generating Unamended: 7 4ºC 12ºC 12ºC 16ºC 16ºC pH Differences with Varying Temperature Determine whether the geochemical characteristics of an AMD attenuating and AMD generating zone are biologically influenced. pH 6 Goal of Study Increasing the incubation temperature resulted in a faster increase in pH measured in the microcosms of the attenuating zone 5 4 3 Does the site possess the capacity for attenuating processes? What microorganisms are potentially responsible for these processes? Is there a difference between the microbial populations in the AMD generating versus the AMD attenuating zone? How do these populations change over time? Conclusions Differences in geochemical trends are biologically influenced 2 0 20 40 Day 60 80 100 An increase in temperature had no significant effect on microcosms from the AMD generating zone pH and ORP data indicate that a larger effect of attenuation processes are present in the AMD attenuating zone Microbial community of AMD attenuating zone possess the capacity for attenuating AMD Microcosms confirm the potential for sulfate and iron reduction and suggest that sulfate and iron reducing bacteria are responsible Community can be stimulated with the addition of organic carbon or an increase in temperature to produce greater attenuation effects Future Work Correlate the changes in the microbial community with geochemical trends observed in the microcosms Determine the microbial sulfate reduction rates within the AMD attenuating and generating zones Isolate acidophilic sulfate-reducing bacteria from Davis Mine Acknowledgements NSF- Biocomplexity of the Environment for Integrated Research and Education in Environmental Systems NSF-Central Microscopy Facility of University of Massachusetts, Amherst Acid Mine Drainage Group at UMass-Amherst (http://www.umass.edu/biocomplexity)
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