Abstract:

The ability of 2 study strains of bacteria - Alcanivorax xenomutans strain (Bead 18) and Shewanella indica strain (Bead 36) to degrade hexadecane was analyzed using gas chromatography mass spectrometry (GC-MS). Each strain was inoculated into ONR7a minimal medium supplemented with 50 mM hexadecane. Incubations were performed in the dark to prevent photo-oxidation of hexadecane. At 48 hours and 120 hours, they were harvested for GC-MS analysis of hexadecane degradation. The negative control consisted of sterile ONR7a with 50 mM hexadecane, with no cell culture. For sample preparation, the methods described in (Marietou et al. 2018, Frontiers in Microbiology, 9:808) were used. An Agilent 5977B Gas Chromatograph-Mass Selective Detector (GC-MSD) instrument equipped with an Agilent DB-5MS UI column was used.

Suggested Citation:

Mullane, Kelli K., Douglas H. Bartlett, and Tianhan Xu. 2020. Hexadecane degradation from bacteria strains Alcanivorax and Shewanella. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/FQBHQBEH

Purpose:

The purpose of this experiment was to ensure the study strains of bacteria can degrade hexadecane.

Data Parameters and Units:

The headers are: Time (Hours), Replicate 1, Replicate 2, Replicate 3 and Average. The various replicate measurements and averages are provided for both bacteria species-Alcanivorax xenomutans (Bead 18) and Shewanella indica (Bead 36).

Methods:

During the growth of the cultures, the remaining hexadecane was extracted with dichloromethane and determined using gas chromatography mass spectrometry (GC-MS) according to the method described by Marietou et al. 2018 with the following modifications. Samples of 0.5 ml were extracted with 5 ml dichloromethane by vigorous mixing at room temperature for 30 minutes. The mixture was allowed to sit for 2 hours and then 0.5 ml of the solvent-oil phase (bottom layer) was transferred to a new glass vial where another 2 ml of dichloromethane were added. Then the new mixture was shaken for 30 minutes and allowed to sit for 2 hours. The solvent-oil phase was transferred to a clean vial where anhydrous sodium sulfate was added and allowed to stand for 30 minutes at room temperature to remove any residual water. Finally, the sample was collected and stored at -20 degree Celsius till further processing. As a control the same concentration of hexadecane oil (1.45% v/v) in ONR7a medium with no bacteria added was used. Experiments were done in the dark to prevent hydrocarbon photooxidation (Widdel 2010). An Agilent 5977B Gas Chromatograph-Mass Selective Detector (GC-MSD) instrument was used for GC-MS analysis. The GC-MSD was equipped with an Agilent DB-5MS UI column (30 m length, 0.25 mm internal diameter). All analyses were carried out with the split ratio of 10:1. Helium was used as the carrier gas with a flow rate of 1.2 ml/minute. The injection temperature was 250 degree Celsius. The oven temperature program was as follows: an initial temperature of 60 degrees Celsius was increased to a final temperature of 270 degrees Celsius at a rate of 15 degrees Celsius /minute, and hold for 2 minutes at 270 degrees Celsius, with a final 2 minute hold at 280 degrees Celsius. The mass spectrometer transfer line temperature was held at 250 degrees Celsius and the ion source temperature at 230 degrees Celsius.

Instruments:

Agilent 5977B Gas Chromatograph-Mass Selective Detector

Provenance and Historical References:

Marietou, Angeliki, Roger Chastain, Felix Beulig, Alberto Scoma, Terry C. Hazen, and Douglas H. Bartlett. 2018. The Effect of Hydrostatic Pressure on Enrichments of Hydrocarbon Degrading Microbes from the Gulf of Mexico Following the Deepwater Horizon Oil Spill. Frontiers of Microbiology. 9:808. doi: 10.3389/fmicb.2018.00808 Widdel F. 2010. Cultivation of anaerobic microorganisms with hydrocarbons as growth substrates, p 3787–3798. In Timmis, Kenneth N., McGenity TJ, van der Meer JR, de Lorenzo V (ed), Handbook of Hydrocarbon and Lipid Microbiology. Springer-Verlag, Berlin, Germany. doi: 10.1007/978-3-540-77587-4_298

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