62nd Annual Report on Research 2017

Over the past year I have worked with three undergraduate students to continue our research examining the degradation of oil by marine fungi. This year has focused primarily on data analysis and synthesis of our three main objectives:

1. Characterize the microbial community growing on weathered oil residues (student Owen Janson ‘18)
2. Determine the flow of oil-derived carbon and other sources of carbon in laboratory incubations with weathered oil (students Owen Janson ’18 and Cicy Geng ’18)
3. Examine the mechanisms of oil degradation (students Cicy Geng ’18 and Miranda Baker ’17)

1. Characterize the microbial community growing on weathered oil residues.

This objective continues our research into exploring how fungi function as part of a microbial community that also includes bacteria. 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. To determine the diversity of the microbial community as well as how the communities change before and after incubation, DNA was extracted from samples at different spatial resolutions including the 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 and sequenced using Illumina MiSeq with our collaborator Dr. Manpreet Dhami at Stanford University. Over the past year, Owen Janson ’18 has focused on analyzing this dataset using bioinformatics approaches. Owen’s analysis has found that mixed populations of fungi and bacteria related to oil degradation are present in all samples and that there is an increase in microbial diversity after the sand patties have been incubated. Based on community composition, there are

no significant differences between the sample types and at different spatial resolutions in the incubations. To expand upon this, Owen’s current research focus is exploring the differences in community function between the different samples in the incubation. Our preliminary analysis has led us to focus on the analysis of genes involved in oil degradation and heterotrophic and autotrophic carbon metabolism. In addition, we have examined oil and dispersant chemicals present in the different samples via gas chromatography mass spectrometry to provide a chemical context for the microbial findings.

2. Determine the flow of oil-derived carbon and other sources of carbon in laboratory incubations with weathered oil.

This objective seeks to determine if oil is the main source of carbon that the microbes grow on, and if not, what other carbon sources can contribute to their growth. To this end we collected samples to measure the natural abundance radiocarbon (C14) content to differentiate carbon derived from oil (contains no C14) from the C14 content of natural organic carbon present as detritus in the sand patty or from carbon fixed from the atmosphere via photosynthesis. We had originally planned to analyze samples via compound specific isotope analysis, but opted for the analysis of bulk samples (fungal hyphae, biofilm, oil, green algae) due to the small size of the samples available. Isotope mass balance was used to determine the amount of carbon derived from oil that is incorporated into biomass. The amount of carbon derived from oil and incorporated into biomass was 100% in fungal hyphae, 80% in the biofilm attached to the sand patties and 15% in the green algae that grew in the incubations. We hypothesize that additional carbon from the atmosphere is fixed and added to the incubation experiments via photosynthesis by bacteria in the biofilm and macroscopic green algae. The green algae biomass contains 20% of carbon that originates from oil. We hypothesize that this 20% is a result of remineralization of oil by bacteria that is converted into the dissolved inorganic carbon pool in the incubations and subsequently used by the algae. Cicy Geng ’18 and Owen Janson ’18 worked collaboratively on this objective, which was completed this summer and is currently being prepared for publication.

3. Examine the mechanisms of oil degradation

To examine the mechanisms of oil degradation by marine fungi, oil-degradation products and secondary metabolites produced by fungal isolates including those from Ascomycota and Basidiomycota Divisions have been examined via gas chromatography mass spectrometry. To compare the ability of nine novel fungal isolates to degrade oil in liquid media we constructed seawater incubations enriched with nitrogen, phosphorous, and crude oil. Miranda Baker ’17 worked on this objective for her senior thesis research and as well as developing and verifying the incubation methods, identified two known oil degradation products from one of the fungal isolates providing insight into its oil degradation mechanism. The analysis of these incubation experiments is ongoing and is being led by Cicy Geng ’18.

Lab work for objectives i) and ii) has been completed and the data is in the last stages of analysis prior to being prepared for publication. Objective iii) is the current focus of lab efforts with the aim that it will be completed by Sept 1, 2018 and written up for publication shortly thereafter. The research described in this report has been presented at an undergraduate poster symposium held at Haverford College by all three students. In addition, Cicy Geng and Owen Janson presented posters in Februrary 2017 at the Association for the Sciences of Limnology and Oceanography Meeting in Honolulu, HI.