Oil Spill Problem - TUC · Definition of the Problem •Accidental crude oil spills are mainly...

Oil spills bioremediation in marine environment-biofilm characterization around oil droplets

PhD Thesis

Maria Nikolopoulou

School of Environmental Engineering Technical University of Crete

Definition of the Problem

• Accidental crude oil spills are mainly caused by operations of extraction, transportation, storage, refining and distribution.

• Between 1.7 and 8.8 million metric tons of oil are released into the world’s water every year.

• About 12% of the oil released into the marine environment is from tanker accidents

The need for action

• Marine shorelines are important public and ecological resources

• Oil spills have posed great threats and caused extensive damage to the marine coastal environments

Loss of species richness, downgraded sediment quality, a negative impact on offshore fish, crustacean fisheries with many long term adverse environmental effects and also on the touristic sector with many socioeconomical side effects.

ENHANCED BIOREMEDIATION

BIOSTIMULATION BIOAUGMENTATION

• Large amounts of oil-degrading bacteria are added to supplement the indigenous microbial population.

• Addition of nutrients or other growth-limiting co-substrates to stimulate the growth of indigenous oil degraders.

Biostimulation vs Bioaugmentation

• Bioaugmentation Research studies have failed to prove conclusively that

seeding is effective (with bioengineered organisms or organisms enriched from different environments and grown in the laboratory to high numbers).

1. The influx of oil causes a relatively fast response in the

hydrocarbon-degrading bacteria; however, in the absence of essential nutrients their growth is limited.

2. Seeded microorganisms usually show a short term benefit; however, over a longer period of time the indigenous populations catch up and the main limiting factor becomes the lack of N & P.

Bioaugmentation appears less effective than

Biostimulation since

Autochthonous Bioaugmentation (ABA)

Isolated single strains or enriched cultures, which are obtained ‘‘before’’ or ‘‘after’’ the contamination of the

target sites, are administered to the sites once contamination occurs

Key idea is to conduct the enrichment of contaminant-degrading bacteria under the same or very similar

conditions to those where bioaugmentation will be performed

Uses exclusively microorganisms indigenous to the sites to be decontaminated

Oil Spill Problem

Open – water (e.g., Oil Spill off the Galicia coast of Spain- Prestige 2002)

• Surface spills

• Deep sea releases

Shoreline (e.g., Oil Spill at Alaska- Exxon valdez 1989)

Major Challenges in Open-waters

Control of the release rates so that optimal nutrient concentrations can be maintained in the sea water over long time periods

Oleophilic Nutrients:

• To overcome the wash out problem, it is recommended to utilize oleophlilic organic nutrients (e.g., Uric acid ,Lecithin)

• The rationale for this approach is that oil biodegradation occurs primarily at the oil-water interface. Since oleophilic fertilizers are able to adhere to oil and provide nutrients at the oil-water interface, enhanced biodegradation should result without the need to increase nutrient concentrations in the bulk sea water

Major Challenges in Open-waters

Biosurfactants: • Biosurfactants are amphiphilic compounds of microbial origin with

considerable potential in commercial applications within various industries.

• The ability of biosurfactants to emulsify hydrocarbon- water mixtures enhances bioavailability of the hydrocarbons to the microbial population thus leads to degradation of hydrocarbons in the environment.

Landfarming High Efficiency and Low Cost

Design Parameters

• Aeration

• Moisture

• pH

• Other constrains

(Nutrients, microorganisms, etc.)

Shoreline cleanup (Sandy Beach)

Main Objective

To examine the effectiveness of novel combined autochthonous bioaugmentation &/or biostimulation

strategies in an oil contaminated environment

EPA’s Bioremediation Agent Effectiveness test:

Designed to determine a product’s ability to biodegrade oil by quantifying changes in the oil composition and microbial activity resulting from biodegradation.

Chemical analyses alkanes and PAHs by GC-MS

Microbiological analyses to determine hydrocarbon degraders by MPN procedure.

Molecular analyses to determine microbial diversity of hydrocarbon degraders

Experimental approaches

1. Autochthonous bioaugmentation and/or biostimulation of seawater microcosm

2. Autochthonous bioaugmentation & biostimulation with isolated hydrocarbon degraders consortium of seawater microcosm

3. Landfarming of oil polluted beach sand through biostimulation

4. Landfarming of oil polluted beach sand through autochthonous bioaugmentation & biostimulation

5. Biofilm investigation on oil droplets & C20 with Confocal Microscopy (UFZ-Magdeburg)

Microcosm Experimental Set Up

1. 20 mL of seawater containing the indigenous microorganisms

2. 0.5 % w/v of weathered (ASTM method D 86) blend Urals-Arabian light crude oil

3. The products were added to the solutions into such amount in order to achieve the optimized nutrient amendment C:N:P (100:10:1)

4. The prepared vials were shaken at 150 rpm at 20 oC until such time that they would be removed for sampling.

5. The entire vial is sacrificed for analysis (a 1-mL aliquot is removed from each vial for the microbiological analysis and the remainder of each flask is used for the chemical analysis).

