Torben Lund Skovhus

Prokaryotic Community Structure and Sulfate Reducer Activity in Water from High-Temperature Oil Reservoirs with and without Nitrate Treatment

ABSTRACT Sulfate-reducing prokaryotes (SRP) cause severe problems like microbial corrosion and reservoir souring in seawater-injected oil production systems. One strategy to control SRP activity is the addition of nitrate to the injection water. Production waters from two adjacent, hot (80°C) oil reservoirs, one with and one without nitrate treatment, were compared for prokaryotic community structure and activity of SRP. Bacterial and archaeal 16S rRNA gene analyses revealed higher prokaryotic abundance but lower diversity for the nitrate-treated field. The 16S rRNA gene clone libraries from both fields were dominated by sequences affiliated with Firmicutes (Bacteria) and Thermococcales (Archaea). Potential heterotrophic nitrate reducers (Deferribacterales) were exclusively found at the nitrate-treated field, possibly stimulated by nitrate addition. Quantitative PCR of dsrAB genes revealed that archaeal SRP (Archaeoglobus) dominated the SRP communities, but with lower relative abundance at the nitrate-treated site. Bacterial SRP were found in only low abundance at both sites and were nearly exclusively affiliated with thermophilic genera (Desulfacinum and Desulfotomaculum). Despite the high abundance of archaeal SRP, no archaeal SRP activity was detected in [35S]sulfate incubations at 80°C. Sulfate reduction was found at 60°C in samples from the untreated field and accompanied by the growth of thermophilic bacterial SRP in batch cultures. Samples from the nitrate-treated field generally lacked SRP activity. These results indicate that (i) Archaeoglobus can be a major player in hot oil reservoirs, and (ii) nitrate may act in souring control—not only by inhibiting SRP, but also by changing the overall community structure, including the stimulation of competitive nitrate reducers.

Problems Caused by Microbes and Treatment Strategies The Effect of Nitrate Injection in Oil Reservoirs – Experience with Nitrate Injection in the Halfdan Oilfield

This chapter deals with the use of nitrate injection for reservoir souring mitigation in an oilfield with seawater injection in the Danish sector of the North Sea. Nitrate impacts on the activity of sulphate-reducing bacteria (SRB) and biofilm redox potential (Larsen et al., 2007), as a result of which corrosion due to SRB activity will be reduced, souring inhibited and previously formed sulphide removed (Larsen, 2002). One important aspect is that the microbiological reduction of nitrate provides approximately three times more energy to SRB than the reduction of sulphate. Therefore, when both nitrate and sulphate are present, nitrate becomes the preferred electron acceptor and SRB capable of growing on nitrate will dominate. Nitrate provides a competitive advantage to nitrate-utilising bacteria (indicated by the general acronym NUB) during competition for available carbon sources as the NUB are capable of much faster growth than SRB.

How Many Microorganisms Are Present? Techniques for Enumerating Microorganisms in Oilfields

The different techniques that exist for enumerating microorganisms will often yield very different results when applied to oilfield samples. For example, enumeration of sulphate-reducing bacteria (SRB) by cultivation may fail to find any microorganisms in samples for which molecular microbiological methods (MMM) indicate levels of thousands or even millions per millilitre or gram. Therefore, it is important to realise the limitations and advantages of the different techniques available to the industry and to take them into account when interpreting data. In oil systems, the most widely used techniques for quantification are based either on culturing, epifluorescence microscopy, or quantitative PCR. In complex samples in which live, inactive, and dead cells are present together with cell material in various states of decomposition, each of these three methodologies enumerates a different subset of microorganisms (Fig. 10.1).

Problems Caused by Microbes and Treatment Strategies: Rapid Diagnostics of Microbiologically Influenced Corrosion (MIC) in Oilfield Systems with a DNA-Based Test Kit

In the past, many operators have encountered failures due to MIC in pipelines and topside facilities contaminated with sulphate-reducing bacteria (SRB). In some cases, severe pitting has resulted in flow lines being either abandoned or replaced (Davies and Scott, 2006). However, there are reports of little or no significant MIC in some systems, despite an apparent significant contamination with SRB (Maxwell, 2006). As most bacterial counts were conducted using serial dilution techniques such as the most probable number (MPN) technique selective enumeration of SRB strains (depending on the type of growth medium used) will inevitably be conducted. Therefore, high bacterial numbers derived from cultivation-based techniques do not necessarily correlate to high SRB numbers causing MIC in the production system (Larsen et al., 2005). In addition, MIC can be caused by other microbes such as sulphate-reducing archaea (SRA), methanogens and fermentative microbes (Larsen et al., 2008, 2009). Also most samples taken by the oil industry are water samples. However, the majority of microbial activity takes place in biofilms that attach to pipeline walls, well tubing and on the inside of topside facilities.

Problems Caused by Microbes and Treatment Strategies: Monitoring Microbial Responses to Biocides; Bioassays – A Concept to Test the Effect of Biocides on both Archaea and Bacteria in Oilfield Systems

The oil and gas industry seeks to reduce the costs of oil and gas production and to minimise the risks of the operation, i.e. to have a high degree of safety for the personnel and protection of the environment. This imposes considerable demands on corrosion inhibition technologies and chemical management. The oil industry has traditionally used cultivation-based methods for microbiological surveillance of oil production facilities to monitor microbiologically influenced corrosion (MIC) risk (Sooknah et al., 2007). However, studies show that it is only possible to cultivate less than 10% of all viable microorganisms and that the population characteristics in a sample may change during the cultivation steps (Maxwell et al., 2004). Therefore, it is obvious that alternative methods are needed that can detect all the microorganisms related to MIC in a sample independent of the cultivation method.

Which Microbial Communities Are Present? Using Fluorescence In Situ Hybridisation (FISH): Microscopic Techniques for Enumeration of Troublesome Microorganisms in Oil and Fuel Samples

Enumeration of microbes involved in souring of oil fields and microbiologically influenced corrosion (MIC) with culture-based methods, usually yield inadequate and contradictory results. Any cultivation step will almost certainly alter the population structure of the sample and thus the results of cultivation analysis are not a good basis for mitigation decisions. The need for methods that are cultivation independent has over the past 10 years facilitated the development of several analytical methods for determination of bacterial identity, quantity, and to some extent function, applied directly to samples of the native population. In this chapter, we demonstrate the features and benefits of applying microscopic techniques to a situation often encountered in the oil and petroleum industry: Control of microbial growth in fuel storage tanks. The methods described in this chapter will focus on direct counts of specific groups of microorganisms with microscopy and these are based on the detection of genetic material and not on culturing.