Manual of Environmental Microbiology, Fourth Edition

Anaerobes are key players in global cycles of elements and nutrition in natural and anthropologic ecosystems and are also causative agents of human and veterinary diseases. Numerous studies have sought to develop culturing techniques for anaerobes to enable the elucidation of their basic physiology, pathogenic mechanisms, and ecological functions. This chapter will describe a brief history of the development of anaerobic culturing techniques from the historical Hungate technique to techniques and apparatuses commonly used in modern laboratories. In addition, recent progress in anaerobic culturing techniques (e.g., single-cell manipulation and isolation, the six-well plating method, the co-culture method, bioreactor-based enrichment, and in situ/in vivo cultivation) will be described, with several examples of the application of these techniques for the isolation of anaerobes from natural and artificial ecosystems.

The advent of new sophisticated spectroscopic and tomographic techniques arise interest in the study of environmental conditions within microbial habitats on a submicroscopic level. These methods are based on electromagnetic radiation and result in either elemental characterization or structure visualization. Both aspects are relevant for the investigation of microbe-habitat interactions why a correlative detection of microbial cells would be useful. Fluorescence in situ hybridization is an ideal technique to identify and localize microorganisms but requires a cell-detection via fluorescence microscopy which has a limited optical and elemental resolution. Therefore the utilization of nanogold as marker for in situ hybridization approaches is of great potential. Gold labels can be visualized with one of the aforementioned techniques on resolutions beyond light microscopy and allow the identification and localization of single microbial cells in their habitat in situ. The basic principal and potential of this method is described in this chapter giving an overview on the development steps of gold-targeted cell detection as well. Selected results exemplarily show applications in environmental microbiology both via fluorescence microscopy and electron microscopy including elemental mapping.

Immunoassay methods for environmental pathogen and toxin detection are well established with formats including lateral flow devices, standard ELISAs, microarray platforms, and biosensor devices. In general, immunoassays provide rapid assay times relative to other conventional methods such as colony counting and PCR approaches. The development of recombinant antibody technologies to produce antibodies with enhanced binding affinities will lead to immunoassays with better sensitivity, specificity and reproducibility. The development of alternative affinity reagents, such as aptamers, engineered proteins and peptides, will provide a greater repertoire of affinity reagents to develop novel immunoassays. Recently, multiplexed assays for the detection of foodborne and waterborne pathogens and toxins have been developed using planar and bead-based microarray approaches. Because environmental pathogens are mostly present in very low numbers, a highly sensitive detection method is necessary. In addition, real-time detection is requisite. Biosensors have the potential to address both of these requirements. Indeed, biosensors are the fastest growing technology for pathogen detection. Integration of biosensors into environmental and food safety monitoring systems is likely to increase in the coming years potentially leading to the development of novel methods that are capable of providing the necessary sensitivity and assay speed to replace the current standards.

Before environmental microarrays can fulfill their diagnostic promise, it is necessary to define an environmental diagnostic. In this chapter, I propose a definition for an environmental diagnostic (EVD) that is consistent with the FDA's definition and meaning of an in vitro diagnostic (IVD), where the emphasis is on the use of the diagnostic to make a decision and take action relative to the effect of a particular condition on human health. In this context, the underlying microarray technologies, methods of manufacture, and intended use can significantly impact the quality and reliability of an environmental diagnosis. Current and future technology should therefore focus on reducing the variability of environmental microarrays during manufacture and use, so that repeatable results can be obtained independent of the user. Analytical process simplification, perhaps through amplification microarrays described in this chapter, may help achieve the objective of repeatability for independent users, but technology per se will not substitute for a clearly defined intended use, effective product design, and objective verification and validation data. At present, the absence of regulatory oversight for EVDs is both a blessing and a curse. It is therefore expected that future technical solutions for realizing microarray-based EVDs will only come with a consensus biological and regulatory opinion regarding the meaning of environmental nucleic acid signatures relative to the real or perceived risks associated with each intended use.

