OTC-26788-MS

Oilfield Microbiology: Detection Techniques Used in Monitoring

Problematic Microorganisms Such as Sulphate-Reducing Bacteria SRB
Douglas G. Bennet, Intertek

Copyright 2016, Offshore Technology Conference

This paper was prepared for presentation at the Offshore Technology Conference Asia held in Kuala Lumpur, Malaysia, 22–25 March 2016.

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Abstract
Microbiologically Influenced Corrosion (MIC) is a well-documented phenomenon that involves micro-
organisms and affects multiple industries with untold economic impact. The most well- known microor-
ganisms within the oilfield, by far, are Sulphate-Reducing Bacteria (SRB). It is thought that through SRB
respiration, corrosion of metals can occur. An exact figure for MIC responsibility in overall corrosion is
currently unknown. However estimates of between 10-50% are not uncommon, when coupled with
estimated costs of metal corrosion in developed countries to be between 2-3% of Gross Domestic Product
(GDP), suddenly the cost implications of MIC gain significance. The technical and economic implications
have gained recognition within the oil and gas industry within the last thirty to forty years and monitoring
techniques to detect microorganisms and corrosion have progressed and developed through increased
interest in microorganisms commonly found within the oilfield. As with human nature, the ability to
predict the future, rather than deal with the consequence is a preferred approach, which is one of the main
driver’s to pro-active monitoring techniques for detection of microorganisms to help determine the risk of
MIC to occur, rather than a reliance on rate of corrosion alone. This approach has led to increased research
in to the subject of oilfield microbiology and development of modern molecular techniques, often
borrowed from other industries such as medical microbiology, that have come to the fore recently.
However a significant focus on cost saving practices within the oil and gas industry has a significant and
direct impact on the type and frequency of monitoring applied (if any).
The objective of this review is to discuss and evaluate the available techniques and review the most
common problems associated with microorganisms within the oil and gas industry. To determine effective
monitoring practices in a practicable and economically viable manner to ensure monitoring can be carried
out effectively, understood and evaluated while implementing control and mitigation strategies with
confidence.
Introduction
Microbiologically Influenced Corrosion (MIC) is corrosion affected by the presence or activity (or both)
of microorganisms in biofilms on the surface of the corroding material (NACE, 2012). The materials
susceptible to MIC are by no means limited to metals only. Nonmetallic materials such as; polymeric
materials, reinforced polymeric composites, concrete, asphalt and wood (Little, 2007) are affected by
2 OTC-26788-MS

specific microbial degradation. Unfortunately, as such, there are no economically viable ‘quick fix’
solutions to avoid the threat of MIC by substituting materials within oil and gas solutions.
Microorganisms are able to survive and grow in extremely harsh environments, for example; under
high pressure, acidic conditions, alkali conditions, low temperatures (-10°C), high temperatures (!99°C),
anaerobic and aerobic conditions and many other ecological limiting parameters. Many oil and gas
reservoirs and process systems are considered to be inhospitable environments for microbiological life to
exist, with some believing it is impossible for microorganisms to survive, however, there have been many
examples of microorganisms surviving and even growing within reservoirs (Vance, 2005) and there is a
real possibility that almost every process system operating will have some form of microbiological
contamination. Microbiological surveys of oilfield systems regularly reveal active, viable bacterial
populations of Sulfate-Reducing (SRB), General-Heterotrophic Bacteria (GHB) and Acid-Producing
General-Heterotrophic Bacteria (APGHB), as well as many other prokaryotes, within reservoir fluids
sampled at the Wellheads. There are many published accounts of microbiological contamination through-
out topside process systems (Larsen et al, 2010). However, problems will only occur when microbiolog-
ical control is not achieved.
The issues associated with uncontrolled microbiological contamination within oil and gas systems are
many and greatly varied, and are not always negative. The consequences can have significant economic
impacts on operations, therefore it is extremely important to understand how microorganisms interact
within each environment. The economic impact of a shutdown due to MIC can cost in the region of
millions of dollars, per failure, a very simple example is a corrosion failure due to MIC within an export
production pipeline;
If Pipeline X produces 10,000 bbls/day of crude, at a market value of $50 per barrel, therefore on a
daily average is generating $500,000 revenue. Pipeline X has a failure due to MIC, for example a pin-hole
leak or pipeline rupture due to corrosion at the 6 o’clock position, severe enough for the pipeline to be
shutdown repaired, the loss of revenue alone for 7 days downtime is $3,500,000. This does not even
account for additional costs for repairing the pipeline; vessel hire, personnel hire, weather delays or
equipment supply and so on, which alone can cost upwards of $1,000,000 for 7 days.
The above scenario is a purely fictional example, used to highlight the potential costs associated with
loss of production time, which in today’s economic climate for the oil and gas industry is not very
welcome.
Therefore, the comparably minor costs conducting microbiological sampling, analysis and interpreta-
tion to understand the risks associated with MIC within a specific production system in detail are well
worth the investment to prevent such failure and knock-on costs.
Reservoir: Microorganisms that are either indigenous (Youssef, 2009) or have been introduced to
reservoirs through drilling activities or secondary recovery methods like water injection are known to
cause problems with hydrocarbon production. These populations of microorganisms tend to be hyper-
thermophilic/thermophilic bacteria and/or archaea able to survive in high temperature and high pressure
conditions due to the environment they exist within. The most common problem associated with
microorganisms within reservoirs is reservoir souring. Reservoir souring is known throughout the industry
as the unplanned appearance and gradual increase in Hydrogen Sulphide (H2S) concentration in the
production phase of a process system which has been produced by Sulphate-reducing prokaryotes (SRP).
Besides the direct risk to personnel due to H2S gas as a poisonous gas, 1000ppm can cause fatality (HSE
EH40, 2005); H2S is an acidic gas which when dissolved in fluids can cause corrosion. Additionally, H2S
can devalue sales gas and crude, or may even be refused by some refineries/export vessels.
Contrary to the problems caused by microorganisms within the reservoir, some select microorganisms
can have a beneficial impact. Nitrate injection with water injection promotes growth of Nitrate-reducing
prokaryotes which can help reduce the potential for reservoir souring by; out-competing SRP (Grigoryan
et al, 2008), producing nitrite which is inhibitive to SRP growth (Hubert and Voordouw, 2007), a shift in
OTC-26788-MS 3