40-mL glass vials were charged with:

Microcosm Experimental Design

Treatment Weathered

crude oil 0.5% w/v

Nutrients (KNO3,

KH2PO4)

Nutrients (uric acid, lecithin)

Rhamnolipid biosurfactant

Preadapted indigenous population

Control ┼

NPK ┼

┼

ULR ┼

┼

┼

NPKM ┼

┼

┼

NPKMR ┼

┼

┼

┼

ULRM ┼

┼

┼

┼

Microcosm Experimental Set Up

1. 50 mL of seawater containing the isolated consortium 2. The inoculum was diluted to have approximately 105

cfu/ml. 3. 0.5 % w/v of weathered (ASTM method D 86) blend

Urals-Arabian light crude oil 4. The products were added to the solutions into such

amount in order to achieve the optimized nutrient amendment C:N:P (100:10:1)

5. The prepared flasks were shaken at 150 rpm at 20 oC until such time that they would be removed for sampling.

6. The entire flask is sacrificed for analysis (a 1-mL aliquot is removed from each flask for the microbiological analysis and the remainder of each flask is used for the chemical analysis).

100-mL glass flasks were charged with:

Microcosm Experimental Design

Treatment Weathered

crude oil 0.5% w/v

Nutrients (KNO3,

KH2PO4)


Isolated Hydrocarbon Degraders Consortium

(Eb8)

Control ┼ ┼

NPKM ┼ ┼ ┼

NPKMR ┼ ┼ ┼ ┼

Soil Microcosm Landfarming Experimental Set Up

1. 2 kg of sieved (<2mm) beach sand containing the indigenous microorganisms

2. 0.5 % w/w of weathered (ASTM method D 86) blend Uralsk light crude oil to reach oil contamination 5000 ppm.


4. water content was adjusted to 60% of the field-holding capacity, i.e. 20%

5. The prepared trays were mixed twice a week and incubated at RT until such time that they would be removed for sampling.

6. Samples were taken from each microcosm by sampling at various points, combining and mixing. 15 gr of sand were removed for the chemical analysis and 5 gr for the microbiological analysis.

7. Experiments were run for 45 days.

Stainless Steal trays were charged with:

Soil Microcosm Experimental Design

Treatment Weathered

crude oil 0.5% w/w

Nutrients (KNO3,

KH2PO4)



Isolated Hydrocarbon

degraders

Control ┼

NPK ┼

┼

ULR ┼

┼

┼

Control Μ ┼

┼

NPKM ┼

┼

┼

ULRM ┼

┼

┼

┼

Soil Microcosm Landfarming Experimental Set Up

1. 1 kg of sieved (<2mm) beach sand containing the isolated hydrocarbon degraders

2. 0.5 % w/w of weathered (ASTM method D 86) Iranian light crude oil to reach oil contamination 5000 ppm.


4. water content was adjusted to 60% of the field-holding capacity, i.e. 20%

5. The prepared trays were mixed twice a week and incubated at 20 oC until such time that they would be removed for sampling.

6. Samples were taken from each microcosm by sampling at various points, combining and mixing. 15 gr of sand were removed for the chemical analysis and 5 gr for the microbiological analysis.

7. Experiments were run for 45 days

Glass trays (Pyrex) were charged with:

Soil Microcosm Experimental Design

Treatment Weathered

crude oil 0.5% w/w

Nutrients (KNO3,

KH2PO4)



Isolated Hydrocarbon

degraders

Control ┼

NPK ┼

┼

ULR ┼

┼

┼

Control Μ ┼

┼

NPKM ┼

┼

┼

ULRM ┼

┼

┼

┼

Biofilm Microcosm Experimental Set Up

1. 100 mL of ONR7 medium containing the isolated consortium Eb8 or E4

2. 0.5 % w/v of weathered (ASTM method D 86) blend Iranian light crude oil


4. The recommended dose dispersant: oil (1:10) by the manufacturer

5. The prepared flasks were shaken at 150 rpm at 20 oC (Eb8) or at 14 oC (E4) until such time that they would be removed for sampling.

6. A few μLs aliquot is removed from each flask were placed to a specifically designed 1,02mm Deep Chamber, stained with Syto9 (bacteria) and immediately examined by CLSM.

7. .

