Salt-tolerant, hydrocarbon-degrading bacteria (Including Dietzia Maris) were isolated from soil samples from a contaminated site in Norman Wells, NT, Canada.

Introduction

Materials and Methods

Results

Discussion Conclusions

References

The bioremediation of petroleum hydrocarbons has been employed as a remedial technolody at comtaminated sites in diverse environments (Kumar, et al., 2010). However, many sites in northern regions in Canada remain contaminated and in situ bioremediation of these sites has not been fully understood due to the effects of colder climates and high salt concentrations associated with petroleum hydrocarbon contamination (Ulrich, et al., 2009, Van Stempvoort and Biggar, 2008). As part of the McGill SURE 2012 program, preliminary pure culture growth experiments as well as one soil slurry microcosm experiment were performed. The objective of the first growth experiment was to determine the optimal type of nutrient amendment for the growth of Dietzia Maris, an indigenous halotolerant, cold-adapted, hydrocarbon-degrading microorganism. The objective of the second growth experiment was to determine the effect of temperature and hydrocarbon type on the growth of Dietzia Maris. Finally, the objective of the soil slurry experiment was to demonstrate that naturally present bacteria in a contaminated soil sample actively degrade hydrocarbons in saline conditions as well as cold temperature scenarios.This project is part of a larger study that seeks to demonstrate the feasibility of bioremediation of petroleum hydrocarbons by naturally present bacteria in contaminated soils in saline conditions and cold climates. The results of this project will contribute to the design of future experiments.

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3) HYDROCARBON DEGRADATION AT COLD TEMPERATURES IN SALINE CONDITIONS:NW soil slurry microcosms were amended with Bushnell Haas nutrient media and 1% v/v diesel fuel to compare complex hydrocarbon biodegradation in saline and non-saline conditions at cold temperatures. 5% and 0% salt concentrations were investigated and killed control samples were prepared by autoclaving for reference. The microcosms were placed on a shaker rotating at 180 rpm for 17 days before being removed from the shaker and stored at 4 °C for 2 months. Samples were analyzed by gas chromatography.

GAS CHROMATOGRAPHY:The soil and aqueous phases were analysed separately for total petroleum hydrocarbons (TPH) by gas chromatography. The non-degraded hydrocarbons were extracted with methylene chloride (DCM). Polar hydrocarbons and remaining water in the solvent samples were removed using silica-gel columns before the samples were concentrated using a nitrogen gas blow-down.

Table 1 - Nutrient types and concentrations

Bioremediation of Petroleum Hydrocarbon- and Salt-Contaminated Soils

1) NUTRIENT OPTIMIZATION FOR THE GROWTH OF DIETZIA MARIS:Different types and concentrations of carbon-free nutrients were investigated in order to determine the optimal nutrient amendment for the growth of Dietzia Maris. Pure Dietzia Maris cultures were grown in shaking flask experiments that were spiked with dodecane. The growth of Dietzia Maris was measured by the optical density (OD) of the aqueous phase. The nutrient types and concentrations investigated are shown in Table 1.

Nicholas Lefrancois, Supervised by Dr. Wonjae Chang and Prof. Subhasis Ghoshal,Department Civil Engineering and Applied Mechanics, McGill University

The results of the nutrient optimization experiment indicate that Bushnell Haas is the optimal nutrient type for the growth of Dietzia Maris. Dietzia Maris is believed to be a key microorganism for biodegradation and this nutrient type may be well suited for the enhancement of biodegradation.Colder temperatures account for a longer lag phase in the growth of Dietzia Maris. They also account for a slower exponential growth phase. The type of hydrocarbon affects the Dietzia Maris population in the stationary phase. Hexadecane, a longer hydrocarbon chain, sustains a larger population than dodecane. The soil slurry experiments performed at low temperatures reveal that the biodegradation is not limited in the two temperature scenarios. This may allow for an extension of the active bioremediation period, which has usually been restricted to short summers at northern contaminated sites (Van Stempvoort and Biggar, 2008).

In addition, the extents of biodegradation in 5% and 0% salt concentrations are similar, which indicates that indigenous microbial populations actively degrade a wide range of hydrocarbons in both non-saline and saline conditions.

Conventional Active Bioremediation period

Extended Active Bioremdiation Period

Foght, J. M., Gutnick, D. L., & Westlake, D. W. S. (1989). Effect of Emulsan on Biodeg-radation of Crude Oil by Pure and Mixed Bacterial Cultures. Applied and Environmental Micro-biology, 55(1), 36-42. Kumar, A.; Chandel, D.; Bala, I.; Muwalia, A.; Mankotiya, L., Managing Water Pollution All the Way Through Well-Designed Environmental Biotechnology: A Review. IUP Journal of Biotechnology 2010, 4 (2), 45-56. Ulrich, A.; Guigard, S.; Foght, J.; Semple, K.; Pooley, K.; Armstrong, J.; Biggar, K., Effect of salt on aerobic biodegradation of petroleum hydrocarbons in contaminated groundwater. Biodegradation 2009, 20 (1), 27-38. Umbanhowar, C. E. 2008-2009 Tundra Coring Pics. http://www.stolaf.edu (accessed August 6, 2012). Van Stempvoort, D.; Biggar, K., Potential for bioremediation of petroleum hydrocarbons in groundwater under cold climate conditions: A review. Cold Regions Science and Technology 2008, 53 (1), 16-41.

- Bushnell Haas commercial media is the optimal nutrient type for the growth of Dietzia Maris.- Colder temperatures lengthen the lag phase and slightly slow the exponential phase of the growth of Dietzia Maris. - Hexadecane sustains larger Dietzia Maris populations in the stationary phase of growth. - Degradation of diesel fuel contamination by indeginous hydrocarbon-degrading bacteria occurs at colder temperatures, which may allow for an extended bioremediation period in cold climates.- Native halotolerant, hydrocarbon degrading bacteria actively degrade hydrocarbons at 5% and 0% salt concentrations.

Figure 2 - Gas chromatography system

25 °C + 4 °C 10 °C + 4 °C

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trol S

ampl

es0%

Sal

t5%

Sal

t

Figure 4 - Chromatograms for non-saline and salt-amended microcosms

2) EFFECT OF TEMPERATURE AND HYDROCARBON TYPE:4 Dietzia Maris pure cultures were prepared with Bushnell Haas media, as per the results of the previous experiment. Two of the cultures were spiked with hexadecane and the other two were spiked with dodecane (Figure 1). One culture with each type of hydrocarbon was then placed on a shaker at 10 °C and the other on a shaker at 25 °C. Figure 1 - Pure Dietzia Maris cultures

Figure 3 - Results for nutrient optimization and temperature

and hydrocarbon type experiments

Optimal Nutrient Type and

Concentration for the Growth of

Dietzia Maris

Effect of Temperature and

Hydrocarbon Type on the

Growth of Dietzia Maris Hexadecane was

solid at 10 °C

A decrease in the peak heights corresponds to the degradation of linear alkanes.

A deacrease in the overall hump area corresponds to the degradation of branched and cyclic alkanes.

Figure 5 - Extension of active bioremediation period

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