1. Abstract In order to study the bioaccumulation of lead by naturally occurring lead-resistant microbes, they were isolated from lead contaminated soils (>1200 ppm bioavailable lead) near a lead-tailings (chat) pile on defined media containing 1 mM PbNO 3 at 25 o C. Isolate Pb3 is a red-pigmented yeast that grows on solid media containing 5 mM total Pb corresponding to 0.35 mM soluble lead. Isolate Pb4 is a bacterium that grew on media containing 2.5 mM total Pb or 0.24 mM soluble lead. When grown in liquid culture isolates extensively removed soluble lead (values in ΔmM OD -1 ) from media containing 0.1 or 1 mM total Pb: Pb3 (0.087, and 0.096) and Pb4 (0.041 and 0.065). Lead uptake was negligible when the concentration was 0.01 mM suggesting that the process was K M - controlled. The growth rate of Pb4, 0.037 h -1, was relatively unaffected by [Pb]. Interestingly, the growth rate of Pb3, 0.076 h -1, was inhibited at 0.1 mM Pb (0.050 h -1 ), and 1 mM Pb (0.033 h -1 ), but stimulated by 48% at 0.01 mM Pb. Escherichia coli was used as control and demonstrated growth inhibition (decrease of k by 91% at 1 mM Pb) and no lead-uptake regardless of concentration. Calcium was inhibitory (12%) to growth of Pb4, exacerbated lead inhibition of growth, and only slightly reduced Pb uptake by the bacterium. On the other hand Calcium was strongly inhibitory to the growth of Pb3 (50%), the inhibition by calcium was partially reversed in the presence of lead, and Ca completely inhibited lead-uptake by the fungus. The fate of bioaccumulated lead in Pb3 is seen to be lead phosphate by EDS spectrum during TEM. The consequence of distinct lead-binding activities of the bacterium and the fungus, and the surprising lead-requirement of Pb3 are discussed. All measurements of growth rate and lead binding were done in quintuplicate, and differences in values cited are justified to p<0.001.

2. Lead Mining in Missouri  East central Missouri is known as the lead-belt region.  Chat is the byproduct of mining lead ore. Large mounds of the material dot the landscape in the lead belt.  Although the lead concentration is considered hazardous, little has been done to contain it.  Hypothesis: Chat represents a natural enrichment for lead- resistant microorganisms.  These organisms may provide insight into: the physiological effects of lead on the cell possible remediation strategies involving bioaccumulation

3. Lead Content of Soil Near a Chat Pile TABLE 1. Bioavailable and Total Lead Concentration in Soil Samples from Park Hills, MO from which Lead-resistant Isolates Were Obtained Portion of soilConcentration of Pb (ppm) found in the following soil samples: #12 a #15 b Exchangeable c 178 Organic d 51635 Mn Oxides e 1984155 Fe Oxides f 673491 Total Bioavailable g 27251289 Total h 40232294 a Source of Pb4 b Source of Pb3 c Pb adsorbed to soil particles; displaced by boiling with 1 M Mg(NO 3 ) 2 at pH 7 for 2 h d Pb liberated by oxidation of soil organic material; room temp extraction with 0.7 M NaOCl at pH 8.5 for 30 min e Pb associated with Mn oxides; extracted with room temp 0.1 M hydroxylamine hydrochloride for 20 min f Pb associated with amorphous Fe oxides; extracted 50 min with ammonium oxalate buffered to pH 3 in the dark g sum of fractions above h Total soil microwave-digestion with 1:1 trace metal grade HNO 3 at 100 psi Method of Shuman, 1985. Soil Science 140:11-22

4. Isolation Strategy  Bacteria were eluted a from chat and applied to the surface of agar plates.  Defined chemical media containing 1 mM Pb lead called “Roane Media” b  This media is formulated to minimize precipitation of lead.  [Pb] soluble = 0.045[Pb] total -0.12 mM a Konopka, et al. 1999. Applied and Environmental Microbiology 65:2256-2259 b Roane 1999. Microbial Ecology 37:218-224

5. Isolates Used in this Study  Pb3: Yeast. No match in FAME database. Web ID a suggests Rhodospora toruloides  Pb4: Gram (–) rod shaped bacterium. Nearest neighbor by RDP b /Phylip Interface Ochrobactrum sp. Str. S1) Pb4 Pb3 TSA Roane Media TSA RM 1mM Pb 2.5mM Pb a www.2.cbs.knaw.nl/yeast.asp b http://rdp.cme.msu.edu/html

6. Approach – Lead Exposure Studies  Grow microbes in presence of Pb. Quintuplicate 50 ml cultures were incubated statically at 25 o C until OD exceeded 0.1. Pb3, Pb4, E. coli (negative) and un- inoculated (abiotic) controls were prepared. Incubations were carried out in media containing 0, 0.01, 0.1, and 1.0 total lead concentrations  Separate cells from media. Cultures were transferred to 50 ml conical tubes, and centrifuged. Pellets were stored at -20 o C, and random pellets were observed under Phase Contrast microscopy to assure purity.  Measure Pb levels by Atomic Absorption Spectrophotometry. All samples were analyzed by ICP-OES at a wavelength of 220.353 nm for soluble lead after acidification (5% nitric acid, ACS Plus) and filtered through 0.2 micron filters.  Evaluate growth by increase in Optical Density (OD).

