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)
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)
Rhamnolipid biosurfactant
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.
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. 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)
Nutrients (uric acid, lecithin)
Rhamnolipid biosurfactant
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.
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. 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)
Nutrients (uric acid, lecithin)
Rhamnolipid biosurfactant
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
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 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
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 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
NPKM_30 NPKM_60 NPKM_90
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
ULRM_30 ULRM_60 ULRM_90
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
Degradation Kinetics of C25 & C30
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
Fluorene Dibenzothiophene Phenanthrene Chrysene
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
Fluorene Dibenzothiophene Phenanthrene Chrysene
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
Fluorene Dibenzothiophene Phenanthrene Chrysene
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
Alcanivorax Thalassolituus Cycloclasticus
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
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
Alcanivorax Thalassolituus Cycloclasticus
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)
Experimental Results
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
Profile of n-alkanes
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
Degradation Kinetics of C15 & C20
C15 C20
μ qs qs/qs0 qs qs/qs0
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
Degradation Kinetics of C25 & C30
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 -
Degradation Kinetics of Pristane & Phytane
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
MPN profile & overall degradation of n-alkanes
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
Fluorene Dibenzothiophene Phenanthrene Chrysene
ng
aro
mat
ics/
ng
ho
pan
e
NPKM
day0 day5
day15 day30
0.00
0.04
0.08
0.12
0.16
Fluorene Dibenzothiophene Phenanthrene Chrysene
ng
aro
mat
ics/
ng
ho
pan
e
NPKMR
day0 day5
day15 day30
Degradation Kinetics of Fluorene & Dibenzothiophene
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
Nucleotide Sequence Analysis Results
Experimental Results
3. Landfarming of oil polluted beach sand through biostimulation
Profile of n-alkanes
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
Profile of n-alkanes
0
2
4
6
8
10
12
14
16
18
20
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
NPK
0 day 15 days 30 days 45 days
Profile of n-alkanes
0
2
4
6
8
10
12
14
16
18
20
22
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
ULR
0 day 15 days 30 days 45 days
Degradation Kinetics of C15 & C20
C15 C20
μ qs qs/qs0 qs qs/qs0
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
Degradation Kinetics of C25 & C30
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
Degradation Kinetics of Pristane & Phytane
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
Fluorene Phenanthrene
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
Fluorene Phenanthrene
ng
aro
mat
ics/
ng
ho
pan
e
ULR
0 day
15 days
30 days
45 days
Degradation Kinetics of Fluorene & Dibenzothiophene
Fluorene Dibenzothiophene
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
MPN profile & overall degradation of n-alkanes
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
Experimental Results
4. Landfarming of oil polluted beach sand through autochthonous bioaugmentation & biostimulation
Profile of n-alkanes
0
10
20
30
40
50
60
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 M
CM_0 CM_7 CM_15 CM_30 CM_45
Profile of n-alkanes
0
10
20
30
40
50
60
70
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
NPKM
NPKM_0 NPKM_7 NPKM_15
NPKM_30 NPKM_45
Profile of n-alkanes
0
10
20
30
40
50
60
70
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
ULRM
ULRM_0 ULRM_7 ULRM_15
ULRM_30 ULRM_45
Degradation Kinetics of C15 & C20
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
Degradation Kinetics of C25 & C30
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
Degradation Kinetics of Pristane & Phytane
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
fluorene phenanthrene
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
fluorene phenanthrene
ng
aro
mat
ics/
ng
ho
pan
e
ULRM 0 day
7 days
15 days
30 days
45 days
Degradation Kinetics of Fluorene & Dibenzothiophene
Fluorene Dibenzothiophene
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
MPN profile & overall degradation of n-alkanes
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
Experimental Results
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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