In this introduction, we briefly survey developments in next-generation sequencing that have been applied to environmental microbiology, consider both major advances and areas where progress has been slow, and highlight chapters in this and other sections of the MEM4 relevant to nucleic acid-based molecular analyses of microbial communities. Experts on various aspects of next-generation sequencing have contributed to this section, including chapters addressing the analysis of microbial communities through the study of (a) ribosomal RNA gene amplicons (Ionescu); (b) shotgun metagenomic sequence data (Lal and Sangwan); and (c) shotgun metatranscriptomic sequence data (Sarode et al.). In addition, this section includes a chapter reviewing functional metagenomics (Marchesi and Morris) and sequencing platforms for environmental microbiologists (Green).

Using metagenomic approaches to interrogate microbial communities is commonly based on sequencing the total DNA extracted from biomass. However, while this approach predominates in the literature, it does not provide an insight into the novel functions within a system and does not provide physical DNA for manipulation. In order to obtain novel functions from a microbial community, functional metagenomics was developed. This approach, which preceded the advent of large-scale sequence based metagenomics coupled with next generation sequencing, revolves around hosting and expressing heterologous DNA in a suitable surrogate host. Coupled with phenotypic screens functional metagenomics provides an alternative approach to obtaining functionally active genes from a microbial system, without the need to culture any organisms.

Analysis of the collective RNA pool from a microbial community - the metatranscriptome - yields valuable information on microbial gene expression patterns and biogeochemical processes in natural environments. Molecular and analytical tools for analyzing metatranscriptomes using high-throughput sequencing have advanced rapidly in recent years and continue to evolve and expand. The technique is increasingly available to individual research projects, even those with a modest budget or lacking an extensive bioinformatics toolkit. A core set of metatranscriptomic practices can now be identified, with key steps including RNA extraction, messenger RNA (mRNA) enrichment, synthesis of complementary DNA (cDNA), shotgun sequencing of cDNA, and bioinformatic analysis of sequence data. This chapter explores key questions that researchers should consider before beginning a metatranscriptomic study and then describes in detail the major steps of a sequencing-based metatranscriptomic analysis, from RNA isolation to functional and taxonomic analysis of sequence data. The questions and methods described here provide an introductory framework for environmental microbiologists interested in using metatranscriptome sequencing to explore microbial community gene expression.

A common assumption for conducting microbiological testing is that the laboratory environment and its equipment are properly operated, cleaned, calibrated, and maintained, that the laboratory is a safe working environment, and that the laboratory has a quality assurance plan that it maintains and adheres to. In order to assure that these requirements are met, a QA program needs to be in place.A goal of the QA program is to give management the opportunity to provide input and take responsibility in the planning, implementation, and assessment stages of the environmental microbiology project. The QA Plan should have sections devoted to laboratory facilities, personnel, and equipment; sampling procedures and handling; and deviations, record keeping and audits.

This is a guidance for the development of an environmental virology laboratory quality assurance document with suggested quality control measures detailed.

Environmental microbiologists collect data, lots of data. However these data are not always the appropriate data to answer the questions the researchers are interested in. In this chapter we discuss what needs to be done to ensure that what you think you are discovering is in fact what the data are saying. We encourage a large amount of statistical thinking prior to the first data point being collected or before the first sample is obtained. Statistical thinking is not generally taught in introductory statistics classes. The nuts and bolts of what will be discussed in the chapter do find their way into statistic classes however not necessarily in a way that prepares scientists for the task of doing science. Here we discuss concepts such as defining the problem, experimental design, the weight of evidence, statistical power, and sources of variation, scope of inference, measurement scale, scale transformation and other topics. We show that environmental microbiology done without careful thinking before, during and after data collection rarely can answer any important question - regardless of how big the spreadsheet is. After reviewing countless papers for over three decades it is our experience that many studies in environmental microbiology are statistically weak and more importantly statistically flawed and that one need read no farther than the methods to decide whether to continue to forward.