redox potential, oxidation of reduced Sulphur compounds by Nitrate-reducing, Sulphur-oxidising bacteria

and most interestingly, some Sulphate-reducing bacteria can actually change to Nitrate-reduction.
Production system: As discussed in the section before, microorganisms can enter a production system
through production wells, which can then lead to microbial contamination within production facilities
(Larsen et al, 2010). Problems encountered from microbial contamination within production systems
include; MIC of well tubing, MIC of flowlines, separation issues due to interface biofilm within separation
and oily water treatments vessels, produced water quality issues due to biomass formation, pump failure
due to MIC of pump seals and MIC within storage tanks.
Export and/or storage: Gas and oil export pipelines can be susceptible to MIC due to a combination of
factors; nutrient supply is not limiting for microorganisms due to the huge presence of carbon source,
surface area for biofilm attachment is plentiful and water will always be present. Confusion can arise when
operator claims that the export oil or gas is ‘dry’. It is extremely rare that an export line will be 100%
‘dry’, and results of 0% moisture content from export samples should be scrutinized in detail if
consistently reported. A very simple example; a platform producing 10,000 bbls/day of crude or gas, with
a moisture content of 0.5%, is often considered to be ‘dry’. The 0.5% moisture content is the equivalent
volume of 7950 liters of water, per day. Water being heavier than oil, together with mixed-phase flow
dynamics, will gather in the low lying areas of the pipeline topography, essentially forming puddles (of
water) within the ‘dry’ line. If microorganisms are present within the export sample, biofilms will become
established in these areas, leading to a risk of MIC and potential failure. The story is the same for storage
tanks, the near stagnant conditions and very large volumes are ideal conditions for water ‘drop-out’, which
will collect in the low areas of the tank. Although, the majority of storage tanks are coated or lined, this
does not guarantee protection from MIC, and very often a minor defect in the coating or lining can result
in an accelerated zone of corrosion.
Water Injection: Problems with microorganisms are a common occurrence within water injection
systems. A normal seawater injection system, ironically, includes all the ingredients for maximizing the
potential for SRB proliferation. SRB are ubiquitous and will therefore always be present within seawater.
This water is usually always treated with a chlorine based treatment to sterilize the water as soon as it is
drawn from the source, before the water is further processed in the water injection system. A very common
issue within offshore seawater injection systems is insufficient chlorine dosing. As the chlorine applica-
tion must be 100% continuous to sterilize the seawater, often this is not achieved. Electrochlorination units
are notoriously unreliable and manual hypochlorination is susceptible to human error. Inevitably, this will
(not if) result in microorganisms passing through to the next stage of the seawater injection system,
filtration and/or heat exchangers, which is an ideal situation for microorganisms to form biofilms; a very
large surface area for attachment, sufficient temperature and nutrients for growth to form a biofilm. The
microorganisms that make it through the filters ("2 microns for fine filters), then enter the deaeration
vessel. Deaerators are the mechanical units to remove dissolved oxygen from seawater (to "50 ppb), and
are remarkably similar in design to fermentation units (large, tall vessel with a very large internal surface
area). Usually the next chemical addition is an oxygen scavenger chemical such as Ammonium bi-sulphite
or Sodium bi-sulphite, to scavenge the remaining dissolved oxygen to a target concentration of "10ppb
or "5ppb in some cases. By this point, any SRB that have made it through are in ideal conditions for
proliferation. SRB are anaerobic microorganisms which can utilise sulphite (Park et al, 2011). By this
point, if an effective biocide chemical is not added, the reservoir will be at a huge risk of microbiological
contamination and problems associated with microorganisms; flowline MIC, well tubing MIC, injection
filter plugging, reservoir souring are just some of the issues to name.
Unfortunately, there are common mistakes made throughout the oil and gas industry in regards to
management of seawater injection systems; little attention is paid to the 100% upkeep of the chlorine
dosage and often defunct electrochlorination units are discarded. Often, filters are bypassed as they are
constantly clogging and restriction injection rates, deaerator towers are run at #100% capacity, vacuum
4 OTC-26788-MS

