TUC Reporting

1. TUC Reporting Maria Nikolopoulou, Eleftheria Antoniou, DanaeVenieri & Nicolas Kalogerakis

2. 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

3. 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

4. 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

5. Growth Curve

6. Growth Curves

7. Isolations • Hydrocarbon degraders • Biosurfactant producers

8. 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 hrs • 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 hrs • A loopfull sample was resuspended in ONR7 medium supplemented with crude oil to perform extensive testing for hydrocarbon degradation on purified cultures.

9. 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 hrs • Randomly picked colony was spread on ONR7 agar platesfollowed by incubation at 20oC for 48 hrs • Picked those colonies of unique appearance and purified them further on the same medium.

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

11. Screening concepts for the isolation of biosurfactant producing m/o BATH TEST MATH TEST 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 crude oil 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. • Harvest cells at the early log phase • Wash them with PUM* Buffer and resuspend them to get an initial OD400 measure of about 0.3 to 0.5 • Set up tubes containing 3 ml of cell suspension and 150μl of C16 or crude oil or any other desirable oil phase. • Incubate for 10 min at 25 oC • 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. *22.2g K2HPO4-H20, 7.26 g KH2PO4, 1.8 g urea, 0.2 g MgSO4 7H20 in 1000ml dH2O, pH 7.1

12. Determination of the degree of hydrophobicity % Hydrophobicity=× 100 • = optical density before mixing • = optical density after mixing

13. M/O may have affinity to the oil phase.Have Biosurfactants been produced?

15. BATH-MATH Tests Results

16. BATH-MATH Tests Results

17. BATH Tests Results on the Isolated Culture

18. Conclusions-Suggestions • BATH test facilitates a qualitative estimation in raw culture and works better with isolated strains cultures. • Biosurfactants producing bacteria or biosurfactants are present in all the enriched cultures. • MATH test instead of BATH • N-C16 instead of crude oil

19. 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.

20. 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.

21. Biosurfactants Structures of common glycolipids types. Sophorolipids (top), rhamnolipids (middle), and mannosylerythritol lipids (bottom).

22. Strategy • Sampling • Screening methods for biosurfactant production • Isolation-purification of biosurfactants • Characterization of biosurfactants

23. Isolation-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

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

25. Future Work • Isolation of hydrocarbon degraders and biosurfactant producers • Identification of possible isolates by conducting DNA extractions and PCR • Producing and Characterizing biosurfactants