Caister Academic Press

Applications of Molecular Microbiological Methods

"A must for scientists in oil field companies" (Fungal Diversity)
Publisher: Caister Academic Press
Edited by: Torben L. Skovhus, Sean M. Caffrey and Casey R.J. Hubert
Det Norske Veritas (DNV), Bergen, Norway; Genome Alberta, Calgary, Canada; Newcastle University, Newcastle, UK; respectively
Pages: xii + 214
Publication date: March 2014
ISBN: 978-1-908230-31-7
Price: GB £199 or US $319Buy book or Buy online
Publication date: January 2014
ISBN: 978-1-908230-69-0
Price: US $319Buy ebook

Innovative, constructive and continually evolving technologies are propelling microbiology into an exciting new era. This new era will witness the harnessing and control of complex microbial communities in a huge variety of applications in the industrial, medical and environmental spheres. State-of-the-art tools are being developed and utilized to analyse the molecules that microorganisms possess and generate, including DNA, RNA, proteins, lipids and cellular metabolites.

This book, written by international experts in the field, presents emerging molecular methods that allow the diversity of a microbial community to be surveyed and its functions to be investigated. The first part of the book provides examples of the application of molecular microbiological methods in various industrial applications. Part two deals with the identification of microorganisms in medical settings while the third part presents case studies that use molecular methods to assess the structure and function of microbial communities in natural environments. The fourth part of the book describes in greater detail the methods and technologies featured in the preceding case studies including metagenomics, stable isotope probing, fluorescence in situ hybridization, quantitative PCR, reverse transcription PCR and single cell methods. These detailed descriptions enable readers to evaluate the applicability of various tools for approaching questions and case studies of their own.

This practical, authoritative and up-to-date volume is a valuable resource for anyone applying or developing molecular methods in any area of microbiology and is a recommended acquisition for all microbiology laboratories.


"This book enlightens the readers with recent and current molecular techniques with case studies which can be used to resolve the challenges encountered in their field of microbiology ... A valuable guide for molecular microbiologists and molecular studies based laboratories. A must for scientists in oil field companies research and development and for quality control sections in other industries." from Fungal Diversity (December 2014)

Table of contents
1. Molecular Methods in Microbiologically Influenced Corrosion: Research, Monitoring and Control
Gerard Muyzer and Florence Marty
Microbiologically Influenced Corrosion (MIC) is an enormous problem in different industrial sectors with large economical consequences. Although research has been performed on microorganisms associated with MIC, the causative microbes have not been identified yet. Traditional microbiological techniques have been used, but they only detect 1% of the microbes in nature and so are not suited for this purpose. Molecular methods, based on DNA and RNA, are more efficient to characterize microbial communities in different environments, as they are sensitive and reliable, and not dependent on cultivation. In this chapter we give an overview of nucleic acid-based molecular methods that have been used to study microbial communities associated with MIC and give an outlook on the use of novel, state-of-the art methods that might be applied in the future.
2. Using the Power of Molecular Microbiological Methods in Oilfield Corrosion Management to Diagnose MIC
Victor V. Keasler and Indranil Chatterjee
It is well established that microbes present in oilfield systems can cause several problems for operators such as hydrogen sulfide (H2S) production, microbiologically influenced corrosion (MIC), and/or biofouling. However, it is still being debated as to exactly what types of organisms actually cause these problems and whether their presence alone is enough to prove that the associated problems are going to be encountered. This chapter highlights two different case histories where microbes were previously believed to be of minimal risk based on culture-based enumeration. However, molecular characterization revealed elevated microbial numbers as well as a potential high risk species in both systems as the predominant sessile organism and a potential significant asset integrity risk. Interestingly, the outcome with regards to localized corrosion was very different between the two systems. This chapter is a reminder that the data obtained through molecular analysis is most valuable when it is correlated back to a system key performance indicator (KPI) to enable smart decisions.
