OTC-27142-MS

Field Studies of Microbiologically Influenced Corrosion of Mooring Chains Devin Witt and Kai-Tung Ma, PhD., Chevron Energy Technology Company; Tim Lee, AMOG Consulting Inc.; Christine Gaylarde, Sukriye Celikkol, Zakari Makama, Iwona Beech, PhDs., University of Oklahoma

Copyright 2016, Offshore Technology Conference This paper was prepared for presentation at the Offshore Technology Conference held in Houston, Texas, USA, 2–5 May 2016. This paper was selected for presentation by an OTC program committee following review of information contained in an abstract submitted by the author(s). Contents of the paper have not been reviewed by the Offshore Technology Conference and are subject to correction by the author(s). The material does not necessarily reflect any position of the Offshore Technology Conference, its officers, or members. Electronic reproduction, distribution, or storage of any part of this paper without the written consent of the Offshore Technology Conference is prohibited. Permission to reproduce in print is restricted to an abstract of not more than 300 words; illustrations may not be copied. The abstract must contain conspicuous acknowledgment of OTC copyright.

Abstract Severe corrosion found on steel mooring components (CSMC) at several sites worldwide has caused

concern in recent years as to whether the components can safely meet their design life. A pilot study

was initiated to understand the underlying corrosion causes with the aim of developing successful

CSMC mitigation methods. In 2014, a field test was conducted offshore at two different locations in

West Africa in order to confirm the contribution of microbiologically influenced corrosion (MIC) to

CSMC. The study provided evidence that MIC is a root cause of the observed severe corrosion in the

form of mega-pits at one of the two test sites. The tests consisted of deploying carbon steel coupons on a

fiber rope, herein referred to as a “microbial baiting kit”, at facilities near the mooring systems to

capture the biofilm forming microorganisms. The kit was submerged approximately three meters below

the water surface for an extended period of time allowing for free swimming microorganisms to

colonize the coupons. The kit was the first of its kind to be used in the industry for investigating MIC of

mooring systems. Upon recovery of the coupons, pitting damage was revealed underneath the fouling

deposits. Following DNA extraction, subsequent analysis of sequences representing fragments of the

bacterial 16S rRNA gene demonstrated that, regardless of the test location, the outer part of the biofilm

formed on coupon surfaces had significantly different microbial community structure when compared to

the surrounding seawater. In both test sites, biofilm DNA analysis confirmed that obtained bacterial

sequences represented the initial colonizers of submerged structures in marine environments. Sequences

identified as belonging to sulfate-reducing bacteria (SRB), which are considered major contributors to

MIC in suboxic/anoxic aquatic environments, were more abundant in biofilms but scarce in water

samples. A higher number of SRB sequences were associated with coupons retrieved from the test

location where pitting attacks were prominent. Sequences indicative of acetic acid-producers and non-

SRB hydrogen sulfide-producing microorganisms, that are also likely MIC contributors, were identified;

however, further work is required to prove the involvement of these prokaryotes in steel deterioration.

The results and finding from this pilot work set the stage for a comprehensive Joint Industry Project

(JIP) launched by DeepStar® and is entitled DeepStar

® CTR12402 Integrity Management of Mooring

Systems Against Corrosion JIP. The aim of the DeepStar® JIP is to determine possible measures of MIC

mitigation.

Key Words: Microbiologically-Influenced Corrosion, Microbial Baiting Kit, Severe Corrosion, West

Africa, Field Tests, Mooring Chain, Mooring Integrity

2 OTC-27142-MS

Figure 1: [A] Recovered Site 1 mooring chain showing extensive corrosion and mega-pitting and [B] a close up

photograph of a mega-pit depicted in Fig. 1.A marked by a red arrow.

1.0 Introduction

Microbiologically influenced corrosion (MIC) has been linked to severe corrosion on steel components

in marine habitats at several locations around the world. It has been identified as responsible for

extensive deterioration of mooring equipment in regions with warm nutrient rich waters and is known to

be a leading contributor to premature mooring system failures and replacements [9], [10], [11]. The

financial losses due to additional integrity management and replacements of prematurely failed mooring

lines of permanent floating production units and marine terminals can be considerable to a company. A

recent example of a major financial loss to a company is the complete replacement of the Serpentina

FPSO mooring system in Equatorial Guinea, West Africa which is a region of suspected MIC activity

[12]. MIC is known to cause both extensive uniform and pitting corrosion on mooring equipment (see

Figure 1) and operators lack an established strategy for mitigating MIC effects. This exploratory study

was the first attempt to investigate MIC as the leading cause of mooring system corrosion in warm

nutrient rich waters using bacterial DNA profiling based on the analysis of 16S rRNA gene fragments,

and employing new generation sequencing (NGS) such as Illumina.