250-mL glass flasks were charged with:

Biofilm Experimental Design

Treatment

With

Eb8(20oC)/E4(14oC)

Weathered

crude oil

0.5% w/v

Nutrients

(KNO3,

KH2PO4)

Rhamnolipid

biosurfactant Corexit marichem S200

Eb8R/ E4R ┼ ┼

Eb8RNP/ E4RNP ┼ ┼ ┼

Eb8C/ E4C ┼ ┼

Eb8CNP/ E4CNP ┼ ┼ ┼

Eb8MNP/ E4MNP ┼ ┼ ┼

Eb8SNP/ E4SNP ┼ ┼ ┼

Biofilm Microcosm Experimental Set Up

1. 100 mL of ONR7 medium containing the isolated consortium Eb8 or E4

2. Droplets of C20 (500000 ppm, 0.4 μl) were placed on a sterile plastic slide


4. The prepared petri dishes were shaken at 100 rpm at 20 oC (Eb 8) or at 14 oC (E4) until such time that they would be removed for sampling.

5. After 5, 7, 11, 14 and 18 days, pieces with 2 C20 droplets per slide were cut off, stained with Syto9 (bacteria) and immediately examined by CLSM.

petri dishes were charged with:

C20 Biofilm Experimental Design

Treatment with

C20

Nutrients

(KNO3,

KH2PO4)

Rhamnolipid

biosurfactant

Consortium

Eb8

Consortium

E4

Eb8 (20oC) ┼ ┼

E4 (14oC) ┼ ┼

Eb8R (20oC) ┼ ┼ ┼ ┼

E4R (14oC) ┼ ┼ ┼

Chemical Analyses (1)

Quantification of the hydrocarbon target analytes was performed by GC-MS according to a modified EPA method (40 CFR Ch. I, Pt 300, App. C)

LIQUID EXTRACTION

1. Flasks contents were sacrificed and extracted by adding approximately 50 mL of DCM spiked with a surrogate recovery standard (4 ng/μL of each d10-anthracene, 5α-androstane in solvent of the final extract)

2. After mixing for several minutes, the flask was set aside to allow the dichloromethane and water layers to partition.

3. The dichloromethane layer was passed through a funnel packed with anhydrous sodium sulfate.

4. The dichloromethane was evaporated in rotavapor concentrator.

Chemical Analyses (1) SOIL EXTRACTION

1. Sand samples were dried with anhydrous sodium sulfate, spiked with a surrogate recovery standard (200 ppm of each d10-anthracene, 5α-androstane) and finally extracted with soxhlet apparatus using 300 mL of DCM

2. The dichloromethane was evaporated in rotavapor concentrator.

3. The volume was blown down to dryness (nitrogen).

Chemical Analyses (2)

6. A known weight of 5-10 mg of the dried oil was dissolved in was transferred onto the Bond Elut TPH SPE cartridge and eluted, under a positive pressure, with 4ml of n-C6 (FI- aliphatics) and 4ml of DCM (FII- aromatics).

7. The two fractions were blown down to dryness (nitrogen)

8. The weight of FI- aliphatics and FII- aromatics was recorded and redesolved in 1 mL n-C6 and 1 mL DCM respectively

9. The final concentration of the internal standards added in each fraction right before the injection is 1 ppm. This solution contained 4 deuterated compounds: d8-naphthalene, d10-phenanthrene, d12-chrysene and d12-perylene.

Depletion of target analytes

• As= Concentration of target analyte in the sample

• Ao= Concentration of target analyte in the initial sample

• Hs= Concentration of 17a(H), 21b(H) C30-hopane in the sample

• Ho= Concentration of 17a(H), 21b(H) C30-hopane analyte in the initial sample

%Depletion =[(𝐴𝑜 𝐻𝑜) − (𝐴𝑠 𝐻𝑠)]

(𝐴𝑜 𝐻𝑜 )× 100

Kinetic Analysis (Batch)

• Cell Growth

• μ specific growth rate (1/h)

• Substrate Utilization

• qS specific degradation rate (μg/cells h)

Xdt

dXrX

Xqdt

dSr sS

Estimation of qs by the integral method

• Integration of Substrate equation yields:

• Average specific degradation rate is obtained by LS

estimation

dtXqSS

t

st 0

0

Microbiological Analyses (1)

Microbial enumerations of hydrocarbon degraders are

performed at each sampling event using a microtiter MPN determination

1. The growth medium was a B-H minimal salts mediun supplemented with crude oil as the hydrocarbon substrate.

2. The MPN plates were 96-well microtiter tissue culture plates

3. Each well contains 180 μL BHS, 5 μL crude oil and 20 μL of the

appropriate dilution of sample

4. The samples were diluted in a 9 mL B-H solution (pH 7).

Microbiological Analyses (2)

5. Tenfold serial dilutions were performed, and the plates were

inoculated by adding 20 μL of each dilution to I of the 12 columns of

eight wells.

