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 HillOct-27-2010

• Molecular microbiological experiment

• Cultivation experiment

Molecular microbiology: Bacterial 16S rRNA gene clone libraries

• Water samples are from 3 cruises:– RV Pelican cruise (May 4th-16th)

• Oily surface water sample– RV Walton Smith cruise (May 26th-June 8th)

• Plume water and water from above and below plume layers

– RV Pelican cruise (September 9th-15th)• 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 4th-16th)--Surface oily water samples

94%

5% 1%

Cycloclasticus

Pseudoalteromonas

Alteromonas

“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 observedrelatives 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 26th – June 8th) --Deep plume water samples

Plume water

1100-1250 m

800 m

1170 m

1210 m

1320 m

Oceanospirillales

Colwellia

Rhodospirillaceae

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)

38%

4%21%

11%

4%

7%

4%

11%Alphaproteobacteria

Gammaproteobacteria

Deltaproteobacteria

Planctomycetes

Bacteroidetes

SAR406

Candidate division SB1

Actinobacteria

16%

16%

5%

9%14%

7%

14%

12%

7%Alphaproteobacteria

Gammaproteobacteria

Deltaproteobacteria

Planctomycetes

Betaproteobacteria

SAR406

Sva0853

Actinobacteria

Acidobacteria

Pelican cruise (Sep 9th-15th)-- Water sample from former plume site

• We went back to old sites where plume was found in

Walton Smith cruise. • No plume any more.

19%

9%

3%

69%

Methylotrophs

Cycloclasticus

Alteromonas

Others

30%

38%

13%

1%

4%

10%2%1%1%

Alphaproteobacteria

Gammaproteobacteria

Deltaproteobacteria

Planctomycetes

Bacteroidetes

Chaldithrix

Verrucomicrobia

cyanobacteria

Gemmatimonadetes

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 ml 50 µl 10 µl 1 µl 0.01 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 & Sequencing Get pure cultures

Direct isolation

Plume Water

100 µl 50 µl 25 µl 10 µl 5 µl

Hexadecane + ONR plates No hexadecane plates

NAME Accession Description Max score Total score Query coverage E value Max identTY1 AY258105.1 Alcanivorax sp. DG813 small subunit ribosomal RNA gene, partial sequence 1471 1471 88% 0 98%

AB055208.1 Alcanivorax sp. Wf-1 gene for 16S rRNA, partial sequence 1471 1471 88% 0 98%EU052714.1 Alcanivorax sp. MH155 16S ribosomal RNA gene, partial sequence 1465 1465 88% 0 97%

TY3 AY258105.1 Alcanivorax sp. DG813 small subunit ribosomal RNA gene, partial sequence 1530 1530 92% 0 98%AB055208.1 Alcanivorax sp. Wf-1 gene for 16S rRNA, partial sequence 1530 1530 92% 0 98%EU052714.1 Alcanivorax sp. MH155 16S ribosomal RNA gene, partial sequence 1524 1524 92% 0 98%

TY4 EU052714.1 Alcanivorax sp. MH155 16S ribosomal RNA gene, partial sequence 1424 1424 87% 0 97%AY258109.1 Alcanivorax sp. DG881 small subunit ribosomal RNA gene, partial sequence 1424 1424 87% 0 97%AY258104.1 Alcanivorax sp. DG812 small subunit ribosomal RNA gene, partial sequence 1424 1424 87% 0 97%

TY5 AB053129.1 Alcanivorax sp. Abu-1 gene for 16S ribosomal RNA, partial sequence 1531 1531 92% 0 97%GQ250604.1 Uncultured gamma proteobacterium clone GOM_WB10-68 16S ribosomal RNA gene, partial sequence1526 1526 92% 0 97%DQ490022.1 Uncultured bacterium clone ODP-18B-02 16S ribosomal RNA gene, partial sequence1526 1526 92% 0 97%

（ http://confrontaal.org/wordpress/?p=7914）

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 ChemicalsAdverse 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