This chapter describes options and considerations for choosing water sampling and processing techniques to enable testing for microbes of public health relevance, including pathogens and microbial indicators of fecal contamination. The framework for the discussion draws relationships between investigation goals, conditions and the selection of sampling techniques. Considerations include identifying target microbes, downstream analytical methods, anticipated water quality, acceptable method detection limits, and application of discrete versus composite sampling. Small-volume and large-volume sampling techniques are discussed for application to a wide range of water types, including drinking water, ground water, surface water, recreational water, and marine water. The chapter describes and compares alternative techniques for sample collection and processing for viruses, bacteria, and parasites, as well as identifying techniques that to capture of multiple microbe types. Field sampling techniques are discussed, as well as laboratory-based sample processing techniques to concentrate water samples for analysis. Issues related to sample quality are addressed as they relate to processing inefficiencies and potential for inhibition of analytical procedures.

Environmental microbiology is wrought with difficult to sample environments, not least of which is soil. Soil is a dynamic environment, ripe with changing viral, prokaryotic, and eukaryotic populations, all awaiting assay. Soil sampling, depending on the assay, experiment, or project can vary from simple probes, deep cores, to soil slurry collections. Atop of this, soils must be collected from a statistically sound perspective as well as considerations made for physiological, cultivation, or molecular analysis. This chapter will focus on the use of soil sampling equipment, schemes, assays, and case studies aimed at introducing the reader to the various caveats and potential pitfalls associated with soil sampling. No one chapter can attempt to cover all potential characteristics of a specific soil sampling situation; therefore, this chapter will focus on general trends in microbial soil sampling.

Fecal contamination of freshwaters and drinking waters may result in serious risks to public health that include gastrointestinal and respiratory illnesses, eye and skin infections, many caused by enteric pathogens. The microbiological quality of freshwaters and drinking waters is usually monitored by the detection of traditional indicators that include total and thermotolerant coliforms, Escherichia coli, and Enterococcus spp. Culture methods are usually employed to detect bacterial indicators, but emerging techniques that include the detection of bacteriophages, as well as PCR-based methods amplifying bacterial 16S or 23S rRNA genes also have been developed. Molecular methods targeting indicator bacteria may reduce the time needed to take action to reduce the impact that fecal contamination of freshwaters and drinking waters represent to public health. In freshwaters used for recreation and consumption, identifying the source of the fecal contamination is important in order to reduce or eliminate its impact to pubic health. Microbial Source Tracking (MST) methods have been developed to identify the possible source (e.g. animal vs human) of the fecal contamination and include amplification of nucleic acids of traditional indicator bacteria. While bacterial indicators have successfully been used to protect public health for the last 100 years, and variations on the theme will be in use for decades to come, the target microorganisms would probably need to be revisited, because of the little information we have about their ecology.

Wastewater treatment is one of the most important of societal commitments to the health of human populations. This summary provides an overview of analyzing how well the processes meet the goal of providing discharges that do not degrade the environment nor put the health of populations dwelling in or affected by that environment. Basic processes of waste treatment are illustrated and both aqueous and solid discharges are addressed with the most important treatment processes and the parameters that are sampled to assess their efficacy presented. In common with most biological systems studied, recent advances in molecular, whole genome or metagenome techniques and procedures have allowed great advances in our understanding of how these microbial systems function. Bioassays of enormously complex means of biological treatment are shown in context. New methods of understanding whole-system microbiomes are noted with seminal publications. Older bioassay techniques are explained and the empirical underpinnings of many processes that are only now understood more fully are reviewed.

Enteric viruses are a leading cause of gastroenteritis and they constitute a diverse group. The viruses commonly implicated in gastroenteritis (cases or outbreaks) are classified into the families Picornaviridae (polioviruses, enteroviruses, coxsakieviruses, and echoviruses), Adenoviridae (adenovirus 40 and 41), Caliciviridae (noroviruses, Sapporoviruses,), and Reoviridae (reoviruses). Although enteric virus infections are mainly associated with diarrhea and self-limiting gastroenteritis in healthy humans, they have also been linked to aseptic meningitis, encephalitis, myocarditis and insulin-dependent diabetes.