6. The inoculated plates were incubated at 20 oC for 2 weeks.

7. At the end of the incubation period, 50 μL of p-iodonitrotetrazolium violet dye (3.5 g/L) was added to each well of the tissue culture

plates.

8. The dye turns from colorless to red (is reduced) in the presence of

actively respiring microorganisms.

Molecular Analyses

Target genes

Primer Sequence (5’-3’) Product

(bp) References

16SrDNA 515F GTGCCAGCMGCCGCGGTAA

291 Kuczynski et

al., 2012 806R GGACTACHVGGGTWTCTAAT

alkB2

ALCalkB2F867 CGCCGTGTGAATGACAAGGG

132 MacKew et

al., 2007 ALCalkB2R999 CGACGCTTGGCGTAAGCATG

alkB

THALalkBF125 GACGTCGCCACACCTGCC

217 MacKew et

al., 2007 THALalkBR342 GGGCCATACAGAGCAAGCAA

phnA

CYCphnAF243 CGTTGTGCGCATAAAGGTGCGG

145 MacKew et

al., 2007 CYCphn-AR388 CTTGCCCTTTCATACCCCGCC

RT-PCR Conditions

Thermal cycling was as follows:

• Initial denaturation for 5 min at 95°C (holding stage)

• 40 cycles of 95°C for 15 s

• 60°C for 1 min.

Reaction volume/well (20 μl) contained:

• 2 μl of sample DNA

• 10 μl of 2× SYBR Green PCR Master Mix

• 100 nM of each primer

Molecular Analyses

Target genes

Primer Sequence (5’-3’)

16SrDNA bacteria

27F AGAGTTTGATCMTGGCTCAG

1492R GGTTACCTTGTTACGACTT

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: 2 μl of sample DNA 5 μl of 1× PCR Buffer solution 5 μl 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

PCR Conditions

Experimental Results

1. Autochthonous bioaugmentation and/or biostimulation of seawater microcosm

n-alkanes overall degradation profile

Control NPK NPKM NPKMR ULR ULRM

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

9060

3015

5

% D

ep

leti

on

n-a

lkan

es

(C1

4-C

35)

Days

Profile of alkanes

0

5

10

15

20

25

30

35

C1

4

C1

5

C1

6

C1

7 Pr

C1

8

Ph

C1

9

C2

0

C2

1

C2

2

C2

3

C2

4

C2

5

C2

6

C2

7

C2

8

C2

9

C3

0

C3

1

C3

2

C3

3

C3

4

C3

5

ng

alka

ne

s/n

g h

op

ane

NPKM

NPKM_0 NPKM_5 NPKM_15


Profile of alkanes

0

5

10

15

20

25

30

35

40

C1

4

C1

5

C1

6

C1

7 Pr

C1

8

Ph

C1

9

C2

0

C2

1

C2

2

C2

3

C2

4

C2

5

C2

6

C2

7

C2

8

C2

9

C3

0

C3

1

C3

2

C3

3

C3

4

C3

5

ng

alka

ne

s/n

g h

op

nae

NPKMR

NPKMR_0 NPKMR_5 NPKMR_15

NPKMR_30 NPKMR_60 NPKMR_90

Profile of alkanes

0

5

10

15

20

25

C1

4

C1

5

C1

6

C1

7 Pr

C1

8

Ph

C1

9

C2

0

C2

1

C2

2

C2

3

C2

4

C2

5

C2

6

C2

7

C2

8

C2

9

C3

0

C3

1

C3

2

C3

3

C3

4

C3

5

ng

alka

ne

s/n

g h

op

ane

ULR

ULR_0 ULR_5 ULR_15

ULR_30 ULR_60 ULR_90

Profile of alkanes

0

2

4

6

8

10

12

14

16

18

C1

4

C1

5

C1

6

C1

7 Pr

C1

8

Ph

C1

9

C2

0

C2

1

C2

2

C2

3

C2

4

C2

5

C2

6

C2

7

C2

8

C2

9

C3

0

C3

1

C3

2

C3

3

C3

4

C3

5

ng

alka

ne

s/n

g h

op

ane

ULRM

ULRM_0 ULRM_5 ULRM_15


Degradation Kinetics of C15 & C20

0

5

10

15

20

25

30

35

0 20 40 60 80 100

ng

alka

ne

s/n

g h

op

ane

t (days)

C15

NPKM NPKMR

ULR ULRM

0

5

10

15

20

25

0 20 40 60 80 100

ng

alka

nes

/ng

ho

pan

e

t (days)