deaerator towers are often under-performing and a greater reliance on chemical oxygen removal is normal.
Over-dosing of bi-sulphite based oxygen scavengers can promote SRB growth (Park, 2011). Biocide
application is very often insufficient and batch applications will never achieve 100% microbiological
control. In most circumstances, the investigation stage and solution to problems associated with water
injection systems are deferred due to the subjective view that these systems are non-revenue generating
systems and preferential focus is paid to the systems directly linked with generating production and
therefore revenue.
Utilities: Similar issues to water injection facilities are encountered within water-based utility systems.
Potable water systems, cooling and heating systems, ballast and fuel displacement water all utilise water
that should be particle free and (microbiologically) clean. Variations in the issues encountered are closely
related to the environmental conditions within the system in question, for example; a potable water supply
for an offshore facility should be frequently tested to ensure there are no pathogenic bacteria such as E.
coli, Coliforms, Enterococci and Legionella bacteria (WHO, 1997) present to ensure no harm to personal.
A cooling or heating water-based system should be tested frequently to ensure microorganism populations
do not proliferate to levels which may result in filter blockages and/or reduced efficiency in heat
exchanger capability. Ballast water is well known to contain microorganisms which can cause environ-
mental issues, particularly with ships which travel worldwide which may contain invasive species and
other non-indigenous species which when introduced to new ecosystems can cause havoc to the
indigenous ecosystem (Molnar, 2008). The last example to discuss but certainly not limit the utility system
issues is; fuel systems. Diesel and petroleum is consumed in the oil and gas industry on a vast scale.
Almost every standalone rotating piece of machinery will consume diesel or petroleum for normal
operations or as a back-up during shutdowns. Emergency generators, jockey pumps, engines, fire water
pumps, motors, heavy machinery, transportation vehicles, to name a few examples store and consume fuel
for everyday operation. These tanks can accumulate free water over the lifespan of the vessel from
condensation, high moisture content of fuel, rainwater etc. which can lead to proliferation of microor-
ganisms. Common issues encountered with these examples are; interface biofilm growth, filter blockages,
biomass accumulation and poor burn quality leading to increased emissions. Again, little attention is paid
to maintaining these types of systems due to the nature of the non-revenue generating system, particularly
during times when market value of oil and gas is low. Tightening budgets mean cost cutting is a priority
and therefore non-essential inspection and maintenance is often deferred or cancelled altogether. In some
circumstances, the result of deferral or cancelling an investigation altogether, can result in increased costs
where a simple inspection or survey may have identified an easily solved issue that would result in less
consumption or optimized use of the system in question, saving costs.

Methodologies

There are many different techniques available for the detection, quantification and identification of
microorganisms and the aim of this review is to discuss some examples which can be used within the oil
and gas industry readily. The techniques discussed are not an exhaustive list by any means, and there are
many more available, but they are an example of techniques which are available from commercial
companies or contractors associated with the industry or can be carried out by specialised personnel or
operators themselves.
A comparative market study was carried out for this review to generate a comparison on; costs, skills
required, turn-around time, data and other applicable information for each technique available. Costs are
based on a ‘per sample’ estimation which does not include any associated costs for skilled personnel to
take the samples or interpret the results of the sample analysis. It is prudent to factor this in if selecting
a particular technique as several of the techniques are highly specialised and require trained microbiol-
ogists to perform the sampling, analysis and/or result interpretation. In some regions of the world where
OTC-26788-MS 5

the oil and gas industry operates, these skills can be very difficult to source or very expensive to import.
Skills required for the various techniques ranges from very simple tasks (rated as 1 in Table 1), such as
filling a clean sample bottle wearing suitable PPE, including nitrile gloves to avoid cross contamination
and provide personal protection (if the sample contains harmful chemicals) to a highly skilled require-
ment, for instance, where a molecular microbiological technique is employed to determine and interpret
the variety of genus of bacteria or archaea within a sample. Turn-around time is an important consider-
ation when selecting a material or technique as it may have a significant impact on determining the
frequency of sampling within a monitoring schedule.