3. Next Generation Sequencing Approach to Understand Pipeline Biocorrosion
Hyung Soo Park, Jaspreet Mand, Thomas R. Jack and Gerrit Voordouw
Use of pyrotag sequencing to determine microbial community compositions associated with corrosion in a pipeline system handling brackish water has shown that different populations are present in different parts of a single pipeline and treatment system. Possible factors influencing the composition of the microbial population include the source of water, flow, time of sampling, mode of growth (planktonic or sessile) and addition of treatment chemicals. Detailed examination of the populations indicated that methanogenic organisms may be responsible for MIC in the upstream water gathering system but downstream of the injection point for sodium bisulfite (an oxygen scavenger) Deltaproteobacteria are more likely the cause of MIC. The mechanism of attack is not necessarily restricted to the widely recognized action of SRB but may also be due to elemental sulfur formed by Desulfocapsa through the disproportionation of bisulfite. These novel insights could not have been obtained through the traditional assay methods currently used in the management of MIC and indicate the promise of molecular methods in future MIC investigations.
4. Molecular Microbiological Methods Applied on Ship Ballast Tank Samples
Anne Heyer, Fraddry D'Souza, Arjan Mol and Hans de Wit
Microbiologically influenced corrosion of steel is a serious problem in the marine environment including offshore and shipping industries. The uses of molecular techniques and electrochemical techniques for evaluating microbial corrosion effects have continued to gain importance. This chapter summarizes a large-scale approach of a simultaneous investigation of corrosion and microbial community within a practical ship ballast tank environment. The corrosion behaviour of ASTM A131 steel, grade EH36 exposed to natural seawater was investigated at two different height levels of the ship ballast tank-sediment and immersion zone. Biofilm samples were scraped from exposed corrosion coupons and subjected to DGGE of PCR-amplified 16S rRNA genes and sequence analysis to characterize the microbial community developed on the sidewall of ship ballast tanks as well as the water phase. In parallel, corrosion was monitored by open circuit potential and linear polarization resistance measurements, indicating a difference in corrosion behaviour at sediment and immersion zone and related to the variation in bacterial community changes. This chapter demonstrates the problem of attached biofilms in the corrosion of ship ballast tanks, which is characterized by a combination of electrochemical and molecular tools.
5. Molecular Characterization of Microbial Communities Associated with Accelerated Low Water Corrosion (ALWC) on European Harbor Structures
Florence Marty, Mark van Loosdrecht and Gerard Muyzer
The microbial communities associated with different corrosion deposit layers retrieved from a harbour steel structure affected by ALWC (Accelerated Low Water Corrosion) were determined by 16S rDNA PCR-DGGE analysis. Comparative analysis of populations associated with ALWC layers and NLWC (Normal Low Water Corrosion) layers evidenced clear differences in the structure and composition of the communities. Dominant phylotypes related to sulphate-reducing bacteria pertaining to Desulfobacteraceae, Desulfobulbaceae and Desulfovibrionaceae were identified in both type of deposits (fraction of 52% and 44% of the total NLWC and ALWC communities, respectively). Phylotypes related to sulphur-oxidizing bacteria belonging to Alpha-, Gamma- and Epsilonproteobacteria were unique to ALWC deposits, while phylotypes related to oxygenic photosynthetic microorganisms (cyanobacteria, diatoms) were only retrieved in NLWC deposits. This suggested that biologically-mediated sulphur cycle was the dominant process within ALWC deposits vs. chemically-mediated sulphur cycle within NLWC deposits. Since most of the members identified are heterotrophs, it is speculated that organic carbon may be available through water pollution and/or proliferation of photosynthetic biomass. Differences in oxygen concentration between ALWC and NLWC areas due to photosynthetic activities in NLWC may be an important factor contributing to the acceleration of corrosion by the mechanism of differential aeration.
6. Application of the qPCR Technique for SRB Quantification in Samples from the Oil and Gas Industries
Mariana Galvão and Márcia Lutterbach
Microorganisms can grow in many different types of fuels and industrial facilities. Microorganisms may grow in the presence of oxygen (aerobic conditions) or in its absence (anaerobic conditions), feeding primarily on hydrocarbon fuel, minerals and other impurities in the water. In practice, the bottoms of fuel storage tanks or even tanks of buses and trucks contain enough water to allow for significant microbial growth. Microbial activity leads to the production of biomass (fouling), which can be deposited at the bottom of the tank. Moreover, the microbial oxidation of hydrocarbons produces corrosive metabolites, such as organic and inorganic acids. Biomass, metabolic products and corrosion products result in problems such as filter and pipeline clogging, production of emulsions, lowered fuel quality and corrosion of metal tanks. The detection and quantification of microorganisms in industrial samples has traditionally been based on culturing techniques such as the most probable number (MPN) method. However, the slow growth of strictly anaerobic sulfate-reducing bacteria (SRB) complicates the rapid detection and isolation of these microorganisms in culture media. In some cases, the extended time for the detection of microorganisms delays urgently required preventive and corrective actions, thus aggravating the corrosive process. Here we present the results of SRB quantification in different samples from the oil and gas industry analyzed by qPCR.