C20

NPKM NPKMR

ULR ULRM

C15 C20

μ qs qs/qs0 qs qs/qs0

NPKM 0.004 65 1 51.3 1 NPKMR 0.023 1239.3 19 854.8 17 ULR 0.024 898.4 14 783.2 15 ULRM 0.010 105 1.6 84.1 1.6


C25 C30

qs qs/qs0 qs qs/qs0

NPKM 15.7 1 8.4 1 NPKMR 257.3 16 131.3 16 ULR 202.3 12 158.3 19 ULRM 47.7 2 24.9 3

0

1

2

3

4

5

6

7

0 20 40 60 80 100

ng

alka

nes

/ng

ho

pan

e

t (days)

C25

NPKM NPKMR

ULR ULRM

0

1

2

3

4

5

0 20 40 60 80 100

ng

alka

nes

/ng

ho

pan

e

t (days)

C30

NPKM NPKMR

ULR ULRM

Degradation Kinetics of Pristane & Phytane

Pristane Phytane

qs qs/qs0 qs qs/qs0

NPKM 39.6 1 42.4 1 NPKMR 760.6 19 807.3 19 ULR 474.7 12 571 13 ULRM 53.4 1.3 59 1.3

0

5

10

15

20

25

0 20 40 60 80 100

ng

iso

pre

no

ids/

ng

ho

pan

e

t (days)

Pristane

NPKM NPKMR

ULR ULRM

0

5

10

15

20

25

0 20 40 60 80 100

ng

iso

pre

no

ids/

ng

ho

pan

e

t (days)

Phytane

NPKM NPKMR

ULR ULRM

Profile of PAHs

0.0

0.2

0.4

0.6

0.8

1.0

Fluorene Dibenzothiophene Phenanthrene Chrysene

ng

aro

mat

ics/

ng

ho

pan

e

NPKM 0 day

5 days

15 days

30 days

60 days

90 days

0.0

0.2

0.4

0.6

0.8

1.0

1.2


ng

aro

mat

ics/

ng

ho

pan

e

NPKMR

0 day

5 days

15 days

30 days

60 days

90 days

Profile of PAHs

0.0

0.5

1.0

1.5

2.0

2.5

3.0


ng

aro

mat

ics/

ng

ho

pan

e

ULR

0 day

5 days

15 days

30 days

60 days

90 days

0.0

0.1

0.2

0.3

0.4

0.5

0.6


ng

aro

mat

cs/n

g h

op

ane

ULRM 0 day

5 days

15 days

30 days

60 days

90 days

Degradation Kinetics of Fluorene & Dibenzothiophene

Fluorene Dibenzothiothene

qs qs/qs0 qs qs/qs0

NPKM 0.28 1 0.79 1.2 NPKMR 0.46 1.6 0.96 1.5 ULR 1.72 6 6.76 11 ULRM 0.29 1 0.62 1

0.0

0.2

0.4

0.6

0.8

1.0

0 50 100

ng

aro

mat

ics/

ng

ho

pan

e

t (days)

Fluorene

NPKM NPKMR

ULR ULRM

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 50 100

ng

aro

mat

ics/

ng

ho

pan

e

t (days)

Dibenzothiophene

NPKM NPKMR

ULR ULRM

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

0

10

20

30

40

50

60

70

80

90

100

0 5 15 30 60 90

MP

N (

cells

/mL)

% D

ep

leti

on

of

n-a

lkan

es

(C1

4-C

35)

Time (days)

% Depletion NPKM % Depletion ULRM MPN/mL NPKM MPN/Ml ULRM

MPN profile & overall degradation of n-alkanes

alkane hydroxylase & PAH dioxygenase genes profile

5 days 15 days

30 days 60 days

1.E+00

1.E+02

1.E+04

1.E+06

1.E+08

1.E+10

1.E+12

1.E+14

NPKM NPKMR ULR ULRM

Gen

e co

pie

s/m

L

Alcanivorax Thalassolituus Cycloclasticus

1.E+00

1.E+02

1.E+04

1.E+06

1.E+08

1.E+10

1.E+12

1.E+14

NPKM NPKMR ULR ULRM

Gen

e co

pie

s/m

L


1.E+00

1.E+02

1.E+04

1.E+06

1.E+08

1.E+10

1.E+12

NPKM NPKMR ULR ULRM

Gen

e co

pie

s/m

L


1.E+00

1.E+02

1.E+04

1.E+06

1.E+08

1.E+10

1.E+12

1.E+14

NPKM NPKMR ULR ULRM

Gen

e co

pie

s/m

L


0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

DS1 NPK30 NPKM30 NPKMR30 S1 ULR30 ULRM30

OTU

s in

Sam

ple

s

Other

Vibrionaceae

Pseudomonadaceae

Moraxellaceae

Oceanospirillaceae

Halomonadaceae

Alcanivoracaceae

Shewanellaceae

Pseudoalteromonadaceae

Idiomarinaceae

Alteromonadaceae

Rhodospirillaceae

Rhodobacteraceae

Flavobacteriaceae

Families Response (Pyrotag-sequencing at Research and Testing

Lab- Lubbock, Texas)