Currently, about 140 waterborne viruses are known to infect humans. The waterborne viruses not only show wide diversity in the illness (including diarrhea, fever, hepatitis, paralysis, meningitis, respiratory and heart diseases, they also show broad diversity in their size, shape, infection process and replication mechanisms. Most of waterborne enteric viruses have icosahedral capsid; however, the structural feature on the surface of capsids varies among different virus groups. The rotavirus capsid is composed of three concentric protein layers and the outermost layer comprises the proteins VP4 and VP7. The PV4 forms spike like structures which are responsible for virus attachment to host cell. Adenovirus has an icosahedral capsid composed of hexon the major capsid protein. In addition, capsid has penton with elongated fibers projecting out of each penton. The distal ends of these fibers have a globular "knob" which functions as the major attachment site for host cell receptors. In general, the members of enteroviruses and caliciviruses / norovirus have capsid with less pronounced surface features; but members of these groups have significantly different annotation of their capsid.

One of the Millennium Development Goals is to halve, between 1990 and 2015, the proportion of the population that does not have sustainable access to safe drinking water, which would increase the coverage from 76% to 88% ( 1 ). This goal was met in 2010, with 116 countries meeting the target ( 2 ). Since 1990, more than two billion people have gained access to improved drinking water sources, with 89% of the population covered. There were only three countries (Democratic Republic of the Congo, Mozambique, and Papua New Guinea) where less than half the population had access to an improved drinking water source, and 35 countries (26 of which are in sub-Saharan Africa) in which only 50–75% of the population has access to an improved source of drinking water. Although this represents a substantial improvement, more than 700 million people still lack access to improved sources of drinking water, almost half of which are in sub-Saharan Africa ( 2 ). It is predicted that there will still be more than half a billion people without access to an improved drinking water supply by 2015.

Microbiologists have confronted the challenges of sampling and analysis of airborne microorganisms since the early 20th century. Today, the concentration and composition of airborne microorganisms are of interest in various areas such as agricultural and industrial settings, hospitals, home and office environments, and military installations. In all of these applications, the term "bioaerosol" is used to refer to airborne biological particles, such as bacterial cells, fungal spores, viruses, and pollen grains, and to their fragments and by-products. A wide variety of bioaerosol sampling and analysis methods have been used, and new methods are being developed. However, no single sampling method is suitable for the collection and analysis of all types of bioaerosols and no standardized protocols are currently available. Therefore, data from different studies are often difficult to compare because of differences in sampler designs, collection times, airflow rates, collection media and analysis methods. In addition, human exposure limits have not been established for bioaerosols because of the lack of exposure, dose, and response data.

The purpose of this chapter is to present various bioaerosol sampling and analysis methods that would allow facilitating an intelligent selection of instrumentation and techniques. The principles of bioaerosol sampling are presented, followed by a review of traditional and emerging sampling methods and techniques, including the results of performance evaluations of the various sampler types. Equipment calibration and air sampling considerations such as collection times and the number of samples are discussed. The advantages and disadvantages of surface sampling methods are also described.

The study of bioaerosol fate and transport has recently gained more attention due to the recognized importance of both indoor and outdoor events that have been shown to impact applicable populations. In this Chapter current thinking and research are presented in the topic areas of hydrodynamic and kinetic forces affecting fate and transport; environmental condition effects on viability; bioaerosol transport and viability modeling; and microorganism inactivation and repair mechanisms.

