Salinivibrio costicola (strain SLC) survives indefinitely in 1% crude oil but the bacterium

does not metabolize crude oil

Clark Oakey, Karl Bischoff, Ruhul Kudus, Ph.D.*Department of Biology, Utah Valley University, Orem UT 84058

Background• Most of the imported and exported crude oil is transported by

supertankers• Some oil tankers collapse and spill crude oil• Every year ~50 different crude oil spills release >100,000 tons of oil

in seas [ITOPF Oil Spill data. http://www.itopf.com/stats.html]• Collapse of marine oil-rigs has added another source of oil spills • Oil spill is a major threat to the well-being of marine and coastal

ecosystems [NOAA http://response.restoration.noaa.gov]

Oil spill-related problems

• Chokes the lungs and gills; kills animals by asphyxiation• Clots feather and fur; kills animals by hypothermia• Prevents phytoplankton from photosynthesis; kills all marine lives• Prevents aeration/oxygenation of water; suffocates marine lives• Blankets bottom mud; kills bottom dwelling organisms• Long-term effects on the marine food chain.

Source: Manual of Oil Pollution. London, International Maritime Organization, 1998

Bioremediation

• Bioremediation is defined as the use of microorganisms to break down pollutants in soil, air or water• Bacteria are the major organisms used for bioremediation• Bacteria used for bioremediation of crude oil are usually soil bacteria

and bacteria that grow in higher temperatures (such as Pseudomonas putida) [Rahman KSM, Thahira-Rahmann JP, Lakshmanaperumalsamy JP, Banat IM. Bioresource Tech 2002; 85:257-261]• Bacteria that naturally grow in salt water and in lower temperatures

could be a good agent for bioremediation of marine oil spills

Isolation of resilient halophiles from SLC

• Samples collected from sand bank, water column and lake floor mud• Grew in a rich medium containing 10% NaCl• Identification made by • biochemical tests• molecular methods- 16S ribosomal DNA sequencing

• Isolated two bacteria that are easy to grow• Halomonas sp.: cannot be stored at -80oC for long time• Slainivibrio costicola: can be stored at room temperature or -80oC for long

time

Some characteristics of S. costicola slcTests ResultsShape and size Curved rods, 0.5-1.0 mM (much longer if grown in ampicillin)

Gram staining G(-)

Mobility Flagellated, highly motile

Respiration Strictly aerobic

Salt-tolerance 1.0-20%, optimum 10% NaCl; obligate halophile

Temperature tolerance 6-40oC, optimum 20oC

Endospore None detected

Antibiotic resistance/sensitivity Resistant to ampicillin, bacitracin, streptomycin, tetracycline and vancomycin/ sensitive to chloramphenicol

Stability Viable at room temperature for six months

Biochemical tests Catalase (+), mannitol (non-fermenter)

Crude oil tolerance of S. costicola slc

• The isolate was grown in five different test mediums (TM 0-5), containing 10% salt and 1.5% agar• TM-0: Meat and yeast extract• TM-1: Essential minerals [N, O, Na, K, Ca, Cl, S, P, NaCO3 and trace

elements• TM-2: Essential minerals plus glucose • TM-3: Essential minerals and 1.0% crude oil• TM-4: Essential minerals plus glucose plus 1.0% crude oil• TM-5: Rich medium (TM-0 and 1.0% crude oil)

TM-0 TM-1 TM-2

TM-3 TM-4 TM-5

Growth of S. colticola slc in different media relative to TM-0

(Gro

wth

x100

)/Gro

wth

in T

M-0

Result of bacterial growth in different media

• The bacterium failed to grow in minimum essential media with or without crude oil • The bacterium formed no colonies but can be recovered from the plate if

transferred on to rich media • The bacterium grew poorly in minimum essential media with glucose

and crude oil• The bacterium grow vigorously in complex organic medium with

crude oil

Metabolism of crude oil by S. costicola (SLC)• Experiment plan• Bacteria was grown in LB broth containing 10% NaCl plus 0.1 % crude oil for

about two months. • The control flasks had the same items except any added bacteria• Bacterial viability was determined every two weeks by plating a 100 mL of the

sample in 10 cm plate (LB agar containing 10% NaCl plus no crude oil)• After 67 days of culture, 250 ml of each of the sample was sent to ALS

Environmental Laboratory, Salt Lake City, UT; for Oil/Grease/TRPH analysis (Test 1664 SGT)

S. costicola (SLC) does not metabolize crude oil• Equally viable bacteria were recovered from the test flask during each

of the six different recovery experiments• The control flasks remained free of any contaminating bacteria during

the entire experiment period • However, there was no difference in the Oil/Grease/TRPH analysis

(Test 1664 SGT) between the control flasks and the test flasks growing S. costicola (SLC)

Summary and conclusions

• S. costicola (SLC) is a highly resilient halophile that can be grown and maintained in large scale • The strain survives in 0.1-1.0 % crude oil for many months but

apparently without metabolizing the hydrocarbon components of the crude oil• Whether the strain metabolized any non-hydrocarbon components of

crude oil remained to be analyzed• The strain can be genetically manipulated to create a suitable crude

oil-bioremediation agent

Acknowledgements

• UVU Presidential Fellowships/Funding to RHK • UVU CSH for SAC funding to CO• Jonathan Oakes (initial isolation) and Noah Kuddus (TM testing)• Dr. Jorma Kirsi and Mr. Ken Slater for various help