2.0 Background

In the summer of 2014, Chevron Energy Technology Company (ETC) set out to conduct an MIC field

study at two permanently moored facilities located in the Gulf of Guinea. The experiments were to be

carried out at two areas significantly differing in water quality. Site 1 is located near a river mouth,

approximately 30 miles (50 km) offshore in a water depth of 138 feet (42 m); it is known to experience

high rates of corrosion related to microbial activity and high levels of marine growth. Site 2, which has

relatively clear water conditions with respect to Site 1, is located approximately 70 miles (113 km)

offshore in a water depth of 4800 feet (1463 m) and has little to no MIC activity with moderate levels of

marine growth. The two sites were selected in order to determine which members of the microbial

community thriving as biofilms on submerged test surfaces were likely key contributors to corrosion.

Figure 2 shows an example of marine growth for each site location.

OTC-27142-MS 3

Figure 2: [A] Marine growth found on the mooring chain after 12 years of service at Site 1 and [B] macro-fouling of

the upper portion of a flowline riser after eight years of service at Site 2.

2.1 Test Kit

The biofilm sampling system was provided in the form of a kit produced at the University of Newcastle,

Australia, under the supervision of Professor Robert Melchers. The system is known as the “bacterial

fishing kit” (BFK), here on referred to as the microbial baiting kit (MBK).

Each MBK consisted of a length of synthetic fiber rope, to which 20 flat steel test coupons (½” wide, 3”

long and 1/16” thick, with one 3/16” hole located ¼” from one end) were affixed. The test coupons

were of the “CO100” specification and were manufactured from commercial light gauge, low tensile

carbon steel sheet. The precise grade of steel was not recorded. The alloying components and heat

treatment process vary from those typical of the higher tensile grades of steel used for manufacture of

mooring components. Although not exactly reproducing the conditions of a mooring system, using light

gauge steel was considered an acceptable tradeoff because of the ready availability of this alloy.

The steel coupons were attached to the fiber rope with a plastic cable tie, which was passed through the

hole in the coupon and then between the strands of the synthetic rope. A second cable tie was also

looped through the coupon to act as a handle during the recovery of the specimen. A photograph of the

assembled MBK and a close up of the steel test coupon is depicted in Figure 3.

4 OTC-27142-MS

Figure 3: A photograph of [A] the assembled Microbial Bating Kit with 20 steel coupons attached, and [B] a steel test

coupon covered with protective plastic and attached to the synthetic rope.

2.2 Microbial Baiting Kit Deployment

The MBKs were installed in the upper water column, with the topmost steel coupon approximately 10

feet (3 m) below the ocean surface. The kit was placed deep enough so as not to break the surface water

at any time during exposure, yet close enough to the water surface to represent the upper water column

which is the area of concern for MIC. The rope had an approximately 30 pound (14 kg) weight secured

to its lower end to keep the MBK in a vertical position at all times.

The MBK for Site 1 was attached directly to the mooring chain of a floating system and allowed to hang

vertically without contacting the mooring chain. At Site 2, the MBK was attached to the railing of a

floating system and was strategically located to not interfere with the daily operation of the floating

facility or contact any underwater structure.

The systems were originally designed to withstand exposure in the field for four to eight weeks (28 to 56

days). However, due to recovery scheduling constraints for Site 1, the MBK was deployed for 154 days.

Upon recovery, it was found that only one coupon was still attached to the fiber rope. The rest of the

steel coupons had been lost over time. The recovered coupon had suffered significant corrosion and was

found to have an enlarged attachment hole with sharp edges. After more detailed inspection, it was

noticed that the plastic cable tie had little to no sign of wear. However, aggressive corrosion attack was

seen along the coupon edge upon the removal of a fouling deposit (Fig. 4 B). The research team

proposes that exceptionally high corrosion rates in the zone near the coupons attachment points was the

most likely cause of losing the coupons at Site 1. It is important to note however, that the focus of

corrosion attack at corners and edges are a commonly observed phenomenon, and not necessarily

associated to MIC attack. Figure 4 displays the recovered steel coupon from Site 1. The photograph

shows the extensive corrosion (red arrows) and considerable fouling deposit on the coupon surface

following its 154 day deployment.

OTC-27142-MS 5

Figure 4: Photographs of the steel test coupon recovered from Site 1 with part of the fouling layer intact [A] and after

the fouling layer was removed [B]. A considerable decrease in the width of the coupon, indicative of material loss, is

apparent around its edges (see red arrows). Note: Ruler dimensions are in cm.

The recovery of the MBK at Site 2 was also a disappointing event. After a severe storm in the area, the

MBK was checked to ensure it was still intact and all of the steel coupons were found to be missing. A

second MBK was developed and shipped to the site where it was installed in the fall of 2014. This latter

MBK was recovered approximately five weeks after its deployment with coupon losses similar to those

observed at Site 1. Of the 20 test coupons, only four were recovered from Site 2. The reason for the

extensive loss of coupons at Site 2 is currently under investigation. A photograph representing Site 2

deployed steel coupon as recovered and from which fouling layers have been removed is displayed in

Figure 5. The extensive pitting of the coupon surface is apparent with distinct regions of coalescing pits

(black arrows) and a “pits within pits” zones (a white arrow).