Airborne bacteria and their associated immunomodulatory ligands, best exemplified by endotoxin, are present in bioactive concentrations in many occupational and some non-occupational environments. Especially high concentrations are found in agricultural settings and in industrial operations handling or processing wet organic matter. Human exposure to these agents induces a variety of respiratory conditions including asthma, asthma-like syndrome, hypersensitivity pneumonitis, organic dust toxic syndrome and pulmonary infections. Endotoxin is an integral component of the bacterial cell membrane and is well-recognized as a potent inducer of lung inflammation. However, low-dose exposure has also been shown to reduce the potential for development of allergic rhinitis and eosinophilic asthma. This chapter presents a critical examination of current knowledge of concentrations and exposures to airborne bacteria and endotoxin in over twenty-five environments. It describes the resulting burden of disease ascertained through epidemiologic studies conducted worldwide. Included in the chapter are detailed methods for the assessment of exposures to endotoxin. New science relating to occupational exposures to the Archaea are also presented along with molecular methods for their identification and quantitation.

Crop plants are subject to diseases caused by viruses, bacteria, oomycetes, and fungi. For many pathogens the aerobiological pathway is important in disease transmission.

Airborne fungal spores and other disease propagules are frequently present in the atmosphere and pose a constant threat to crops as well as plants in the natural environment. This chapter reviews mechanisms of pathogen dispersal with the focus on wind and rain splash and also discusses both local and long distance transport. Various examples of pathogens from the groups above are described with details on the dispersal phase as well as disease forecasting and aspects of control. For many plant diseases, forecasting models are valuable tools alerting the grower regarding the likelihood of disease spread and when to apply control measures. Development of forecasting models requires an understanding of all stages of dispersal from spore takeoff to transport to deposition within the crop. Knowledge of the influence of meteorological conditions on spore development and the ability of spores to survive long-distance transport are also needed for accurately modeling disease spread. In addition to descriptions of well known plant pathogens, several emerging plant diseases are described along with the potential use of plant pathogens in anti-crop biological warfare

Soil is a reservoir for a diversity of transient human pathogenic viruses and protozoa that include some species with truly edaphic lifestyles. The ability to detect these pathogens in soil has increased in tandem with concerns about the impact of human activity on soil quality. Separation from the sample matrix and concentration in solution free of interfering contaminants are key preliminary steps to the detection and/or identification of specific species, which is increasingly performed with sensitive molecular methods. The present chapter describes procedures applied in the separation of viruses from soil by desorption from soil particles, their concentration and detection/quantification in laboratory-grown mammalian cell cultures using Quantal methods or plaque assays. Methods for extraction of nucleic acids to permit detection of viruses that are presently difficult or impossible due grow in vitro due to a lack of suitable cell lines (noroviruses, astroviruses, sapoviruses, rotaviruses, hepatitis A and E viruses) by polymerase chain reaction (PCR), conventional reverse-transcription PCR (RT-PCR) or real-time PCR and RT-PCR are also presented. The detection of protozoa against the large background microflora and extraneous components in soil requires a series of variable preliminary elution, concentration and purification steps that are determined by the properties of both sample and target protozoa. Classical methods that rely on microscopic examination for detection/identification of Acanthamoeba spp, Entamoeba histolytica, Naegleria fowleri, Giardia spp., Balantidium coli, Toxoplasma gondii, Cyclospora cayetanensis and Cryptosporidium parvum are presented along with alternative molecular approaches based on the recognition of specific antigens or DNA sequences.

Section X of the Manual of Environmental Microbiology addresses the burgeoning area of microbial source tracking (MST), a collection of methodologies and approaches whose aim is to determine the dominant source(s) of fecal contamination of water bodies. Many health- and management-related areas can benefit from MST analyses, including total maximum daily load (TMDL) determinations, beach and water resource management, quantitative microbial risk assessment (QMRA), and epidemiology. This chapter provides a brief overview of the rationale for and theory of MST, followed by chapters that detail MST methodologies and practice. This chapter also suggests future directions for a field that has changed considerably over the last two decades - as it has evolved from an emphasis on building large databases of bacterial phenotypes or genotypes toward the use of culture-independent methods that focus on single genetic targets or microbial community structure. The field is in the process of adopting high-throughput DNA sequencing methods that allow consideration of genomic and metagenomic data to identify host-associated microorganisms and/or patterns in communities that may contribute to accurate identification of fecal pollution sources in the environment.

