Land and sea polluted with petroleum-based synthetic and natural substances have led to the development of techniques using biological organisms for clean-up. The technique is called bioremediation. “Bio” refers to biology and “remediation” to clean-up. The types of microorganisms used are bacteria fungi and actinomycetes. Bacteria can be used for bioremediation under anaerobic or aerobic conditions. Actinomycetes are generally anaerobic and grow from spores forming filaments and branches. The applications and environment for optimum bioremediation varies depending on the pollutant and the biological organisms. Different combinations work better depending on the temperature, substrata and other environmental variables. The goal is to find the combination that allows the organisms to work the best to “break down” the organic pollutant by degrading so it will be suitable for an energy source for the biological organism.
Consortia are microbes that are known to be specialized in breaking down particular types of organ pollutants. This technique is called bioaugmentation because the consortia “augment” that is increase the size, number or strength of the micro-organisms. Biostimulation is used on an in-situ pollutant where the natural micro-organisms are available but they need to be “stimulated” by nutrients to speed up the process of degrading the pollutant and the effect of a surfactant to break up the kerosene so the microorganisms have more surface area available which should also speed up the process.
The lab was designed to determine the effect on two types of soil – pristine and contaminated – while varying temperature and addition of biostimulants in the form of the nutrients – NH4Cl (ammonium chloride), KCl (Potassium chloride), NaH2PO4.2H2O (Sodium phosphate) and MgSO4 (Magnesium sulfate) and in the form of a surfactant (Tween 80). Agar was used as the substrata. The kerosene degrader was not incubated before the solutions were made; the soils pristine or contaminated were added to the solution without an incubation step. If incubation had taken place we would expect the kerosene degrading bacteria to acclimate to the kerosene which would be expected to improve their rate of growth.
Ten treatments were used after two weeks incubation to compare the amount of degradation of Kerosene. The results follow.
(1) Kerosene and Water Treatment (with no soil added). No growth was observed which was expected since the source of the microorganisms is the soil.
(2) Water and Soil and BMS Treatment plus contaminated soil showed good growth measuring an AGI of 5 at both temperatures of 10°C and 20°C. The growth at these two temperatures exceeded the optimum growth range. At 37°C growth was reduced suggesting that 37°C is outside the optimal growth range of the organisms.
(3)Kerosene Treatment plus (2) Water and Soil and BMS Treatment plus contaminated soil showed good growth at 10°C but no growth at the other temperatures.
Considering (4) Water and Soil and BMS and Glucose Treatment and (5) Glucose and Kerosene Treatment. We found the addition of the carbon source whether as glucose or kerosene at a temperature of 37°C improved the growth of the microorganisms.
Comparisons of experimental salts addition. Treatment (3) and Treatment (8) demonstrated that with the agar substrata the salts, BMS, were very important in stimulating the microorganisms for growth. When salts were not included, then temperature played an important role in growth with maximum growth at 25°C.
(6) Water and Soil and BMS and MgSO4 Treatment compared to (7) Water and Soil and BMS and MgSo4 and Kerosene Treatment growth at 25°C suggests that MgSO4 does not inhibit growth.
(9) Water and Soil and BMS and Tween80 did not demonstrate improved degradation amounts.
(10) Water and Soil and BMS and Tween80 and Kerosene also demonstrated no improvement: the results were the same without Tween80 except at 37°C which showed reduced growth.
The experimental results demonstrate overall that glucose and BMS are important biostimulants. When the agar substrata results were compared to the BMS results they were equal or close to equal except for Treatment 8 in which the agar nutrient substrate measured an AGI of 1 compared to an AGI of 5 for the BMS demonstrating the importance of BMS for a successful treatment.
On the second day no change in Carbon source or Phosphorus nutrient was observed except for two of the treatments. The Mg2SO4 slightly reduced growth with the BMS (nutrient agar) although the amount may not be statistically significant. (8) Water and Soil and Kerosene Treatment when streaked on the agar substrata not produce significant growth.
Kerosene is hydrophobic which causes it to partition on to soil and float on top of water. The Volatile components of kerosene could escape more easily into the atmosphere as well as be more in contact with Oxygen (O2) which would mean more degradation in the water than in the soil. Air sparging is a technique done at the site of the contaminant by injecting air into the groundwater; when the contaminant comes in contact with the air, contaminant is volatized, forming bubbles and is removed by the bubbles. Air sparging might be a technique which would stimulate volatilization, increasing growth in order to degrade the kerosene.
The experiment counted the number of kerosene degrading microbes in order to determine “success” which assumes bacterial abundance gives the best rate of degradation. An experiment could measure CO2 production compared to bacterial abundance because measuring it provides a more accurate measurement of carbon degradation.