Metal Bioremediation Bioreactor With Bacillus L Bacteria.

Hello.

I am trying to breakdown metals like copper, manganese dioxide, and Nickel chloride with a bacteria called Bacillus L which spores and has been successful in 2 out of my 3 metal bioremediation projects. The key here is safe sporing bacteria with Maltose, Glucose, Sucrose, Corn starch, Potato Starch, etc with Sodium formate in small amounts to only encourage that bacteria from growing (selective medium).

I have grown the Nickel chloride reactor for 3 weeks and I don't see any change probably the Nickel chloride which attacks RNA and DNA killed the bacteria or significantly slowed the growth down.

Warning:

Copper powder is an Irritant, Sodium formate is toxic it acts as an irritating base and it is harmful if inhaled or swallowed. Nickel (II) chloride is toxic if ingested or inhaled and can cause cancer with high repeated exposure please use proper PPE and discard waste as Toxic waste as well. Thanks.

Sodium Metabisulfite is toxic and acids release toxic Sulfur dioxide gas. It a strong acid salt. Potassium Permanganate is a strong Oxidizer and will stain your skin brown be prudent while working with it. Corrosive to eyes and skin.

Vanadium oxide is very toxic by inhalation and toxic by ingestion wear PPE. Lye (KOH) is corrosive.

Supplies

Copper metal Powder

Corn Starch

Sodium formate

Potato Starch

Potassium Permanganate

Sodium metabisulfite.

Nickel (II) Chloride.

Maltose sugar.

Glucose sugar.

Potassium Hydroxide

Vanadium Oxide (V2O5).

4 percent Acetic acid in this case.

Agar.

Supplies:

Hot plate

Jewelry Scale up to 1000 g plus or minus 0.001 g error.

Air bubbler for an Aquarium.

Beakers or glass jars to pour in the completed agar gels.

Rubber hoses (distillation type) (Yellow) for the Air bubbler.

Step 1: Making the Copper Bioreactor.

About 7 g of copper powder (3.7 g initially up to 7 g) was added to a clean beaker, 2.45 g of sodium formate was added to 600 ml water, 3.78 g of Potato Starches and 7 g of Agar was added it was heated up to 80 degrees C. This is important to make sure it does not BOIL. The temperature should be kept below 90 degrees C with a thermometer.

This can take depending on your hotplate 30-45 minutes.

We want the agar to be fairly soft so air and the bacteria can easily penetrate it can fail if a too hard set of agar.

Good manual stirring is required too. This helps a lot.

You can at 70 degrees C pour the Agar mix into a big Jar or Plastic bag to allow more air and moisture plus CO2 to enter the Agar (this is Optional and It a way to enhance the growth of the bacteria.).

In period of a 4-6 weeks, the solution shall turn black, grey, purple, blue then emerald green (Copper formate at the end) due to the bacteria.

Step 2: Making the Manganese Bioreactor.

I don't have MnO2 so I need to add carefully Potassium Permagnate to a solution of sodium metabisulfite. Manganese dioxide a black solid is formed but the reaction MUST be done very carefully since it generates steam and lots of heat!

20 g of KmnO4 and 20 g of 600 ml of sodium metabisulfite is added slowly to mix. See above. It must be washed with very cold distilled water to get Pure MnO2. Then water and corn starch 5 g, Potato starch 4 g and Agar and 3 grams of sodium formate are added and heated to 80-90 degrees C. Some Maltose and Glucose were added to the water mix. (4 g each).

The yellow color in pic 4 is what happens when either sodium carbonate (forms outside the cell when formic acid is used up ) and reacts with air to form Manganese (III) hydroxide which is less toxic than other forms of Manganese salts. This yellow appearance only occurs in basic conditions pH 8 to 9. Again 4-6 weeks are required for this reaction to be complete.

Step 3: Nickel Chloride Bioreactor Prep.

Weighing out 2 g of Nickel (II) Chloride 6 Hydrate, 5 g of Maltose, 1 g of Sodium formate, and 7 g of Agar, as well as 3 g of Corn starch, was added to 200 ml of water. Air bubbler hoses were added to the beaker with 100 ml of water with 4 g of extra Maltose sugar. The air bubbler was set to High to get as much Oxygen into the system. The bacillus bacteria can grow in low oxygen and high oxygen environments. Sometimes after 5 days, the Air pump is turned Off to save the pump and activate the high oxygen environment. and then air from the bubblers was used to increase airflow to the agar.

However, even after 4 months the Nickel salt was too toxic and interfered with the bacteria's ability to reproduce and the bacteria were unable to be Reduced or Oxidized into other forms of metals. Like MnO2 in +2 was converted into +3 which was less toxic which made it survive more.

Step 4: Vanadium Oxide Prep.

Please see the end of Instructables for Pic.

We add to a distiller flask 5 g V2O5 to 13 g of KOH (Potassium Hydroxide) and most expect for 3.78 g KOH is used up so a solution of 4 percent Acetic acid 15 ml is added to the 300 ml solution until the pH is 6.2 roughly. This will allow the bacteria to grow.

If the pH is 8 or above prob the bacteria will have a difficult time growing.

To 400 ml of water about 1 g of K3VO3 is added with 5 g Potato starch, 5 g Maltose, 2 g Sodium formate, and 7 g of Agar then it is heated carefully to 90 degrees C for 30-40 minutes and allowed to cool to a solid. About 100 ml of water with a concentrated solution of Bacillus L is added with 5 g of extra Maltose for the bacteria. It is sent with air bubbler two rubber hoses for 5 days approx.

A change in color did occur!

Step 5: Final Thoughts in Manganese, Copper and Nickel Reactions With Bacillus L Bioremediation.

The Copper even tho it was toxic was not as toxic to this sporing of the bacillus bacteria. However, the bacteria could partly resist the copper even if it was toxic (disrupted enzymes, slowed down the metabolism of the cell of that bacteria.

The air control for 2-3 months was critical and so was Soft agar gels. It worked very well for the Copper and Manganese reactors since MnO2 is low toxicity even with sodium formate.

On the other hand Nickel salts like Nickel (II) chloride are toxic and cancer-causing and damage DNA and induce free radical O2 in the cells (bacteria) thus they cannot be used to Neutralize the bacteria (1-month trial).

Other salts like Vanadium may be used since it can convert into less toxic salt forms so if I had extra time I may wanted to use it or buy Salts of Vanadium could potentially be used.

This shows that Solid and other metal salts have the potential to be converted into less toxic metals by bacteria while being trapped in the agar. Solid agar waste. Nickel will have to be delt as dangerous solid waste when it is done.

Thin plastic bags PE could be used instead of glass since they are more permeable to air and oxygen as well as CO2 (Carbon dioxide) so they could be more effective.