Human fecal waste is thought to contain the largest number of human pathogens representing the greatest public health risk in comparison to other fecal sources. Human fecal pollution can be introduced into water resources from damaged sewer lines, faulty septic systems, combined sewer overflows, illicit dumping activities, and even recreational bathers themselves. Ensuring public safety and adequate water quality therefore requires methods that can confirm the presence or absence, as well as, determine the concentration human fecal pollution sources. This chapter provides an overview of human-associated fecal source identification methods commonly used to assess water quality. In addition to describing various methods, this chapter will also discuss factors to consider for method selection.

Field study design and implementation are critical to the success of fecal source tracking (FST) studies. Significant advances in the field of microbial and chemical source tracking (MST or CST) provide access to a variety of analytical tools, beyond traditional culture-based measurements of fecal indicator bacteria (FIB), in the identification and apportionment of pollution sources adversely impacting water quality and of public health concern. Execution of investigative studies employing these tools requires a phased approach: defining the questions and desired outcome, site assessment, field sampling/laboratory analysis, confirmatory testing, statistical analysis, interpretation of results, and translation of results into actionable items. A sound study design addresses spatial and temporal variability as well as geographic distribution of markers or target organisms used to develop statistically robust data sets from which sound conclusions are drawn. While FST tools may be cutting edge, and informative in their own right, multiple lines of evidence are necessary to adequately characterize pollutant source, loading, and human exposure risk. Assessments of environmental, meteorological and hydrological parameters may increase accuracy in the assignation of relative contributions (from multiple sources) in complex environments. In the absence of strong correlation between FIB, physical attributes and MST or CST markers, a weight of evidence approach may be used to target human exposure interventions, which may take the form of engineered, naturalized, or educational measures. A multi-barrier approach to protecting public health, cognizant of confounding factors impacting the analytical or remediation process, should be stressed and include stakeholder engagement throughout the process.

An overarching goal of biology is to understand how evolutionary and ecological processes generate and maintain biodiversity. While evolutionary biologists interested in biodiversity tend to focus on the mechanisms controlling rates of evolution and how this influences the phylogenetic relationship among species, ecologists attempt to explain the distribution and abundance of taxa based upon interactions among species and their environment. Recently, a more concerted effort has been made to integrate some of the theoretical and empirical approaches from the fields of ecology and evolutionary biology. This integration has been motivated in part by the growing evidence that evolution can happen on “rapid” or contemporary time scales, suggesting that eco-evolutionary feedbacks can alter system dynamics in ways that cannot be predicted based on ecological principles alone. As such, it may be inappropriate to ignore evolutionary processes when attempting to understand ecological phenomena in natural and managed ecosystems. In this chapter, we highlight why it is particularly important to consider eco-evolutionary feedbacks for microbial populations. We emphasize some of the major processes that are thought to influence the strength of eco-evolutionary dynamics, provide an overview of methods used to quantify the relative importance of ecology and evolution, and showcase the importance of considering evolution in a community context and how this may influence the dynamics and stability of microbial systems under novel environmental conditions.

Marine planktonic microorganisms, including Bacteria, Archaea, Protozoans (Protists), and Viruses, all play critically important roles in marine ecosystems and global biogeochemical cycles. This chapter focuses on the non-photosynthetic marine plankton organisms, and summarizes information on what organisms are present in the ocean, their diversity, distributions, activities and interactions.

Extremely acidic environments, defined having a pH of <3, are found in locations as diverse as the Arctic and the Tropics. While these be natural phenomena, human activity, most notoriously mining of metals and coals, are often responsible for the severe acidification of localized environments. The indigenous microflora in extremely acidic environments include species of prokaryotes and eukaryotes, many of which are obligately acidophilic. Acidophiles are widely distributed throughout the "tree of life", and include species of Bacteria, Archaea and Eukarya that are often only very distantly related to each other. Various mechanisms are used by acidophiles to adapt to the challenges they face, which include contending with elevated concentrations of transition metals and metalloids, and severely limited bioavailability of macronutrients such as phosphate.