6 OTC-27142-MS

Figure 5: A photograph of a steel coupon exposed at Site 2 in the as received condition [A] and after the fouling layer

has been removed revealing extensive pitting of the surface [B].

Upon comparison of the steel coupons recovered from the two sites, it is apparent that corrosion damage

at Site 1 is greater than that seen at Site 2. This was anticipated, based on previous inspections of

mooring components at the two floating facilities. The use of molecular ecology techniques allowed

comparison of the biofilm community structure between the two sites, thus gaining a better

understanding of which microorganisms are the likely key contributors to corrosion.

At the time of recovery of the test coupons at Site 2, location-specific water samples were also collected.

A DNA profile of the planktonic (free-swimming) microbial population in the water samples was

obtained, along with the profile of biofilm communities associated with the fouling deposits.

The loss of a large number of coupons from the test rig over the exposure period indicates that a

thorough redesign of the “bacteria fishing kit” test rig is required if the test is to be conducted again.

Lessons learned from this experience have been incorporated into the design of the “microbiological

fishing kit” being tested for DeepStar® CTR12402 Integrity Management of Mooring Systems Against

Corrosion JIP.

3.0 Analysis of Steel Coupons

Each of the test coupons, as well as the water samples, were analyzed at the University of Oklahoma

(OU) to obtain the DNA profile of the microbial community based on the sequence of the 16S rRNA

gene fragment (253 kb). The 16S rRNA gene (1.5 kb of total length) is a section of DNA found in the

ribosome of all bacteria and archaea (prokaryotes), for which the first 'r' in rRNA stands for ribosomal.

The ribosome is composed of two subunits, the large subunit (LSU) and the small subunit (SSU).

Generally, in bacteria, the SSU is coded for by the 16S rRNA gene, and the LSU is coded for by the 23S

rRNA and 5S rRNA genes. The 16S rRNA gene is a commonly used tool for identifying bacteria and

archaea. While traditional characterization of prokaryotes depended upon phenotypic traits like Gram

positive or Gram negative and bacillus or coccus, taxonomists today consider analysis of organism DNA

more reliable than classification based solely on phenotypes. The 16S rRNA gene is a useful tool for

extracting and identifying bacteria and archaea DNA from plant, animal, fungal, and protist DNA within

the same sample.

OTC-27142-MS 7

The fouling deposits were removed from the coupons by scraping with a sterile scalpel blade under

aseptic conditions. The bioinorganic deposit removed from the coupons included cells and metabolites

of the biofilm organisms, as well as corrosion products. After the initial removal of the fouling deposit,

the microorganisms residing in the tightly adhered layer still present on the surface were also identified

based on DNA profiling. The material obtained from the scraping is referred to as the outer biofilm, and

the layer which remained adhered to the coupon after scraping as the inner biofilm. Note that from the

Site 1 specimen, which was received and analyzed first, only the outer biofilm was examined and that

this coupon was not subject to the subsequent DNA extraction process. The latter procedure was

performed on the Site 2 specimens, when the success and value of the inner biofilm processing was

established. A detailed explanation is provided in Section 4.2 of this communication.

Water samples were passed through a 0.1µ sterile filtration system, to collect free-swimming

(planktonic) microorganisms. DNA was then extracted from each of the steel samples and from the 0.1µ

filters using an in-house developed protocol based on a combination of commercial DNA recovery kits.

Analysis of the DNA sequences result in classifications of the microbial community into Operational

Taxonomic Units (OTU) which represent groupings of related biological organisms. In this context,

OTUs are used to group functionally and genetically similar microorganisms, where each grouping may

be at different levels of the taxonomic rank (i.e. domain and kingdom, down to the level of genus), as

defined by the International Code of Nomenclature for algae, fungi, and plants [1].

4.0 Results

The results of this study are divided into five sections detailing different parts of the MIC investigation.

4.1 Principal Component Analysis

Principal Component Analyses (PCAs) were performed on the recovered samples from both Site 1 and

Site 2 using data derived from the DNA analysis of the recovered coupons.

Principal component analysis (PCA) is a technique used to emphasize variation and bring out strong

patterns in a dataset. It can be used to make data easy to explore and visualize. Principal components

(PC) are labeled 1, 2, 3, etc. in order of decreasing importance, as determined by the statistical software.

Each PC is associated with a percentage value for the fraction of the total variance that is explained by

the PC. Thus, PC1 always has the highest percentage value. Each sample is associated with a number

(“weight”) for each of the principal components. In a PCA graph, in which the x-axis represents PC1

and the Y-axis PC2, each sample is represented by a point determined by the respective PC weight

factors.

With the help of a PCA plot, the relative similarities (or differences) between biofilm populations can be

assessed. Figure 6 represents a PCA plot obtained based on the analysis of the entire population of

bacteria identified in biofilms on coupons retrieved from the two test sites and the water sample

collected at Site 2. Two coupons were analyzed for Site 2, while for Site 1, a duplicate analysis of the

outer biofilm was carried out. The grouping or scatter of the points represents the similarity or difference

of the biofilm and seawater populations relative to each other.