Table 1—A comparison of selected microbiological techniques commonly used for microbiological analysis at oil and gas
locations.
Estimated Skills required (1-3,
Cost per Low to Highly trained)
Sample Turn
Technique (USD) Sampling Analysis around time Data return Additional requirements

Adenosine Triphosphate $100 1 2 10 minutes Microbiological activity Luminometer, consumables,

(ATP) refrigerator
Most Probable Number $50 1 2 28 Days Viable cell enumeration Bacterial growth media,
(MPN) salinity of sample,
consumables, incubator
Immunology (antibody $50 1 2 20 minutes Bacterial activity Test kit, consumables
test kits)
qPCR $500 1 3 10 Days Bacterial and Archaea Sterile sample bottles,
differentiation and molecular microbiology
enumeration laboratory, Primers
DGGE $500 1 3 2 months Microorganism Sterile sample bottles,
community molecular microbiology
representation laboratory
FISH $800 2 3 5 Days Bacterial and Archaea Prepared sample preservation
differentiation of bottles, molecular
selected probes and microbiology laboratory,
enumeration artificial DNAprobes
Sequencing $2000 2 3 2 months Bacterial and Archaeal Sterile sample bottles,
differentiation and molecular microbiology
enumeration to genus laboratory
level

Introduction to selected techniques for review

Adenosine Triphosphate (ATP) There are commercial ATP test kits which can be easily purchased on
the internet or from laboratory suppliers. These test kits have been designed to be portable so that sample
analysis can be performed at site. This allows for sample analysis to be carried out immediately after
sample collection, at site. Therefore an indication of microbiological activity (ATP is present in all living
cells) can be quantified. This is a particularly efficient tool when basic indication of microbiological
activity is required at very short notice.
The method is based on the measurement of ATP, which is a direct and interference-free indicator of
total living biomass. ATP is present in all living cells, being involved in energy metabolism. It quickly
disappears upon the death of the cell and can thus be used as an indicator of the amount of living
organisms present in a sample. ATP is measured using the firefly Luciferase assay, where a sample
containing ATP is introduced to a solution containing the enzyme Luciferase, which naturally occurs in
the tails of fireflies, to produce light. The light is detected in a photomultiplier or luminometer, the output
being proportional to the amount of ATP, units are usually recorded as Relative Light Units (RLU). The
reaction can be explained below;
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Although these test kits are convenient and quick, it should be noted that there are some drawbacks to
this method; there is no differentiation between the various types of microorganisms within a sample as
all living cells contain ATP. The ATP content of a cell is dependent on cell volume therefore larger
bacterial cells will return a higher number of ATP than the equivalent number of smaller cells. The method
may also encounter interference from chemicals such as biocides and high sulphide content.
Most Probable Number (MPN) Sometimes referred to as serial dilution to extinction, triplicate series is
a commonly used technique to enumerate viable bacterial cells. This technique is a microbiological
culturing technique described in the internationally recognized test method NACE TM0194:2004.
Triplicate MPN inoculations can be carried out to enumerate viable bacterial populations of many types
of bacteria including but not limited to; Sulphate-reducing bacteria (SRB), Nitrate- reducing bacteria,
General-heterotrophic bacteria (GHB) and acid-producing general- heterotrophic bacteria (APGHB).
As this technique requires bacterial growth media, which is formulated to selectively grow a specific
group of bacteria, certain information is required before selecting the growth media. It is essential to know
the water chemistry and operating conditions of the sample location prior to selecting the growth media.
As this technique detects viable cultures of bacteria contained within the system under sampling,
replicating the conditions as closely as possible is essential for the bacterial cells to survive and grow
within the growth media. Due to this fact, many cells will die upon removal from the environment (sample
point), and only as little as 1-10% of the populations will continue to grow within the growth media.
Samples are taken at site and inoculated directly in to boxes of bacterial growth media containing
sealed vials of media. Following triplicate serial dilution, three 1mL aliquots of the sample are inoculated
in to the first three vials of media, where after, serial dilutions are carried out in triplicate series for the
rest of the dilutions. This methodology enables the user to enumerate bacterial populations to cells per mL
quantity using the MPN technique. It is not uncommon for some to practice single-series dilutions,
whereupon only one vial of media is inoculated with the original sample and the dilution series continues
for the rest of the dilutions. If this method is used, it will become difficult to replicate results due to the
increased inaccuracy of single series against triplicate. It will also be impossible to enumerate the results
beyond one logarithmic factor, for example, single series results will be presented as; SRB at 103 or 104
cells per mL. It is good practice to increase the number of replicates of dilution series to increase the
accuracy of the test method, which will also produce results with greater accuracy, for example, triplicate
MPN results will be presented as; SRB at 2.5 $ 103 or 7.5 $ 104 cells per mL.
Immunology Techniques There are commercially available field test kits which employ the use of
antibodies to detect the presence of certain species of bacteria. The idea behind the technology is borrowed
from immunology whereupon a foreign body (antigen) is introduced to an organism which then identifies
the antigen and produces antibodies and an immunoresponse which then attach on to the antigen and
disable it. The same idea is used for SRB, which is introduced into and organism as a pure culture to
produce an immunoresponse, releasing antibodies which can then be isolated and purified from the
organism’s blood. If an aliquot of the antibody medium is then injected in to a pure culture of the same
bacteria, the antibodies will attach on to the cells, resulting in coagulation and precipitation. As the
antibody will be specific to the particular type of bacteria, it will not do the same in a different culture
of another type of bacteria, therefore being specific to certain bacteria.
Field kit’s based on antibody technology are available, which uses antibodies raised to the APS
reductase enzyme, present in all SRB. The kit allows enumeration of SRB in a variety of samples to be
determined in minutes. As the kits avoid the step to grow bacteria the results are available quickly.
Limitations to these types of kits however include; due to the specific nature of the antibodies developed
against pure strains of SRB, the sample to be analysed will contain mixed microbial consortia and it will
OTC-26788-MS 7