Inorganic energy sources (reduced iron and sulfur) are highly abundant in many extremely acidic environments. Chemolithotrophic acidophiles are the basis of food webs in subterranean and also contribute to net primary production in deep submarine geothermal vents. However, where solar energy is available phototrophic acidophiles, predominantly species of acidophilic eukaryotic microalgae, proliferate and assume the dominant role of primary producers. Acidophilic microorganisms interact with each other in various ways, including via redox transformations of iron and sulfur, generating electron donors and acceptors for prokaryotic metabolisms, and via provision of organic compounds (supporting heterotrophic species) or inorganic carbon (supporting autotrophs). Acidophiles have long been used to extract metals from ores (biomining) and biotechnologies are emerging that harness their abilities to remediate polluted waters and recover metals.

The capability to (1) systematically collect, process, and archive nucleic acids from "extremely low-biomass" spacecraft-related environments, and (2) effectively assess the diversity of microorganisms present on spacecraft and associated cleanroom surfaces was developed, and validated. This capability enabled generation of the most comprehensive (bacterial, archaeal, and fungal) assessment of spacecraft-associated biodiversity to date. The capability to provide a passenger list of the microorganisms associated with flight hardware developed and validated in this study bridges a significant gap in technology and dramatically increases NASA's ability to explore and verify the scientific findings of both in-situ life detection and sample-return missions.

Although there is a growing understanding of the biodiversity associated with low biomass surfaces, it remains challenging to provide a comprehensive inventory of microbes present. In this study three molecular approaches were attempted: conventional cloning techniques, PhyloChip DNA microarrays, and 454 tag-encoded pyrosequencing, together with a methodology to systematically collect, process, and archive nucleic acids, to assess the phylogenetic breadth of microorganisms present on spacecraft and associated surfaces. The analysis methods yielded very different results; traditional approaches provided the least comprehensive assessment of microbial diversity, while PhlyoChip and pyrosequencing detected more diverse microbial populations. The findings of this pioneering study provided new and important insights into the benefits and limitations of modern molecular approaches for assessing the microbial diversity associated with samples extremely low in total biomass. These are of particular relevance to current and future NASA endeavors, as well as homeland security, medical, pharmaceutical, and semiconductor applications.

The last decades have seen a development of the research on animal-microbe interactions that is showing that animals, and in general higher organisms, are embedded in a microbial world. The recent developments in the research of the associations of microbes with the invertebrate gut are confirming that also in these animals the interactions with symbiotic microorganisms had a major role in the host evolution with important consequences, among others, on physiology, behavior, stress adaptation and ecology of the host. In this chapter such kind of interactions are analyzed and an overview of the different roles that gut-microbe symbiosis plays on the host fitness is presented. The profound effects of symbionts on the invertebrate biology are also discussed in the light of their potential exploitation for controlling invertebrate related problems such as, for instance and among others, controlling agriculture insect pest and transmission of vector borne diseases.

Any organism with a gut system will make use of microbes to enable the most efficient digestion of food that it ingests. The gut microbiota is a complex community of many different bacteria, archaea, viruses and lower eukaryotes that are required to act in unison to break down a wide variety of foodstuffs and even associated toxins (see below) to maintain the health of their host. The structure of this microbial community is influenced by many factors, including host (immune) response to the non-host cells, food intake and interactions between and competition within the members of the microbiota themselves. Understanding these interactions and the processes that lead to the establishment of a stable gut microbiota is a rapidly expanding area of microbial ecology. Whilst initially focused on understanding the human gut microbiota, researchers are now turning to the study of animal gut microbiota, and in particular those animals that we rely on for food, such as ruminant livestock (cattle and sheep) and poultry.

