Post on 16-May-2015
description
Cara M. Santelli
Department of Mineral SciencesSmithsonian Institution, National Museum of Natural History
Email: santellic@si.edu; Twitter: @biominerals
Microorganisms contributing to manganese (Mn) remediation in passive treatment technologies
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
Passive Remediation Systems
Fe Attenuation
Mn Attenuation
Neutral pH
Low metals
(pH 6-8)
Mn(II) Mn(IV)(dissolved) (solid)
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?
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)
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
• 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
Location of MRBs
DS – De Sale (Slippery Rock Creek Watershed)DR – Derry Ridge (Saxman Run)FV – Fairview
Which microbes promote Mn(II) oxidation?
Microbial Mn oxidation
WHY??? No physiological reasonNo “gain” for converting Mn(II) to Mn(IV)
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
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)
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
Plectosphaerella cucumerina
500 µm
5 mm
5 mm
hyphae
oxides
-Most commonly isolated
-Found in almost all sites
green algae (Chlorophyta)
diatoms (Bacillariophyta)
*Species ID underway*
Mn(II) oxidizing algae
Dominique Chaput - postdoctoral fellow (SI)
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
What influences growth and oxidation of fungi?
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
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
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
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!!!
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?
But microbes don’t grow individually in the environment…
What are the overall community dynamics?
Community effects on Mn removal?
Positive interactions
Negative interactions
Community effects on Mn removal?
Positive interactions
Negative interactions
Enhanced oxidation
Inhibited oxidation
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)
“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
High efficiencyLow efficiency
Distribution of Mn(II)-oxidizers
Mn(II) oxidizers are already in the MRBs!
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)