Microbial Response to Macondo Oil and Dispersant
Leads: D. Jay Grimes and Kevin Dillon
Introduction
We have used our initial Gulf Research Institute funding administered by the Northern Gulf
Institute to ask three fundamental microbiological questions: (1) how prevalent are dioxygenase
genes in the DH microbial community at sea and along the Mississippi shorelines (both the coast
itself and the barrier islands); (2) what is the involvement of Vibrio spp. in the microbial
response to oil and dispersant; and (3) what is the effect of the oil/dispersant mixture and the
dispersant alone on heterotrophic microbial activity in surface waters? To date, we have
developed methods and have started gathering data on the microbial response to MC252 and we
will briefly describe some of these findings.
Initial Results
The Grimes lab has optimized polymerase chain reaction (PCR) conditions
for using a 78-base pair DNA primer that targets the Rieske Fe-S center
common to all dioxygenases. The gel to the right shows the results for a run
using two DNA size ladders (far left and right in gel), five Vibrio isolates
from the DH blowout site and a pure culture of Pseudomonas aeruginosa
(lane 5). The five Vibrio strains have common banding patterns at the 78-bp
location and the P. aeruginosa in lane 5 gave equivocal results. We are
continuing the optimization of this primer for PCR use and we are now in the process of
conjugating the primer to the fluorescent dye Cy3 for use in a new fluorescence in situ
hybridization (FISH) technique called RING (recognition of individual genes)-FISH. Finally, we
have begun laboratory microcosm studies that will examine the fate of MC252 oil and/or
dispersant (Corexit 9500) in Davis Bayou water.
The Grimes collaboration with Drs. Sandra McLellan (UW – Milwaukee) and Mitch Sogin
(Marine Biological Laboratory, Woods Hole) has allowed us to integrate our functional gene
assessments into the context of comprehensive community profiles. Sand samples have been
collected across the coastline of three states (MS, AL and FL) every 6 to 8 weeks since the
begining of June. This archive of samples provides a valuable record of changes that have
occurred over this time period and across a wide geographic span. Thus far, sequencing work
(454 sequencing) by McLellan and Sogin has produced comprehensive profiles of the microbial
community at 6 of the 21 sites sampled. In baseline samples, the microbial community across
these sites was fairly consistent, which was important to establish in support of the hypothesis
that oil impacts will shape microbial communities similarly across coastal beaches. The
sequencing work has already identified potential microbial community members that are
elevated at oil-impacted beaches. The overall profile of the numerous taxa (>200 groups) present
in beach sand and water is shown below. We observed a similar overall population structure for
sand samples, and a
distinct one for water.
We found key taxa
increased at oilimpacted beaches. One
taxon, the
Alcanivoracaceae
family [a group of known hydrocarbon degraders – at the green arrow] increased approximately
10 fold at the heavily impacted Gulf Shores State Park, AL (GSP) compared to the non-impacted
St. George Island, FL (StG).
The Dillon lab at USM has conducted short term (48 hours) bacterial incubation experiments
examining bacterial growth and respiration in whole seawater and in 1-micron filtered seawater
(bacterial size fraction) with different treatments (unamended control, 1% oil+dispersant
addition, and 1-5% dispersant alone) to examine the effects of these mixtures on in situ whole
water and bacterial communities. Bacterial growth in these experiments is still being analyzed;
however, results to date show that the oil/dispersant (Corexit 9500) mixture had no discernable
effect on bacterial communities in any treatment on short timescales. The dispersant treatments
have shown variable results between nearshore and offshore waters. In nearshore waters from
Mississippi Sound, the dispersant greatly increased respiration in whole water as well as in the
bacterial size fraction. Oxygen concentrations in the dispersant treatments decreased from
approximately 7mg L-1 to 5.8 mg L-1 in the bacterial size fraction, while the whole water
treatment decreased to a much lower concentration of 2.7 mg L-1 in 48 hours (see figure below).
Conversely, no such stimulatory effect was observed in incubations using offshore waters.
Follow up experiments to be conducted before December 2010 will examine whole water and
bacterial responses over the same time period as well as longer timescales (1 month) to
determine if responses differ due to bacterial community shifts over this longer time frame.
Bacterial Size Fration
Whole Water
230
240
220
220
O2 conc (uM)
O2 conc (uM)
200
210
200
control
190
disp only
180
180
160
140
control
120
oil/disp
disp only
oil/disp
100
170
80
0
10
20
30
time (hrs)
40
50
0
10
20
30
40
50
time (hrs)
Proposed Research
We will continue to gather data that will answer the three fundamental questions posed in the
Introduction of this pre-proposal. Specifically, we propose to continue examining whole water
and bacterial community responses using the current incubation methods while adding additional
parameters to better characterize the bacterial response in whole and size-fractioned seawater.
Radiolabled thymidine and leucine incorporation experiments will be added to the experimental
design to provide an independent measure of bacterial growth and growth efficiencies as the
uptake of these compounds is directly related to DNA and protein synthesis within cells. These
experiments will be conducted over short (48 hrs) and longer timescales (3-6 months) to examine
immediate and long term effects on the bacterial populations in whole and size fractioned
seawater. Finally, the percent dioxygenase positive bacteria in these populations will be
determined by using the Cy3-labeled 78-bp primer in the RING-FISH procedure.
We will examine archived samples (collected and frozen at -80oC before the initial GRI-NGI
funding became available) for total bacteria (DAPI) and for the presence of dioxygenase-positive
bacteria (using RING-FISH) and for the presence of Vibrios (using a FISH primer for 16S
rRNA). This will allow us to calculate the percent Vibrios in the total DAPI count, the percent
dioxygenase-positive bacteria in the total DAPI count and the percent dioxygenase-positive
Vibrios. We will do this for archived DH samples collected over three cruises on the R/V Ferrel
and one cruise on the R/V Tommy Munro. Finally, we will extract bulk DNA from all of these
samples and PCR amplify the dioxygenase genes using our 78-bp primer. These amplicons,
along with others collected since the DH blowout, will be sequenced and subjected to
phylogenetic analysis.
We will continue to work with McLellan and Sogin to sample the 21 beach sites across FL, AL
and MS and further develop the comprehensive community profiles of water, submerged sand
and bermed (beach) sand. In the initial samples, several differences in the community profiles
were noted between clean samples and oiled samples; we would expect these differences to
become less pronounced with time.
This proposed research will address the three fundamental questions. The research will provide
information about functional and community genomics at clean and oiled sites, microbial activity
in whole and size-fractioned seawater, and the role of Vibrios in the microbial response to the
MC252 incident.