Encouraging High School Students To Learn About Bioremediation

This paper presents a laboratory activity for high school students used to stimulate their interest in environmental engineering and the role of bioremediation in cleaning up the environment. The proposed laboratory activity utilized six, 2-L plastic bottles that contain 100-grams of indigenous soil in each to serve as bioreactors. Varying amounts of glucose are added to the reactors, which are monitored with time for a period of five to ten days. Questions for assessing the exercise along with sample laboratory results are provided. Introduction The challenges of environmental engineers have historically focused on the design of drinking water treatment facilities, municipal and industrial wastewater treatment, solid waste collection and disposal systems, and air pollution control equipment. In recent years, these challenges have expanded to include the identification, removal, and treatment of hazardous chemicals and wastes that have resulted from inadvertent spills or illegal discharges to the land, water, and air. This paper presents a laboratory activity for high school students used to stimulate their interest in environmental engineering and the role of bioremediation in cleaning up the environment. Bioremediation is a natural process in which indigenous microorganisms found in soil and water are utilized for treating primarily toxic organic compounds such as solvents, pesticides, herbicides, and precursors for industrial processes. These microorganisms transform the toxic organic compounds into less harmful products such as carbon dioxide and water. It can be used to treat contaminated media, excavated soil, soil in situ, groundwater, surface water, and gases emanating from soil. Bioremediation requires the control and manipulation of microbial processes, therefore, requiring the integration of scientific principles with engineering. The proposed laboratory activity utilizes six soil bioreactors to measure the concentration of glucose over time to simulate how heterotophic bacteria in the soil would consume and transform gasoline or oil into innocuous products. Different glucose concentrations are added to the soil in five of the bioreactors and the sixth serves as a control. Parameters that may be measured over time include dissolved oxygen (DO), pH, glucose concentration, and chemical oxygen demand (COD) or biochemical oxygen demand (BOD). Colony forming units (CFU) and turbidity analyses may also be conducted to quantify the microbial growth rate. P ge 772.1 Proceedings of the 2002 American Society for Engineering Education Annual Conference & Exposition Copyright 2002, American Society for Engineering Education Background Consider a scenario that leads to a hazardous material being released into the environment. For example, when a tanker truck overturns and gasoline percolates through the soil and into the groundwater, how does this impact the ecosystem? Some of the gasoline volatizes and is released to the atmosphere contributing to smog production. Gasoline that reaches the groundwater contaminates it making it unfit for human consumption. Aerobic, heterotrophic bacteria that proliferate in the soil are capable of using the organic components in the gasoline for energy and the synthesis (growth) of more bacteria. Engineers capitalize on this phenomenon and attempt to further stimulate the microbial degradation process. Microorganisms are used in environmental engineering for treating industrial and municipal wastewater; contaminated groundwater; digestion of sludges; and remediation of toxic and hazardous wastes. Aerobic, heterotrophic bacteria require oxygen, carbon, nitrogen, and phosphorus in order to flourish in the environment. Heterotophic microorganisms use organic carbon for two purposes; synthesis of cellular components and the production of energy. The following equation denotes the synthesis of biomass assuming that the composition of a typical microorganism can be expresses as C5H7O2N: O H CO N O H C NH O O H C 2 2 2 7 5 3 2 6 12 6 4 + + → + + Eq [1] A portion of the organic carbon is also oxidized for the production of energy. The stoichiometric equation for the oxidation of carbon is as follows: 2 2 CO O C → + Eq [2] Bacteria typically grow at an exponential growth rate according to the following equation: ( ) t K O e X X − = Eq [3] Xo, and X = Microorganism concentration initially and at the end of the monitoring period, mg/L, K = Microorganism growth rate constant, days, and t = Monitoring time period, days. Substrate such as glucose or sucrose is typically consumed by bacteria at an exponential rate according to the following equation: ( ) t k O e S S − = Eq [4] So, and S = Substrate concentration initially and at the end of the monitoring period, mg/L, P ge 772.2 Proceedings of the 2002 American Society for Engineering Education Annual Conference & Exposition Copyright 2002, American Society for Engineering Education k = Substrate removal