Download Interaction of Lead and Calcium with Lead

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 PbNO3 at 25 oC. 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 KMcontrolled. 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.
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 leadresistant microorganisms.
 These organisms may provide
insight into:
 the physiological effects of
lead on the cell
 possible remediation strategies
involving bioaccumulation
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 soil
Concentration of Pb (ppm) found in the
following soil samples:
#12a
#15b
Exchangeablec
17
8
Organicd
51
635
Mn Oxidese
1984
155
Fe Oxidesf
673
491
Total Bioavailableg
2725
1289
Totalh
4023
2294
a
Source of Pb4
Source of Pb3
c Pb adsorbed to soil particles; displaced by boiling with 1 M Mg(NO ) at pH 7 for 2 h
3 2
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 at 100 psi
3
b
Method of Shuman, 1985. Soil Science 140:11-22
Isolation Strategy
 Bacteria were eluteda
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
Isolates Used in this Study
Pb4
Pb3
RM
TSA
TSA
1mM
Roane Media Pb
2.5mM
Pb
 Pb3: Yeast. No match in FAME
database. Web IDa suggests
Rhodospora toruloides
 Pb4: Gram (–) rod shaped
bacterium. Nearest neighbor
by RDPb /Phylip Interface
Ochrobactrum sp. Str. S1)
a
www.2.cbs.knaw.nl/yeast.asp
b http://rdp.cme.msu.edu/html
Approach – Lead Exposure
Studies
 Grow microbes in presence of Pb.
Quintuplicate 50 ml cultures were incubated statically at 25 oC
until OD exceeded 0.1. Pb3, Pb4, E. coli (negative) and uninoculated (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 oC, 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).
Growth Rate of Microbes at
Different [Pb]
0.2
0.19
0.18
0 mM
0.17
0.18
0.01 mM
GROWTH RATE hr -1
0.16
0.14
0.1 mM
0.11
0.12
0.1
1.0 mM
0.08
0.08
0.05
0.06
0.03
0.04
0.04
0.04
0.04 0.04
0.02
0.02
0
E. coli
Pb3
ORGANISM
Pb4
Statistical Test a
Significant differences (p<0.001), Comment
2-Way ANOVA
Growth 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.coli
Growth rate changed with each [Pb], decreases in k were significant
ANOVA of Pb3
Growth 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 Pb4
Growth 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
Lead Uptake, ΔmM per OD,
by Microbes
0.10
0.10
0.09
0.01 Pb
0.08
0.1 Pb
Relative Pb uptake
0.08
1.0 Pb
0.07
0.07
0.06
0.04
0.05
0.04
0.03
0.02
0.01
0.00
0.00 0.00
0.00
0.00
0.00
E.coli
Pb4
Pb3
ORGANISM
Statistical Test
Significant differences (p<0.001), Comment
2-Way ANOVA
Uptake 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.coli
No uptake, ergo no differences, Pb depostion not a factor in toxicity
ANOVA of Pb3
Uptake rate changed with each [Pb], uptake of Pb showed a threshold
value perhaps KM controlled
ANOVA of Pb4
Uptake rate changed with each [Pb], uptake of Pb showed a threshold
value perhaps KM controlled
Growth Rate of Microbes as
Influenced by Calcium
0.20
0.19
0 mM Pb ; 0 mM Ca
0.17
0.18
0.1 mM Pb
Growth rate hr-1
0.16
Pb + Ca
0.14
0.12
Ca
0.10
0.08
0.06
0.08
0.05
0.06
0.04
0.03 0.03
0.04
0.04
0.04
0.03
0.03
0.02
0.00
E.coli
Pb3
Pb4
Organism
 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.
Influence of Calcium on Lead
Uptake
0.09
0.08
0
0.1 Pb
0.09
PbCa
0.07
0.06
0.04
0.05
0.04
0.04
0.03
0.02
0.00
0.01
0.00
Pb4
Pb3
Isolate
 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.
Evidence for Cell
Association of Lead in Pb3
and Pb4
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.
a – Brightfield TEM image of Pb3 @ 25,000X
magnification. Specimen was grown on Pbfree media and is not stained. No ultrastructure 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.
Electron Diffraction
Spectra
2.5E+03
Major Peak Pb3(PO4)2
Signal Intensity
2.0E+03
1.5E+03
1.0E+03
5.0E+02
Minor peaks
are variety of
Pb minerals
0.0E+00
Electron Diffraction Spectra of the black granular
material in TEM image b confirms the material is Pb,
with at least 4 crystalline forms.
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.
Acknowledgements
 This poster was originally to be
presented by Jennifer Seabaugh, but
she was not able to attend. Her coauthors 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.