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Biocorrosion in the Diesel Fuel Infrastructure: Impact of Ultra Low
Sulfur Diesel, Fa;y Acid Methyl Esters and Select AlternaAve Fuels
Joseph M. Suflita University of Oklahoma
BIOCORROSION WORKSHOP 2013 Boulder, CO
Microbial biodegrada-on of fuels is associated with decreased product quality, compromised equipment performance, and the biocorrosion of metal surfaces
Changing fuel formula-ons are rou-nely used in the exis-ng carbon steel infrastructure.
Many reports of increased corrosion problems.
Background
We hypothesize that fuel-‐induced metal corrosion is at least a func-on of:
i) the chemical composi-on of the fuel
ii) its inherent suscep-bility to biodegrada-on
iii) the contact of the fuel with microorganisms that catalyze biodegrada-on/biocorrosion processes.
Hypothesis
PerspecAve
Important consequence onboard
Navy ships
PerspecAve
The use of different different diesel fuel blends can lead to a host of biologically-catalyzed problems: • Biocorrosion Cross contamination • Biofouling of sensors Clogging of fuel lines • Deterioration fuel quality Coalescer performance
1
2
3
4 Adapted from: Heyer et al., 2013. Ship ballast tanks a review from microbial corrosion and electrochemical point of view Ocean Engineering 70:188–200.
Different Biocorrosion Zones in a Ballast Tank 80% of the world's trade volume is transported by ships
Ballast Tank Model O
xygen concentration [%]
≥ 65% of ships have sediment in their ballast
0
1
2
3
4
5
6
7
8
9
10
0 5 10 15 20
Depth (mm)
O2 C
oncentra-o
n (ppm
)
X 8 X 1
Interface JP5 -‐ Fuel
Steep Gradients in Oxygen in Fuel/Filter Sterilized Seawater IncubaAons Inoculated with Marinobacter
Ballast Tank
107 cells/ml Fuel Seawater
mid sec-on
fully submerged zone
-‐ measurement by Wolfenden and Avci
0
1
2
3
4
5
6
7
8
9
10
0 5 10 15 20
Depth (mm)
O2 C
oncentra-o
n (ppm
)
X 8 X 1
Interface JP5 -‐ Fuel
Steep Gradients in Oxygen in Fuel/Filter Sterilized Seawater IncubaAons Inoculated with Marinobacter
Ballast Tank
107 cells/ml Fuel Seawater
mid sec-on
fully submerged zone
-‐ measurement by Wolfenden and Avci
Fuels of Interest Traditional petroleum-based fuels:
HSD, LSD, ULSD, F76, JP5
First generation biofuel: FAME-Biodiesel
Second generation biofuels: Algal-based F76,
Camelina-based JP5
1) Composition of the fuels? Gas chromatography-mass spectrometry 2) Can fuel support microbial growth? Anaerobic incubations; but oxygen exposure has important implications 3) What can a targeted assay of the metabolome tell us? GC-MS 4) What microbes and activities should be monitored? Host of procedures
5) Impact of fuel formulations on microbiologically induced metal corrosion?
Questions and Approaches
First-Generation Biodiesel
World-wide annual biodiesel production, 1991-2009
0
1,000
2,000
3,000
4,000
5,000
6,000
1985 1990 1995 2000 2005 2010 2015
Mill
ion
Gallo
ns
Source: F.O. Licht; Worldwatch
Ear
th P
olic
y In
stitu
te -
ww
w.e
arth
-pol
icy.
org
Note: 2012 is a projection.
World-wide annual biodiesel production, 1991-2012
(The rates are corrected for biodiesel-unamended controls)
0
200
400
600
800
1000
1200
0
50
100
150
200
250
300
Fresh Sediments Methanogenic consorAum
USS Arizona USS Ge;ysburg
Sulfate Con
sumpA
on (µM
/day)
Metha
ne Produ
cAon
(µM/day)
Contaminated aquifer
Oil-degrading methanogenic
consortium
Enrichment USS Arizona
Ballast Water USS Gettysburg
Biodiesel (BD) -‐Supported Anaerobic Microbial Metabolism with Various Inocula
Fate of Biodiesel vs. Other Fuels
Biodiesel and Carbon Steel Corrosion
Odd # C-atom Methyl Esters
Even # C-atom Methyl Esters DetecAon of the same suite of fa;y acids in mulAple incubaAons (freshwater/marine; exposed to HC/BD or not)
First Generation Biodiesels are Very
Labile
What About Other Fuels?
