By: Jason Jean and Janice Wing Davis Mine Project By: Jason Jean and Janice Wing

Davis mine

Drainage below tailings pile

Tailings pile

Drainage below tailings pile

Drainage below another tailings pile

Sample of pyrite at Davis Mine

Stream confluence upstream from A4

Stream confluence up close

Presentation Outline Reasons for our research project Methods Findings Obstacles Research continuation Impact on us

Reasons for our research project Personal Professional Enrichment media

Methods Sterile, anaerobic technique pH adjustment Inoculation Incubation Observations and Microscopy

Acquiring mine water from well

Collection bottles for Davis Mine groundwater

Measuring well water depth

Time to think

Sterilize bottles in the acid wash

Preparing enrichment media under anoxic conditions.

pH adjustment

Sterilize Postgate and Pfennig media before inoculation in the autoclave. Davis Mine Well media was not sterilized in the autoclave before inoculation.

Sediment from Well 1, 3, 4 were used

Opening sediment jars and inoculating sediment into media bottles under anoxic conditions

Incubation and observation

Storage and incubation of transfer bottles

Findings Sulfur-reducing bacteria grew in Postgate pH 7.0 Sulfur-reducing bacteria grew in Davis Mine Well 1 Groundwater media at pH 7.0 Thread-like filaments were visible in sample jars that developed a black precipitate, but in no others. Desulfobulbus was successfully cultivated in Postgate media but was not successful in Pfennig. Orange film developed in Pfennig enrichment bottles.

Enrichment bottles with Postgate’s medium at pH 3.0 4.0 5.0 7.0

Enrichment bottles with Pfennig’s medium at pH 3.0 4.0 5.0 7.0

Davis Mine Well 1 media at pH 3.0 4.0 5.0 7.0

Davis Mine Well 3 media at pH 3.0 4.0 5.0 7.0

Davis Mine Well 4 media at pH 3.0 4.0 5.0 7.0

Black precipitate formed in Postgate media of pH 7 Black precipitate formed in Postgate media of pH 7.0 inoculated with Desulfobulbus

Black precipitate formed in transfers of Postgate media at pH 7.0

Black precipitate formed in transfers of Davis Mine Well 1 media at pH 7.0

Microscopy using a live/dead bio-stain with this fluorescent microscope. Pictures were taken.

Microscopy of Postgate pH 3.0 sample inoculated with Well 1 sediment

Microscopy of Postgate pH 4.0 inoculated with Well 4 sediment

Postgate at pH 5.0, sediment from Well 4 Cluster

Postgate pH 7.0 sediment from Well 4 Thread-like filament

Postgate pH 7.0 sediment from Well 4 Dark spots of pyrite forming

What is the orange film and sediment? (Seen in all Pfennig media, here the sample is pH 3.0)

Obstacles Time Equipment Anaerobic technique is rigorous

Presence of oxygen

Contamination by a fungus

Tough going

Research continuation Dilution transfers and DNA extraction Continued incubation Postgate media with a variety of donors DM Well media with a variety of donors Mercy Iron-reducers

Impact on us Better understanding of long-range research Lab and project research experience Integrate into classroom curriculum aligned with state frameworks Biology Environmental Science

Questions ??

The End