be difficult to isolate pure strains of SRB. Large numbers of cells are required for the coagulation to be
successful a minimum of at least 103 cells per mL must be present within the sample. Obviously, as these
antibodies are specific to SRB and may not detect low numbers ("103 cells per mL), results may provide
false security in that SRB levels are considered insignificant if this is the only technique applied, as
bacterial growth may continue further downstream of a sample point once conditions become favourable
for exponential growth.
Polymerase Chain Reaction (PCR) PCR is a DNA based technique to quantify specific microorgan-
isms, often referred to as quantitative PCR (qPCR). The aim of PCR technology is to specifically increase
a target from an undetectable amount of starting material within a sample. As with most DNA based
molecular methods, the first step requires extraction of the DNA from the sample. The next step is to
amplify the amount of DNA through PCR with a primer which has a fluorescent tag. During thermocy-
cling the fluorescent marker accumulates, which can be used to quantify the starting number of the target.
The target copied during the process depends on the primer used and can be tailored to specifically
quantify certain microbial groups. The most common molecule (target) used to identify micro-organisms
is 16S rRNA or the gene (the DNA) itself. 16S rRNA has proved to be a reliable molecule to use in order
to identify micro-organisms because of its universal distribution (ribosomes are present in all living cells)
and information content (rRNA is a highly conserved molecule).
Specific primers have been designed to detect a range the most commonly found genus of SRB. The
primers have been used to demonstrate the presence of SRB in a wide range of diverse habitats. Where
viable counts of the SRB populations provide an estimate of the total population size, the application of
primers allows the diversity of the SRB population to be assessed. This helps to differentiate between the
different types of SRB and highlights those that are able to survive in specific environments, for example,
only specialised SRB may survive after the application of a biocide dose or within an extremely hot vessel
(#80°C). Once this information in known to the user; specific treatments can be tailored for the process
system in order to obtain microbiological control.
However, due to the specific nature of the technique, the user must know what target they are looking
for. This also means results may return negligible results if the target is not detected from the analysis,
if the primer does not find a target.
Denaturing Gradient Gel Electrophoresis (DGGE) DGGE is another technique which utilises DNA
and PCR technology to differentiate between the different species present within a community from a
single sample. Again, the first step of the technique requires extraction of DNA from the sample, followed
by amplification by PCR. The DNA fragments are then transferred on to a denaturing gradient gel,
separated based on their DNA sequence. The DNA migrates across the gel, which has a gradient of
denaturing conditions, the different DNA strands present within the sample will deposit at different
intervals across the gel. DNA contains four nucleotide bases (Guanine, Cytosine, Adenine and Thymine)
which pair across the two strands of the double-helix molecule. The extent to which the DNA strand will
denature depends on its nucleotide sequence composition (G-C base pairs are stronger than A-T base
pairs) and the partial denaturing results in a decrease in mobility through the gel with a band of DNA
forming. The amount of bands forming at different intervals across the gel indicates the different DNA
present within the sample, which represents the community present within a sample. A gel with very few
bands, and a thick single band, indicate a community dominated by a single species, and vice versa, a gel
with many bands of similar intensity indicates a diverse community within a species.
Comparison of the communities within different samples, from different locations throughout a process
system provides valuable information on the environments throughout the process and how it affects the
microbial populations. Pairing this information with operational data and information will help to
understand what affects microbial populations. This technique alone does not provide information on the
species of microorganisms contained within the samples.
8 OTC-26788-MS