• Focus on ULSD • Many reports of problems
with ULSD • Several hypotheses can be
advanced
• To make ULSD, refineries must treat it severely • Molecules are broken apart to release the organosulfur moieties • This changes several fuel properties:
density, viscosity, lubricity, etc • Additives used to maintain performance characteristics (e.g. often up to 2% biodiesel)
ULSD: Is It Really Different?
Oil Desulfurization
Deagglomeration
Depolymerization
(from Mochida and Choi, 2004)
Need to Compare the Biological Stability of HSD,
LSD & ULSD Obtained Fuel Samples Directly From the Refinery - Before Any
Additives Incubated with Several Inocula –
Marine and Freshwater
Passaic River assA OTU 4 Passaic River assA OTU 5
Arthur Kill assA OTU 4 Arthur Kill assA OTU 5
Arthur Kill assA OTU 2 Arthur Kill assA OTU 3
Fort Lupton OTU 1 SDB Consortium assA OTU 2
SDB Consortium assA OTU 3 Gowanus Canal assA OTU 2
Passaic River assA OTU 6 Gowanus Canal assA OTU 4 Passaic River assA OTU 7 Passaic River assA OTU 8 Passaic River assA OTU 3
Newtown Creek assA OTU 1 Gowanus Canal assA OTU 5 Passaic River assA OTU 9
Passaic River assA OTU 1 Passaic River assA OTU 2
Desulfoglaeba alkenexedens ALDC assA (GU453656) SDB Consortium assA OTU 5
Strain HxN1 masD (AM748709) Ballast Water assA OTU 2
Desulfatibacillum alkenivorans AK-01 assA1 Desulfatibacillum alkenivorans AK-01 assA2 Ballast Water assA OTU 1 Arthur Kill assA OTU 1 Newtown Creek assA OTU 2
Propane-degrading consortium assA OTU 1 Ballast Water assA OTU 3
Gowanus Canal assA OTU 1 Gowanus Canal assA OTU 3 SDB Consortium assA OTU 1 SDB Consortium assA OTU 4
Fort Lupton assA OTU 2 Fort Lupton assA OTU 3
Fort Lupton putative glycyl radical enzyme OTU 1 Fort Lupton putative glycyl radical enzyme OTU 2
Delta proteobacterium NaphS6 nmsA (CU466269) Delta proteobacterium NaphS2 nmsA (CU466266)
Desulfotomaculum sp. Ox39 bssA (EF123665) Desulfobacterium cetonicum bssA (EF123662)
Thauera aromatica DNT-1 bssA (AB066263) Thauera aromatica T1 tutD (AF113168) Azoarcus sp. T bssA (AY032676) Azoarcus sp. DN11 bssA (AB285034)
Magnetospirillum sp. TS-6 bssA (AB167725) Thauera aromatica bssA (AJ001848) Aromatoleum aromaticum EbN1 bssA (CR555306) Fort Lupton bssA OTU 2 Sulfate-reducing bacterium TRM1 bssA (EF123667) Casper bssA OTU 6 Sulfate-reducing bacterium PRTOL1 bssA (EU780921)
Desulfobacula toluolica bssA (CU466268) Geobacter sp. FRC-32 bssA (NC_011979) Geobacter metallireducens GS-15 bssA (CP000148) Geobacter grbiciae bssA (EF123664)
Geobacter sp. TMJ1 bssA (EF123666) Casper bssA OTU 5
Casper bssA OTU 4 Fort Lupton bssA OTU 1
Casper bssA OTU 3 Casper bssA OTU 1 Casper bssA OTU 2
100
100
100
100
100
100
81
98
90
100
100
100
100
100
100
78
99
82
88
100
100
100
100
79 91
100
92 98
100
100
100
74 89
99
97 100
97
100
98
89
94
92
68
65
75
90 70
84
100
0.1
bssA
assA
nmsA Putative uncharacterized glycyl radical enzymes
Anaerobic HC biodegradation gene probes described
Passaic River assA OTU 4 Passaic River assA OTU 5
Arthur Kill assA OTU 4 Arthur Kill assA OTU 5
Arthur Kill assA OTU 2 Arthur Kill assA OTU 3
