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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 Protocols There 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.

Microbiology. --- Biochemical engineering. --- Biochemical Engineering. --- Microbial biology --- Biology --- Microorganisms --- Bio-process engineering --- Bioprocess engineering --- Biochemistry --- Biotechnology --- Chemical engineering

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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 Protocols There 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.

Microbiology. --- Biochemical engineering. --- Biochemical Engineering. --- Microbial biology --- Biology --- Microorganisms --- Bio-process engineering --- Bioprocess engineering --- Biochemistry --- Biotechnology --- Chemical engineering

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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 Protocols There 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.

Microbiology. --- Biochemical engineering. --- Biochemical Engineering. --- Microbial biology --- Biology --- Microorganisms --- Bio-process engineering --- Bioprocess engineering --- Biochemistry --- Biotechnology --- Chemical engineering

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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 Protocols There 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.

Microbiology. --- Biochemical engineering. --- Biochemical Engineering. --- Microbial biology --- Biology --- Microorganisms --- Bio-process engineering --- Bioprocess engineering --- Biochemistry --- Biotechnology --- Chemical engineering

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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 Protocols There 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.

Microbiology. --- Biochemical engineering. --- Biochemical Engineering. --- Microbial biology --- Biology --- Microorganisms --- Bio-process engineering --- Bioprocess engineering --- Biochemistry --- Biotechnology --- Chemical engineering