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Columbia Copper Experiment Notebook

June

June 22- June 25

June 22

• Met with Jason, who showed us how to make A. ferrooxidans liquid media, prepared Copper 1 and 2 samples (with and without bacteria) and placed on shaker. Will be periodically checked until June 25 twice a day for activity.
• Showed us recipe for solid media, and made enough for two samples, 20 mL (one control with no bacteria). Copper samples were submerged in agarose (see Steven’s pictures). Also learned protocols listed below.

Starting Sample masses includes: Copper 1: (liquid media only) Sample 1: 0.0092g, Sample 2: 0.0107g and Copper 2:(liquid media only): Sample 1: 0.1318g, Sample 2: 0.1286g. For Copper 2: (solid media) the masses for sample 1 was 0.1272g and mass for sample 2 was 0.1282g. |}

Liquid media and flasks were placed in shaker at 11:35 am, 225 rpm, 30C. Solid media and petri dishes were placed in incubator at 12:42 pm, 30C.

June 23

• Checked the progress of all copper samples that included Copper 2 samples in solid media with and without bacteria, Copper 1 and 2 samples in liquid media each containing two samples of with and without bacteria.
• Observations were made for all samples. Optical density was measured for all of the samples submerged in liquid basal AFM low pH solution. Mass was weighed only copper 2 in liquid media with and without bacteria.

Solid Media Samples: Copper 2- Sample 1 (fully submerged, without bacteria)

• Observations: Solid media mostly clear except for the outer edges of the Copper which appeared to be a light golden yellow color (note picture below). Copper turned to a dark brown color. No observable size change to the copper.

Copper 2- Sample 2 (half submerged, with bacteria)

• Observations: Solid media turned from clear to a light golden yellow color (similar to that of the outer edges of the Copper from sample 1 mentioned above). Copper turned to a dark brown color. No observable size change to the copper.

Liquid Media Samples: Copper 1- Sample 1 (with bacteria)

• Observations: Noticeable 7+ small pieces of copper sample within liquid media (note picture below). Conclude to not take mass for this sample.
• Cuvette reading2: 0.003 A
• Current mass: N/A, Previous mass: 0.0092g, Initial mass: 0.0092g
•  % Mass Lost: N/A

Copper 1- Sample 2 (without bateria)

• Observations: No significant copper pieces visible in liquid media (note picture). Conclude to not take mass for this sample.
• Cuvette reading: 0.004 A
• Current mass reading: N/A, Previous mass: 0.0107g, Initial mass: 0.0107g
•  % Mass lost: N/A

Copper 2- Sample 1 (with bacteria)

• Observations: No observable change (see photo below).
• Cuvette reading: .004A
• Current mass: 0.1063g, Previous mass: 0.1318g, Initial mass: 0.1318g
•  % Mass Lost: 19.35%

Copper 2- Sample 2 (without bacteria)

• Observations: No observable change to copper (see picture below).
• Cuvette reading: 0.040 A**
• Cuvette reading: 0.018 A***
• Current mass reading: 0.0953g, Previous mass: 0.1286g, Initial mass: 0.1286g*
•  % Mass lost: 25.89%

June 24

• Took out all four samples for standard check of OD and mass of copper 2 samples. Solid media was also examined. All samples (liquid and solid) were photographed.
• No visible copper 1 samples present in either sample (see Observations)

Solid Media Samples: Copper 2- Sample 1 (fully submerged, without bacteria)

• Observations: A tint of green color on copper surface, the immediate radius of the copper has brown color, outside of the previously mentioned radius is a yellow tinge to the solid media. The rest of the solid media was clear. No observable size change.

Copper 2- Sample 2 (half submerged, with bacteria)

• Observations: A darker more present shade of green on copper, compared to that of sample 1 mentioned above. Outer edges of the copper appeared black. Media was a uniformed amber color. No observable size change.

Liquid Media Samples: Copper 1- Sample 1 (with bacteria)

• Observations: Liquid media copper brown, that may include ions that are not copper. No visible copper pieces, and conclude to not take mass for this sample. During procedure, centrifuged for another 1 minute to original 2 minutes at sane speed (due to the lack of expected visible pellets of some kind*)
• Cuvette reading1: 0.025 A
• Current mass: N/A, Previous mass: N/A, Initial mass: 0.0092g
•  % Mass Lost: N/A

Copper 1- Sample 2 (without bateria)

• Observations: Slight light green color to the media, and media appeared slightly cloudy. No significant copper pieces visible in liquid media. Conclude to not take mass for this sample.
• Cuvette reading: 0.033 A
• Current mass reading: N/A, Previous mass: N/A, Initial mass: 0.0107g
•  % Mass lost: N/A

Copper 2- Sample 1 (with bacteria)

