25th International Meeting on Organic Geochemistry IMOG 2011

O-66
Investigation of metagenomic clone library in biosurfactant
synthesis and hydrocarbon biodegradation processes
Suzan Vasconcellos 1 , Bruna Dellagnezze 2 , Isabel Natália Sierra Garcia 2 , Valéria
Oliveira 2 , Philip Hendry 4 , Carol Nichols 5 , Eugenio Santos Neto 3 , Herbert Volk 4
1 Federal University of São Paulo - UNIFESP, São Paulo, Brazil, 2 Campinas University - UNICAMP,
Campinas, Brazil, 3 PETROBRAS R & D Center, Rio de Janeiro, Brazil, 4 CSIRO, Sydney, Australia, 5 CSIRO,
Hobart, Australia (corresponding author:supantaroto@yahoo.com.br)
Many studies of physiologically diverse bacterial
communities, present in petroleum reservoirs have
been reported. These include thermophilic and
methanogenic archaea, hydrocarbon-oxiding bacteria,
sulfate reducing bacteria, manganese and iron
reducing bacteria and many fermenting
microorganisms. These metabolically different
microorganisms can be responsible for petroleum
biodegradation, which decreases the economic value
and increases loss and difficulties in oil exploration
and refining. In this context, through projects linked to
Petrobras, a metagenomic library of 31,000 clones
was constructed from the microbiota present in a
Brazilian petroleum reservoir, located at Potiguar
Basin (Mossoró, RN) (Vasconcellos et al., 2010).
From this metagenomic library, biocatalytic and
biosynthetic investigations are being developed and
8,000 clones were screened to assess their ability to
biodegrade and emulsify hydrocarbon. From these,
10 clones were selected as candidates in a more
detailed investigation. The present study sought to
evaluate these clones for their ability to (1)
biodegrade different hydrocarbons and (2) to produce
exopolymers (EPS) with emulsifying activity.
The selected clones were pre-cultured, individually
or in a consortium (300 mL LB media, 37 o C, shaking
at 150 rpm for 24 h), according to methods described
by Vasconcellos et al (2010). The culture assays were
developed by combining the following ingredients: 10
mL microbial suspension (OD ≥ 1.0, absorbance at
600 nm); 100 mL Bushnell Haas mineral medium; 50
µL chloramphenicol (12.5 µg/mL); 200 µL vitamin
solution. In addition, 1 mL or 0.1 g of hydrocarbon:
hexadecane, naphthalene or 0.33 mL of each of three
hydrocarbon, n-alkanes mix (dodecane, tetradecane
and hexadecane) was added. The assays were run in
triplicate. After 10 days of incubation (37 o C), the EPS
from the cultures assays were extracted. according
methods described by Klock et al. (2007). It was
observed that the mixture of n-alkanes resulted in a
better performance of the clones, with respect to EPS
yields (>1 g EPS/ g cells). The partial structural
elucidation of the extracted EPS was determined
through colorimetric analysis for total carbohydrate,
protein and uronic acid content. The results
demonstrated that the EPS fractions were composed
mainly of proteins. The ability of the extracted EPS to
emulsify hydrophobic compounds was evaluated
using hexane and hexadecane (Fleck et al., 2000).
The results were compared to Triton 100 as positive
control. EPS solutions from 4 clones showed
emulsification higher than for Triton X (65 to 80%).
GC-MS analyses were performed to evaluate the
potential of the clones to biodegrade the
hydrocarbons, added as carbon sources. The
obtained results experiments demonstrated that 1
clone could be considered as a potential hydrocarbon
biodegrader. It was able to degrade, to some extent
(43 to 99%), all of the evaluated substrates during the
10 day experiment. Results of this study
demonstrated that the clones that gave the highest
EPS yields and emulsification indexes were not the
same as those that showed the highest hydrocarbon
biodegradation indexes. By combining clones that
presented the highest biodegradation index for each
substrate of interest, it may be possible to achieve
effective biodegradation when aiming their application
towards a bioremediation processes. If the application
of these clones in MEOR is a desired goal, a
combination of clones that produced high amounts of
EPS with others that demonstrated optimum
emulsification indexes would be a good environmental
friendly solution.
References
[1] Fleck, L.C., Bicca, F.C., Ayub, M.A.Z. (2000) Biotech.
Lett. 22: 285-289.
[2] Klock, J-H, Wieland, A., Seifert, R., Michaelis, W.
(2007). Mar. Biol. 152:1077-1085.
[3] Vasconcellos, S.P., Figueiredo Angolini, C.F., Sierra
García, I.N., Dellagnezze, B.M., Silva, C.C., Marsaioli,
A.J., Santos Neto, E.V., Oliveira, V.M. (2010) Org.
Geochem. 41:675-681.
127