8 OTC-27142-MS

Figure 6: PCA plot for total bacterial diversity

Figure 6 presents a comparison of the inner and outer DNA profiles of microbial biofilm populations

and seawater population from Site 2 versus the outer biofilm at Site 1. This PCA plot reveals that the

DNA sequences representing microorganisms in the outer biofilm at the two test sites vary significantly

from one another, as shown by the PC2 axis difference between the two locations.

The PCA plot for sulfate-reducing bacteria (SRB), considering just the subset of the total population

identified as belonging to taxa which represent SRB, is shown in Figure 7. This plot displays a greater

variance between Site 1 and Site 2 populations along the PC1 axis, reflecting the difference in the

biofilms, with the more developed Site 1 biofilm having progressed to the anoxic conditions favored by

SRB. Nevertheless, SRB OTUs were also detected in the less mature and less abundant Site 2 biofilm,

demonstrating that these organisms were present at this earlier stage of development. This is not

surprising as, owing oxygen depletion due to microbial respiration, suboxic/anoxic areas are known to

exist within biofilms comprising mainly aerobic organisms.

Figure 7: PCA plot for sulfate-reducing bacteria at two test sites

OTC-27142-MS 9

When considering the populations of the sulfide oxidizing group (Chromatiales order), a clear difference

between the Site 2 inner biofilm compared to the other samples can be observed (see Figure 8). This

group is related to the oxidation of sulfides to thiosulfates, which is implicated in the pitting corrosion of

steels, and may be related to the corrosion pattern observed on the Site 2 coupons.

Figure 8: PCA plot of Sulfur oxidizing bacteria group

A further group to consider is the Clostridia, a group of anaerobic bacteria associated with the

production of hydrogen sulfide (H2S), which are presented in Figure 9. The clear variance of the data

point for the Site 1 specimens from the Site 2 specimens is significant in indicating a difference between

the specimens. There is a higher influence of hydrogen sulfide in Site 1.

Figure 9: PCA plot for bacteria of the genus Clostridium, known for hydrogen sulfide production

10 OTC-27142-MS

4.2 Site 1 Microbial Communities

Results of the DNA profiling for the samples collected from Site 1 are shown in Table 1. As noted

earlier, only the outer biofilm of the Site 1 specimen was recovered, since at the time of analysis there

was no evidence that there was a thin layer of bioinorganic deposit left on the surface of the coupon after

mechanical removal of the fouling layer contained microbial DNA of sequencing quality. Independent

laboratory experiments conducted by the OU team demonstrated that on carbon steel surfaces exposed to

seawater such a layer was indeed present. Therefore, upon receiving Site 2 specimens and scraping off

fouling deposits for outer biofilm profiling, an additional DNA extraction process was carried out using

the entire coupon to obtain the inner biofilm profile.

The microbial community on the Site 1 specimens is dominated by Gammaproteobacteria (67.5% of

OTU), of which Vibrionales are preponderant. The Gammaproteobacteria also include the

Oceanospirillales group which is associated with the breakdown of hydrocarbons, including oil. Also

present are Desulfobacter, Desulfovibrionaceae and Desulfovibrio genera, which represent sulfate

reducing bacteria known to contribute to MIC in the marine environment, and Clostridiales,

Fusobacteriaceae and Shewanella groups, which are associated with the production of hydrogen sulfide

(H2S). Other bacterial groups, such as Epsilonproteobacteria, were relatively abundant, and are

associated with the ocean sulfur cycle, and may play a role in the take-up from seawater of sulfur (in the

form of sulfates), making this available to SRB and other sulfur metabolizing microorganisms.

OTC-27142-MS 11

Operational Taxonomic Unit Outer Biofilm

Archaea 0.08%

Bacteria

Alphaproteobacteria 9.11%

Betaproteobacteria 0.01%

Gammaproteobacteria 67.50%

Oceanospirillales 1.30%

Vibrionales 62.80%

Chromatiales 0.20%

Deltaproteobacteria 2.51%

Epsilonproteobacteria 4.04%

Zetaproteobacteria 0.01%

Acidimicrobiales 0.41%

Bacteroidetes 6.23%

Clostridiales 2.12%

Fusobacteriaceae 4.50%

Phycisphaerales 0.09%

Pirellulaceae 0.52%

Pelagibacteriaceae 0.02%

Thalassiosira 0.01%

Sphingomonadales 2.25%

Desulfobacter 0.42%

Desulfovibrionaceae 0.09%

Desulfovibro 1.66%

Arcobacter 3.60%

Sulfurimonas 0.40%

Shewanella 0.14%

Oceanospirillales 1.30%

Pseudomonadaceae 0.01%

Cyanobacteria 0.26%

Algae 0.18% NOTE: columns will not sum to 100% due to double counting populations at different taxonomic rank

Table 1: Prevalence of Microbial OTU for Site 1 Specimen

4.3 Site 2 Microbial Communities

Results of the genetic analysis and classification of microbial OTU for the samples collected from Site 2

are listed in Table 2.