• Observations: Visible brown copper color to liquid media. Pellets formed when the media was centrifuged. Many fluctuations in mass was seen, possible sources of error maybe water droplets or cleanness of the tweezers.
• Cuvette reading: .667A
• Current mass 1: 0.0550g, Current mass 2: 0.0388g, Current mass 3: 0.0285g
• Previous mass: 0.1063g, Initial mass: 0.1318g
•  % Mass Lost 1: 48.26%,  % Mass Lost 2: 63.50%, % Mass Lost 3: 73.19%

Copper 2- Sample 2 (without bacteria)

• Observations: Copper is visible in faint blue-green liquid media. No observable change to copper. Observable pellets after centrifuging (Unknown origin). Note the varied measurements in the masses, although not of a significant drop as in Copper 2 liquid media sample 1.
• Cuvette reading: 0.040 A
• Current mass reading1: 0.0907g, Current mass reading2: .0842g
• Previous mass: 0.0953g, Initial mass: 0.1286g
•  % Mass lost 1: 4.83%, % Mass lost 2: 11.65%

June 25

• Took out all four samples for standard check of OD and mass of copper samples. Solid media was also examined. All samples (liquid and solid) were photographed.
• Only sample 2 of copper 2 in liquid media was able to be measured for the other samples were either etched by bacteria or physically torn apart by shaking or by the AMF media.

Solid Media Samples:

Copper 2- Sample 1 (without bacteria) Removal: 9:51, Return: 9:55 Observations: Green copper sample on surface. No clear media visible. (see photo below).

Copper 2- Sample 2 (with bacteria) Removal: 9:51, Return: 9:55 Observations: A light shade of green on copper. Outer edges of the copper appeared black. Media was a uniformed amber color. No observable size change. (see photo below).

Note: One or both of the solid media samples dried up, therefore requiring another set of experiments.

Liquid Media Samples:

Copper 2- Sample 1 (with bacteria) Removal1: 9:56, Return1: 10:23, Removal2: 10:41, Return2: 10:54* Observations: Visible brown and murky copper color to liquid media. Pellets formed when the media was centrifuged. Copper appeared to turn into little powder, pellet form, and conclude mass is difficult to measure and may alter data. (see photo below). Cuvette reading1: .034A, Cuvette reading2: .057A* Today’s mass (fourth day): N/A Third day’s mass1: 0.0550g, Third day’s mass2: 0.0388g, Third day’s mass3: 0.0285g Second day’s mass: 0.1063g, Initial mass: 0.1318g % Mass Lost: N/A (All of the copper was etched)

• Note: Since the spectrophotometer reading was significantly lower than that of yesterday (.667 A), the sample’s OD was read twice

Copper 2- Sample 2 (without bacteria) Removal: 10:24, Return: 10:58 Observations: Copper is visible in muddy but faint blue-green liquid media. No observable change to copper (see picture below). Observable pellets after centrifuging (Unknown origin again like yesterday). Note the varied measurements in the masses, although not of a significant drop as in Copper 2 liquid media sample 1 which turned to nothing. Cuvette reading: 0.118 A Current (fourth day) mass1: .0400g Current (fourth day) mass2: .0324g Third day’s mass reading1: 0.0907g, Third day’s mass reading2: .0842g Second day’s mass: 0.0953g, Initial mass: 0.1286g

•  %Mass change1: 55.90%, % Mass change2: 64.28%
  •  %Mass change1: 52.49%, % Mass change2: 61.52%

• Note: Based on first reading from yesterday
  • Note: Based on second reading from yesterday

Copper 1- Sample 1 (with bacteria) Removal1: 10:55, Return: 11:04 Observations: Liquid media copper brown, that may include ions that are not copper (note picture below). Some visible tiny fragments of original sample, but conclude to not take mass for this sample. After centrifuging there were visible pellets (bacteria). Cuvette reading1: 0.094 A Current mass: N/A, Previous mass: N/A, Initial mass: 0.0092g % Mass Lost: N/A

Copper 1- Sample 2 (without bateria) Removal: 10:24, Return: 11:19 Observations: Slight light green color to the media, and media appeared slightly cloudy. No significant copper pieces visible in liquid media (note picture). Conclude to not take mass for this sample. Cuvette reading: 0.022 A Current mass reading: N/A, Previous mass: N/A, Initial mass: 0.0107g

% Mass lost: N/A

July

July 5 - July 19

July 5

• Cut 50 pieces of 2x2 cm copper pieces
• Made Liquid Media

July 7

After removing the flasks containing AFM from the shaker, we noticed a rather obvious layer of whitish-yellow precipitate sitting at the bottom of the flasks. The flasks without AFM—the controls with the distilled water and sulfuric acid—did not contain this white precipitate. The source of the precipitation is unknown.

August

July 29 - August 4

After a failed attempt at setting up solid media copper etching experiments to test for control and efficiency, we were able to correct our mistakes by adjusting for the firmness of our solid media solutions and proceed with experimental setup.