rate constant, days, and t = Monitoring time period, days. Materials and Methods This section presents the materials that are necessary for performing the laboratory exercise along with a step-by-step procedure. Table 1 presents the materials required to complete the laboratory exercises. A turbidimeter and materials for performing CFUs are not necessary unless microbial growth rates are going to be determined. The cost per student is approximately $2.05 with exception to the BOD or COD analysis. Table 2 lists the 5 steps for performing the study. Glucose stock solutions may be eliminated and the glucose added directly to the 2-L bottles or 1L flasks. The bottles or flasks must be mixed for 1 to 2 minutes after adding the glucose. Table 1. Materials necessary to complete laboratory exercise. Six, 2-L plastic bottles with caps or six, 1-L Erlenmeyer flasks 600-grams of soil Glucose stock solution of 100 g/L. 3-liters of tap water and funnel Analytical balance Dissolved oxygen (DO) meter and probe pH meter and probe Glucose determination reagent strips or equipment necessary to measure biochemical oxygen demand (BOD) or chemical oxygen demand (COD) Fish pump compressor, tubing, and diffuser stone for aeration (Optional) Heterotrophic plate count media and petri dishes and turbidimeter (Optional) Table 2. Procedures for performing laboratory exercise. 1. Prepare six, 2-L plastic bottles by adding 100 grams of indigenous soil and 500 ml of distilled or tap water to each. 2. Prepare glucose stock solution of 100 g/L or weigh appropriate amounts of glucose to add directly into each 2-L bottle. 3. Add 2.5-ml of the 100 g/L stock glucose solution to bottle #1; add 5-ml of the 100 g/L stock glucose solution to bottle #2; add 10-ml of the 100 g/L stock glucose solution to bottle #3; add 20-ml of the 100 g/L stock glucose solution to bottle #4; 20-ml of the 100 g/L stock glucose solution to bottle #5, and no substrate is added to bottle #6 which serves as the control. Mix gently for 1 to 2 minutes. Aerate bottle #5 constantly (Optional). 4. Measure the initial pH, DO, glucose, and/or COD of the liquid portion of the combined mixture of soil, tap water and glucose solutions. Optional measurements include: turbidity and CFU, which also may be measured daily. 5. Incubate the 2-Liter bottles at room temperature; measure the DO, pH, glucose, and/or COD of the liquid portion daily or every other day for at least five days. P ge 772.3 Proceedings of the 2002 American Society for Engineering Education Annual Conference & Exposition Copyright 2002, American Society for Engineering Education Table 3 lists the parameters for evaluating bioreactor performance and Table 4 provides several questions to be considered by the students. Table 3. Parameters to evaluate. 1. Plot each of the above parameters (DO, pH, glucose, COD) as a function of time for each reactor. 2. Determine the growth rate of the microorganisms for each reactor. (Optional) 2. Determine the COD or glucose removal rates for each reactor. Table 4. Questions and data analysis. Which reactor yielded the highest microbial growth rate and which one had the highest rate of glucose removal? Did the microorganism concentration in any of the soil bioreactors decrease with time? Explain why this might have happened. How does the DO concentration affect the microorganism growth rate? How does pH affect microbial growth rate? How would you modify your experiment to improve the glucose removal rates? Using the maximum glucose removal rate determined from the above experiment, how long would it take to reduce an initial glucose concentration of 1000 mg/L? Sample Runs Six, 2-L soil bioreactors were operated from December 17, 2001 through December 27, 2001. One hundred grams of indigenous soil from outside the engineering building at Mercer University and 500-mL of tap water were added to each 2-L container. One reactor served as the control and, varying amounts of glucose were added to the five remaining soil bioreactors. Reactors 1, 2, 3, 4, and 5 had theoretical glucose concentrations of 0.5 g/L, 1.0 g/L, 2.0 g/L, 4.0 g/L, and 4.0 g/L of glucose. Bottle #5 was aerated constantly for the first five days of the experiment. Aeration was omitted on day 6 and resumed on day 7. The remaining bioreactors were aerated for approximately 5 minutes daily and the following parameters were measured: DO, pH, COD, and glucose concentration. DO was measure using a Orion Model 810 dissolved oxygen meter and probe. The meter was calibrated in air according to the manufacturer’s recommendations. pH was measured with a Fischer Scientific Accumet Model 25 pH meter with plastic electrode. The pH meter was calibrated with pH buffer solutions at pH values of 4, 7, and 10, respectively. COD was performed using a HACH COD Reactor with a colorimetric reading. Colorimetric measurements were made with a HACH DR/2000 direct reading spectrophotometer. Glucose was measured using Keto-Diastix Reagent Strips manufactured by Bayer. The Ketos