61st Annual Report on Research 2016

Over the past year I have worked with four undergraduate students to continue our research examining the degradation of oil by marine fungi. This year we are working with laboratory incubations comprised of marine fungi and oil with three main objectives in mind:

i) Characterize the microbial community responsible for oil degradation (students Owen Janson ‘18 and Cicy Geng ’18)

ii) Determine the flow of oil-derived carbon in the incubation system (student Alana Thurston ’16)

iii) Examine the mechanism of oil degradation (student Miranda Baker ’17)

i) Characterize the microbial community responsible for oil degradation

This objective acknowledges that microbes function as part of a community, and that to understand oil degradation in a more environmentally-relevant context, it is important to characterize the microbial community as a whole. To this end, oil-soaked sand patties containing weathered oil from the Deepwater Horizon oil spill, and collected from Gulf coast beaches were incubated in water in the lab. Approximately one week after incubation, biofilms become visible on the surface of the sand patty. Confocal microscopy performed by Owen Janson and Cicy Geng, indicated that fungal hyphae dominated the biofilms. To determine the diversity of the biofilm community and the sand patty to which it was attached, DNA was extracted from the following samples: sand patty prior to incubation, incubated sand patty attached to the biofilm, the center of the incubated sand patty, the biofilm, and the water in the incubation. The DNA extracted from these samples was amplified for both bacteria (16S) and fungi (ITS1f/ITS2) regions. The resulting amplicon pools are currently being sequenced using Illumina MiSeq with our collaborator Dr. Manpreet Dhami at Stanford University. We anticipate receiving and analyzing the work in the coming months. We hope to determine what the dominant microbes are, and if these are known to degrade oil.

14 microbes with different morphologies were also isolated from the biofilm. To examine the ability of the isolates to degrade oil they have been grown on a mixture of crude oil and sand prepared in the laboratory and incubated for ~60 days. Based on gas chromatography (GC) of the oil remaining after this time, all, but two of the isolates are able to degrade crude oil. Preliminary sequencing data of DNA extracted from these isolates indicates that in addition to obtaining a wider diversity of Ascomycota (including Fusarium, Purepureocillium, Scopulariopsis and Aspergillus) we have isolated fungi from the Basidiomycota Division (including Pseudozyma and Rhodotorula). We are pleased to have increased the diversity of oil-degrading fungi in our laboratory isolate collection as this will enable us to potentially compare different mechanisms of oil degradation between fungi from different divisions.

Cicy Geng and Owen Janson presented this work at an undergraduate poster symposium held at Haverford College. Both students are continuing to work on this project throughout the academic year. Once the high throughput sequencing data is obtained we will be able to place our isolates in context and hypothesize how the microbial community present in these samples may be interacting to degrade oil.

ii) Determine the flow of oil-derived carbon in the incubation system

To determine the flow of oil-derived carbon we wanted to specifically monitor the uptake of oil into bacterial and fungal lipids. Phospholipid Fatty Acids (PLFAs) are commonly used as biomarkers for bacteria and can also be used for fungi. We sought to determine if there were any lipids in addition to PLFAs that were unique to fungi. Considering this, Alana Thurston, examined the lipid profile of the three marine fungi that we had already isolated for her senior thesis research. Alana presented her findings internally at Haverford and also presented her work at the Ocean Sciences Meeting in New Orleans in February 2016, with a poster titled, “Exploring the lipid profiles of three marine fungi.” The major finding of this work was that biomarkers of quantitative significance that were identified and unique to the fungal isolates included ergosterol, some distinctive PLFAs as well as some unsaturated hydrocarbons including alkenes and alkynes. These biomarker compounds are useful to determine if fungi are present in a sample and their abundance could provide information about the relative quantitative significance of fungi compared to bacteria. In terms of their utility to determine the uptake of oil into fungal lipids, we are currently assessing the scale of the laboratory incubation that would be necessary to obtain sufficient biomarkers for carbon isotope analysis. We are continuing to explore the utility of fungal biomarkers in our laboratory incubations by extracting the sand patty before incubation, the sand patty during incubation, and the biofilm to investigate the types of PLFAs that are present and what can be learned from differences in the lipid composition between these samples. Considering the mixed microbial communities discovered in i), the ability to detect biomarkers from different microbes (bacteria versus fungi and potentially between different fungi) is a challenge that we are actively pursuing. This work is a collaboration between Cicy Geng, Owen Janson and Miranda Baker who is focusing on this objective for her senior thesis research project.

iii) Examine the mechanism of oil degradation

Once sequencing data for the mixed microbial communities in the biofilm as well as for the lab isolates has been obtained, we will probe the literature and genomic databases for information for the mechanisms of oil degradation that the fungi we are working with have. This genomic work will be coupled to metabolomic work i.e. analyzing the chemicals that the fungi excrete during oil degradation as well as the oil transformation products that are formed as a result of oil degradation. Preliminary analyses of the metabolomics of individual isolates incubated with oiled sand has begun to analyze the polar and non-polar pools of metabolites. So far these extracts have been analyzed via gas chromatography mass spectrometry, but this will be expanded to analysis via liquid chromatography mass spectrometry in the coming year. Miranda Baker has begun working on the analysis via GC-MS and will continue with this throughout her senior year.