Kenneth N. Timmis – författare

The development of powerful new techniques and refmements of tech­ niques in molecular genetics in recent years, and the surge in interest in biotechnology based on genetic methods, have heralded a new golden age in molecular genetics, and stimulated in diverse disciplines much interest in the technologies themselves and their potential uses in basic and applied biomedical sciences. Although some excellent specialist laboratory manuals (especially the Cold Spring Harbor Laboratory manuals by I. H. Miller; R. W. Davies et al. ; and T. Maniatis et al. ) on certain chapters of molecular genetics exist, no general text that covers a broad spectrum of the sub­ ject has thus far been published. The purpose of this manual is to pre­ sent most, though of necessity not all of the important methods of molecular genetics, in a series of simple experiments, many of which can be readily accomplished by the microbiologist, biochemist or biotechnologist that has had only limited exposure to genetics. The remainder of the experiments require either greater familiarity with the subject, or guidance by someone with such experience. The book should, therefore, not only enable individuals to acquire new proce­ dures for ongoing projects, but also serve as a basis for the teaching of molecular genetic techniques in formal predoctoral and postdoctoral laboratory courses.

This Volume describes methods for cultivating hydrocarbon-producing and -consuming microbes, covering compounds in a range of states – gaseous (e.g. methane), liquid (e.g. alkanes of intermediate molecular weight) and solid (e.g. many PAHs and asphaltene). It also examines the cultivation of aerobic and anaerobic hydrocarbon degraders using a range of electron acceptors (e.g. oxygen, nitrate, sulphate, metals, (per)chlorate), and a separate chapter is devoted to explaining the cultivation of methanogens. Special attention is given to: high-throughput cultivation, growing microbes as biofilms, and cultivating fastidious microbes, as well as the preservation of microbial pure cultures and consortia. Accordingly, this Volume will be of value to anyone embarking on the selective enrichment and cultivation of novel microorganisms.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume presents protocols for systems and synthetic biology applications in the field of hydrocarbon and lipid microbiology. It complements another Volume that describes generic protocols for wet experimental and computer-based systems and synthetic biology studies. The protocols in this Volume demonstrate how to employ systems and synthetic biology approaches in the design of microbes for the production of esters, isoprenoids, hydrophobic polymers, rhamnolipid biosurfactant, and peptide antimicrobial and thioether-stabilised molecules. Also presented is a protocol for the engineering of transcription factor-based biosensors for intracellular products, and another for the creation of a synthetic hydroxylase with novel activity for the selective oxyfunctionalisation of linear alkanes.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivativesand lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume presents relevant single-cell and single-molecule approaches in the study of microbes producing and utilizing hydrocarbons and lipids. While generically applicable for all microorganisms, the approaches described are, wherever possible, adapted to the field of study of hydrocarbon and lipid microbiology. The methods include basic procedures for isolating single cells by means of microfluidics and flow cytometry, and their cultivation in arrays as pure clones; for isolating, amplifying and sequencing single-cell genomes and transcriptomes; and for analysing single-cell metabolomes by means of Raman spectroscopy. Single-molecule approaches include the use of protein:fluorescent dye fusions for protein localization and methods for the production of cell division protostructures and lipid monolayers. Methods for the functional analysis of single cells include detection of metabolically active (protein-synthesizing) cells in environmental samples by bioorthogonal non-canonical amino acid tagging, Raman spectroscopy combined with stable isotope labelling and fluorescent in situ hybridisation, and visualization of single cells participating in gene transfer activity. Lastly, protocols are presented for single-cell biotechnological applications, including biofuel production.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume presents key microscopy and imaging methods for revealing the structure and ultrastructure of environmental and experimental samples, of microbial communities and cultures, and of individual cells. Method adaptations that specifically address problems concerning the hydrophobic components of samples are highlighted and discussed. The methods described range from electron microscopy and light and fluorescence microscopy, to confocal laser-scanning microscopy, and include experimental set-ups for the analysis of interfacial processes like microbial growth and activities at hydrocarbon:water interfaces, biofilms and microbe:mineral interfaces. Three forms of fluorescence in situ hybridization - CARD-FISH, MAR-FISH and Two-pass TSA-FISH - are described for the ecophysiological analysis of functionally active microbes in samples. The methods presented will enable readers to obtain an ultrastructural picture of, and identify the key functional microbes in, samples under investigation. This in turn will constitute a key framework for the interpretation of information from other experimental approaches, such as physicochemical analyses and genomic investigations.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to respondingto these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume provides protocols for the biochemical analysis of hydrocarbon- and lipid-relevant products, cell components and activities of microbes that interact with hydrophobic compounds. They include methods for the extraction, purification and characterisation of surface tension-reducing bioemulsifiers and biosurfactants that increase the surface area and hence bioavailability of hydrophobic substrates. Protocols for the isolation and biochemical analysis of lipids and polyhydroxyalkanoates, food storage products made during nutrient abundance that represent important biotechnological products, are presented. The extraction of membrane lipid rafts, sub-organelles that fulfil important functional roles for the cell membrane, and the isolation and characterisation of membrane phospholipid biomarkers, are also described. The purification and characterisation of integral membrane hydrocarbon-oxidising enzymes are addressed. Lastly, two generic methods for the genetic analysis of catabolic pathways and analysis of ligand binding are presented.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume features protocols for investigating the hydrocarbon- and lipid-specific activities of microbes. They include methods for studying chemotaxis, the colonisation of hydrocarbon surfaces, hydrocarbon uptake, respiration, nitrogen fixation, sulphate reduction, membrane stabilisation through cis-trans isomerisation of membrane fatty acids, and the production of biosurfactants and biopolymers in response to the presence of hydrocarbons. A protocol for studying the ability of microbes to control the concentration of hydrocarbons in their aqueous environment is also described, and phenotyping methods to reveal microbes’ more general metabolic activities are presented. Several protocols for investigating acid production in connection with oil souring and biocorrosion by microbes in oil well, oil transportation and storage settings are presented. Lastly, protocols for measuring methanogenesis, as an example of microbial hydrocarbon production, are described.

