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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.

July 18 - July 23

July 18

170 pieces of 2 x 2 cm copper pieces were cut using scissors. The samples were then rinsed with 70% ethanol and dried with KimWipes. We made 1L AFM for later use. Labeled 12 petri dishes that had solid media(6 for bacteria and 6 for no bacteria, in which each dish has 4 pieces of copper). The masses of each copper sample were measured and recorded. Refer to the Summary of Tables for the masses of the copper samples. We could not finish set up because we do not have bacteria ready. Instead, we took a 1.2 mL OD 25.7 bacteria and diluted it to 10 mL of OD 3.08 bacteria. We placed 100 mL of the fresh AFM we made into each of our 9 flasks, and inoculated 1 mL of OD 3.08 bacteria into each flask for them to grow. We then placed the flasks in the shaker. It will only take one day for the bacteria to deplete the media because of the high starting OD.

July 19

Preparing Liquid and Solid Media:

At the start of the lab session 1 L of liquid AFM and 150 mL of solid AFM were created according to protocol provided by Jason. Using the radius of the petri dish (5 cm), it was determined that in order to create a layer of solid media of about 1 mL high, 8 mL of solid media would be placed in each petri dish. The solid media was pipetted into pre-labeled dishes containing copper pieces. The temperature of the solid media dropped too quickly upon pipetting, however, and the media solidified before it could be spread evenly on the dish. In order to submerge the copper pieces in media, the solid media was pipetted onto the copper pieces, then the dish was tilted to promote spreading. The parts of some of the copper pieces that were not submerged (specifically 2.3, 2.4, 4.3, 5.2) began oxidizing only two hours after the solid media was added to the dishes. All the copper pieces in the petri dishes also had air bubbles trapped underneath them. 100 mL of liquid media was pipetted into each of six prelabeled flasks.

Preparing Cells: The cell cultures were retrieved from the undergraduate lab, then placed into centrifuge bottles and then centrifuged with a water balance for five minutes at 12,000 g. Once removed from the centrifuge, the supernatant was poured out.

Masses of 9 microcentrifuge tubes were taken, labeled, and recorded. About 1 mL of liquid media was added to the pellet and the solution was transferred to the labeled microcentrifuge tubes. The tubes containing solution were than massed. The tubes were then microcentrifuged for 2 min at 12,000 g to better isolate the cells from the supernatant. We removed as much of the liquid as possible from the centrifuge tubes, leaving behind the pellet of cells. The tubes containing the pellet only was massed in order to determine the approximate mass of the bacteria in each tube (See Table 1 for masses).

Preparing Experiments: 1 mL of AFM was added to the microcentrifuge tubes and the pellets were mixed into the media. One solution was pipetted into each of three liquid media flasks (see Table 1 for corresponding bacteria and flasks). Copper pieces were massed and one was placed into each of the six liquid media flasks. The remaining six solutions were pipetted into six petri dishes and then spread around the plate using beads. The petri dishes were placed into the incubator at 7:28PM.

July 20

July 21

July 22

We took measurements of the masses of copper in the liquid media and the two samples of the copper in solid media (see past trials for copper cleaning procedures). When the flasks containing AFM and bacteria were removed from the shaker, they appeared a dark muddy brown. The copper samples in the flasks were completely etched away, leaving trace amounts of copper pieces which we did not mass. The masses of the copper pieces in these flasks were assumed to be 0. The solutions in the AFM flasks not containing bacteria were a cloudy white. The copper samples in one of the Petri dishes, that had the solid media without bacteria, were coated in a uniform blue-green layer which may have been copper that was etched away. The copper samples in Petri Dish #4, which did contain bacteria, were covered in a patchier green-white layer. Note that sample 4.3 experienced some oxidizing even before bacteria was added to the dish during setup on Thursday. The distilled water and iron sulfate flasks had transparent green-tinted solutions.

July 23

July 24 - July 27

July 24

We washed and autoclaved 9 flasks. Also, made 1L liquid media and grew cells in 9 flasks of 100 mL fresh AFM, while shaking, with initial OD of 0.5.

July 26

We cut 5x5 cm piece of copper, put nail polish on several 2x2 cm copper pieces, and centrifuged cells. We made 1L liquid media and used pH paper to test for pH. The pH was around 3-4 and so we added in more sulfuric acid. The pH still did not change. We put our cells back in the 9 flasks, each containing 100 mL of just made liquid media (AFM). The flasks were placed into a shaker.

July 27

The flasks were still clear from one day of shaking. We centrifuged one of the flasks and found out that there was no pellet. We used a pH meter and found out that the liquid media pH was a little below 1, which meant that all our cells, from July 24, died. We discarded the contents of all 9 flasks, cleaned them, and autoclaved them.

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

The last of the copper pieces from the last experiment were massed this week. A new set of experiments were started, making use of the fact that the hybrid setup (a layer of liquid AFM over the copper pieces preserved in solid media) from last week was effective in etching. We wanted to test how thin we could make the layer of liquid media so that etching would still occur. See our Data and Conclusions Page for a summary of our data.