Within the OTU of Oceanospirillales are the Oleiphilaceae, the “oil loving” bacteria. These were

identified as 0.26% of the inner biofilm, but not detected in the outer biofilm and the seawater samples.

It is speculated that this may be due to the contamination of the coupon surface by oil prior to exposure.

However, no definite reason could be determined.

12 OTC-27142-MS

Saprospiraceae were also identified as being more abundant in the inner biofilm (6.7%) relative to the

outer biofilm (3.2%). These OTU are associated with metabolism of complex carbon sources. This again

may be due to coupon contamination prior to exposure however, no definite reason could be determined.

Operational Taxonomic Units Seawater Outer Biofilm Inner Biofilm

Archaea 0.1% 3.7% 0.1%

Thermoplasmata - 3.6% 0.1%

Bacteria 98.5% 68.5% 80.0%

Alphaproteobacteria 30.1% 13.8% 24.6%

Rickettsiales 15.4% 3.9% 6.3%

Rhodobacteriales 5.4% 4.8% 14.2%

Betaproteobacteria - 1.0% <0.1%

Gammaproteobacteria 35.9% 17.2% 18.2%

Oceanospirillales 16.0% 2.9%

Vibrionales 2.7% 2.5% 3.2%

Chromatiales2 <1% 5.3% 3.0%

Deltaproteobacteria - 3.1% 2.5%

Acidimicrobiales / Actinobacteria1 19.2% 3.4% 0.7%

Flavobacteriales 5.9% 10.5% 14.5%

Rhodospirillales 2.9%

Alteromonadales 4.6%

Methylococcales 5.8%

Verrucomicrobiales (Pedosphaerae) 1.2%

Unclassified 0.7%

Cyanobacteria 0.3% 20.0% 1.3%

Chloroplasts3 1.1% 5.7% 18.8%

Stramenopiles 1.7% 17.8%

Unclassified OTU 0.2% <0.1%

Notes:

1. Includes iron-oxidizing bacteria

2. Purple Sulphur Bacteria

3. Includes algae. Eukarya Kingdom

Abundance of each OTU is expressed as a percentage of the OTU within that taxonomic group. As such,

certain OTUs will be counted as an abundance of the total out of their domain, class, order or other

taxonomic rank. For example, Archaea, Bacteria and Eukarya (the latter represented principally by

Chloroplasts) are at the rank of domain, and with the unclassified OTU represent the entire population.

Below this level, abundance is expressed as abundance within that taxonomic group. The practical effect

is that columns will not sum to 100% due to double counting of populations at each taxonomic rank. Table 2: Prevalence of Microbial OTUs for Site 2 Specimens

4.4 Comparison of Site 1 and site 2 Biofilms

A comparison of the relative abundance of different OTUs between Site 1 and Site 2 is given in Table 3,

with notes on the functional behavior of members of each OTU.

OTC-27142-MS 13

Group

Site 1

(%)

Site 2

(%) Comments

Alphaproteobacteria 9.11 13.75

Betaproteobacteria 0.01 0.95

Gammaproteobacteria 67.5 17.24 More numerous in environments with hydrocarbons present

Deltaproteobacteria 2.51 3.07 Includes sulfate- and sulfur-reducing bacteria

Epsilonproteobacteria 4.04 0 Important in sulfur cycling in the ocean.

Zetaproteobacteria 0.005 0.005 Only described member is Mariprofundus ferrooxidans, an

iron oxidizer.

Archaea 0.08 3.73 Very diverse group. Some oxidize reduced sulfur compounds.

Acidimicrobiales 0.405 3.16 Aero or microaerophilic actinobacteria. May oxidize or reduce

iron.

Bacteroidetes 6.23 10.5 Very diverse group.

Algae 0.18 5.65 Eukaryotic phototrophs. Primary Colonizers

Cyanobacteria 0.26 20 Prokaryotic Phototrophs.

Clostridiales 2.12 0.09 Anaerobes. May Produce H2S

Fusobacteriaceae 4.5 0.03 Anaerobes. May Produce H2S. Found to act as an anchor for

biofilm formation [8]

Phycisphaerales 0.09 2.93 Facultative anaerobes

Pirellulaceae 0.52 5.1 Aerobic. Contain high levels of sulfatases

Pelagibacteriaceae 0.015 1.18 Aerobic. One of the most abundant uncultivated groups in

ocean surface.

Thalassiosira 0.01 1.05 Centric diatom

Sphingomonadales 2.25 0.45 Mainly aerobic. May be involved in biodiesel degradation [5]

Desulfobacter 0.42 0 Anaerobes. Sulfate-reducing bacteria (SRB)

Desulfovibrionaceae 0.09 0 Anaerobes. SRB

Desulfovibrio 1.66 0 Anaerobes. SRB

Arcobacter 3.6 0 Microaerophilic. May oxidize sulfur compounds. Can degrade

aromatic hydrocarbons [6]. Capable of denitrification [7].

