Project scientific advancements at TUC

1. Project scientific advancements at TUC TUC Research Team: EleftheriaAntoniou, EmmanouelaKorkakaki, Maria Nikolopoulou, DanaeVenieri & Nicolas Kalogerakis ULIXES – 1st Year Meeting Agenda, Capo Granitola, Mazara del Vallo, Italy January 22, 2012

2. ULIXES project - TUC is involvement: • WP2 – Task 2.1: Sampling organization and geochemical characterization of the coastal target sites • Sampling at site (2) The Elefsina Gulf, Attika, Aegean Sea • Sampling at site (3) the salt wetlands of Keri in Zakynthos, Ionian Sea • WP4 – Task 4.1: Collection 1: Hydrocarbondegrading microbes • Collection 1A – Biofilm forming prokaryotes • Collection 1B – Biosurfactants forming prokaryotes • WP8 – Task 8.2: Treatment at site 2 - Elefsina Gulf, Attika, Aegean Sea, Greece

3. Elefsina bay - ELPE Oil Refinery Area Sampling Points 5 m 0,5 m 5 m 0,5 m 5 m 10 m 0,5 m 5 m 10 m

4. Island of Zakynthos – Keri Bay area Sampling Points 0,5 m 5 m 10 m H (Herodotous oil seeps) 0,5 m 5 m 10 m

5. Enrichment of the samples • In 250 ml flasks at first we added 10ml of each sample to 90 ml of ONR7 medium (artificial seawater) • 0.5%w/v crude oil • Incubation at 20 oC • Orbital shaker at 200 rpm

6. Establish reliable growth curves • Performed 5 re-inoculations each time 1 ml of culture taken from the early exponential phase and added to 99ml of ONR7 medium • Plate counts on marine agar • OD Measurements at 600nm • We concluded that many of the bacteria measured with OD still viable weren’t countable on marine agar plates

7. Growth Curve (Elefsina bay)

8. Growth Curves (Elefsina bay)

9. Growth Curve (Zakynthos island)

10. Growth Curves (Zakynthos island)

11. First Task: Isolations • Hydrocarbon degraders • Biosurfactant producers

12. Isolations of Hydrocarbon Degraders • Samples from the enriched cultures taken at the early exponential phase were spread on ONR7 agar plates supplemented with crude oil followed by incubation at 20oC for 48 h • Distinguish among different colonies wasn’t possible • Randomly picked colonies of unique appearance were spread on marine agar plates followed by incubation at 20oC for 48 h • A loopfull sample was resuspended in ONR7 medium supplemented with crude oil to perform extensive testing for hydrocarbon degradation on purified cultures.

13. Isolations of Hydrocarbon Degraders • Sample taken from the latest purified enriched cultures spread on ONR7 agar plates supplemented with crude oil followed by incubation at 20oC for 48 h • Randomly picked colony was spread on ONR7 agar platesfollowed by incubation at 20oC for 48 h • Picked those colonies of unique appearance and purified them further on the same medium.

14. Isolations of Biosurfactant Producers • Sample taken from the oil phase of the MATH test spread on ONR7 agar plates followed by incubation at 20oC for 48 h • Randomly picked colony was spread on ONR7 agar plates followed by incubation at 20oC for 48 h • Picked those colonies of unique appearance and purified them further on the same medium.

15. MATH TESTfor the isolation of biosurfactant producing microbes Harvest cells at the early log phase Wash them with Phosphate Buffer and resuspend them to get an initial OD600measure of about 0.4 to 0.6 Set up tubes containing 3 ml of cell suspension and 150μl of C16 or any other desirable oil phase. Vortex for 120 s Let the phases separate for 15 min Carefully remove the lower aqueous phase with a Pasteur pipette and transfer it to a cuvette and determine the absorbance after mixing.

16. Determination of the degree of hydrophobicity % Hydrophobicity=× 100 • = optical density before mixing • = optical density after mixing The higher the degree of hydrophobicity (H%), the higher amount of hydrophobic cells that transfer to the C-16 (oil) phase

17. MATH Tests Results (Elefsina Bay)

18. MATH Tests Results on the Isolated Cultures (Zakynthos samples)

19. Molecular Analyses DNA extractions from isolated cultures were performed according to a modification of mini-preparation Method described by Moore et al. (2004)

20. PCR Conditions Thermal cycling was as follows: • Initial denaturation for 5 min at 94°C (holding stage) • 30 cycles of 94°C for 1 min • 55°C for 1 min • 72°C for 2 min • Final extension at 72°C C for 10 min Reaction volume/well (50 μl) contained: • 2μl of sample DNA • 5μl of 1× PCR Buffer solution • 5μl of 0.5 μM of each primer • 0.05 μl of 0.01 mM of dNTPs • 0.3 μl of 0.03U/ μl Taq Polymerase

21. Identification of Isolates -Nucleotide Sequence Analysis

22. Identification of Isolates -Nucleotide Sequence Analysis

23. Identification of Isolates -Nucleotide Sequence Analysis

24. Biosurfactants A structurally diverse group of amphiphilicbiomolecules with both hydrophilic and hydrophobic moieties (glycolipids, lipopeptides, lipoproteins, phospholipids or lipopolysaccharides) Biosurfactant producing bacteria are isolated from hydrocarbon contaminated areas and screened for biosurfactant - bioemulsifier production. The most promising biosurfactant producing strains are used for surfactant growth and production.

