60th Annual Report on Research 2015

For the past year, I have engaged five undergraduate students and a postdoctoral fellow at Haverford College in research investigating the degradation of oil by marine fungi. Fungi were isolated from oiled sand samples that we collected in 2012 from beaches in the Gulf of Mexico that had been impacted by the Deepwater Horizonoil spill. To isolate the fungi, fungal mycelia were dissected and adapted to grow on an Artificial Seawater (ASW) minimal medium, supplemented with crude oil. In order to identify the fungi, the large subunit 28S rRNA gene and the Internal Transcribed Spacer (ITS) region was PCR-amplified using primers to target a wide range of fungal species (ITS-1F and ITS-4). Sequencing of the ITS-region of the fungal ribosomal RNA operon and the large subunit 28S rRNA gene, confirmed that all fungal isolates belong to the Division Ascomycota, with the closest match of isolate DI_I1 to Fusarium solani (100% match in BLAST for the ITS region and 100% match for the 28S rRNA gene), GS_I1 to Scopulariopsis brevicaulis (81% match for the ITS region and 100% match for the 28S rRNA gene) and GS_I2 to Aspergillus terreus (98% match for the ITS region and 100% match for the 28S rRNA gene). The isolation, culturing and identification of fungi was led by postdoctoral fellow Rachel Simister who trained undergraduate student Miranda Baker in these molecular techniques.

To determine the optimal conditions for fungal degradation of oil, the three isolates of ascomycete fungus were adapted to grow on an Artificial Seawater (ASW) minimal medium, supplemented with crude oil. These incubations were performed by undergraduate student Carolyn Poutasse who worked to determine the optimal temperature for oil degradation, and by Miranda Baker who assessed the affect of different salinities on oil degradation. In these experiments, the extent of oil degradation was examined by analyzing changes in oil via gas chromatography coupled to flame ionization detection (GC-FID) to examine oil quantity, and gas chromatography coupled to mass spectrometry (GC-MS) to examine the composition of the oil. Undergraduate student Alana Thurston supported these studies by assisting with oil extraction, clean-up and GC-MS analysis. All experiments were run in triplicate to check the reproducibility of the results. Statistical analyses of total oil degradation, total PAH degradation, C17/pristane and C18/phytane ratios were performed. Differences within the data were determined statistically using two-way analysis of variance (ANOVA) and a Tukey's Honestly Significant Difference (HSD) test at p = <0.05. Significant trends were observed for the temperature experiment, but not for the salinity experiment. The results from the temperature study indicated that consistent trends in oil degradation were not related to fungal species or temperature and the three isolates degraded variable quantities of oil (32-65%). Fungal isolates preferentially degraded short (<C18; 90-99%) as opposed to long (C19-C36; 7-87%) chain n-alkanes and straight chain C17- and C18-n-alkanes (91-99%) compared to their branched counterparts, pristane and phytane (70-98%). Polycyclic aromatic hydrocarbon (PAH) compounds were also degraded by the fungal isolates (42-84% total degraded), with a preference for low molecular weight over high molecular weight PAHs. This study provides evidence that Ascomycota fungi are able to degrade structurally diverse oil-derived compounds. Carolyn presented the results of this study at the 249th American Chemical Society National Meeting in Denver, CO in March 2015. The results have since been written up and accepted for publication in Marine Pollution Bulletin with Rachel Simister as the lead author and the aforementioned undergraduate students as coauthors.

The previously described research completes the first of our two proposed study aims to 1) Isolate and culture fungi in conditions typical of marine environments and 2) Determine the degradation efficiency of structurally diverse organic compounds. We have recently moved onto the next stage of our proposal where we are focusing on examining the mechanism of oil degradation and specifically the flow of oil carbon in the system. We are currently working to determine the relative amounts of oil that is incorporated into fungal biomass versus the amount of oil that is respired. With funding from this grant, three undergraduate students (Brenna Boehman, Zexi Geng and Miranda Baker) began this work this past summer, and student Alana Thurston is now continuing this work for her senior thesis. We have begun by examining the lipid profile of the three marine fungi to quantify biomarkers of interest that could be used in future experiments to determine the uptake of oil into fungal biomass. In this instance, the uptake of oil would be determined by measuring the carbon isotopic composition of these fungal biomarkers relative to the carbon isotopic composition of the available carbon sources. To date, the three species of marine fungi have been cultivated in the lab prior to drying and extracting with a mixture of methanol and dichloromethane (2:1 ratio) to extract their lipids. The lipid extract is subjected to silica gel chromatography and separated into three fractions by eluting sequentially with dichloromethane, acetone, and methanol to obtain non-polar, neutral, and polar fractions respectively. All fractions are then derivatized prior to analysis via GCMS. Preliminary analysis has revealed the presence of phospholipid fatty acids (PLFA), ergosterol and pyrrolo[1,2-a]pyrazine-1,4-dione, a potential antimicrobial. Quantification of these biomarkers and further analysis of additional biomarkers of interest is ongoing. Once this method has been developed we plan to apply this approach to fungal biofilms growing on oiled sands to determine how much oil is incorporated into the fungi and if there is significant variation between fungal isolates and varying environmental conditions.