7. Molecular Microbiological Methods Applied to Microbial Methane Production in Oil Reservoirs, Coal Beds, and Shales
Lisa M. Gieg and Karen Budwill
Research in recent decades has revealed that microbial communities thrive in subsurface fossil energy deposits such as crude oil, coal, and shale reservoirs. While numerous microbes have been cultivated from such environments, molecular microbiology methods (MMM) such as 16S rRNA gene sequencing has revealed an even broader microbial diversity that includes organisms associated with aerobic, anaerobic, and fermentative metabolic abilities. Other MMM, such as functional gene analysis and metagenomics, are starting to be applied to samples from subsurface fossil energy environments to more fully describe or predict the functions associated with the extant microbes. Such information can help inform the development of sustainable energy recovery technologies by stimulating in situ biomethane production. In this chapter, we briefly overview the key features of crude oil reservoirs, coal seams, and shale deposits that support methanogenic communities, the MMM that are being used to describe the associated microbial members and functions, and highlight two case studies as examples wherein MMM were used for microbial community analysis in fossil energy reservoirs.
8. Characterization of Bacterial Communities in Suspected Prosthetic Joint Infections
Yijuan Xu, Henrik C. Schønheyder, Lone Heimann Larsen, Mogens Berg Laursen, Garth D. Ehrlich, Jan Lorenzen, Per H. Nielsen, Trine R. Thomsen and the PRIS Study Group
Steadily increasing numbers of persons have received prosthetic joints to help them relieve pain and restore function associated with damaged joints. Infection of joint prostheses occurs rarely but represents very serious complication. Diagnosis of prosthetic joint infections (PJI) remains difficult. Microbiological culture methods have hitherto been regarded as the reference standard. This case study was designed to assess which molecular tools can most effectively be implemented in a new diagnostic strategy for diagnosing PJIs. We evaluated a 78-year old man with an acute infection following total hip replacement with microbiological culture methods and molecular methods including: Sanger sequencing of cloned 16S rDNA, 454 Lifesciences-based 16S rDNA pyrosequencing, Ibis T5000 biosensor analysis, and bacterial 16S fluorescence in situ hybridization (FISH). A preoperative joint aspirate was evaluated by culture methods and 16S rRNA gene PCR which both revealed the same microorganism, Streptococcus dysgalactiae. However, most likely due to the start of antibiotic therapy, perioperative surgical samples obtained two days later were culture-negative, but remained positive by all applied molecular methods. This study suggests that culture-independent molecular methods can be useful for clinical microbiological diagnosis, and it is important for all these methods to achieve short turnaround time, clinical validation and cost-effectiveness to become feasible for diagnostic use.
9. Using the Core and Supra Genomes to Determine Diversity and Natural Proclivities among Bacterial Strains
Laura Nistico, Josh Earl, Luisa Hiller, Azad Ahmed, Adam Retchless, Benjamin Janto, J. William Costerton, Fen Z. Hu and Garth D. Ehrlich
The realization at the beginning of the current millennium that there are very substantial differences in gene content among the component strains of bacterial species together with the observation that there exists profound phenotypic heterogeneity amongst these strains led directly to the development of the field of comparative bacterial genomics, and the concepts of the core and supra (pan) genomes. The core genome is composed of the set of genes shared by all members of a given species (or other taxonomic grouping), and the supragenome is the set of all genes contained by the same grouping. Those genes not present in the core genome, but present in the supragenome, are referred to as distributed (accessory) genes, and it is this last group which is responsible for much of the intraspecies heterogeneity. In the current work we provide comparative genomic evidence for both the core genome hypothesis and the distributed genome hypothesis, and describe a testable means to determine if a strain or particular group of strains belongs within an extant genomically-defined species (or other taxonomic) grouping.
