biocorrosion(in(the(diesel(fuel( infrastructure:impactof

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

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


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


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




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


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



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

biocorrosion(in(the(diesel(fuel( infrastructure:impactof

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