7. Growth Rate of Microbes at Different [Pb] Statistical Test a Significant differences (p<0.001), Comment 2-Way ANOVAGrowth rates differ between each organism, expected Growth rates differ between each treatment, expected Interaction between treatment and organisms, organisms respond differently to Pb ANOVA of E.coliGrowth rate changed with each [Pb], decreases in k were significant ANOVA of Pb3Growth rate changed with each [Pb], decreases in k were significant from 0.01 – 1.0 mM, but increase in k at 0.01 mM is meaningful (and wholly unexpected!) ANOVA of Pb4Growth rates changed only between 0.01 and 0.1 mM [Pb], interaction with Pb showed a threshold value a Results identical with SASS and SPSS software packages. Each ANOVA was followed by Tukey HSD Post Hoc Test to investigate nature of differences

8. Lead Uptake, ΔmM per OD, by Microbes Statistical TestSignificant differences (p<0.001), Comment 2-Way ANOVAUptake rates differ between each organism, expected Uptake rates differ between each treatment, expected Interaction between treatment and organisms, organisms take up lead in different manners ANOVA of E.coliNo uptake, ergo no differences, Pb depostion not a factor in toxicity ANOVA of Pb3Uptake rate changed with each [Pb], uptake of Pb showed a threshold value perhaps K M controlled ANOVA of Pb4Uptake rate changed with each [Pb], uptake of Pb showed a threshold value perhaps K M controlled

9. Growth Rate of Microbes as Influenced by Calcium  Calcium was inhibitory to each organism tested.  Lead was not able to reverse the inhibition of E. coli Ca, therefore they probably are not competing for similar cell sites.  Calcium exacerbated inhibition of Pb4 by Lead suggesting that their mode of action may be similar.  Calcium did reverse some of the inhibition of Pb3 in the presence of Lead. Remember at low concentrations Pb stimulates the growth of the yeast.

10. Influence of Calcium on Lead Uptake  Lead uptake by Pb3 was completely inhibited by Ca. This strongly suggests that the two divalent are competing for a similar site. The Ca may prevent the deleterious effects of excess Pb.  Lead uptake was only modestly affected by Ca, again suggesting that there is no interaction between the two metals.

11. Evidence for Cell Association of Lead in Pb3 and Pb4 a – Brightfield TEM image of Pb3 @ 25,000X magnification. Specimen was grown on Pb- free media and is not stained. No ultra- structure is visible within the cell. Bar = 0.5 µm. d – Brightfield TEM image of Pb4 @ 20,000X magnification. Isolate was cultured on Pb-free media and is not stained. Unidentified curved regions are visible inside the cell (arrows). Bar = 0.25 µm. b – Brightfield TEM image of Pb3 @ 15,000X magnification. Isolate was cultured on media augmented with 1.5 mM Pb. Specimen was not stained. Densely stained regions are visible inside the cell (arrows). These regions are likely Pb. Bar = 1 µm. e – Brightfield TEM image of Pb3 @ 25,000X magnification. Isolate was cultured on media enriched with 1.5 mM Pb. Specimen was not stained. Again, curved regions are visible inside the cell (arrows). Densely stained regions (Figure 3.4b) are not present. Bar = 0.25 µm. c – Brightfield TEM image of Pb3 @ 50,000X magnification. Isolate was cultured on media enriched with 1.5 mM Pb. Additionally, specimen was stained with OsO4, Pb citrate, and uranyl acetate. Densely stained regions are visible inside the cell (arrows), as are various membranes (arrowheads) and membrane bound organelles (asterisks). Bar = 0.25 µm. f – Brightfield TEM image of Pb3 @ 25,000X magnification. Isolate was cultured on media enriched with 1.5 mM Pb. Specimen was stained with OsO4, Pb citrate, and uranyl acetate. Densely stained regions are not visible inside the cell. However, light regions (nucleoid region) existed inside the cells (asterisk), and unidentified honeycomb structures (arrowhead) were observed inside many of these regions. Bar = 0.25 µm. Transmission electron microscopy images of ~70 nm thick sections visualized on a Jeol 100S TEM microscope at 100 keV accelerating voltage with objective aperture 4. Figures 3.4a – 3.4c were visualized using condenser aperture 1 and Figures 3.4d – 3.4f were visualized using condenser aperture 1. Pictures were taken onto Kodak 4489 electron microscope film (Eastman Kodak company, Rochester, New York) and the negatives were scanned at 700 dpi using an AGFA Duoscan scanner.

13. Electron Diffraction Spectra Major Peak Pb 3 (PO 4 ) 2 Minor peaks are variety of Pb minerals Electron Diffraction Spectra of the black granular material in TEM image b confirms the material is Pb, with at least 4 crystalline forms.

14. Summary and Conclusion  Bacteria and yeast were isolated from a chat pile and they showed resistance to the effects of lead relative to control bacteria like E. coli.  Pb4 morphologically resembles Rhodospora toruloides, but could not be identified by FAME analysis. More importantly the yeast showed the ability to bio-accumulate lead, removing 0.9 mM of soluble Pb per OD unit of growth. Small amounts of Pb, 0.01 mM actually stimulated growth. Further the Pb was seen to deposit inside the celluls by TEM and EDS analysis. This organism may be of substantial value in the removal and recovery of Pb from contaminated waters.  On the other hand, an Ochrobactrum strain removed lead less efficiently, and the cellular fate was not apparent.  The influence of Calcium underscores the differences in the interaction of Pb with the cells. In the yeast there is clearly interaction, where the Ca can reverse some of the toxic effects, and prevent uptake. In the bacterium, Ca has little influence on uptake, and the action of the two toxicants appear to be additive.

15. Acknowledgements  This poster was originally to be presented by Jennifer Seabaugh, but she was not able to attend. Her co- authors want to recognize that she was the driving force behind this research.  Ms. Seabaugh would like to thank members of her MNS Thesis committee, Drs. Christina Frazier and Philip Crawford for their guidance.  Dr. Edelman of Miami University, Oxford, Ohio, provided the electron microscope facility and his expertise.  Dr. Steven Overman was a valuable resource in identifying and obtaining chat.  The authors would like to thank Maija Bluma for her technical assistance.