2. Autochthonous bioaugmentation & biostimulation with isolated hydrocarbon degraders consortium of seawater microcosm

Concentration profile of alkanes at time 0 & Control at 0, 15, 30 days

0

1

1

2

2

3

3

4

4

5

5

6

6

7

7

C14 C15 C16 C17 Pr C18 Ph C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 C29 C30 C31 C32 C33 C34 C35

ng

alka

ne

s/n

g h

op

ane

CM_0 CM_15 CM_30 NPKM_0 NPKMR_0

Profile of n-alkanes

0

1

2

3

4

5

6

7

8

ng

alka

ne

s/n

g h

op

ane

NPKM

day 0 day 5 day 15 day 30


0

1

2

3

4

5

6

7

8

ng

alka

ne

s/n

g h

op

ane

NPKMR

day 0 day 5 day 15 day 30


C15 C20


CM 0.011 9 1 24 1

NPKM 0.031 250 28 218 21

NPKMR 0.083 196 23 197 20

0

1

2

3

4

5

6

7

0 10 20 30 40

ng

alk

an

es

/ng

ho

pa

ne

t (days)

C15

NPKM NPKMR

0

1

2

3

4

5

6

7

0 10 20 30 40

ng

alk

an

es

/ng

ho

pa

ne

t (days)

C20

NPKM NPKMR


0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 10 20 30 40

ng

alk

an

es

/ng

ho

pa

ne

t (days)

C25

NPKM NPKMR

0.0

0.5

1.0

1.5

2.0

2.5

0 10 20 30 40

ng

alk

an

es

/ng

ho

pa

ne

t (days)

C30

NPKM NPKMR

C25 C30

qs qs/qs0 qs qs/qs0

CM 12 1 ~0 -

NPKM 121 10 31 -

NPKMR 128 11 41 -


0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 10 20 30 40

ng

is

op

ren

oid

s/n

g h

op

an

e

t (days)

Pristane

NPKM NPKMR

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 10 20 30 40

ng

is

op

ren

oid

s/n

g h

op

an

e

t (days)

Phytane

NPKM NPKMR

Pristane Phytane

qs qs/qs0 qs qs/qs0

CM 10 1 6 1

NPKM 29 3 21 3

NPKMR 116 12 92 12


1E+02

1E+03

1E+04

1E+05

1E+06

0

10

20

30

40

50

60

70

80

90

100

5 15 30

MP

N (

cell

s/m

L)

(%)

De

ple

tio

n o

f n

-alk

ane

s (C

14-C

35)

Time (days)

NPKM NPKMR NPKM NPKMR

Profile of PAHs

0.00

0.04

0.08

0.12

0.16


ng

aro

mat

ics/

ng

ho

pan

e

NPKM

day0 day5

day15 day30

0.00

0.04

0.08

0.12

0.16


ng

aro

mat

ics/

ng

ho

pan

e

NPKMR

day0 day5

day15 day30


0.00

0.01

0.01

0.02

0.02

0.03

0.03

0.04

0.04

0.05

0 10 20 30 40

ng

aro

ma

tic

s/n

g h

op

an

e

t (days)

Fluorene

NPKM NPKMR

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0 10 20 30 40

ng

aro

ma

tic

s/n

g h

op

an

e

t (days)

Dibenzothiophene

NPKM NPKMR

Fluorene Dibenzothiophene

qs qs/qs0 qs qs/qs0

CM ~0 - ~0 -

NPKM 0.97 - 3.94 -

NPKMR 1.04 - 3.06 -

Nucleotide Sequence Analysis Results


3. Landfarming of oil polluted beach sand through biostimulation


0

2

4

6

8

10

12

14

16

18

C12 C13 C14 C15 C16 C17 Pr C18 Ph C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 C29 C30 C31 C32 C33 C34 C35

ng

alka

ne

s/n

g h

op

ane

Control

0 day 15 days 30 days 45 days


0

2

4

6

8

10

12

14

16

18

20


ng

alka

ne

s/n

g h

op

ane

NPK



0

2

4

6

8

10

12

14

16

18

20

22


ng

alka

ne

s/n

g h

op

ane

ULR



C15 C20


Control 0.005 61.3 1 50.6 1

NPK 0.014 201.1 3.3 161.6 3.2

ULR 0.017 202.7 3.3 145.9 2.9

0

5

10

15

20

25

0 10 20 30 40 50

ng

alkn

es/n

g h

op

ane

t (days)

