Acid mine drainage is the main pollutant of water in the mid-Atlantic region. It is caused when water flows over materials containing sulfur. This causes a solution that has net acidity in it. Acid mine drainage occurs from coal mines currently been used or mines that have been abandoned. Water flows through these mines and deposits the materials in streams. This degrades the stream which results in loss of aquatic lives and contaminates the water so it cannot be used for other activities. Acid mine drainage (AMD) is a solution formed from chemical reaction between water, and rocks containing sulfur bearing materials. The water coming from coal mining areas are usually acidic and have been exposed to materials like rock containing pyrite. Acid is formed when pyrite, an iron sulfide is reacted with water, and air. It forms sulfuric acid and dissolves iron which begins to form red, yellow, or orange sediment. This occurs at the bottom of streams containing acid mine drainage.
This environment is perfect for iron oxidizing bacteria to create a thriving habitat. As acid mine drainage proceeds, the bacteria present increases the rate of action for the acid mine drainage. Although acid mine drainage degrades aquatic lives, microbes such as bacteria and archaea flourish in this region. They live in extreme areas with high temperature and also survive in regions with low pH, which can be as low as 4. Iron oxidizing bacteria derive off the energy they need to survive by oxidizing the iron present in these mine areas. These iron concentrations can be as low as 0.1 mg which is still enough for this bacteria to prosper in.
Background and Rationale
The bacteria used in this experiment was able to oxidize the sulfide found in pyrite to sulfate. They utilize carbon dioxide as their carbon source and sulfide in pyrite as their energy source. Redox reactions is used in this process for the formation of ATP used by the bacteria. Chemolithoautotrophs are the bacteria that undergo this kind of reaction because they oxidize inorganic compounds and use carbon dioxide as their carbon source. The chemical formula for this experiment is: 4FeS2 + 15O2 +14H2O ? 8SO42- +4Fe (OH)3 +16H+. Bacteria with lower pH levels enhance this reaction, because of their iron-oxidizing power resulting in: Fe2+ > Fe3+, S- > S6+. Acidithiobacillus ferrooxidans, is an iron oxidizing microbe that was used to stimulate this reaction and speed up the oxidizing reaction of sulfide in the experiment. It is gram negative, ?-proteobacterium that optimally grows in low pH and temperatures. It was found to be a heterotroph, because it requires planktons to survive, and it also lowers the pH of the solution after a certain period of time. It is known to be one of the few microorganisms able to gain energy from oxidation of ferrous iron in acidic environments. This bacteria is an essential member of microbial consortia because it is used to recover copper using a process known as bioleaching. This bioleaching process is the beginning to how this bacteria oxidizes iron. Ferrooxidans plays a key role when this bacteria is reoxidizing the Fe (II) to Fe (III) because it completes its cycle. Iron oxidizing bacteria are among the first microbes discovered for carrying out the fundamental geological process involving material containing iron. This bacteria could be called acidophiles because of their pH for optimal growth. Also their response to oxygen based on their physiology contributes to how they react in this solution.
Materials and Methods
At the beginning of this experiment, our team initially took the steps to create microorganisms that are autotroph. This was the goal because of our understanding that our chosen bacteria used inorganic carbon as its carbon source. With this in mind, we began the experiment using the sources to create acid mine drainage which is oxygen, iron pyrite, and CO2. We took 3 empty bottles and filled it with 1 gram of pyrite, 40 ml of acid water, and 10 ml of water into each bottle. Before going further, we ensured that each bottle is equal to one another. 1 ml of microbe was added only into the first bottle and after we used the serial dilution method to transport to the other two bottles. The serial dilution method is a series of sequential dilutions used to create a reduction in the culture of cells to a more usable concentration level. Finally we sealed each bottle using the crimper to allow CO2 and oxygen to enter. After a couple days of incubation, our team repeatedly did the Gram-staining method to find any new growth in the bottles. After repeated incubation periods and staining, our results remained null with little to no life found. With these results, we changed our goal from creating an autotroph to heterotroph assuming dormant cells were present. To do this we added 5 ml of yeast into each bottle and left for another incubation period. At the end of this period, we repeated the Gram-staining method and checked each under the microscope for hopes of life.
Results and Discussion
At the beginning of this experiment we expected to grow microorganisms that are autotroph. This attempt resulted in little to no life found which lead us to a different objective which was to create a heterotroph microorganism. With the intent that dormant cells are within the solution, we assumed growth will occur with the aid of yeast inside the solution. The results were a little more optimistic but they were almost identical to the first attempt being very little life. With this information, our team determined that the source we retrieved microbes from may have been outdated or the environment it initially adapted in changed. These two factors may play a role in why our solution bottles had very little life in them. We could also conclude that our solution may have not had enough iron present if any at all. Iron oxidizing bacteria can only multiply in its ideal habitat which contains high levels of iron.
Iron?oxidizing bacteria produce their energy by oxidizing the iron Fe (II) to Fe (III), which is how this bacteria survives. If iron is not naturally present, the chance of bacteria growth occurring is slim to none which may have occurred in this experiment. In the future, we can change the way we perform this experiment and how we extract microbes. We will need to ensure these microbes are fresh and are maintained within their known environment. This known environment will consist of maintaining low temperatures and pH levels and also ensuring enough iron is in the solution. This will start off the experiment strong and ensure growth is occurring because the microbes will be multiplying. With these corrections made, an optimal environment and microorganisms that thrive will be successful.