The Science Behind It: A ‘Cultural’ Change in Detecting Microbiological Activity

In addition to existing endogenously in petroleum reservoirs, microbes often can be introduced into oilfield systems at the initial stages of drilling and well completion.

Editor’s note: Learn more about microbiologically influenced corrosion in this Materials Performance quarterly special feature, “The Science Behind It.” After you’ve read the MP article on molecular microbiological methods, which are becoming more commonly used as an alternative to bacteria culturing in artificial growth media, explore the science behind the corrosion problem, which is presented in several related CORROSION articles listed at the end of this article.

The presence of microorganisms and their associated activity is increasingly recognized as a major concern in the oil and gas industry. In addition to existing endogenously in petroleum reservoirs, microbes often can be introduced into oilfield systems at the initial stages of drilling and well completion; during shut-ins; and during secondary and tertiary oil recovery, when fluids are injected into the formation to maintain reservoir pressure and force the recovery of hydrocarbons.

Microbiological activity can adversely affect oilfield systems in several ways. General heterotrophic bacteria are directly involved in biofouling (biofilm formation), which can contribute to reduced efficiency of production and associated equipment operation. For example, the formation of biofilms can provide favorable environments for anaerobic sulfate-reducing bacteria (SRB), which produce hydrogen sulfide (H2S). The production of H2S not only reduces the quality of the oil and gas produced (souring of production fields), but also increases the risk of corrosion. The process by which corrosion is initiated and/or accelerated by the activities of microorganisms is commonly known as microbiologically influenced corrosion (MIC). A comprehensive overview of MIC can be obtained from Little and Lee’s book, Microbiologically Influenced Corrosion.1

Detecting microbiological activity from oilfield samples (in deposits, scales, biofilms, and fluids) is an important initial step in evaluating a system’s risk of MIC. Detection methods focus on the application and implementation of biological techniques that make use of the bacteria’s features. In the oil and gas industry, detecting microbiological activity is still primarily based on cultivation techniques.

Viable Culture Techniques

Culture-based methods involving bacteria culturing in specific artificial growth media have long been established as the standard technique in the oil and gas industry for the identification and enumeration of bacteria.2 The most commonly used culture-based method is the serial extinction dilution technique, with the test consisting of a serial dilution step followed by an incubation step using a growth medium selected for the bacteria of interest.

For example, if the bacteria of interest are SRB, vials containing a specifically formulated SRB growth medium will be used in the test. If possible, the serial dilution part of the test should be carried out within a few hours of obtaining the subject samples (typically done on site). Following the incubation period, the SRB population density in the original sample is determined to the nearest order of magnitude by the number of vials in each dilution series that turn black because of bacterial sulfide production.

According to current standards,2 the prescribed incubation period for SRB is a minimum of 28 days; however, a very good indication of the SRB count usually can be obtained after 10 to 14 days of incubation. To improve the accuracy of such methods, threefold or fivefold replicate serial dilution enumeration is typically carried out to allow a mean probable number (MPN) of bacteria to be determined from standard MPN tables.3

A common criticism of the culture-based methods, such as serial extinction dilution MPN, is their inherent bias regarding strain isolation and growth, meaning that only a small proportion (usually <1%) of the microorganisms in a sample are cultivable and, therefore, analyzed.4-5 Consequently, these techniques may not be adequate for detecting all the microorganisms potentially involved in the corrosion processes. Within a biofilm, only some of the bacteria directly cause MIC, while other bacteria in the biofilm community indirectly contribute to MIC.6 The culture-based methods may severely misinterpret/misrepresent the actual system condition7 by underestimating the population size of the bacteria and failing to reflect the role of the uncultivated and, therefore, undetected bacteria potentially involved in MIC.4 Conversely, field situations have been reported where significant levels of SRB have been enuenumerated but the system shows little or no MIC, which has been attributed to the selective enrichment of SRB not heavily involved in MIC.7-8

Molecular Microbiological Methods

The limitations associated with the culture-based methods have led to the development of other culture-independent methodologies, which are gaining increased acceptance in the oil and gas industry.9 There are several different types of culture-independent methods, such as the measurement of adenosine triphosphate levels and specific enzyme activity, and the powerful molecular microbiological methods (MMM).10 MMM involve the extraction of deoxyribonucleic acid (DNA) directly from the microorganisms present in a sample. The polymerase chain reaction (PCR) is then used to amplify copies of a particular gene in the sample to such an extent that the DNA fragments can be used to identify the bacteria. A progression of the traditional PCR method is the real-time quantitative PCR (qPCR) technique, which can provide more accurate and quantitative reproductive enumeration data regarding microbial communities, in addition to bacterial characterization.11

Microbiological monitoring based on advanced molecular microbiological analysis has successfully been applied to offshore production pipelines,12 which led one North Sea operator to discontinue traditional MPN culturing methods as part of its microbiological monitoring programs.10 These molecular techniques have been demonstrated to be a faster, more accurate means of microbiological monitoring;4 and they accommodate the rapid identification and quantification of all microorganisms present in a sample, including the clear majority that cannot be cultured.13 Further improvements over culture-based methods include the increased quality of data obtained from culture-independent methods, and the fact that the molecular analyses can be conducted directly on the samples, which results in a faster response time (within a few days vs. one to two weeks).

Is It Time to Change?