Aug 19

See procedure for August 17. Four petri dishes were returned to the incubator (two bacteria, two without) at the end of the day. The copper pieces in the petri dish with bacteria that were matched lost, on average, about four times as much mass due to etching as the pieces in the dish without bacteria.

Aug 20

See procedure for August 17. Two petri dishes were returned to the incubator (one with bacteria, one without) at the end of the day. Again, the copper pieces in the petri dish with bacteria that were matched lost, on average, about four times as much mass due to etching as the pieces in the dish without bacteria.

Aug 21

The copper pieces in the last two petri dishes were massed. See procedure for August 17.

Aug 22

Setup for a new set of experiments took place today. More 2cm x 2cm copper pieces were cut and cleaned. The cells and supernatant were spun and the pellet was resubmerged in 10 mL of fresh AFM. We prepared the following petri dishes in triplicate (see solid media protocol for making solid media):

• Hybrid dish with 1 mL OD 1.708 bacteria + 9 mL liquid AFM (10 mL liquid media layer in total).
• Hybrid dish with 1 mL OD 1.708 bacteria + 4 mL liquid AFM (5 mL liquid media layer in total).
• Hybrid dish with 1 mL OD 1.708 bacteria (1 mL liquid media layer in total).
• Hybrid dish with 10 mL AFM only.
• Hybrid dish with 5 mL AFM only.
• Hybrid dish with 1 mL AFM only.

The dishes were placed in the incubator in the lab.

Aug 23

Flasks from yesterday were cleaned and autoclaved. The copper pieces from last week were viewed under a microscope.

Aug 25

One of each type of petri dish setup from Aug 22 was taken out of the incubator so that the copper pieces could be removed and weighed. A 'C' was painted on the face of several pennies with nail polish and thrown into a flask with 100 mL of AFM an 1 mL of OD 0.35 A. ferooxidans and put on a shaker.

August 26 - September 1

The last of the copper pieces in the hybrid setup petri dishes from last week were massed this week. Another experiment was set up to test if we could decrease the amount of liquid media in these dishes even further and still have etching occur. See our Data and Conclusions Page for a summary of our data.

Aug 26

One of each type of petri dish as was set up last week was taken out of the incubator, and the copper pieces in them were weighed.

Aug 27

One of each type of petri dish as was set up last week was taken out of the incubator, and the copper pieces in them were weighed.

The liquid media in the petri dishes that did not contain any bacteria was tinted a slight blue, most likely due to cupric ions that had diffused through the solid media into the liquid media solution. More cells were also grown for our next set of experiments.

Aug 29

A new set of experiments was set up today. The cells that were set to grow two days prior were isolated via the centrifuge, then resuspended in 10 mL of fresh AFM. The components of the solid media were also prepared for

Eighteen petri dishes were labelled. The following hybrid petri dish setups were prepared in triplicate, with two pieces of pre-massed copper submerged in a thin layer of solid media in each dish:

• Hybrid dish with 1 mL of OD 1.648 bacteria + 3 mL of liquid AFM (4 mL of liquid media total)
• Hybrid dish with 1 mL of OD 1.648 bacteria + 2 mL of liquid AFM (3 mL of liquid media total)
• Hybrid dish with 1 mL of OD 1.648 bacteria + 1 mL of liquid AFM (2 mL of liquid media total)
• Hybrid dish with 4 mL of liquid AFM only
• Hybrid dish with 3 mL of liquid AFM only
• Hybrid dish with 2 mL of liquid AFM only

A 'C' was painted on pennies and thrown into a flask containing 100 mL of liquid media and 100 microliters of OD 25.2 bacteria for etching.

The more heavily stained flasks from last week were filled with acid for cleaning and set on the shaker overnight to clean.

September

September 2 - September 8

Sept 2

One of each dish from the experiment set up on August 29th was taken out of the incubator. The copper pieces were taken out, cleaned with distilled water an 70% ethanol, and massed.

Sept 3

See procedure for September 2nd.

The painted pennies were removed from the flask. The pennies weren't etched correctly, which was likely a result of using a different type of nail polish compared to the first time.

Sept 4

See procedure for September 2nd.

The AFM solutions in the control dishes (not containing bacteria) were tinted a light blue, indicating that some of the cupric ions might have diffused through the solid media and into the liquid media.

In the cases of the hybrid dishes containing bacteria, the greatest etching occurred in the cases where there was more AFM (liquid media). This is likely due to the fact that the increased amount of ferric ions in solution supported greater cell growth, which in turn spurred on faster etching.

In the cases of the dishes that did not contain bacteria, except for one case (copper piece 7.2), the etching remained fairly consistent, regardless of the amount of liquid media in the petri dishes.

September 23 - September 29

Pennies were painted on with nail polish to form the letters 'C' and/or 'U,' placed a flask with liquid media and A. ferooxidans and left to shake over several days. They were then taken out and the nail polish was removed from them using acetone to reveal still-shiny patches of copper underneath the areas not exposed to the liquid media. These pennies were made in anticipation of the Maker Faire at the New York Hall of Science in Queens as proof of concept and as souvenirs for people who stopped by our table.