Fort Lupton OTU 1 SDB Consortium assA OTU 2
SDB Consortium assA OTU 3 Gowanus Canal assA OTU 2
Passaic River assA OTU 6 Gowanus Canal assA OTU 4 Passaic River assA OTU 7 Passaic River assA OTU 8 Passaic River assA OTU 3
Newtown Creek assA OTU 1 Gowanus Canal assA OTU 5 Passaic River assA OTU 9
Passaic River assA OTU 1 Passaic River assA OTU 2
Desulfoglaeba alkenexedens ALDC assA (GU453656) SDB Consortium assA OTU 5
Strain HxN1 masD (AM748709) Ballast Water assA OTU 2
Desulfatibacillum alkenivorans AK-01 assA1 Desulfatibacillum alkenivorans AK-01 assA2 Ballast Water assA OTU 1 Arthur Kill assA OTU 1 Newtown Creek assA OTU 2
Propane-degrading consortium assA OTU 1 Ballast Water assA OTU 3
Gowanus Canal assA OTU 1 Gowanus Canal assA OTU 3 SDB Consortium assA OTU 1 SDB Consortium assA OTU 4
Fort Lupton assA OTU 2 Fort Lupton assA OTU 3
Fort Lupton putative glycyl radical enzyme OTU 1 Fort Lupton putative glycyl radical enzyme OTU 2
Delta proteobacterium NaphS6 nmsA (CU466269) Delta proteobacterium NaphS2 nmsA (CU466266)
Desulfotomaculum sp. Ox39 bssA (EF123665) Desulfobacterium cetonicum bssA (EF123662)
Thauera aromatica DNT-1 bssA (AB066263) Thauera aromatica T1 tutD (AF113168) Azoarcus sp. T bssA (AY032676) Azoarcus sp. DN11 bssA (AB285034)
Magnetospirillum sp. TS-6 bssA (AB167725) Thauera aromatica bssA (AJ001848) Aromatoleum aromaticum EbN1 bssA (CR555306) Fort Lupton bssA OTU 2 Sulfate-reducing bacterium TRM1 bssA (EF123667) Casper bssA OTU 6 Sulfate-reducing bacterium PRTOL1 bssA (EU780921)
Desulfobacula toluolica bssA (CU466268) Geobacter sp. FRC-32 bssA (NC_011979) Geobacter metallireducens GS-15 bssA (CP000148) Geobacter grbiciae bssA (EF123664)
Geobacter sp. TMJ1 bssA (EF123666) Casper bssA OTU 5
Casper bssA OTU 4 Fort Lupton bssA OTU 1
Casper bssA OTU 3 Casper bssA OTU 1 Casper bssA OTU 2
100
100
100
100
100
100
81
98
90
100
100
100
100
100
100
78
99
82
88
100
100
100
100
79 91
100
92 98
100
100
100
74 89
99
97 100
97
100
98
89
94
92
68
65
75
90 70
84
100
0.1
bssA
assA
nmsA Putative uncharacterized glycyl radical enzymes
-Callaghan, et al., 2010. ES&T. 44(19), 7287-7294.
0
10
20
30
40
0 100 200 300
HSD LSD
ULSD SHIP
BD
control
SO4 (
mM
) Sulfate Reduction by Incubations Containing
Seawater from a Navy Ballast Tank and Refinery Cuts of Diesel Fuel
No Difference in Rates
2ppmS
250-450ppmS
1530ppmS
Sulfate Reduction by Incubations Containing Contaminated Aquifer Sediments and Refinery
Cuts of Diesel Fuel
0
1
2
3
4
5
6
7
8
9
10
0 50 100 150 200
BD HSD LSD
ULSD
control
SO4 (
mM
)
No Difference in Rates
Methane Production from a Methanogenic Consortium Capable of Hydrocarbon
Biodegradation
0
5
10
15
20
25
30
0 50 100 150
BD
HSD LSD
ULSD
control
CH
4 (m
M) No Difference in Rates
DGGE profile of NAVY ballast tank communities incubated with ship fuel, diesel fuels
with different sulfur status, or biodiesel
Bacillus sp.
control
Navy ship fuel
ULSD LSD HSD SOY Fuel-free
DGGE profile of NAVY ballast tank communities incubated with ship fuel, diesel fuels
with different sulfur status, or biodiesel
Bacillus sp.