Sulfurimonas 0.4 0 Grow on reduced sulfur and oxidized nitrogen.

Shewanella 0.14 0.93 Facultative anaerobes, dissimilatory metal reducers. May

produce H2S

Chromatiales 0.2 5.3 Includes purple sulfur bacteria. Some strictly anaerobic.

Oceanospirillales 1.3 3.89 Aerobes. Potential sulfur oxidizers and hydrocarbon

degraders

Pseudomonadaceae 0.01 0.26 Mainly obligate aerobes.

Vibrionales1 62.8 2.51

Facultative anaerobes. Produce Extracellular Polymeric

Substances (EPS) that help other microorganisms attach

themselves. Have been associated with metal corrosion [2].

Notes:

1. The genus Vibrio itself comprised 33.0% and 1.8% of OTUs in Site 1 and Site 2 biofilms, respectively.

2. Columns will not sum to 100% due to double counting of populations at different taxonomic rank. Table 3: Comparison of DNA profiles between outer biofilm microbial communities from Site 1 and Site 2

The comparison of the outer biofilm microbial communities DNA profiles at the two sites needs to be

considered in light of the following points:

14 OTC-27142-MS

Site 1 has a well-documented history of high rates of corrosion and formation of macro pits

associated with MIC, while Site 2 has been much less affected by corrosion.

The coupons deployed at Site 2 were exposed for a period of approximately 35 days, compared

to 154 days for the coupons at Site 1. As such, the biofilm is less developed and the fully

anoxic conditions associated with the onset of anaerobic corrosion and/or MIC may not have

developed in biofilms on Site 2 specimens.

DNA profile of the Site 1 microbial communities comprise of sequences representing populations of

Desulfobacter, Desulfovibrionaceae, and Desulfovibrio, all of which belong to a group of SRB, and are

relatively absent in the Site 2 communities. As such, the corrosion of the Site 2 coupon cannot be

attributed to SRB alone. The increased population of SRB in the Site 1 biofilm is consistent with the

proposed role of SRB in the formation of the corrosion mega-pits observed in mooring chains recovered

from the Site 1 facility [9], [10].

In contrast, the lower abundance of sequences from phototrophic bacteria and algae in the Site 1 outer

biofilm communities relative to Site 2 indicates that photosynthesis and production of oxygen in the

biofilm is much less significant at Site 1, and is less likely to play a key role in the corrosion process. At

Site 2, the role of the phototrophic bacteria and algae may also decrease over time as the biofilm

develops.

The microbiological community at Site 1 contains a high population of the sequences derived from

anaerobic Vibrionales, a group that has been associated with localized metal corrosion by Gaylarde and

Videla [2]. With the high abundance of Vibrionales relative to SRB, further investigation is required to

understand the relative roles and importance of each group in development of localized corrosion.

Lastly, the test coupon at Site 1 was found to have more marine growth on the coupon than that of Site

2. From Table 3, it is shown that Site 1, when compared to Site 2, has a higher concentration of DNA

sequences representing Vibrionales and Fusobacteriaceae, both of which are reported to act as very

good biofilm anchors [8]. From the observations, it is hypothesized that these two microorganism OTUs

could be heavily involved in promoting abundant biofilm development at Site 1; however, further

research is needed to confirm this hypothesis.

4.5 Comparison of Seawater Composition at Site 1 and Site 2

A comparison of the chemical analysis of seawater samples collected on 12 December 2010 and 18 July

2013 from Site 1 and Site 2 respectively, is presented in Table 4. The analysis of each of these samples

was conducted by the oil company’s in-country laboratory.

OTC-27142-MS 15

Parameter Units Site 1 Site 2

pH - 7.96 6.94

Anions

Nitrite (As N) mg/l <0.001 Not Analyzed

Nitrate (As N) mg/l 12.079 Not Analyzed

Nitrate (total) mg/l 53.5 <0.001

Sulfate (total) mg/l 2024.0 2960.3

Sulfite (total) mg/l <0.001 Not Analyzed

Thiosulfate mg/l Not Analyzed Not Analyzed

Ammonia (As N) mg/l <0.001 Not Analyzed

Phosphates mg/l Not Analyzed <0.001

Cations

Phosphorus (total) mg/l 0.224 0.158

Phosphorus (reactive) mg/l Not Analyzed Not Analyzed

Ammonium (total) mg/l <0.001 28.45

Other

Dissolved Oxygen mg/l 5.27 Not Analyzed Table 4: Comparison of seawater chemistry between exposure Site 1 and Site 2

The result of analysis of seawater collected at Site 1 constituted a key piece of evidence used in the

SCORCH JIP to identify the correlation between high dissolved inorganic nitrogen (DIN) levels in

seawater and the occurrence of high corrosion rates and localized mega-pit formation associated with

MIC. The DIN level has been the key parameter of water samples that have been used to determine MIC

susceptibility. For the Site 1 data, whilst total DIN is not explicitly reported, a high total DIN is

indicated by the high concentration of dissolved inorganic nitrogen available in the form of nitrates

(12.079mg/l); even considering that levels of nitrogen in the form of nitrites, ammonium and ammonia

were negligible, therefore, do not add to the total DIN, a DIN of 12mg/l is a high level.