C15

NPK ULR

Control

0

2

4

6

8

10

12

14

16

0 20 40 60

ng

alka

ne

s/n

g h

op

ane

t (days)

C20

NPK ULR

Control


C25 C30

qs qs/qs0 qs qs/qs0

Control 27.6 1 13.3 1

NPK 98.2 3.5 47.7 3.6

ULR 89.5 3.2 44 3.3

0

2

4

6

8

10

0 10 20 30 40 50

ng

alka

ne

s/n

g h

op

ane

t (days)

C25

NPK ULR

Control

0

1

2

3

4

5

0 10 20 30 40 50

ng

alka

ne

s/n

g h

op

ane

t (days)

C30NPK ULR

Control


Pristane Phytane

qs qs/qs0 qs qs/qs0

Control 30.2 1 34 1

NPK 88.4 3 99.3 3

ULR 89.4 3 102.5 3

0

2

4

6

8

10

0 10 20 30 40 50

ng

iso

pre

no

ids/

ng

ho

pan

e

t (days)

Pristane NPK

ULR

Control

0

2

4

6

8

10

12

0 10 20 30 40 50

ng

iso

pre

no

ids/

ng

ho

pan

e

t (days)

Phytane NPK

ULR

Control

Profile of PAHs

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Fluorene Phenanthrene

ng

aro

mat

ics/

ng

ho

pan

e

Control

0 day

15 days

30 days

45 days

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9


ng

aro

mat

ics/

ng

ho

pan

e

NPK

0 day

15 days

30 days

45 days

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8


ng

aro

mat

ics/

ng

ho

pan

e

ULR

0 day

15 days

30 days

45 days



qs qs/qs0 qs qs/qs0

Control 0.46 1 2.10 1

NPK 2.29 5 6.44 3

ULR 1.74 4 4.21 2

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0 10 20 30 40 50

ng

aro

mat

ics/

ng

ho

pan

e

t (days)

Fluorene

NPK ULR

Control

0.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 40 50

ng

aro

mat

ics/

ng

ho

pan

e

t (days)

Dibenzothiophene

NPK ULR

Control


1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

0

10

20

30

40

50

60

70

80

90

100

0 7 15 21 30 38 45

MP

N c

ells

/g d

ry s

and

% D

ep

leti

on

of

n-a

lkan

es

(C1

2-C

35)

Time (days)

NPK-ULR

% Depletion NPK % Depletion ULR

MPN/g NPK MPN/g ULR


4. Landfarming of oil polluted beach sand through autochthonous bioaugmentation & biostimulation


0

10

20

30

40

50

60


ng

alka

ne

s/n

g h

op

ane

Control M

CM_0 CM_7 CM_15 CM_30 CM_45


0

10

20

30

40

50

60

70


ng

alka

ne

s/n

g h

op

ane

NPKM


NPKM_30 NPKM_45


0

10

20

30

40

50

60

70


ng

alka

ne

s/n

g h

op

ane

ULRM


ULRM_30 ULRM_45


C15 C20

qs qs/qs0 qs qs/qs0

Control M 160.7 1 133.7 1

NPKM 1203.8 7.5 1096.1 8.2

ULRM 1252.1 7.8 1097.2 8.2

0

10

20

30

40

50

60

0 10 20 30 40 50

ng

alka

ne

s/n

g h

op

ane

t (days)

C15

CM NPKM

ULRM

0

10

20

30

40

50

60

0 10 20 30 40 50

ng

alka

ne

s/n

g h

op

ane

t (days)

C20

CM NPKM

ULRM


C25 C30

qs qs/qs0 qs qs/qs0

Control M 86.6 1 36.9 1

NPKM 751.9 8.7 324.3 8.8

ULRM 745.5 8.6 302.7 8.2

0

5

10

15

20

25

30

35

40

0 10 20 30 40 50

ng

alka

ne

s/n

g h

op

ane

t (days)

C25

CM NPKM

ULRM

0

2

4

6

8

10

12

14

16

0 10 20 30 40 50

ng

alka

ne

s/n

g h

op

ane

t (days)

C30

CM NPKM

ULRM


Pristane Phytane

qs qs/qs0 qs qs/qs0

Control M 59.2 1 36.4 1

NPKM 700.9 12 598.3 17

ULRM 621 10.5 512.6 14

0

5

10

15

20

25

30

35

0 10 20 30 40 50

ng

iso

pre

no

ids/

ng

ho

pan

e

t (days)

Pristane

CM NPKM

ULRM

0

5

10

15

20

25

30

0 10 20 30 40 50

ng

iso

pre

no

ids/

ng

ho

pan

e

t (days)