This Volume covers protocols for in-silico approaches to hydrocarbon microbiology, including the selection and use of appropriate statistical tools for experimental design replication, data analysis, and computer-assisted approaches to data storage, management and utilisation. The application of algorithms to analyse the composition and function of microbial communities is presented, as are prediction tools for biodegradation and protein interactions. The basics of a major open-source programming language, Python, are explained. Protocols for calculating reaction kinetics and thermodynamics are presented, and modelling the environmental fate of hydrocarbons during bioremediation is explained. With the exception of molecular biology studies of molecular interactions, the use of statistics is absolutely essential for both experimental design and data analysis in microbiological research, and indeed in the biomedical sciences in general. Moreover, studies of highly varying systems call for the modelling and/or application of theoretical frameworks. Thus, while two protocols in this Volume are specific to hydrocarbon microbiology, the others are generic, and as such will be of use to researchers investigating a broad range of topics in microbiology and the biomedical sciences in general.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide rangeof microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume addresses the pros and cons of oligonucleotide probes, primers and primer combinations, and importantly considers how to design the best tools for the microbial taxa and/or processes being investigated. Individual chapters focus on the design of primers targeting genes that code for enzymes associated with the following functions: degradation of aromatic, aliphatic and chlorinated hydrocarbons under aerobic and anaerobic conditions, methanogenesis, methane oxidation, and the nitrogen cycle.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume presents generic protocols for wet experimental and computer-based systems and synthetic biology approaches relevant to the field of hydrocarbon and lipid microbiology. It complements a second Volume that describes protocols for systems and synthetic biology applications. The wet experimental tools presented in this Volume include protocols for the standardisation of transcriptional measurements, application of uracil excision-based DNA editing for, inter alia, multi-gene assembly, the use of fluxomics to optimise “reducing power availability”, and the incorporation of non-canonical amino acids into proteins for optimisation of activities. Phenome-ing microbes, using a combination of RNA-seq and bioinformatic algorithms, is presented, as is an illustration, using methylotrophs as an example, of how the different key omics approaches constitute a pipeline for functional analysis, acquisition of a systems overview, and metabolic optimisation. Complementary computational tools that are presented include protocols for probing the genome architecture of regulatory networks, genome-scale metabolic reconstruction, and bioinformatic approaches to guide metabolic engineering. The Volume also includes an overview of how synthetic biology approaches can be used to improve biocontainment. Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume presents a comprehensive series of generic protocols for the genetic and genomic analysis of prokaryotic isolates. Genetic methods for functional analyses employ the latest cloning vectors, gene fusion methods and transposon mutagenesis systems, as well as systems for introducing protease-cleavage sequences into permissive sites in proteins under investigation. Genomic methods described include protocols for transcriptomics, shotgun proteomics, interactomics, metabolic profiling, and lipidomics. Bioinformatic tools for genome annotation, transcriptome display and the integration of transcriptomic data into genome-scale metabolic reconstructions are described. Protocols for 13C-based metabolic flux determinations and analysis of the hierarchical and metabolic regulation of fluxes through pathways are included. The Volume thus enables investigators to functionally analyse an isolate over the entire cellular range spanning the gene, the genome, the transcript repertoire, the proteome, the interactome, the metabolic network with its nodes and their regulatory hierarchies, and the metabolic fluxes and their physiological controls.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume presents protocols for investigating the genetic, metabolic and ecological potential and functional analysis of microbial communities. Methods are described for the creation and bioinformatic assessment of metagenomic and metatranscriptomic libraries, and for metaproteomic analyses, which provide important insights into the metabolic potential and interactions of community members. These in turn lead to specific hypotheses concerning the functional contributions of individual populations in the community, which may be investigated by the stable isotope probing approaches described in this Volume, making it possible to identify those community members primarily responsible for particular functions. Methods for the direct extraction of proteins from environmental samples for sequencing and activity tests are presented, providing a broad overview of prevailing metabolic activities and of the types of microbe involved in them. Protocols for the analysis of nutrient flow through microbial communities and for the modelling of dynamic physiological interactions in communities are also provided. Lastly, the book presents a protocol for the quantitative assessment of permissiveness for the transfer of conjugative plasmids, important agents of physiological change and evolution in microbial communities.