The Site 2 seawater analysis data unfortunately does not include results for total DIN or for nitrite or

ammonia. Measured concentration of nitrates was negligible; however, the concentration of total

ammonium was very high (28.45mg/l). Conversion of this concentration to ammonium as nitrogen

equates to a reduced but still high figure of 22.1mg/l, much higher than Site 1. However, despite this

high level of nitrogen in the Site 2 seawater, corrosion has not been observed at this site to the same

extent as at Site 1. This could indicate that ammonium-driven microbial nitrogen metabolism is not a

major contributor to MIC.

5.0 Discussion of Results

With reference to the Site 2 specimen, based on the thin corrosion layers present and the DNA make-up

of the biofilm community, the corrosion process was likely to be under an Oxygen Concentration

Control mechanism (Phase 1 according to phenomenological model proposed by Melcher’s) [4]. The

prevailing anoxic conditions required to support development of a large SRB population had not yet

developed, which agrees with the part of the model that predicts corrosion experienced during relatively

short exposure time. It is noteworthy that the phototropic bacteria and algae in the biofilm while

respiring CO2 and releasing oxygen, would provide an additional oxygen source, in surplus to that

present in bulk seawater. It is, thus, conceivable that under favorable oxygen transport conditions,

localized microbial oxygen production within biofilm matrix would contribute to an overall oxygen-

driven aerobic corrosion process.

16 OTC-27142-MS

The Site 1 specimen represents a later stage of the corrosion process, where suboxic or anoxic

conditions have developed at the lowest layers of the biofilm (the inner biofilm), and anaerobic

microorganisms, including SRB and other H2S producers, can be established and drive anaerobic

corrosion. A biotically produced H2S-instigated MIC mechanism is consistent with that proposed as the

main cause of the formation of mega-pits in the upper reaches of the mooring chains. This model is

outlined in the SCORCH JIP report [3] and by Melchers [4].

Further investigation is required to understand details of the mechanism of transition from the oxygen-

driven initiation of pitting to the development of sulfidogenic conditions and the development of mega-

pits observed on the recovered mooring chain. However, as a working hypothesis, the following is

proposed.

(i) Pitting is initiated as an abiotic corrosion process.

(ii) This abiotic corrosion process is driven by a ready supply of oxygen dissolved in seawater, and/or

transport of oxygen generated by phototropic bacteria and algae within biofilm.

(iii) The increased energy flux as a result of the corrosion in the biofilm niches, will facilitate the

proliferation of other oxygen-tolerant as well as strictly anaerobic microbial populations able to

metabolize seawater sulfates, as well as other available forms of sulfur into hydrogen sulfide.

(iv) Hydrogen sulfide is oxidized by microorganisms such as photosynthetic, anaerobic Chromatiales, or

Purple Sulfur Bacteria, into elemental sulfur or sulfuric acid. OTUs of Chromatiales were detected in

relatively high abundance within the biofilm on the coupon recovered from Site 2.

(v) Biogenically produced sulfur / sulfuric acid may be involved in corrosion of steel specimens.

Alternatively, the sulfur compounds may accumulate within biofilm regions, providing a focus for

development of sulfur reducing, H2S producing, organisms in these regions at later stages in the

corrosion process (once truly anoxic or alternating suboxic/anoxic conditions develop), causing the

localized MIC that drives merging of the individual corrosion pits into mega-pits.

Furthermore, the secondary DNA recovery, performed on a speculative basis, was extremely successful

in extracting DNA at the steel/biofilm interface, including DNA found within the tightly adhered

corrosion product layers. Microorganisms present in the inner biofilm are likely the most significant to

the MIC process, as the metabolites of these inner biofilm microorganisms are positioned to interact

with the metal surface without requiring extensive transport to reach it. This technique of DNA

extraction is a new development in the investigation of MIC, and should be considered in future studies.

Acidimicrobiales OTUs were relatively more abundant in the water at Site 2 than the outer biofilm, and

decreased further in abundance in the inner biofilm which is most closely associated with MIC

processes. These OTUs, which encompass iron-oxidizing bacteria, indicate that such bacteria may not

play a significant role in MIC occurring at the site.

The Gammaproteobacteria (including Oceanospirillales and its subgroup Oleiphilaceae) are known to

play a role in the degradation of organic carbon compounds [14], as are Saprospiraceae. The relative

abundance of these microorganisms in the biofilm may be associated with exposure of the Site 1 and

Site 2 coupons to hydrocarbons, possibly due to contamination of the coupons prior to deployment.

However, no relationship between these bacteria and MIC can be established from the existing data.