Fluorescence in situ Hybridisation (FISH) FISH is another molecular microbiological technique that
can be used to identify and quantify microorganisms within a sample. The technique requires the fixation
of samples at site, which essentially preserves the microorganisms information, as a fixation stage is
required, DNA can be damaged therefore the more robust molecule rRNA is the target for this technique.
The second step of the technique is a hybridisation step, where an artificially-made DNA probe which is
labelled with a fluorescent tag is introduced to the target rRNA within a cell, which will bind to one
another if the match is perfect. This results in hybridised microbial cells which fluoresce under ultra-violet
(UV) light. The technique is very specific, as there is a washing stage which removes any un-bound
artificial DNA probes, which will not be in the sample when placed under a UV light. As the DNA probes
are artificially manufactured, different probes, with different coloured fluorescent tags can be introduced
to the sample to detect the corresponding target and quantified by specialised fluorescent microscopy.
However, due to the specific nature of the artificial DNA probes, the user must know what target they
are looking for. This also means results may return negligible results if the target is not detected from the
analysis, if the probe does not find a target.
DNA Sequencing DNA sequencing technology has improved in recent years as databases containing
DNA libraries continue to expand and knowledge of the individual DNA sequences increases. These
techniques have become more readily available in the commercial market however still remain by far the
most expensive of the techniques under review within this paper. These techniques are similar to PCR
technology in that DNA is extracted from a sample, and sorted in to a library of small DNA segments prior
to amplification. The DNA segments are amplified sequentially and identified by a light signals emitted
which is compared to a DNA library for identification. These methods can reveal unknown microorgan-
isms if they are not included in the DNA library.
The data generated by these techniques can be difficult to interpret as knowledge of microorganisms
to the genus level is extremely detailed information which in the majority of cases of oilfield investiga-
tions is beyond any historical records data set. The level of subject expertise for these types of techniques
is required to be at a very detailed level. In fact, there are only a handful of commercial laboratories
available at this moment in time offering these types of analyses to the oil and gas industry. Therefore,
turn-around times and costs are substantial.
Technique comparison;
● ‘Cost per sample’ are estimated figures based on average market values and some of the techniques
are based on a bulk analysis basis (i.e. 10 or 100 tests per kit) or minimum order quantities.
● ‘Skills required’ indication: 1 % Filling a bottle wearing correct PPE, 3 % Capability to run
sophisticated analytical microbiological equipment and aseptic analyses. Note; anything above a
level 1 skill level would require a trained field scientist (microbiologist) to attend site for sample
collection, preparation and/or analysis.
● ‘Turn-around time’ does not include sampling or transportation time, this is based on the analysis
time only.
● ‘Data return’ this is a very brief summary of the type of information returned from each technique,
‘Bacterial activity’ being the most basic data return and ‘Bacterial and Archaea differentiation and
enumeration to genus level’ being the most detailed data return.
● ‘Additional requirements’ is listed to include any information which is important to consider when
selecting an appropriate technique, this includes brief information on equipment and conditions for
the analysis to be carried out.
Results
A microbiological survey of an oilfield located within South East Asia was requested by a client that
wishes to remain anonymous, with a request for part of the investigation to include a comparison of
OTC-26788-MS 9

microbiological techniques. The oilfield consisted of production facilities and seawater injection (treated
with nitrate chemical for reservoir souring control), which included several wellhead platforms and
mixed-phase flowlines that tied back to a central processing platform. The central processing platform
included the separation and seawater injection facilities. Subsea seawater injection flowlines flowed to the
wellhead platforms where some of the injection wells were located. Production was exported via an export
pipeline to an onshore terminal.
The data set was limited as only one microbiological survey was conducted however it provided a
valuable insight in to the type of results obtained from the various techniques discussed within this review.
It should be noted that not all of the techniques discussed were selected for the survey due to cost
constraints and only one set of samples were able to be sampled and analysed. In an ideal situation, repeat
samples from the same sample points would have been taken to increase the data set (for instance 10
samples from each point) and would be continued to be monitored over a longer period of time (6-12
months) at frequent time intervals (monthly).

