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Cara M. Santelli
Department of Mineral SciencesSmithsonian Institution, National Museum of Natural History
Email: [email protected]; Twitter: @biominerals
Microorganisms contributing to manganese (Mn) remediation in passive treatment technologies
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Colleen Hansel (WHOI)
Funding
Dominique Chaput (Smithsonian)
Collaborators
Bill Burgos (PSU)
Fubo Luan – PSUDonald Pfister – HarvardSam Webb – SSRLAlice Dohnolkova – EMSLStream Restoration, Inc.Brent Means - OSM
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Passive Remediation Systems
Fe Attenuation
Mn Attenuation
Neutral pH
Low metals
(pH 6-8)
Mn(II) Mn(IV)(dissolved) (solid)
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Passive Remediation Systems
Fe Attenuation
Mn Attenuation
Neutral pH
Low metals
(pH 6-8)
Only partially effective
What is promoting Mn(II) oxidation and subsequent Mn oxide mineral formation?
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Mn(II) Mn(IV)Dissolved MnO2
Minerals
Lab experiments show abiotic factors only partially account for Mn oxidation in AMD treatment systems
Luan et al., 2013, Applied. Geochem.
pH >9
O2
Promoters:
(Mineral surfaces)(microbes)
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Objectives:
• Identify Mn-oxidizing microbes
• Identify conditions that induce growth/oxidation
• Determine key players
• Identify mechanisms for oxidation
• Characterize biomineralization products
Basic science research informs bioremediation strategy
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• Sampled variety Mn treatment systems in PA
Comparative study of passive MRBs
• High dissolved Mn (II) concentrations
A geologist doing what geologists do best….banging on rocks and digging holes in the ground.
• Varying attenuation efficiency
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Location of MRBs
DS – De Sale (Slippery Rock Creek Watershed)DR – Derry Ridge (Saxman Run)FV – Fairview
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Which microbes promote Mn(II) oxidation?
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Microbial Mn oxidation
WHY??? No physiological reasonNo “gain” for converting Mn(II) to Mn(IV)
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Culture-based Approach:
15 different media types
> 1000 cultures started from sediment, water, rocks, “goo”
DNA sequenced for identification
Non-oxidizing culture
Mn-oxidizing microbes
Dana Lazarus – undergrad intern
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Santelli et al., 2010, AEMChaput et al., in prep.
>95% Mn-oxidizing isolates were Fungi and Algae!!!!
~150 isolates - 9 different Ascomycete fungal species - 4 different bacterial species
- >14 algal species (more being isolated)
Culture based approach
Species = “types”(e.g., Homo sapiens)
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Santelli et al., 2010, AEM
Mn(II) oxidizing fungi
• Most are very common soil fungi• Found all over the world• All systems had fungi
Plectosphaerella cucumerina
Stilbella aciculosa Pithomyces chartarum
500 µm
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Plectosphaerella cucumerina
500 µm
5 mm
5 mm
hyphae
oxides
-Most commonly isolated
-Found in almost all sites
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green algae (Chlorophyta)
diatoms (Bacillariophyta)
*Species ID underway*
Mn(II) oxidizing algae
Dominique Chaput - postdoctoral fellow (SI)
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Culture based approach
Necessary and informative:• Diverse fungi & algae (and some bacteria) promote
Mn removal in passive treatment systems• Species types do not correlate with system efficiency
Drawbacks:• Not all organisms are culturable (understimate)• Can not determine cell abundance or activity in situ
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What influences growth and oxidation of fungi?
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Physiological Growth Experiments
• pH 5 – 8 • Light/Dark• Mn tolerance• Nutrient source (e.g., C and N)• Nutrient concentration
Conditions:
Measured mycelia radial growth rates
pH and light/dark had no impact on growth rates
Oxidation rates are difficult to measure
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Plectosphaerella cucumerina
Mn(II) tolerance > 10 mM for 7 of 9 species
Mn (II) tolerance
10,000
5,000
1,000
750
Microdochium bolleyi
500
250
0
Mn(II) mM
Growth at 10 mM
High metal
tolerance
Mn(II) toxic at 5 mM
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Plectosphaerella cucumerina
Growth rates influenced by Mn(II) concentration
Mn (II) tolerance
10,000
5,000
1,000
750
Acremonium strictum
500
250
0
Mn(II) mM
Growth rates
Growth rates
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Nutrient (C) source and concentration
Stagonosporanodorum
Plectosphaerella cucumerina
Pyrenochaeta inflorescentiae
0.5mM 5.0mM 50.0mM
-
+
+-
- + +
+ -
Glucose
Fungal isolate
Acetate
+
+
-
- -
-
5.0mM 50.0mM
• No consistent trends• Mn oxidation can be turned on or off!!!
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Nutrient (C) source and concentration
Stagonosporanodorum
Plectosphaerella cucumerina
Pyrenochaeta inflorescentiae
0.5mM 5.0mM 50.0mM
-
+
+-
- + +
+ -
Glucose
Fungal isolate
Acetate
+
+
-
- -
-
5.0mM 50.0mM
• Next: Test “realistic” sources: • Mushroom compost, corncobs, woodchips?
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But microbes don’t grow individually in the environment…
What are the overall community dynamics?
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Community effects on Mn removal?
Positive interactions
Negative interactions
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Community effects on Mn removal?
Positive interactions
Negative interactions
Enhanced oxidation
Inhibited oxidation
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Extract total DNA
DNA Community diversity
Species Identification
Molecular Biological approachMRB Samples Soil Sample
4 Manganese Removal Beds (MRBS) Sampled
2 high efficiency2 low efficiency
Amplicon pyrosequencing/Illumina(bacteria, archaea, fungi,
algae)
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“Known” Mn oxidizers account for < 0.15% of bacterial community
High efficiencyLow efficiency
~1-9% of fungal communityGreatest proportion of fungi in high efficiency MRBs~ algae ? (in progress)
Distribution of Mn(II)-oxidizers
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High efficiencyLow efficiency
Distribution of Mn(II)-oxidizers
Mn(II) oxidizers are already in the MRBs!
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Complex microbial communities in MRBs
Next steps:• More growth experiments (“realistic” C sources,
algae, community competition assays)
• Laboratory mini-MRBs (best “realistic” C sources, track total community dynamics)