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume presents methods for quantifying microbial populations and characterising microbial communities by extracting and analysing biomarkers such as RNA, DNA and lipids. The chapters cover a wide range of topics, including: cell separation from oil-rich environments, enumeration of hydrocarbon degraders and sulphate reducers using most-probable-number techniques, and quantification by means of real-time PCR. A variety of molecular methods are described for microbial community profiling, such as phospholipid fatty acid analysis, DGGE, T-RFLP and SSCP. One chapter examines high-throughput sequencing, and provides important information on the associated procedures required for thorough data analysis. A further chapter is devoted to the characterisation of protistan communities, while the closing chapter describes multiplex fluorescent antibody microarrays for detecting microbial biomarkers.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume presents methods for analysing and quantifying petroleum, hydrocarbons and lipids, based on their chemical and physical properties as well as their biological effects. It features protocols for extracting hydrocarbons from solid matrices, water and air, and a dedicated chapter focusing on volatile organic compounds. Several approaches for separating and detecting diverse classes of hydrocarbons and lipids are described, including: (tandem) gas chromatography (GC) coupled with mass spectrometry (MS) or flame-ionisation detection, Fourier-transform induction-coupled-resonance MS, and fluorescence-based techniques. The book details high-performance liquid chromatography MS for microbial lipids, as well as a combination of techniques for naphthenic acids. Two chapters focus on quantifying bioavailable hydrocarbon fractions by using cyclodextrin sorbents and bacterial bioreporters, respectively, while a closing chapter explains how compound-specific stable-isotope analysis can be used to measure the fate of hydrocarbons in the environment. Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume describes methods for simulating natural environments by using reproducible and controllable meso- and microcosm experiments to analyse hydrocarbon-degrading microorganisms and to test hypotheses. It presents important aspects of the preparation of experimental set-ups, hydrocarbon application and sampling, and features protocols for experiments with different types of samples, such as biofilms, aquatic systems (e.g. groundwater, streams), and sediments, including oscillating oxic-anoxic mesocosms, as well as for in-situ experimentation in subtidal and deep sediments. Two chapters are dedicated to cultivation under high-pressure conditions, and several chapters include protocols for processing samples for downstream chemical, microbial or activity analyses. Several of the approaches presented are generic and will benefit anyone embarking on designing meso- and microcosm experiments. < Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume presents applications of hydrocarbon microbiology in the context of environmental pollutant degradation, covering pollutants such as petroleum and related wastes (i.e. oil sludge), biofuels, lipid-rich wastes, chlorinated solvents and BTEX, in several environments (marine, soil, groundwater). The approaches presented range from laboratory experiments and treatment in reactors to field applications. Two chapters highlight innovative approaches to address relevant questions in pollutant degradation, such as low environmental concentrations of pollutants, and the biodegradation of complex pollutant mixtures using biofilms. Rather than presenting the applications in the form of protocols, some of the chapters in this Volume include detailed practical information on the opportunities offered by and limitations of the different approaches, providing valuable information for researchers planning to perform bioremediation experiments.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume covers protocols for various applications in hydrocarbon microbiology, including those of interest for industrial processes, biocatalysis, lipid and biofuel production, bioproducts, or the human microbiome. It presents detailed protocols for the functional screening of enzymes acting on greasy molecules (i.e. lipases, esterases), including assays for enantioselective biocatalysts, as well as approaches for protein display technologies. Protocols for improving fuel quality and production of biofuel and lipids in different hosts (bacteria, algae, yeast) are also provided. The production of biogas from organic waste and its fermentation into value-added products such as polyhydroxyalkanoates is covered, as well as an in-vitro model of the gut microbiome for short-chain fatty acid metabolism and microbial diversity analyses. The applications presented are examples of the many potential applications in hydrocarbon and lipid microbiology, and many (i.e. protein-display technologies) will also be of interest in other research fields. Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitationof useful activities of such microbes.

