1. Oil Cleanup Bacteria Richard Wolfe

2. Introduction A wide variety of micro-organisms are known to degrade petroleum. Batch culture A technique used to grow micro-organisms or cells. A limited supply of nutrients for growth is provided; when these are used up, or some other factor becomes limiting, the culture declines. Cells, or products that the organisms have made, can then be harvested from the culture. [2] A flow-through culture was used. The bacteria were enriched by a flow of sea water that included crude oil. This method enables the bacteria to grow.

3. Methods Crude oil was heated to remove the volatile fraction, the part susceptible to evaporation. The oil was mixed with fresh seawater at a ratio of 1:5 making a chocolate mousse. 2 strains of bacteria were purchased: Alcanivorax borkumenus – found in natural marine environments worldwide and known for the degradation of aliphatic hydrocarbons, flammable hydrocarbons that do not contain a benzine ring. Oceanobacter kriegii – NOT known as an oil degradation bacteria

4. 2 cultures of Indonesian seawater were made: Culture 1 –1L seawater and a pumice stone with 3 grams of chocolate mousse crude oil applied. Non-sterilized water was exchanged for first 10 days then sterilized seawater was supplied for the next 6 days Culture 2 – 1L seawater and a pumice stone with 3 grams of chocolate mousse crude oil applied. Non-sterilized water was exchanged for 3 days then sterilized seawater was supplied for the next 13 days. After 16 days: 60 isolates removed from chocolate-mousse crude oil in culture 1 70 isolates removed from chocolate-mousse crude oil in culture 2 20 isolates removed from aqueous portion of culture 1 20 isolates removed from aqueous portion of culture 2 These 170 isolates were incubated on an agar medium covered with crude oil for 6 weeks

5. GC-MS (gas chromatography-mass spectrometry) was used to determine which isolates exhibited: 1. n-alkane degrading activity 2. n-alkane degrading and oil-emulsifying activities 40 of the 170 isolates were chosen. PCR amplification and sequence analysis of the 16S rRNA genes was used to determine the Taxonomic affiliation of these 40 isolates. (Table 1) The sequences were compared to existing sequences using the BLAST bioinformatics tool.

6. Rep-PCR – (repetitive extragenic palindromic sequence PCR) a highly reproducible and simple method to distinguish closely related strains, to deduce phylogenetic relationships between strains and to study their diversity in a variety of ecosystems. (https://www.msu.edu/~debruijn/)https://www.msu.edu/~debruijn/ Rep-PCR was used to determine the strain of the 40 isolates.

7. Results Alcanivorax and Marinobacter were expected to be found.

8. Results The 2 nd closest relative to 201, 1014, and 1018 is Oceanobacter kreigii which is NOT petroleum-hydrocarbon-degrading but 201, 1014, and 1018 are. O.kreigii was verified by GC-MS.

9. The same tests were used except Japanese seawater was used. Alcanivorax strains found in Indonesia are not closely related to the strains found in Japan. This suggests that Alcanivorax strains related to A.borkumensis SK2 prefer temperate seawater and the other types prefer tropical seawater.

10. Crude oil contaminated sand was submerged in Indonesian seawater simulating sand on a beach. 4 cultures were made, 3 had different fertilizers and 1 no fertilizer. Cultures were left for 38 days. Microcosms of each culture were analyzed by T-RFLP (Terminal restriction fragment length polymorphism). Restriction enzymes cut the 16S rRNA gene from the many organisms. These genes of various lengths are amplified by PCR. The various genes are sorted by length using gel electrophoresis. The lengths are quantified and sequenced. Blast was used to find the closest relative in GenBank.

11. Of the 82 clones that were analyzed, 31 had a closest relative of Oceanospirillum and the 2 nd closest relative was Oceanobacter kriegii.

12. Conclusion 1. Oceanobacter-related strains could dominate in the natural tropical environment after an oil spill. 2. Oceanobacter-related bacteria could be major degraders of petroleum n-alkanes spilt in tropical seas.

13. References 1. Teramoto M, Suzuki M, Okazaki F, Hatmanti A, Harayama S: Oceanobacter-related bacteria are important for the degradation of petroleum aliphatic hydrocarbons in the tropical marine environment. Microbiology (2009), 155:3362- 3370 2. A Dictionary of Biology. 2004. Encyclopedia.com. 5 Apr. 2011..http://www.encyclopedia.com