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)