Microbial community dynamics of the oil leak: time series study, microbial water column stratification, hydrocarbon substrate response Tingting Yang, Lisa Nigro, Luke Mckay, Tony Gutierrez, Andreas Teske UNC Chapel Hill Oct

Molecular microbiological experiment Cultivation experiment

Molecular microbiology: Bacterial 16S rRNA gene clone libraries Water samples are from 3 cruises: –RV Pelican cruise (May 4 th -16 th ) Oily surface water sample –RV Walton Smith cruise (May 26 th -June 8 th ) Plume water and water from above and below plume layers –RV Pelican cruise (September 9 th -15 th ) Same site and depth as Walton Smith cruise, after plume has gone. All water samples (500 ml) were filtered through 0.22 µm Anodisc filters or ISOPORE membrane filters.

Pelican cruise (May 4 th -16 th ) -- Surface oily water samples

Gulf of Mexico Cycloclasticus strains had broad substrate ranges which included naphthalene, substituted naphthalenes, phenanthrene, biphenyl, anthracene, acenaphthene, and fluorene. (Geiselbrecht et. al, 1998) Cycloclasticus is a special polycyclic aromatic hydrocarbon (PAH)- degrading bacteria

We suggest that the observed relatives of Cycloclasticus and/or Colwellia are blooming as a result of their capacity to consume propane, ethane, and potentially butane, although not at the exclusion of other bacteria or metabolisms. While Cycloclasticus is known for its ability to degrade aromatic compounds, sequences observed here are 90% similar to putative ethane and propane oxidizers identified by stable isotope probing, indicating the capability in this evolutionary lineage.

Walton Smith cruise (May 26 th – June 8 th ) --Deep plume water samples Plume water m

800 m 1170 m 1210 m 1320 m

Phylogenetic tree of plume samples (1170 m and 1210 m) The close relatives are aerobic alkane oxidizers of the genera Oleispira and Thalassolituus.

Near-bottom water sample below plume (1320 m) Sea water sample above plume (800 m)

Pelican cruise (Sep 9 th -15 th ) -- Water sample from former plume site We went back to old sites where plume was found in Walton Smith cruise. No plume any more.

It looks like the microbial diversity at the plume site has recovered. To be checked with statistical comparison of above-plume sample (May 31) and post-plume sample (Sept 9-16)

Conclusion of molecular microbial experiment Effect of oil spill on microbial community Surface (0 m) - Dominated by Cycloclasticus group (Gammaproteobacteria); Medium sea water (800 m) - High diversity Alphaproteobacteria are the main group Plume water (1170 m & 1210 m) - Dominated by Oceanospirillales group (Gammaproteobacteria) Deep water (1310 m) - High diversity, high proportion of Alphaproteobacteria and Deltaproteobacteria Time series study shows the microbial diversity at the plume site has increased (methylotrophs, aromatic oxidizers and typical marine bacteria).

Cultivation experiment Two methods –Series dilution –Direct isolation

Series dilution Plume Water 0.5ml 0.1 ml50 µl10 µl1 µl0.01 µl 100 µl Hexadecane 3 ml ONR medium+ Room temperature Shake in dark

3rd day

Check whether hexadecane is the only C source of these cultures. PCR & SequencingGet pure cultures

Direct isolation Plume Water 100 µl50 µl25 µl10 µl5 µl Hexadecane +ONR platesNo hexadecane plates

Alcanivorax spp alkane degrader

First RV Pelican cruise w. Arne Diercks: Luke Mckay RV Walton Smith cruise w. Mandy Joye: Lisa Nigro and Tingting Yang Second RV Pelican cruise w. Ken Sleeper: Tingting Yang, Howard Mendlovitz and Dan Hoer RV Cape Hatteras cruise w. Kevin Yaeger: Kelly Speare (just came back with oily sediment samples!) Thanks Andreas Teske, Mandy Joye, Tony Gutierrez, Kai Ziervogel, Lisa Nigro, Luke Mckay, Barbara MacGregor, Kelly Speare!

Hazardous Chemicals Adverse Health Effects Benzene (crude oils high in BTEX, benzene, toluene, ethylbenzene, and xylene) Irritation to eyes, skin, and respiratory system; dizziness; rapid heart rate; headaches; tremors; confusion; unconsciousness; anemia; cancer Benzo(a)pyrene (a polycyclic aromatic hydrocarbon reproductive [see below], formed when oil or gasoline burns) Irritation to eyes and skin, cancer, possible effects Carbon dioxide (inerting atmosphere, byproduct of combustion)Dizziness, headaches, elevated blood pressure, rapid heart rate, loss of consciousness asphyxiation, coma Carbon monoxide (byproduct of combustion) Irritation to eyes, skin, and respiratory Dizziness, confusion, headaches, nausea, weakness, loss of consciousness, asphyxiation, coma Ethyl benzene (high in gasoline)Irritation to eyes, skin, and respiratory system; loss of consciousness; asphyxiation; nervous system effects Hydrogen sulfide (oils high in sulfur, decaying plants and animals) Irritation to eyes, skin, and respiratory system; dizziness; drowsiness; cough; headaches; nervous system effects Methyl tert-butyl ether (MTBE) (octane booster and clean air additive for gasoline, or pure MTBE) Irritation to eyes, skin, and respiratory system; headaches; nausea; dizziness; confusion; fatigue; weakness; nervous system, liver, and kidney Polycyclic aromatic hydrocarbons (PAHs) (occur in crude oil, and formed during burning of oil) Irritation to eyes and skin, cancer, possible reproductive effects, immune system effects Sulfuric acid (byproduct of combustion of sour petroleum product) Irritation to eyes, skin, teeth, and upper respiratory system; severe tissue burns; cancer Toluene (high BTEX crude oils)Irritation to eyes, skin, respiratory system; fatigue; confusion; dizziness; headaches; memory loss; nausea; nervous system, liver, and kidney effects Xylenes (high BTEX crude oils)Irritation to eyes, skin, respiratory system; dizziness; confusion; change in sense of balance; nervous system gastrointestinal system, liver, kidney, and blood effects