control
Navy ship fuel
ULSD LSD HSD SOY Fuel-free
indistinguishable
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Tota
l OTU
%
Major Phylogenic Groups Identified With Pyrosequencing
Epsilonproteobacteria
Deferribacteres
Cyanobacteria
Alphaproteobacteria
Synergistetes
Desulfovibrionales
Gammaproteobacteria
Spirochaetes
Firmicutes
Bacteroidetes
Thermotogae
Deltaproteobacteria
Ballast Water
0%
10%
20%
30%
40%
50%
60%
70%
80%
Total O
TU %
CompensaAon Water Filter 1
CompensaAon Water Filter 2
CompensaAon Water Filter 3
No Substrate Control
USS Ge;ysburg Diesel
High Sulfur Diesel
Low Sulfur Diesel
Ultra Low Sulfur Diesel
Soy Based BioDiesel
Dominant Deltaproteobacteria
LSD
HSD
ULSD
Ballast Water
No Diesel
Gettysburg
Biodiesel
~ half of these sequences have 100% similarity to the genus Desulfobacter, an acetate-utilizing SRB previously implicated in corrosion
Camelina JP-‐5 aZer
Camelina JP-‐5
before
5 min 76 min
JP-‐5 petro before JP-‐5 petro aZer
5 min 76 min
GC-‐MS Comparison of TradiAonal and Alternate Military Fuels
-‐ before and afer incubaAon in anaerobic seawater
0
10
20
30
40
50
60
0 50 100 150 200 250
Sulf
ate
(mM
)
Time (days)
Anaerobic Fuel/Seawater Incubations
Algal F76, CamJP5, ULSD
Un-amended, JP5, F76
Biodiesel
Similar results regardless of inoculum (fresh or marine) or
prior exposure history to hydrocarbon/methylesters
0
10
20
30
40
50
60
0 50 100 150 200 250
Sulf
ate
(mM
)
Time (days)
Anaerobic Fuel/Seawater Incubations
Algal F76, CamJP5, ULSD
Un-amended, JP5, F76
Biodiesel
Lee and Little Experiments Exhibited Corrosion and
Ended in 3 Months WHY??
Branched
tetradecane (C14)
Tridecane (C13)
Tetradecane (C14)
Pentadecane (C15)
Strict anaerobic incubations
Less stringent anaerobic incubations
JP-5 petro before
JP-5 petro after
JP-5 petro before
JP-5 petro after
20 min 40 min
5 min 76 min
Straight chain alkanes
Branched alkanes
Notable Fatty acids
Metabolites or other observations
JP5 C10-C15 A few Alkylated catechols,
benzoates, and phenols/a few low MW alcohols and
acids
Camelina-based JP5 C10-C18 C9-C20
Alkylated catechols,and benzoic acid / a few low MW
alcohols and acids
F76 C11-C15 and C22 - Alkylated benzoates
Algal-based F76/petro F76
C10-C20 and C25 C11-C21
Catechol and alkylated benzoates / a few low MW
alcohols and acids
Soy-based BD (20%)
C11-C14 and C22-C25 A few C5-C18 and
C20 C:16, C18:0/1/2 removed;
Catechols
ULSD C11-C22 - Alkylated catechols, benzoate, alkylated phenol
Less Stringent Anaerobic Incubations
Straight chain alkanes
Branched alkanes
Notable Fatty acids
Metabolites or other observations
JP5 C10-C15 A few Alkylated catechols,
benzoates, and phenols/a few low MW alcohols and
acids
Camelina-based JP5 C10-C18 C9-C20
Alkylated catechols,and benzoic acid / a few low MW
alcohols and acids
F76 C11-C15 and C22 - Alkylated benzoates
Algal-based F76/petro F76
C10-C20 and C25 C11-C21
Catechol and alkylated benzoates / a few low MW
alcohols and acids
Soy-based BD (20%)
C11-C14 and C22-C25 A few C5-C18 and
C20 C:16, C18:0/1/2 removed;
Catechols
ULSD C11-C22 - Alkylated catechols, benzoate, alkylated phenol
Less Stringent Anaerobic Incubations
Phenols & Catechols and Low
MW Fatty Acids
Catechol