25. Biosurfactants Lack of availability of economic and versatile biosurfactants Principal aim • Finding new structures with: • Strong interfacial activity • Low critical micelle concentration (CMC) • High emulsion capacity • Good solubility in a broad pH-range • Economically competitive: good production strains with high yields Discovery of new biosurfactant producing microbes by applying different screening methods.

26. Biosurfactants Glycolipids • Rhamnolipids • Sophorolipids • Trehalose lipids Structures of common glycolipids types. Sophorolipids (top), rhamnolipids (bottom). Lipopeptides • Bacillus Lipopeptides

27. Strategy to isolate biosurfactant producers: • Sampling • Screening methods for biosurfactant production • Extraction-purification of biosurfactants • Characterization of biosurfactants

28. Extraction-Purification • Extraction : • Acid precipitation (higher yield)- Solvent Extraction crude extract free from the aqueous culture medium Ethyl acetate and hexane, chloroform:methanol (2:1, v/v) • Detection • Thin Layer Chromatography TLC Simple method – detection & structural information • Purification • Silica gel Column Chromatography polar head + hydrophobic tail

29. Rhamnolipids • Extractions • Cell-free culture is obtained after centrifugation • Acid precipitation (pH~3) along with solvent extraction (ethyl acetate) ~8 g extract/L of culture broth Detection-Thin Layer Chromatography Development solvent: CHCl3:CH3OH:CH3COOH Visualization reagent: Anthrone (Green Color) Solvent front Rf = 0.49

30. Rhamnolipids • Purification • Silica gel Column Chromatography: • Gradient Solvent system: • CHCl3 (neutral lipids & non-polar pigments) 1 • CHCl3 : CH3OH (5:0.3 v/v) followed by CHCl3 : CH3OH (5:0.5 v/v) (mono-rhamnolipids) 2 • CHCl3 : CH3OH (5:5 v/v) (di-rhamnolipids) 3 2 3 1 • The column chromatography yield is small, at a range of 0.3% for both mono- and di-rhamnolipids TLC shows high concentration of mono and di-rhamnolipids in both fraction 2 & 3 Rf = 0.75

31. Sophorolipids • Extractions • Whole culture broth • Ethyl-acetate ~5 g extract/L of culture broth Detection-Thin Layer Chromatography Development solvent: CHCl3:CH3OH:H20 Visualization reagent: p-Anisaldehyde (Purple color) Solvent front Rf = 0.53 Rf = 0.40 Rf = 0.13 Rf = 0.26

32. Sophorolipids • Purification • Silica gel Column Chromatography: • Gradient Solvent system: • CHCl3 (neutral lipids & non-polar pigments) 1 • CHCl3 : CH3OH increments from 9.8:2 to 6:4 (all sophorolipids present) 2 Purification yielded a higher amount of sophorolipids ~4% of the extracted sample 2 1 Rf = 0.6 The high yields of sophorolipids obtained from the column chromatography are confirmed by the TLC analysis Rf = 0.36 Rf = 0.5 Rf = 0.2 Rf = 0.08

33. Trehalose Lipids • Extractions • Whole culture broth • Chloroform:Methanol (2:1 v/v) ~8 g extract/L of culture broth Detection-Thin Layer Chromatography Development solvent: CHCl3:CH3OH:H20 Visualization reagent: p-Anisaldehyde (Green color) Solvent front Rf = 0.65-0.78 Rf = 0.08 Rf = 0.2

34. Trehalose Lipids • Purification • Silica gel Column Chromatography 1: • Gradient Solvent system: • n-Hexane (excess hydrocarbon substrates) • CHCl3 : CH3OH 2:1 v/v (trehalose and other lipids) • Column Yield in Biosurfactant mixture 44% • Silica gel Column Chromatography 2: • Gradient Solvent system: • CHCl3 (triglycerides) • CHCl3 : CH3OH (10:1 v/v) (fatty acids-alcohols) 1 • CHCl3 : CH3OH: CH3COOH (5:1:0.01 v/v/v) (fatty acids) 2 • CHCl3 : CH3OH (5:1.5 v/v) (trehalose lipids) 3 1 2 3 Rf = 0.7 TLC shows the presence of biosurfactants in all three column fractions. Further enhancement of the purification (solvent polarity) is needed. Rf = 0.51 Rf = 0.13

35. Micelles Polymers + micelles Characterization The biosurfactants self-association is of high interest especially when it comes to their association with hydrophobic particles like hydrocarbons. • Surface tension • UV-Vis absorbance, electric conductivity (CMC) • Dynamic Light Scattering (structure, CMC) • Small angle X-ray scattering (SAXS)(shape and size)

36. Future Work • Continue on isolation of Biosurfactant producers • Identification of possible isolates by conducting DNA extractions and PCR • Improvement of the Biosurfactant production and isolation procedures • Biosurfactant characterization