Executive Summary

          The purpose of this study is to investigate the bioremediation, or the introduction of microorganisms to break down environmental pollutants, of BTEX compounds using bacteria in a bioreactor. BTEX is a combination of chemicals that is found in flowback fluid. These chemicals share the shape of the benzene ring, making them non-polar  volatile, and aromatic. Often, soil near areas where hydraulic fracturing ("fracking") takes place is contaminated with BTEX, causing concern environmental concern. The study was divided into two parts: bioreactor construction and bacteria culture, using models found in previous research.

          The purpose of a bioreactor is to catalyze a biochemical reaction using a living organism. An ideal bioreactor is made of inexpensive materials, utilizes natural resources such as sunlight and wind, and produces chemicals that decrease pollution. We focused on a batch reactor, a vessel where reactants are introduced, a chemical process takes place, and products are removed. Yeast was used to simulation the bioreactor process using plastic bottles. Carbon dioxide, dissolved oxygen, and temperature were measured and recorded using Vernier Logger Pro software. These results were then used to determine the efficiency of two different sized bottles. Because they had similar efficiencies, the smaller was used to model how bacteria metabolism in a warm and cold environment to find the optimum temperature for the bioreactor. Because the rate of reaction occurred too suddenly in the warm environment--the levels of carbon dioxide and dissolved oxygen level off after about 25 minutes--and too slowly in the cold environment--after 50 minutes the level of production of carbon dioxide decreased--the optimum temperature was not found.

          In the later component, Pseudomonas putida was chosen as the bacteria to use in the bioreactor because the F1 strain contains the necessary enzyme to break the aromatic ring in two chemicals in BTEX and turn them into less harmful compounds. To obtain toulene-digesting bacteria, toluene, the “T” in BTEX, was introduced to colonies of P. putida that were cultured using both agar plates and nutrient broth tubes. Colonies of P. putida were sustained in these media and manipulated through changes in the percentage of toluene to the nutrient broth. Optical density was used to measure bacterial growth, and gram staining was used to determine if the bacteria was indeed P. putida by checking if it was gram negative and rod-shaped. The Folin-Ciocalteu reagent test was used to confirm the digestion of toluene. We found a significant amount of growth of bacteria in increasing percentages of toluene that some bacteria survived in a 50% v/v toluene, nutrient broth mixture. Gram staining concluded that the bacteria were rod-shaped, but because of possible contamination, the bacteria did not produce significant results for either gram positive or negative. The Folin-Ciocalteu reagent test was not successful in confirming the digestion of toluene by the bacteria grown.

Future Research

        In order to maximize the potential of the bioreactor and assess its full capability, further research must be conducted. It would be beneficial to employ a different test in lieu of Folin’s reagent in order to determine whether or not the bacteria were metabolizing the toluene. In addition, to test and understand how the bioreactor functions and what it is fully capable of, it must actually be put to use with bacteria. Though the process was simulated in a research lab setting using water, glucose, and yeast, it would be most beneficial to apply it to the P. putida, and to do this accordingly, the batch reactor would be assembled and conditions of temperature, time of exposure, carbon dioxide, and dissolved oxygen would be closely monitored throughout via Vernier Logger Pro software and changed accordingly. With a longer time period and greater funding, higher quality and better functioning bioreactors could be developed.

Results

Bioreactor

Initial Bioreactors

Conclusion:

          Because the graphs of temperature, dissolved oxygen, and carbon dioxide were similar between the two sized bioreactors (2 L and 20 oz), the smaller size was chosen to be used for testing temperature environments for efficiency.

Warm Bioreactors

Conclusion:

          The rate of carbon dioxide production and dissolved oxygen intake increased and then level off by 25 minutes. This reaction occurred too quickly for ideal conditions of a bioreactor.

Cold Bioreactors

Conclusion:

          The rate of carbon dioxide production increased much more slowly than the warm reactors and then decreased after 50 minutes, indicating a slow metabolism and finally a decrease in metabolism. The dissolved oxygen increases unlike the warm reactors, and then slowly decreases as the bacteria use the oxygen. The bacteria, however, use it more slowly than the warm reactors and level off at a higher rate of dissolved oxygen.

Bacteria

Optical Density

Conclusion:
          Though there was inconsistency, the typical value of the control plates was higher than that of the toluene plate. A higher absorbance reading means that there was more bacteria growth. This indicates that because of the addition of toluene, there was less bacteria growth.

Gram Stain

Conclusion:
          Because P. putida is a gram negative bacteria, the predicted result was pink rods. However, due to a possible contamination of the materials for the procedure, the results between gram negative and gram positive were inconclusive. However, the test confirmed that the bacteria were rod-shaped.

Folin's Reagent

Conclusion:

          The data proves inconclusive because of the great dissimilarity between the absorbance values for each trial accounting for the lack of precision. These values were expected to be close because each trial followed the same procedure and used the same materials as the next. This can be attributed to many errors, including those made by the individual such as not properly following the procedure. An abnormally high value in absorbance could be the result of a dirty cuvette since it would absorb more of the light than the clean cuvette. A high value could also be caused by an increased density of bacteria for the same reason of more absorption of light. A significantly lower value in absorbance could be the result of a too low level of liquid in the cuvette so that no or little light would be absorbed. Lastly, a low absorbance could be accounted for by an error in extracting the nutrient broth from the test tubes. If extracted incorrectly, toluene, which does not contain the stained hydrocarbons, could be present in the solution placed in the spectrophotometer.