OTC-27142-MS 17

The high prevalence of Cyanobacteria and Chloroplasts in the Site 2 communities indicates that

photosynthetic respiration is occurring in the biofilm. This process will result in the production of

additional oxygen during the day and CO2 at night, which will diffuse through the biofilm. The biofilm-

generated oxygen has the potential to play a role in accelerating the corrosion process, due to interaction

with H2S producing thiosulfates, creating a positive feedback loop where thiosulfate reducing bacteria

(rather than sulfate-reducing bacteria) become more numerous and increase their production of H2S, a

portion of which reacts directly with the iron to produce FeS. The impact of CO2 generated within the

biofilm on corrosion requires elucidating. Noting that the specimens were exposed in the photic (light-

exposed) zone, and that the SCORCH JIP identified a correlation between depth and the level of

corrosion in the (permanently submerged) near-surface zone, further investigation of the relationship

between light exposure of upper mooring lines and the level of corrosion is warranted and will be

investigated in the DeepStar® CTR12402 project.

The classification of OTUs from the Site 2 specimens did not identify a significant population of groups

associated with SRP. This indicates that the exposure period may have been too short for the conditions

required to support a SRP population to develop (i.e. the inner biofilm had yet to become sufficiently

anoxic). This hypothesis will be further investigated in the DeepStar®

CTR12402 project.

Microbial community at Site 1 includes Desulfobacter, Desulfovibrionaceae and Desulfovibrio, which

represent genera of sulfate-reducing bacteria (SRB) and are often considered key contributors of MIC in

a marine environment. The abundance of these OTU is, however, relatively low, at 0.42%, 0.09% and

1.66% respectively. With the help of the DeepStar® CTR12402 project, the abundance of the SRB DNA

sequences can be compared between different test sites. Moreover, both relative abundance and structure

of SRB community can be correlated with the aggressiveness of MIC. OTU of Clostridiales,

Fusobacteriaceae, and Shewanella groups, which are associated with the production of Hydrogen Sulfide

(H2S) as well as, Epsilonproteobacteria that are involved in the ocean sulphur cycle, were also relatively

abundant. All of the above groups can contribute to the anaerobic corrosion process by creating zones of

H2S production and sulfide, sulfur accumulation.

6.0 Conclusions

The findings of this investigation provide evidence that the corrosion of carbon steel coupons at both test

sites can be classified as MIC. The presence of DNA sequences representative of SRB in the Site 1

biofilm community, and their comparative absence in the DNA profiles of Site 2 consortia, as well as

the prevalence of localized corrosion in form of mega-pits at Site 1 compared to lesser extent of pitting

corrosion at Site 2, supports the hypothesis that SRB are likely contributors to MIC and mega pit

formation. Against this hypothesis, the following points ought to be considered:

1. The coupons at Site 2 were exposed for a period of approximately 35 days, compared to 154

days for the coupons exposed at Site 1. As such, the biofilm is less developed and suboxic/anoxic

conditions required for SRB to reach their full metabolic potential may not have developed

within the Site 2 biofilms.

2. The low abundance of SRB may indicate that activity derived solely from sulfate reduction is not

a key driving mechanism of localized mega-pit localized corrosion and that H2S-production by

other anaerobic or sub-oxic biofilm residents may be of considerable importance to corrosion.

Further investigation is required to better understand the role of H2S-producing bacterial

consortia comprising SRB in MIC and mega-pit formation.

18 OTC-27142-MS

Overall, the findings of the study tend to support the conclusions of the SCORCH JIP [9] as to the likely

involvement of SRB in MIC of mooring chains in the upper water column, leading to the formation of

mega-pits. However, noting the low levels of OTUs representing SRB in the Site 1 microbiological

communities, and the earlier proposed key role of SRB in the formation of mega-pits, further

investigation is required to better understand whether such a low abundance of SRB sequences can

support the corrosion process that results in mega-pit formation, and to determine whether other

sulfidogenic microorganisms are more central to the MIC process.

7.0 Future Work

As already stated, future work into the subject of MIC is being conducted. DeepStar®

CTR12402

Integrity Management of Mooring Systems Against Corrosion [13] is a JIP that is currently carrying out

a more detailed investigation using an MBK and qualitatively and quantitatively analyzing microbial

biofilm communities and corrosion rates in selected regions at different geographical locations. The

participants of this JIP anticipate that MIC mitigation methods will be developed that can aid in

controlling corrosion in MIC prone regions.

8.0 Acknowledgments

The authors would like to acknowledge the Chevron marine superintendent, Angelo Merolla, and his

team of mooring masters for their support with the deployment and recovery of the MBKs. The authors

would also like to thank Prof. Robert Melcher’s and AMOG Consulting for the development of the

MBKs that were deployed and Prof. Robert Melcher’s valuable input into the corrosion analysis.

Additional thanks and acknowledgement should be given to Chevron ETCs Technology Development

program for funding of the study and the University of Oklahoma Biocorrosions Center for travel

support for Christine Gaylarde.

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