Figure 1—Comparative results of quantitative SRB analysis by; ATP, MPN and QPCR within production wells at the wellhead platforms
10 OTC-26788-MS

Figure 2—Comparative results of quantitative SRB analysis by; ATP, MPN and QPCR within seawater injection system

Table 2—454 Pyrosequencing results from production well samples. NB. Halo (Halomonadaceae), Alter (Alteromonadaceae),
Pseudo (Pseudomonadaceae), Desulfo (Desulfobulbaceae), Eub (Eubacteriaceae), Chrom (Chromatiaceae)
Halo (%) Alter (%) Pseudo (%) Desulfo (%) Eub (%) Chrom (%) Others (%)

Well 3 85.6 "1 "1 "1 "1 "1 11.1

Well 4 36.2 13.7 11 "1 "1 "1 32
Well 6 40 8 11 "1 "1 6 31
Well 8 32 4 9 11 5 "1 32

Although all samples yielded sufficient DNA for further analysis, only four samples qualified for 454
pyrosequencing after pre-PCR amplifications (Wells; 3, 4, 6 and 8).
The communities mostly belonged to the bacteria kingdom with most of the bacteria belonging to the
phylum of the proteobacteria (between ~84.0% and ~99.0%). On a class level the communities detected
in the samples taken from the wells belonged primarily to the y- proteobacteria (ranging from ~69% to
~97%, respectively).
At the family level most of the locations were dominated by Halomonadaceae, a marine and moderately
halophilic microorganism that are phenotypically rather diverse. The sample taken from Well 3 detected
around 85.6%, which was the highest percentage detected across the well samples analysed.

Table 3—454 Pyrosequencing results from seawater injection samples.

Alcanivoracaceae (%) Rhodobacteraceae (%) Vibrionaceae (%) Others (%)

DA Tower Pump Outlet 58.5 10.5 4 22.4

Upstream WHP A Filter "1 91.2 "1 5.8
Downstream WHP B Filter "1 72.4 "1 21
OTC-26788-MS 11

Although all samples yielded sufficient DNA for further analysis, only three samples qualified for 454
pyrosequencing after pre-PCR amplifications (DA Tower Pump Outlet, Upstream WHP A Filter and
Downstream WHP B Filter).
The communities belonged primarily to the bacteria kingdom with most of the bacteria belonging to
the phylum of the proteobacteria (between 90.3% and 97.9%). The community detected within the sample
from DA Tower Pump Outlet belonged primarily to the y- proteobacteria (82.3%), whereas the popula-
tions in the samples from Upstream WHP A Filter and Downstream WHP B Filter belonged predomi-
nantly to the a-proteobacteria (92.2% and 81.2%, respectively).
At a family level; both Upstream WHP A Filter and Downstream WHP B showed a high proportion
of Rhodobacteraceae (~91% and ~72%, respectively), these microorganisms are known to be dominant
bacteria in marine biofilms and belong to the sulfur-oxidizing bacteria.
In contrast, the population detected from the sample taken at DA Tower Pump Outlet showed that only
~10.5% belonged to the family of Rhodobacteraceae, whereas ~58.5% belonged to the family of
Alcanivoracaceae. Some members of the Alcanivoracaceae are halophilic, aerobic, oil-degrading marine
bacteria that are found in low abundance in unpolluted environments in the upper layer of the ocean, they
can quickly become predominant in oil- contaminated open oceans and coastal waters when nitrogen and
phosphorus are not limiting.
The two systems in comparison: The seawater injection system communities, in general showed a more
dominated population (apart from the sample from DA Tower Pump Outlet) with 91.2% and 72.4%
belonging to Rhodobacteraceae. However, the communities detected at the wells showed more diverse
populations (apart from location Well 3, which was dominated by Halomonadaceae with 85.6%) with
many different microorganisms present at each of the different locations.