          In order to prevent these errors from occurring in the future, there should be a uniformity in the cuvettes, in size and cleanliness, to prevent a difference in absorbance readings. Also, all sample should be filtered, mixed, or centrifuged before being placed in the spectrophotometer in order to remove the possibility of bacteria interfering with the reading. Finally, the containers used when extracting the nutrient broth should be easily accessible by the pipettes, in order to prevent toluene from being mixed with the nutrient broth.

Procedures

Bacteria

Pouring an Agar Plate:

• Melt bottled agar at 95o in a hot water bath.
• Pour the hot, liquid agar into a sterile petri dish cracked open slightly.
• Let the agar cool and set, then flip and store upside down to avoid contamination.

Plating the P. putida:

• Retrieve a sample from either a plate or test tube of bacteria with a wire loop.
• Streak the loop onto a third of the plate and rotated and repeated to spread out colony growth.
• Replace the lid and store upside down in an incubator.

Transferring P. putida from Plate to Broth:

• Retrieve a sample from a plate with a wire loop.
• Immerse the loop into a sterile test tube of nutrient broth and move the loop back and forth to dislodge the bacteria into the broth.
• Seal tightly and place into an incubator for growth.

Introducing Toluene to P. putida:

• Grow bacteria until enough sustained growth and obtain a sample.
• Immerse the bacteria in a test tube of nutrient broth and toluene.
• Incubate and grow the bacteria.
• Plate, incubate, and grow the bacteria.
• Repeat with a gradually increasing percentage of toluene.

Measuring Optical Density:

• Find the optimum wavelength for measuring nutrient broth in the spectrophotometer .
• Create a "blank" sample by pouring nutrient broth (without any bacteria) into a cuvette and zeroing the spectrophotometer to this absorbance reading.
• Put the sample of nutrient broth with bacteria into a cuvette and put it inside the spectrophotometer to read its absorbance.

Gram Staining Bacteria Growth:

• Place a sample of bacteria onto a clean slide with a sterilized wire loop.
• Allow the slide to dry and pass it through a Bunsen flame breifly to set the bacteria.
• Flood the slide with crystal violet solution, Gram's Iodine solution, alcohol, and safranin solution in rounds with washing in between.
• Blot the slide dry, place a drop of oil over the bacteria, and examine underneath a microscope.

Carrying Out a Folin's Reagant Test:

• Combine nutrient broth, water, Folin-Ciocalteau reagent.
• Wait for at least 30 seconds, then add sodium carbonate solution, and shake to mix.
• Let the mixture sit for 2 hours and then measure the absorbance in the spectrophotometer.

Bioreactor

Constructing the Bioreactor:

• Pick a container and lay out with a stir plate and clamp.
• Locate places for the probe and air holes and drill.
• Clean the bioreactor, set up, add stir bar.
• Fill with reactants and record results.

Measuring the Results of Bioreactor:

• Set up the probes inside the bioreactor.
• Attach to a computer with Vernier Logger Pro software.
• Choose an interval to record data and a length to run.
• Start the software immediately after the reactants are added.

Setting up Warm and Cold Environments:

• Warm: setup the bioreactor inside of an incubator.
• Cold: setup the bioreactor inside of a refrigerator.
• Record results as before.

Introduction

Hydraulic fracturing, or fracking, is the extraction of shale gas from below the ground. The extraction of shale gas has many potential economic benefits as a source of fossil fuel, and has provided many new jobs to the Western Pennsylvania region. There are environmental risks involved in fracturing, including the pollution of water and air. One product that poses a risk is BTEX, a combination of chemicals found in flowback fluid. These chemicals share the shape of the benzene ring, making them nonpolar, volatile, and aromatic. The bonds in the aromatic ring structure create strength, making it difficult to break apart the molecule. Soil containing these compounds is often contaminated, causing concern in areas where fracturing takes place. Only 0.56 of a gram of Toluene, the “T” in BTEX, is soluble in one liter of water. It is toxic to humans and other mammals and is lethal to fish. It is commonly used as an industrial solvent, extraction agent, and synthetic medium to produce high-order aromatics.

            Bacteria are asexual, sometimes rapidly growing organisms. Both archaebacteria and eubacteria have a rod-like structure. Obligate aerobes respire with oxygen while facultative anaerobes respire in the presence of oxygen and obligate anaerobes cannot respire with oxygen at all. Pseudomonas putida is a rod-shaped bacterium with flagella that is gram negative and generally non-pathogenic. The F1 strain is able to break the aromatic ring in toluene and ethylbenzene and turn them into less harmful compounds. There are several methods of obtaining the bacteria: to buy the F1 strain of P. Putida directly and cultivate it; to buy a general culture of P. Putida and expose it to increasing concentrations of toluene to produce only bacteria that will digest toluene; and to retrieve a soil sample, extract any bacteria through filtration or centrifugation, and repeat the exposure procedure. To cause P. putida to metabolize, bacteria were cultured using agar plates and nutrient broth tubes. Colonies of P. putida were sustained and further, manipulated by the addition of toluene (BTEX). In the bottled bioreactors, yeast was used to stimulate the bioreactor process combined with glucose, ammonium sulfate, potassium phosphate monobasic, magnesium sulfate heptahydrate, and calcium chlorate dehydrate.