The widespread use of organochlorine pesticides (OCPs), mainly in the past, has caused serious environmental problems. Many OCPs were recently categorized as persistent organic pollutants (POPs) that should be controlled as toxic environmental contaminants. On the other hand, many bacterial strains and consortia have been identified that can degrade OCPs, including man-made ones, and various pathways for the biodegradation of OCPs have been clarified. Especially, aerobic OCP-degrading bacteria have been analyzed in detail as an excellent model for studying the bacterial adaptation and evolution in the environment. In fact, most such degradation pathways are thought to be established by the assembly of preexisting and newly evolved pathways, involving enzymes whose functions are thought to have evolved during relatively short period. Furthermore, a large amount of bacterial genomic information is now available, and the appearance and evolution of bacteria capable of degrading man-made OCPs can be discussed on the basis of such genomic information and mobile genetic elements. These accumulating knowledge on the biodegradation of OCPs will also be useful for practical bioremediation.

Waste organic matters such as organic compounds in wastewater and waste biomass from agricultural practices contain tremendous amount of energy. Recently microbial electrochemical technology (MET) receives great attention as promising technology to harvest energy from waste organics and produce directly electricity and valuable chemicals. MET use the bioelectrochemical system (BES) where microorganisms are used as catalyst for various electrochemical reactions. Two main mechanisms of extracellular electron transfer (EET), i.e. direct EET and indirectly mediated EET, from bacteria into anode or from cathode to bacteria have been reported. Microorganisms, which can transfer electrons into anode or receive electrons from cathode, are designated as electron transfer microorganisms (ETMs). The activity of ETMs directly and substantially affects the BES performance to produce electricity in MFCs and valuable products in MECs. Tremendous variety of ETMs has been reported and the variety seems to be depending on substrate types, substrate concentrations, poised electrode potentials, and electron acceptors. Most progress of MET in BES has been achieved from researches on application for wastewater treatment to produce electricity. MET is also used for biosensors, bioremediation, producing biofuels and industrial chemicals, and reverse electrodialysis. The present chapter will summarize recent reports of MET focusing on the developments of microbial aspects such as detailed EET mechanisms and diversity of ETMs. In addition, the newest various applications of MET will be briefly introduced.

Environmental Systems Microbiology is well positioned to move forward in dynamic complex system analysis probing new questions and developing new insight into the function, robustness and resilience in response to anthropogenic perturbations. Recent studies have demonstrated that natural bacterial communities can be used as quantitative biosensors in both groundwater and deep ocean water, predicting oil concentration from the Gulf of Mexico Deep Water Horizon spill and from groundwater at nuclear production waste sites (16, 17, 25). Since the first demonstration of catabolic gene expression in soil remediation (34) it has been clear that extension beyond organismal abundance to process and function of microbial communities as a whole using the whole suite of omic tools available to the post genomic era. Metatranscriptomics have been highlighted as a prime vehicle for understanding responses to environmental drivers (35) in complex systems and with rapidly developing metabolomics, full functional understanding of complex community biogeochemical cycling is an achievable goal. Perhaps more exciting is the dynamic nature of these systems and their complex adaptive strategies that may lead to new control paradigms and emergence of new states and function in the course of a changing environment.

Geomicrobiology involves the study of microbes that rely on certain geochemical conditions and substrates for growth and survival. Field and laboratory methods need to be carefully considered in order to thoroughly understand unique biogeochemical environments. Choices of field site as well as culture-dependent and culture-independent approaches are central to the study of systems where geomicrobiological processes such as metal oxidation, reduction, and adsorption predominate. We describe general considerations for fieldwork including choice of site, safety issues, and sampling options. Development of powerful ‘omics’ approaches such as metaproteomics and metagenomics now allow researchers to more fully understand complex geomicrobiological phenomena from the molecular to ecosystem scales. Examples are presented where combined 'omics' analyses have shown what biogeochemical processes are occurring and how these influence the geochemical environment. When combined with geochemical analyses, microbiological data from a given system can reveal what key metal transformation processes are occurring, their relative importance in the environment, and the ultimate impact that microbes have on the geochemistry of a system. This chapter serves as a practical guide for initiating and developing a variety of geomicrobiology projects, and can be used in conjunction with microbiology courses and teaching laboratories where questions regarding microbial transformation of metals are being explored.