This Volume describes methods for investigating microbes in their natural environment and how to obtain representative samples and preserve them for subsequent analyses. Chapters are arranged according to the environments under investigation, which include: oil reservoirs, fracking fluids, aquifers, coal beds, oil sands and their tailing ponds, lakes, rivers, leaves, polar seas and ice, the sea-surface microlayer, mud flats, microbialites, and deep-sea fauna. A variety of downstream analytical procedures are described, including: nucleic-acid extraction and preparation for high-throughput sequencing, fluorescence in-situ hybridisation, and cultivation of aerobic and anaerobic hydrocarbon-degrading microbes. Though most chapters focus on hydrocarbon-rich environments, many of the approaches used are generic, and as such will be of value to researchers embarking on studies of microbes and their processes in the field.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume describes methods for cultivating hydrocarbon-producing and -consuming microbes, covering compounds in a range of states – gaseous (e.g. methane), liquid (e.g. alkanes of intermediate molecular weight) and solid (e.g. many PAHs and asphaltene). It also examines the cultivation of aerobic and anaerobic hydrocarbon degraders using a range of electron acceptors (e.g. oxygen, nitrate, sulphate, metals, (per)chlorate), and a separate chapter is devoted to explaining the cultivation of methanogens. Special attention is given to: high-throughput cultivation, growing microbes as biofilms, and cultivating fastidious microbes, as well as the preservation of microbial pure cultures and consortia. Accordingly, this Volume will be of value to anyone embarking on the selective enrichment and cultivation of novel microorganisms.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume presents protocols for systems and synthetic biology applications in the field of hydrocarbon and lipid microbiology.