The evolution of microbiological monitoring techniques from culture-dependent methods to more state-of-the-art and advanced culture-independent molecular techniques has undoubtedly yielded improvements in detecting and monitoring microbiological activity in oilfield systems. For oilfield systems where implementation of a new microbiological monitoring program is required, MMM are the obvious choice. When used to estimate the total amount of bacteria in a specific environment, MMM give a better estimation than their culture-based counterparts.14

Before deciding whether a change to culture-independent monitoring methods is beneficial for existing microbiological monitoring programs, it is important to have a comprehensive understanding of the system under threat from MIC. For example, the use of viable culture techniques would be sufficient for microbiological monitoring in systems where the control of microbial population and biofilm growth has been proven to be successfully achieved through a biocide treatment. Typically, biocides are nonselective, affect a broad spectrum of bacteria, and are applied to control all possible microbiological activity. Therefore, even monitoring only the culturable bacteria will allow the time point and trend measure data required to optimize a treatment in the field.5

When a biocide treatment proves to be unsuccessful, a more detailed analysis of the system using MMM can provide the additional information required to both characterize and quantify which specific bacteria present in the system are responsible for MIC (both directly and indirectly). The application of MMM, therefore, allows for improved optimization of remedial actions (e.g., adjusting biocide treatments to target the specific bacteria involved in corrosion processes) and MIC monitoring procedures.15 To tackle new challenges in the industry, new solutions are required. Any advances in our understanding of MIC and the effects of microbes will undoubtedly come from improvements in the ways we can detect and monitor microbiological activity. The introduction of culture-independent molecular techniques has already provided more detailed information regarding the specific bacteria involved in MIC.9,13

In the long term, the widespread application of molecular techniques throughout the industry could improve understanding of MIC and the specific microbes involved in the varied operational environments where bacteria can be present and cause problems. MMM is potentially the new standard by which we can improve our knowledge and understanding of how to effectively manage microbiological activity in oilfield systems.


1 B.J. Little, J.S. Lee, Microbiologically Influenced Corrosion (Hoboken, NJ: John Wiley & Sons, Inc., 2007).

2 NACE TM0194-2014, “Field Monitoring of Bacterial Growth in Oil and Gas Systems” (Houston, TX: NACE International, 2014).

3 J.F.D. Stott, “Corrosion in Microbial Environments,” Shreir’s Corrosion, Vol. 2 (Amsterdam, The Netherlands: Elsevier, 2010), pp. 1,169-1,190.

4 J. Larsen, et al., “Identification of Bacteria Causing Souring and Biocorrosion in the Halfdan Field by Application of New Molecular Techniques,” CORROSION 2005, paper no. 05629 (Houston, TX: NACE, 2005).

5 S. Maxwell, et al., “Monitoring and Control of Bacterial Biofilms in Oilfield Water Handling Systems,” CORROSION 2004, paper no. 04752 (Houston, TX: NACE, 2004).

6 T. Gu, D. Xu, “Why Are Some Microbes Corrosive and Some Not?” CORROSION 2013, paper no. 2336 (Houston, TX: NACE, 2013).

7 J. Larsen, et al., “Significance of Troublesome Sulfate-Reducing Prokaryotes (SRP) in Oil Field Systems,” CORROSION 2009, paper no. 09389 (Houston, TX: NACE, 2009).

8 K.B. Sørensen, et al., “Cost Efficient MIC Management System Based on Molecular Microbiological Methods,” CORROSION 2012, paper no. 1111 (Houston, TX: NACE, 2012).

9 R. Eckert, “Using Molecular Microbiological Methods to Investigate MIC in the Oil and Gas Industry,” MP 50, 8 (2011): p. 50.

10 A. Price, et al., “Detection of SRP Activity by Quantification of mRNA for Dissimilatory (bi) Sulfite Reductase Gene (dsrA) by Reverse Transcription Quantitative PCR,” CORROSION 2010, paper no. 10253 (Houston, TX: NACE, 2010).

11 B.P. Lomas, R. de Paula, B. Geissler, “Proposal of Improved Biomonitoring Standard for Purpose of Microbiologically Influenced Corrosion Risk Assessment,” SPE International Oilfield Corrosion Conference and Exhibition 2016, paper no. SPE-179919-MS (Richardson, TX: SPE, 2016).

12 U.S. Thomsen, R.L. Choong Meng, J. Larsen, “Monitoring and Risk Assessment of Microbiologically Influenced Corrosion in Offshore Pipelines,” CORROSION 2016, paper no. 7194 (Houston, TX: NACE, 2016).

13 J. Larsen, et al., “Molecular Identification of MIC Bacteria from Scale and Produced Water: Similarities and Differences,” CORROSION 2008, paper no. 08652 (Houston, TX: NACE, 2008).

14 B. Geurkink, et al., “Value of Next Generation Sequencing as Monitoring Tool for Microbial Corrosion: A Practical Case from Bioprophyling to Tailor Made MMM Analysis,” CORROSION 2016, paper no. 7764 (Houston, TX: NACE, 2016).

15 L.H. Hansen, et al., “The Application of Bioassays for Evaluating In-Situ Biocide Efficiency in Offshore Oil Production in the North Sea,” SPE International Symposium on Oilfield Chemistry, SPE 121656 (Richardson, TX: SPE, 2009).

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