Discussion
The results from the microbiological survey conducted, although limited to a small selection of samples
do highlight the difference in results obtained from the various microbiological analysis techniques
discussed within this report.
A common misconception related to microbiological results, which are usually reported in cells per mL
or cfu (planktonic) format, is the question of; what is a low result and what is a high result? Often, a limit
of "102 cells/mL is considered low. This can be misrepresented if additional critical operational
information is missing. For example; if two pipelines have a relatively low fluid velocity ("3 m/s), which
is considered to be slow enough for planktonic bacterial cells to attach on to the internal surface of the
pipeline and colonise to form a biofilm, operational information should be considered carefully. If pipeline
X was producing 10,000 bbls/day, sampled for SRB returned a result of 2.5 $ 102 cells/mL, would be
considered a low result, compared with pipeline Y, producing 1000 bbls/day, sampled for SRB returned
a result of 2.5 $ 103 cells/mL, which would be considered a moderate result, when in actual fact, the
number of planktonic SRB in both examples is exactly the same;
● Pipeline X, was producing 250 cells/ml at a flow rate of 10,000 bbls/day, in theory, a total of 3.975
$ 1011 bacterial cells entered the pipeline in one day.
● Pipeline Y, was producing 2500 cells/ml at a flow rate of 1000 bbls/day, in theory exactly the same
amount of bacterial cells entered the pipeline in one day.
ATP results when converted to a Microbial Equivalent (ME) value can be directly compared to
quantitative results from MPN and QPCR techniques which are reported in cells per mL of sample. The
results from the production wells sampled during the microbiological survey suggest that ATP and MPN
results are relatively similar. The ATP and MPN results are generally low ("102 cells per mL - with one
exception, Well 9 ATP count). The QPCR results however are consistently higher, with only one result
at 101 cells per mL and the rest at 102 or higher. The most likely explanation for this difference is the
12 OTC-26788-MS

sensitivity of the techniques. QPCR is able to detect dead, inactive and active cells within a sample,
therefore, an assumption could be drawn that the ATP and MPN results were lower than QPCR due to a
portion of the microbiological cells within the sample being inactive or dead.
The results of the seawater injection system can be analysed as a pathway rather than individual
samples from a single sample point as the flow of seawater passes through the system with sample points
located through the system (DA Tower Outlet to the DA Tower Pump Outlet onwards to the sentinel
points; Downstream WHP A and B Filters), the only difference between the upstream filter sample points
being the length of flowline between the central processing platform and the wellhead platform. An
additional factor to this system is that nitrate chemical was being injected to the sweater injection system
for the control of reservoir souring. The results from MPN analysis suggest that microbiological
populations decrease between the CPP and WHP, whereas ATP results suggest a decrease only between
the CPP and WHP B. QPCR results suggest that there is no decrease between the sample points with all
of the results returning similar counts. Again, this could suggest an operational factor is impacting the
results. Was the nitrate chemical achieving the desired microbiological control of increasing NRB activity
preferentially over SRB activity? QPCR may have detected inactive and dead SRB cells at the WHP A
and B sample points where MPN only detected viable SRB cells at much lower values. A further influence
is the potential interference of NRB populations which would very likely be at high levels due to the
addition of nitrate chemical, with the ATP technique, as the ATP technique cannot differentiate between
groups of bacteria as it is estimating bacterial activity through the measurement of ATP (present in all
living cells). Therefore, the results obtained could be skewed and potentially indicate three separate
scenarios.
The example of the microbiological survey highlights the importance of gathering as much information
as possible to interpret microbiological data in conjunction with other physicochemical/operational data
that may have an influence on the results. This also highlights the importance of skilled labour (qualified
microbiologists or related science) available to interpret results, particularly data produced from the
molecular microbiological techniques discussed within this review. Microbiological techniques are
essential tools for determining the presence and activity of microbiological populations within oil and gas
systems which can have significant impacts on oil and gas production and therefore revenue generation.
It is prudent that an effective monitoring program is selected and put in place. The monitoring should be
continued on a frequent basis to generate as much relevant data as possible within acceptable limitations,
in order for accurate interpretation of scenarios and to allow mitigation strategies to be applied effectively
and efficiently as and when required.

Glossary
MIC – Microbiologically Influenced Corrosion
SRP – Sulphate-Reducing Prokaryotes
SRB – Sulphate-Reducing Bacteria
SRA – Sulphate-Reducing Archaea
GHB – General Heterotrophic Bacteria
APGHB – Acid-Producing General Heterotrophic Bacteria
NRB – Nitrate-Reducing Bacteria
NRA – Nitrate-Reducing Archaea
ATP – Adenosine T riphosphate
MPN – Most Probable Number
H2S – Hydrogen Sulphide
Planktonic – Free-swimming microorganisms in liquid samples
Sessile – Surface attached microorganisms in solid samples
OTC-26788-MS 13

Aerobic microorganisms – Microorganisms that require Oxygen for growth

Anaerobic microorganisms – Microorganisms that do not require Oxygen for growth
DNA – Deoxyribonucleic Acid
RNA – Ribonucleic Acid
DAPI – 4’,6-diamidino-2-phenylindole
qPCR – Quantitative Polymerase Chain Reaction
DGGE – Denaturing Gradient Gel Electrophoresis
FISH – Fluorescence In Situ Hybridisation
PPE – Personal Protective Equipment
VFA – Volatile Fatty Acids
PPM – Parts Per Million
PPB – Parts Per Billion

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