July 31

A. ferroxidans were left to grow overnight and Solution C (pH 2.35 and 2 w% iron, or ~10g/500 mL) was made.

Aug 2

The A. ferroxidans pellets were separated from its supernatant using a centrifuge. Solution D, an agar solution necessary for making the solid media, was made to have a concentration of 1%. The following experiments were set up twice in solid media (unless otherwise stated)--one dish for each experiment contained cells, and one dish did not:

• One 5x5 cm copper piece, with cardstock placed on top of the solid media
• Four 2x2 cm copper pieces with nail polish (one fully painted on both sides, one half painted on both sides, one half painted on one side, and one not painted) with cells on each copper for the petri dish containing cells
• Four 2x2 cm in 15 mL of solid media with 15 mL of AFM on top
• Solid media enclosing copper in liquid media in shaker

Aug 3

The agar concentration was found to be too low for the experiments set up on 8/2, as the solid media did not solidify overnight. The experiments were scrapped and restarted with solid media of a higher agar concentration (4%).

Aug 4

4% agar was a more successful alternative. The experiments listed above for August 2nd were set up once again.

August 5 - August 11

Observations were made of the experiments set up the past week. Firmer solid media, greater % of agar in solution, may have also contributed to the reduced oxidation we saw in the copper pieces. The bacteria in most of our setups were also consistently etching more copper in the same period of time compared to the dishes that did not contain bacteria.

Aug 5

The copper pieces were massed and more experiments were set up.

Aug 7

Observations were taken of the flasks filled with liquid media containing the copper piece in a solid media "shell." The solution in flasks containing no bacteria appeared unchanged. Copper in solid media in these flasks appeared darker compared to Day 1. In the flasks containing bacteria, the solutions turned a deep amber color. The solid media was still intact. The flask labelled Bac 1 developed a yellow residue and its solution was more opaque than flasks Bac 2 and 3. The yellow residue also seemed to be coating solid media.

Aug 8

The copper pieces in the flasks were massed and it was determined that the average mass of copper etched away by the flask containing copper was greater than the average mass of copper etched in the case where there was no bacteria (11% mass loss versus 1%, respectively). Biological etching was considered a success.

Aug 9

Observations were made of the petri dish setups.

1. Big Copper with Cardstock:

   - w/ bacteria: bacteria seems to promote oxidation of Cu (Copper turned green) w/o bacteria: copper turned a very dark brown. solid media in bac and no bac have turned yellow.

2. Nail Polished Copper:

   - no visible etching seem through layer of nail polish for both bacteria and no bacteria. - etching where Cu is exposed is still occurring.

3. Solid and Liquid Media:

   - w/ bacteria: liquid media turned orange-brown and there is some oxidation on copper pieces - w/o bac: copper pieces not shiny anymore and turned dark brown. Liquid media is still transparent.

Aug 10

The copper pieces were removed from all the petri dish experiments. It was determined that more copper was etched from the petri dishes containing bacteria in all cases.

August 12 - August 18

Since the experiment with the solid media encased copper shaking in the liquid media proved successful last week, we decided to repeat the experiment with much less liquid media--the AFM solution would only be a thin layer, with and without bacteria sitting on top of a petri dish with copper submerged underneath a layer of solid media. Setup for this experiment was done this week. The AFM solution was continually replenished throughout the week to ensure the bacteria would always have a source of ferrous ions for continuous growth.

Aug 14

Setup for the hybrid experiment (solid media with a layer of liquid media on top) took place. Solution B for solid media production had to be made again, due to bacterial contamination in our existing batch. Ten petri dishes with four copper samples in each were prepared--five with 15 mL of AFM and 1 mL of OD 27.5 bacteria, and five with only 15 mL of AFM. Dishes were placed in an incubator.

Aug 15

The liquid media in the petri dishes containing bacteria was tinted brown, but remained clear in the dishes not containing bacteria.

New AFM was added to replace the old liquid layer in the petri dishes containing bacteria. The cells were first separated using a centrifuge, and then put back in solution in 1 mL of fresh AFM, then transferred back to the petri dishes. There was a lot of brown precipitate in our pellet, much more than there were cells, that we couldn't separate, so we added it back to our dish for minimal loss of cells.

Aug 16

See Aug 15. Additionally, the copper pieces had a dark brown layer building up on its surface within the solid media where they were exposed (not covered by nail polish).

Aug 17

The same procedure for replacing the AFM from 8/15 was used for all but one of the dishes containing cells. The copper pieces from this dish were extracted from the solid media, cleaned, and then massed. This process was repeated for one petri dish not containing bacteria. On average it was determined that the copper in the petri dish with bacteria lost more mass on average than the pieces without bacteria. From this data, the surface area of the exposed copper, and the known density of the copper, we attempted to measure the depth of copper etched as well. The rest of the dishes were returned to the incubator.
The liquid media in the bacteria dishes was an orange-brown (precipitate buildup), while the liquid media in the dishes without bacteria remained clear.

Aug 18

See previous day. Even more etching had occurred in both cases, with the biological etching system proving to be the faster system yet again.

August 19 - August 25

Aug 19

Aug 20

Aug 21

Aug 22

August 26 - September 1

Aug 29

September

September 2 - September8

Sept 4