10. qPCR and RT-qPCR Applied to Methane-cycling Archaea in the Marine Sediments of the White Oak River Estuary
Karen G. Lloyd
Quantitative PCR (qPCR) and reverse transcribed quantitative PCR (RT-qPCR) are powerful tools for quantifying the DNA and RNA, respectively, of specific groups of microorganisms in marine sediments. They can also be used to identify potential environmental functions of uncultured microorganisms by correlating microbial abundance and activity to geochemistry. An example of this usage is the present case study of the White Oak River estuary. Here uncultured anaerobic methane-oxidizing archaea (ANME) were shown to change in abundance (inferred from DNA quantified by qPCR) and activity (inferred from RNA quantified by RT-qPCR) in response to methane-oxidizing conditions, as well as methanogenic conditions. This non-culture-based method therefore raises the hypothesis that ANME archaea are capable of reversing their metabolism to methanogenesis. qPCR and RT-qPCR in marine sediments require careful checking to ensure maximal DNA and RNA extraction efficiencies, and minimial co-extraction of PCR inhibiting substances. If these pitfalls are avoided, qPCR and RT-qPCR can be used in other applications to develop hypotheses about the physiology of uncultured microorganisms in environmental samples.
11. The Metabolic Function of Uncultured Microorganisms Assessed Through Single Cell Genomic Techniques
Dorthe Groth Petersen
Most microorganisms resist cultivation, which prevent detailed descriptions of their physiological properties. Recent advances in single cell genomics have begun to allow analysis of the genetic content of single cells from hitherto uncultured and inaccessible lineages. This chapter focuses on methods for analyzing single microbial cells such as whole genome amplification, fluorescent activated cell sorting and optical tweezer technologies, and highlights case studies based on these methods. Furthermore, methods for combining isotope labeling and molecular identity within single cells such as MAR-FISH, Nano-SIMS and Raman spectroscopy analyses are also introduced.
12. Assessing Microbial Activity and Degradation Pathways in the Environment by Measuring Naturally Occurring Stable Isotopes in Organic Compounds
Martin Elsner, Christian Griebler, Tillmann Lueders and Rainer U. Meckenstock
Assessing microbial degradation of organic compounds in the environment currently faces two challenges; (a) detecting and quantifying not only microbial populations, but also in situ activity in the environment; (b) transferring mechanistic insight into (bio)chemical transformation mechanisms from the laboratory to the environment. Compound-specific isotope analysis (CSIA) measures stable isotope ratios (e.g. 13C/12C, 2H/1H, 15N/14N, 18O/16O, 34S/32S) in single target compounds (e.g. anthropogenic pollutants, sulfate). Since light isotopes (e.g. 12C) are typically degraded faster, heavy isotopes (e.g. 13C) become enriched in the remaining target compound. CSIA can analyze this enrichment as a way to demonstrate and quantify in situ degradation. Since analysis is conduced at natural isotopic abundance, no label is necessary and information can be obtained directly in natural systems. This book chapter gives three illustrative examples of the power of CSIA. (i) High resolution sampling at a hydrocarbon-contaminated aquifer can relate maximal abundances in degrader genes (microbial potential) to steep gradients in 13C/12C isotope values (evidence of activity). (ii) Isotope analysis of multiple elements in the pesticide atrazine is able to elucidate the underlying bacterial transformation mechanism. (iii) Isotope fractionation can serve as a novel concept to demonstrate ongoing natural attenuation across scales at contaminated sites.
13. Metagenomic Analysis of Microbial Communities and Beyond
Lars Schreiber
From small clone libraries to large next-generation sequencing datasets - the field of community genomics or metagenomics has developed tremendously within the last years. This chapter will summarize some of these developments and will also highlight pitfalls of current metagenomic analyses. It will illustrate the general workflow of a metagenomic study and introduce the three different metagenomic approaches: (1) the random shotgun approach that focusses on the metagenome as a whole, (2) the targeted approach that focuses on metagenomic amplicon sequences, and (3) the function-driven approach that uses heterologous expression of metagenomic DNA fragments to discover novel metabolic functions. Lastly, the chapter will shortly discuss the meta-analysis of gene expression of microbial communities, more precisely metatranscriptomics and metaproteomics.