This Volume presents relevant single-cell and single-molecule approaches in the study of microbes producing and utilizing hydrocarbons and lipids. While generically applicable for all microorganisms, the approaches described are, wherever possible, adapted to the field of study of hydrocarbon and lipid microbiology. The methods include basic procedures for isolating single cells by means of microfluidics and flow cytometry, and their cultivation in arrays as pure clones; for isolating, amplifying and sequencing single-cell genomes and transcriptomes; and for analysing single-cell metabolomes by means of Raman spectroscopy. Single-molecule approaches include the use of protein:fluorescent dye fusions for protein localization and methods for the production of cell division protostructures and lipid monolayers. Methods for the functional analysis of single cells include detection of metabolically active (protein-synthesizing) cells in environmental samples by bioorthogonal non-canonical amino acid tagging, Raman spectroscopy combined with stable isotope labelling and fluorescent in situ hybridisation, and visualization of single cells participating in gene transfer activity. Lastly, protocols are presented for single-cell biotechnological applications, including biofuel production.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume presents key microscopy and imaging methods for revealing the structure and ultrastructure of environmental and experimental samples, of microbial communities and cultures, and of individual cells.

This Volume provides protocols for the biochemical analysis of hydrocarbon- and lipid-relevant products, cell components and activities of microbes that interact with hydrophobic compounds.

This Volume features protocols for investigating the hydrocarbon- and lipid-specific activities of microbes. They include methods for studying chemotaxis, the colonisation of hydrocarbon surfaces, hydrocarbon uptake, respiration, nitrogen fixation, sulphate reduction, membrane stabilisation through cis-trans isomerisation of membrane fatty acids, and the production of biosurfactants and biopolymers in response to the presence of hydrocarbons. A protocol for studying the ability of microbes to control the concentration of hydrocarbons in their aqueous environment is also described, and phenotyping methods to reveal microbes’ more general metabolic activities are presented. Several protocols for investigating acid production in connection with oil souring and biocorrosion by microbes in oil well, oil transportation and storage settings are presented. Lastly, protocols for measuring methanogenesis, as an example of microbial hydrocarbon production, are described.

This Volume covers protocols for in-silico approaches to hydrocarbon microbiology, including the selection and use of appropriate statistical tools for experimental design replication, data analysis, and computer-assisted approaches to data storage, management and utilisation. The application of algorithms to analyse the composition and function of microbial communities is presented, as are prediction tools for biodegradation and protein interactions. The basics of a major open-source programming language, Python, are explained. Protocols for calculating reaction kinetics and thermodynamics are presented, and modelling the environmental fate of hydrocarbons during bioremediation is explained. With the exception of molecular biology studies of molecular interactions, the use of statistics is absolutely essential for both experimental design and data analysis in microbiological research, and indeed in the biomedical sciences in general. Moreover, studies of highly varying systems call for the modelling and/or application of theoretical frameworks. Thus, while two protocols in this Volume are specific to hydrocarbon microbiology, the others are generic, and as such will be of use to researchers investigating a broad range of topics in microbiology and the biomedical sciences in general.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide rangeof microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume addresses the pros and cons of oligonucleotide probes, primers and primer combinations, and importantly considers how to design the best tools for the microbial taxa and/or processes being investigated. Individual chapters focus on the design of primers targeting genes that code for enzymes associated with the following functions: degradation of aromatic, aliphatic and chlorinated hydrocarbons under aerobic and anaerobic conditions, methanogenesis, methane oxidation, and the nitrogen cycle.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume presents generic protocols for wet experimental and computer-based systems and synthetic biology approaches relevant to the field of hydrocarbon and lipid microbiology.

This Volume presents a comprehensive series of generic protocols for the genetic and genomic analysis of prokaryotic isolates. Genetic methods for functional analyses employ the latest cloning vectors, gene fusion methods and transposon mutagenesis systems, as well as systems for introducing protease-cleavage sequences into permissive sites in proteins under investigation. Genomic methods described include protocols for transcriptomics, shotgun proteomics, interactomics, metabolic profiling, and lipidomics. Bioinformatic tools for genome annotation, transcriptome display and the integration of transcriptomic data into genome-scale metabolic reconstructions are described. Protocols for 13C-based metabolic flux determinations and analysis of the hierarchical and metabolic regulation of fluxes through pathways are included. The Volume thus enables investigators to functionally analyse an isolate over the entire cellular range spanning the gene, the genome, the transcript repertoire, the proteome, the interactome, the metabolic network with its nodes and their regulatory hierarchies, and the metabolic fluxes and their physiological controls.Hydrocarbon and Lipid Microbiology ProtocolsThere are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

This Volume presents protocols for investigating the genetic, metabolic and ecological potential and functional analysis of microbial communities.