Clear Indication of Aerobic Hydrocarbon
Metabolism
O2
O2
O2
Bacterial Communities in Less Stringent Anaerobic Incubations
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Seawater ULSD Biodiesel 5%
Biodiesel 20%
JP5 F76 Camelina JP5
50:50 mix JP5
50:50 mix F76
Unclassified
Chloroflexi
Gammaproteobacteria
Epsilonproteobacteria
Deltaproteobacteria
Alphaproteobacteria
Firmicutes
WS3
Bacteroidetes
Planctomycetes
Verrucomicrobia
Lentisphaerae
*Deltaproteobacteria: SRBs Firmicutes: Clostridia
Desulfoglaeba alkanexedens strain ALDC • Reduces sulfate
• Degrades hydrocarbons via fumarate addition
• Mineralizes C6-C12 n-alkanes
• Known to cause localized corrosion of
carbon steel
• Syntrophic growth 2 um
A Model Hydrocarbon-‐Degrading SRB
Mass Spectra of Alkylsuccinic Acid Metabolites
2 8 5 5
7 3
1 4 7
3 8 7 Rel
ativ
e ab
unda
nce
Decane
271
CH CH COOSi(CH3)3
CH 2 COOSi(CH3)3
CH 3
(CH 2 ) 7 CH 3
271
(M-15)+ = 387
2 8
4 3
7 3
1 2 9
1 4 7
7 2 4 3
3 5 9
5 5
Rel
ativ
e ab
unda
nce
Octane CH CH
COOSi(CH3)3
CH 2 COOSi(CH3)3
CH 3
(CH 2 ) 5 CH 3
243
(M-15)+ = 359
4 3
5 5 7 3
1 4 7
2 1 5 331129
Mass/charge
Rel
ativ
e ab
unda
nce
1 7 2
2 1 7
2 6 2
1 2
2 1 7
2 6 2
1 7 2 2 6 2
2 1 7
Hexane CH CH
COOSi(CH3)3
CH 2 COOSi(CH3)3
CH 3
(CH 2 ) 3 CH 3
215
(M-15)+ = 331
succinyl moiety
di-silyl moiety
0
5
10
15
20
25
30
35
40
0 50 100 150 200 250 300
Sulfa
te (m
M)
Time (days)
Unamended
BD
ULSD
Key West Seawater/Sediments + Desulfoglaeba alkanexedens
Anaerobic BiodegradaAon of ULSD
25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00
25.00 30.00 35.00 40.00 45.00 50.00 55.00
C10 C11
C12
C13
C14
C15 C16 C17 C18 C19
Time (min)
Respon
se
TIC overlay before and aZer incuba-on with ULSD
Red boxes denote the degraded por-ons of alkane peaks ***Desulfoglaeba
is known to degrade C6-‐C12 n-‐alkanes
Extracted Ion Chromatograms Of CnH(2n-‐2)O4 Signature Hydrocarbon Metabolites Detected by
LC-‐QToF Analysis succinate metabolites Unknowns
14 16 18 20 22 24 26 28 30 32 34
ULSD from NAVY ULSD from refinery
Time (min)
Respon
se
C10 C11
C12 C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
Two ‘different’ ULSDs were used…….. -‐ One directly from a refinery the other from NAVY -‐ Slightly different when analyzed by GC/MS
-‐addi-ves? -‐ H2S? -‐ Signaling?
But Why??
Summary 1) FAME biodiesel is more labile than other fuel components
and can exacerbate metal biocorrosion 2) No difference in the suscep-bility of HSD, LSD or ULSD to
anaerobic biodegrada-on in marine or freshwater environments; addi-ves in fuel likely important
3) Whenever fuel biodegrada-on was coupled to sulfate reduc-on, the corrosion impact was high
4) Not all ULSDs are created equal; slight differences in chemical composi-on can be important
5) Small amounts of oxygen can have a large impact on both biodegrada-on and biocorrosion
6) The presence of the requisite microorganisms is important; inocula-on with a hydrocarbon-‐degrading sulfate reducing bacterium
Acknowledgements
OU Biocorrosion Center Chris Lyles Deniz Aktas Tiffany Lenhart Irene Davidova Kathleen Duncan Iwona Beech Jan Sunner Brad Stevenson
Naval Research Laboratory
Brenda Liole Jason Lee
ConocoPhillips
Paul Ryder
Crew of the USS Geoysburg