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Project scientific advancements at TUC. TUC Research Team: Eleftheria Antoniou, Emmanouela Korkakaki , Maria Nikolopoulou , Danae Venieri & Nicolas Kalogerakis. ULIXES – 1st Year Meeting Agenda, Capo Granitola, Mazara del Vallo, Italy January 22, 2012.

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project scientific advancements at tuc

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

ulixes project tuc is involvement
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
slide3

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

slide4

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

enrichment of the samples
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
establish reliable growth curves
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
first task isolations
First Task: Isolations
  • Hydrocarbon degraders
  • Biosurfactant producers
isolations of hydrocarbon degraders
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.
isolations of hydrocarbon degraders1
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.
isolations of biosurfactant producers
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.
math test for the isolation of biosurfactant producing microbes
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.

determination of the degree of hydrophobicity
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

molecular analyses
Molecular Analyses

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

pcr conditions
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
biosurfactants
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.

biosurfactants1
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.

glycolipids

Biosurfactants

Glycolipids
  • Rhamnolipids
  • Sophorolipids
  • Trehalose lipids

Structures of common glycolipids types. Sophorolipids (top), rhamnolipids (bottom).

Lipopeptides

  • Bacillus Lipopeptides
strategy to isolate biosurfactant producers
Strategy to isolate biosurfactant producers:
  • Sampling
  • Screening methods for biosurfactant production
  • Extraction-purification of biosurfactants
  • Characterization of biosurfactants
extraction purification
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

rhamnolipids
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

rhamnolipids1
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

sophorolipids
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

sophorolipids1
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

trehalose lipids
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

trehalose lipids1
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

characterization

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)
future work
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