This project is supported by the European Union under the Food, Agriculture and Fisheries and Biotechnology theme of the 7th Framework

Programme for Research and Technological Development under Grant Agreement no. 312139

Integrated biotechnological solutions for combating marine oil spills

Prof. Nicolas Kalogerakis, Coordinator School of Environmental Engineering Technical University of Crete

3rd Conference on "Ocean of Tomorrow" Projects, 11 November 2015 , Brussels

Weathering processes & period of activation The essence of

KILL•SPILL

To turn bioremediation from a weathering process into an attractive technology, several “problems” must be overcome (by human intervention):

Speed up the growth of indigenous hydrocarbon degraders or add (previously grown & lyophilized degraders) - bioaugmentation.

Add missing nutrients (primarily N&P) in oligotrophic environments - biostimulation.

Disperse oil into fine droplets (surfactants or biosurfactants)

Oxygen limitations can be a real challenge.

Can we make bioremediation a Green Technology?

Kill•Spill Project Focus / Objectives

1. Review State-of-the-Art & identify technology gaps

2. Develop techniques (biosensors & chemical) to monitor hydrocarbon degraders & hydrocarbon degradation

3. Develop novel bio-based dispersants

4. Develop novel bioremediation agents

5. Develop solutions for sediments

6. Develop multi-functional bioremediation agents

7. Test their toxicity

8. “Mandatory” Field Testing of Technologies Emergency Response Companies as Kill•Spill partners: EPE (Greece) & MMB (Norway)

Development of biosensors for the in-situ monitoring of hydrocarbons at contaminated sites

KIL

L•SP

ILL

Sen

sors

Construction of plasmids encoding sensor/promoter::gfp pairs that, when introduced into a suitable host, respond to the presence of hydrocarbons by producing Green Fluorescent protein.

Sevilla, E., Yuste, L., and Rojo, F., 2015; Marine hydrocarbonoclastic bacteria as whole-cell biosensors for n-alkanes; Microbial Biotechnology, 8: 693-706

Gas CO2

Biodegradation is associated with an enrichment of 13C in the remaining contaminant pool increasing 13C

Compound Specific Isotope Analysis

First KillSpill FISH kits are now

commercially available …

FISH protocols adapted to easy and standardized VIT®

format allows fast and specific on-site monitoring

Identification and quantification of various hydrocarbon-

degrading bacteria using developed test kits:

o Alcanivorax borkumensis

o Cycloclasticus pugetii

o Marinobacter sp.

o Oleispira antarctica

o Oleiphilus messinensis

o Thalassolituus oleivorans

KIL

L•SP

ILL

FISH

kit

s

Green: Alcanivorax borkumensis, Yellow: Oleispira antarctica; Total magnification 1000 x

Green: Alcanivorax borkumensis, Yellow: Oleispira antarctica; Total magnification 1000 x

Microbial Biosurfactants: Rhamnolipids

Sophorolipids

Biosurfactants + Oxygel (O2 delivery) + treated CaCO3

(adsorption/sinking agent) + hydrocarbon degrading bacteria.

Several plant surface active extracts/oils from BioBased Europe Ltd (a Scottish company).

Surface active products to date K

ILL•

SPIL

L b

iosu

rfac

tan

ts

Strain Biosurfactants (main congener) Yield (g/L)

Pseudomonas (aeruginosa PAO1) Rhamnolipids (Rha-Rha-C10-C10) 2-3

Burkholderia (Thailandensis E264) Rhamnolipids (Rha-Rha-C14-C14) 2-4

Candida bombicola Sophorolipids 20-100

Enhancing aerobic biodegradation with Oxygen releasing compounds: Gel form

Oxygel™

With additives (solvents & surfactants

from Bio-Based):

Suitable for free oil absorption into its matrix

& Oxygen release for enhanced degradation.

Without additives:

Suitable for injection into

sediments

Oil desorption & degradation

stimulation by oxygen release

Slow O2 release in H2O

Slow-release microparticles for the slow release of HC-degrading cells, biosurfactants, fertilizers, enzymes and e-acceptors

Polycaprolactone (PCL) COREs

PCL

Alginate HC-deg MOs

Nutrients (SRFs)

Kill•Spill Snorkel Proof of concept: a novel bio-electrochemical approach to

accelerate in situ the degradation of petroleum hydrocarbons in anoxic marine sediments

Large scale application considerations: Radius of influence & geometry

Development of a “smart gate” system allowing the release of the nutrient in the contact of an organic phase

P

P

PP

P

P

P

P

P

P

P

PGATEOPENING

AQEUOUSPHASE ORGANICPHASE=

SELF-ASSEMBLY

SILICAPOLYCONDENSATION

CALCINATION

C16H33

N+

Biostimulation of crude oil contaminated beach sand

Biostimulation with N & P Biostimulation with N & P + rhamnolipids

Kill•Spill Sediment Cleaner OXIC (inside)

ANOXIC (outside)

Project co-funding by the European Union within the 7th Framework Programme under Grant Agreement No. 312139 is gratefully acknowledged

www.killspill.eu

Kill•Spill - Integrated Biotechnological Solutions for Combating Marine Oil Spills

Thank you for your attention!

All 28 Kill•Spill products developed or currently under development can be viewed at