14. Stable Isotope Probing in Environmental Microbiology Studies
S. Jane Fowler and Lisa M. Gieg
Traditional microbiological methods involving the isolation of microbes from the environment in pure culture have been shown to be ineffective at accessing the majority of microbial diversity. Methods that allow the study of microbes in their native environment or in mixed cultures have been gaining in popularity in recent years. Stable isotope probing is a method that allows the identification of the taxonomic groups that are actively metabolizing a substrate, typically in enriched cultures but also in in situ communities. This technique involves incubations with an isotopically- labelled substrate (e.g. 13C-labelled) during which time the isotope label is incorporated into the biomolecules of organisms actively degrading the substrate. This is followed by the extraction and analysis of these biomolecules in order to identify the organisms incorporating the isotope label. Stable isotope probing has been refined for the analysis of all classes of biomolecules, and has been used to identify the key microbes involved in a number of biological processes.
15. Fluorescence in situ Hybridization (FISH) for the Identification and Quantification of Microorganisms
Cristina Moraru and Elke Allers
Fluorescence in situ hybridization (FISH) targeting ribosomal RNA (rRNA) has become a standard method in molecular ecology. FISH allows identification and quantification of microorganisms. The main methodological variations are the use of fluorochrome labeled probes (monolabeled FISH, DOPE-FISH) or Horse Radish Peroxidase (HRP) labeled probes, known as Catalyzed Reporter Deposition-FISH (or CARD-FISH). The first section of this chapter provides an overview of (i) the two methodological variations, (ii) sample evaluation by microscopy and image analysis, and (iii) probe design and principles of specific hybridization. The second section discusses pros and cons of rRNA FISH, including a comparison of the monolabeled and CARD-FISH variations. The final section describes different applications and new developments in rRNA FISH, emphasizing the wide range of questions and samples it can be applied to.
16. Quantitative Real-time Polymerase Chain Reaction (qPCR) Methods for Abundance and Activity Measures
Vibeke Børsholt Rudkjøbing, Tine Yding Wolff and Torben Lund Skovhus
Quantitative real-time polymerase chain reaction (qPCR) is a method by which DNA and RNA target molecules can be analyzed in real-time and quantified. The method has a wide range of applications, and its use has become increasingly popular in different scientific and commercial fields. This method is based on amplification of target molecules in the presence of fluorescent dyes that enable detection. Despite this relatively simple principle there are many options for customizing and optimizing the reaction, both in terms of overall experimental setup and choice of dye and DNA/RNA target. Besides an introduction to the fundamentals of qPCR, this chapter contains a description of theory and strategy and demonstrates how to use qPCR to determine microbial abundance and activity. Abundance measurements are generally performed by targeting DNA and typically include use of standard dilution series in order to obtain quantitative measurements. Conversely, the activity of microorganisms is determined by gene expression profiling, and the target is typically cDNA synthesized from RNA molecules. Such experiments generally do not provide absolute measurements, but instead are performed as a relative quantification, where the measurements of the target gene are related to a reference gene. The qPCR method has many advantages; chiefly it is a culture-independent method that offers great simplicity and flexibility along with a rapid turnaround time of a few hours. Additionally, the method has relatively low instrumentation demands. However, there are some critical limitations and considerations to the method, which are described in the chapter.
17. Investigation of Microorganisms at the Single-Cell Level using Raman Microspectroscopy and Nanometer-scale Secondary Ion Mass Spectrometry
Stephanie A. Eichorst and Dagmar Woebken
The field of single-cell ecophysiology has taken an exciting turn with the introduction of two powerful techniques, nanometer-scale secondary ion mass spectrometry (NanoSIMS) and Raman microspectroscopy. These techniques allow the investigation of microorganisms and their associated activity at the single-cell level. When combined with stable isotope tracers and/or identification of the targeted cell using fluorescence in situ hybridization (FISH), they have the potential to link the identity of a microorganism with its in situ activity. Raman microspectroscopy detects the scattering of light due to interaction with chemical bonds of cell constituents thereby providing compound specific information, which can also be used for bacterial identification. NanoSIMS permits highly sensitive analysis of multiple elements or isotopes with sub-micrometer spatial resolution, allowing the measurements of microbial activity when used in stable-isotope tracer experiments. In this chapter we present the principle for each technique, discuss their strengths and weaknesses, and document their applicability with particular emphasis on microbial ecology research. The integration of these single-cell techniques in the field of microbial ecology will improve our understanding of the ecophysiology of (novel) microorganisms across a multitude of environments.

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(EAN: 9781908230317 9781908230690 Subjects: [microbiology] [bacteriology] [molecular microbiology] [environmental microbiology] )