25th International Meeting on Organic Geochemistry IMOG 2011

P-149
Targeted chemical and physical characterisation of a biosurfactant
produced by the novel Actinobacterium
Ina Hvidsten 1 , Gunhild Bødtker 2 , Tanja Barth 1
1 Petroleum and Colloid Chemistry Research Group, Depatment of Chemistry, University of Bergen, Bergen,
Norway, 2 Uni CIPR, Uni Research AS, Bergen, Norway (corresponding author:ina.hvidsten@kj.uib.no)
The research findings of the last two decades have
already proved that chemical compounds of biological
origin can be both valuable for industrial applications
and environmentally friendly [1]. Identification of such
compounds produced by prokaryotic microorganisms,
referred to as metabolites, is one the major tasks of
metabolomics, see Table 1 for the summary of
approaches and techniques.
Major metabolomic approaches
(applicable also for extracellular metabolites)
Metabolic Semi-quantitative, pre-defined metabolite type.
profiling Analytical Techniques (AT): TLC, FT-IR.
Targeted Quantitative, structure analysis, extensive sample
analysis preparation.AT: HPLC-UV/ELCD, LC/GC-MS,NMR.
Metabolic High throughput, global analysis pattern recognition
fingerprinting for sample classification. AT: FT-IR, NMR.
Metabolic Analysis of chemical/biochemical alterations
footprinting produced by an organism on its environment.
Table 1: Summary of metabolomics approaches and techniques,
those used in this project are highlighted. Modified after [2].
These structurally diversified compounds are seconddary
metabolites of wide range molecular mass
produced by microorganisms either in the late log
phase and/or as a response to different stresses
imposed on the cell, though carbon source (cs) is the
major regulative factor [3]. BSs exhibit interesting
properties in respect to MEOR [4, 5] due to their
amphiphilic nature.
Our project addresses the characterisation as specified
in Table 1 of surface active compounds. These
bio-surfactants (BSs) are produced by a novel Grampositive
mesophilic aerobic chemo-organotrophic
prokaryote, phylum Actinobacteria, family Corynebacteriaceae.
Experimental:
Samples: Pure culture incubations were set up with
a). different carbon sources (sugar; n-dodecane n-
C12) in 2L round flasks and b). different volumes in 2L
and 10L flasks, added n-C12 as the only cs. The
cultures were incubated at optimal temperature 30 o C
w/t agitation and harvested at the early stationary
phase - after 4 weeks. Two types of samples were
obtained: 1. aqueous medium 2. bacterial cell
biomass.
Pre-treatment and –separation: centrifugation
(7000rpm, 25 min.); sample-type 1: quenching of
enzymatic activity; sample-type 2: lyophilisation.
Preliminary screening: oil-spreading test;
determination of cell-surface charge. Fractionation
and purification for sample-type 2: Soxhlet extraction:
a crude extract; Solid Phase Extraction (SPE):
fractionation of the crude extract; purity check by
normal phase/NP- and reverse phase/RP-TLC; FT-IR.
Surface-active fractions: Reverse Phase High
Pressure Liquid Chromatography (RP-HPLC); LC-
ESI/MS. Structure identification by NMR was
attempted.
Results and Discussion:
The results show that production of BS in this microorganism
is regulated by the carbon source, and that
it is coupled to utilisation of certain water-immiscible
substrates (here, a straight chain alkane). A large
area of water-to-carbon-source interface is vital for
high BS-yield. Further, both sample pre-separation
and oil-spreading tests indicate that the BS is both
strongly cell-associated and partially released into the
aqueous medium by the end of the log phase. Cellsurface
charge tests, along with oil-spreading tests,
indicate production of a potent anionic BS. The SPE
polar fractions that exhibited highest activity in oilspreading
test were analysed further by FT-IR: which
identified presence of aliphatic (Cn), ether (C-O-C),
carbonyl (C=O) and amine moieties, TLC indicated
peptide moieties. Further characterisation indicates
production of lipopeptide-type BSs. Method
reproducibility is challenged by matrix interferences
due to the complexity of samples. Therefore, we also
present a flowchart of the reliable and detailed
analysis protocol and suggestions for calibration
methods for complex biological samples in targeted
metabolomics analysis.
References:
[1] Banat et al. (2010) Appl. Microbiol. Biotechnol. 87,
427-444.
[2] Shulaev, V. (2006) Brief. Bioinf. 7:2, 128-139.
[3] Ruiz et al. (2010) C.Rev.Microbiol. 36:2,146-167.
[4] Bødtker et al. (2009)Ant. van Leeuw.96,459-469.
[5] Kowalewski et al. (2005) 13 th Europ. Sym. on IOR.
Acknowledgments: 1. Statoil, Norway; 2. Terje
Lygre, UoB, Norway.
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