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.