Phytane

CM NPKM

ULRM

Profile of PAHs

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

fluorene phenanthrene

ng

aro

mat

ics/

ng

ho

pan

e

Control M 0 day

7 days

15 days

30 days

45 days

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8


ng

aro

mat

ics/

ng

ho

pan

e

NPKM 0 day

7 days

15 days

30 days

45 days

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4


ng

aro

mat

ics/

ng

ho

pan

e

ULRM 0 day

7 days

15 days

30 days

45 days



qs qs/qs0 qs qs/qs0

Control M 0.96 1 2.74 1

NPKM 2.86 3 6.38 2.3

ULRM 1.52 1.6 2.89 1

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 10 20 30 40 50

ng

aro

mat

ics/

ng

ho

pan

e

t (days)

Fluorene

CM NPKM

ULRM

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 30 40 50

ng

aro

mat

ics/

ng

ho

pan

e

t (days)

Dibenzothiophene

CM NPKM

ULRM


1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

0

10

20

30

40

50

60

70

80

90

100

0 7 15 21 30 38 45

MP

N c

ells

/g d

ry s

and

% D

ep

leti

on

of

n-a

lkan

es

(C1

4-C

35)

Time (days)

NPKM-ULRM

% Depletion NPKM % Depletion ULRM

MPN/g NPKM MPN/g ULRM

n-alkanes overall degradation profile

ControlNPK

ULRCM

NPKMULRM

0

10

20

30

40

50

60

70

80

90

100

45

30

15

7

% D

ep

leti

on

of

n-a

lkan

es

(C1

4-C

35)

Days

Pristine Elefsina


5. Biofilm investigation on oil droplets&C20 with Confocal Microscopy (UFZ-Magdeburg)

2nd day 1 week 1 week

Crude oil from ONR7 Culture

Crude oil from ONR7 Culture

1 week 1 week 10 days

Crude oil from ONR7 Culture with Rha (Eb8 & E4)

Crude oil from ONR7 Culture with corexit (Eb8 & E4)

5 days 7 days 11, 14 days

Crude oil from ONR7 Culture with S200 (Eb8 & E4)

5 days 11 days 14 days

Crude oil from ONR7 Culture with marichem (Eb8 & E4)

5 days 7 days 11 days

Crude oil from ONR7 Culture with marichem (Eb8 & E4)

14 days 18 days

C20 from ONR7 Culture (Eb8 & E4)

5 days 7 days 11-18 days

C20 from ONR7 Culture with Rha (E4)

5 days

7 days

11-18 days

C20 from ONR7 Culture with Rha (Eb8)

5 days

7 days

Concluding Remarks • Degradation kinetics: saturated fraction is degraded faster & more

extensively than the aromatic fraction

– Alkanes: C15>C20>C25>C30>C35

– PAHS: fluorene > dibenzothiophene > phenanthrene > chrysene

• Pristane and Phytane use as conserved internal markers in biodegradation index is unreliable as they are also degraded.

• Control treatment had no significant hydrocarbon degradation. Microbial growth and hydrocarbon breakdown are dependent on nutrients & other additives.

• An increase in qs in agreement with an increase in μ implies a growth associated degradation rate.

• The ability of biosurfactants to emulsify oil and making it available to hydrocarbon degraders is crucial as a bioremediation strategy.

Concluding Remarks • ineffectiveness of inorganic nutrients (NPK) in enhancing microbial

growth and thus facilitate hydrocarbons degradation has been proved in Seawater 1 experimental set. On the contrary inorganic nutrients usually being washed out when applied in seawater perform better when applied to sand almost equally to ULR performance (Sand 1&2 experimental sets).

• It needs to be stressed out that although Alcanivoracaceae as investigated was the dominant family at early stage of the experiment especially in treatments with inorganic nutrients, Pseudomonadaceae was the dominant family at the late stage of the experiment especially in treatments with biosurfactant (rhamnolipids).

Concluding Remarks • CLSM investigation revealed that bacteria are organized into

clusters forming strings, star and grape like shapes of bacteria and fine oil droplets bridging each other with EPS.

• Moreover gradual dissolution of C20 droplet is encouraged in both consortia (especially in the presence of rhamnolipids).

• The combination of ABA-bioaugmentation with biostimulation could be a promising strategy to speed up bioremediation in cases where there is lack of both nutrients and indigenous degraders (at early times).

Acknowledgments

“Unravelling and exploiting Mediterranean Sea microbial diversity and ecology for Xenobiotics’ and pollutants’ clean up” – ULIXES FP-7 PROJECT No. 266473

Research Funding Program: Heracleitus-II.

PROGRAMME IKYDA 2012 Program for the promotion of the

exchange and scientific cooperation between Greece and Germany

State Scholarships Foundation

Thank you for your attention!

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