Oil Cleanup Bacteria

Richard Wolfe

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.

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

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

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.

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/)
Rep-PCR was used to determine the strain of the 40 isolates.

Results
Alcanivorax and Marinobacter were expected to be found.

Results
The 2nd 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.

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.

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.

Of the 82 clones that were analyzed, 31 had a closest relative of

Oceanospirillum and the 2nd closest relative was Oceanobacter
kriegii.

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.

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), 155:33623370
2. A Dictionary of Biology. 2004. Encyclopedia.com. 5 Apr. 2011
http://www.encyclopedia.com>. .

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