Manual of Environmental Microbiology, Third Edition

The origin of scientific research in environmental microbiology rests in the observations of Antony van Leeuwenhoek that were published in 1677. During the intervening centuries, the expansion of our knowledge regarding environmental microorganisms has been based on increasingly detailed observations and experimentation, in which we have been aided by advancements in microscopy and the development of biochemical and mathematical tools. Microorganisms literally cover our planet, and can be found as deep as several kilometers both in glacial ice sheets and in bedrock. The microorganisms chemically interact with their physical environment, and their most notable effect has been the creation of an oxidizing atmosphere on this planet. By way of these chemical interactions, microbes remain crucial to the biogeochemical cycling which supports the continuation of life on our planet, turning over the elements that represent the basic ingredients of life such as carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur. Microorganisms are also used as tools to help us intentionally degrade both natural and anthropogenic materials in wastewater digestors, composters, landfills, natural terrestrial environments, and natural or artificial aquatic environments. The subject area of microbially mediated chemical transformations bridges the hydrosphere and lithosphere due to the relatedness of the involved microbial metabolic processes, which often are performed by either the same or related genera of organisms.

The most important thing which must be understood about microorganisms in their environments is that no microbe exists by itself. Microbes form the understructure which supports what we perceive as being the macrobial realm. The fact that this assemblage of microbes and macrobes has evolved together binds it together. No single species selected out of this assemblage could survive for very long on its own in the wild without the biological activity provided by those species which occupy the connecting niches. The degree of species diversity within communities can vary enormously. Communities typically may have several, many, or innumerable species. The indigenous populations that make up the community are responsible for the biotic balance that is maintained. They regulate the population densities or biomass of the individual component species of the community, and they act to prevent the establishment of invading species. Many types of microorganisms feed upon other microorganisms. Often, the same microbes which did the feeding then serve as prey and hosts for other microorganisms and macroorganisms in elaborate food chains. Climate is an important consideration for microbes, and there is a microbial geography. An obvious need that must be satisfied for an organism to become established is the presence of all nutrients that it requires. Knowledge of microbial community structure and community function has a key role in improving our lives. Furthermore, this knowledge will aid in understanding ways of maintaining the environment and its microbial communities.

This chapter provides a descriptive outline of the Bacteria, the Archaea, and some new chemical boundaries of their habitats in the more usual environmental sense (e.g., association with soils, waters, and some extreme environments) and also includes a consideration of microbes associated with macrobes. The recognition that beneath the widely disparate nutritional and environmental needs for the growth and sustenance of different microbes there was an underlying unity in their physiological attributes was a major conceptual contribution that had a marked practical influence on the development, nature, and extent of one's understanding of the significance of prokaryotic diversity. A widely accepted phylogenetic tree that classifies life in three major categories, Archaea, Bacteria, and Eucarya, is based on the inferences that Archaea and Eucarya diverged from ancestors of the Bacteria, first as a single lineage and only later diverging and becoming separately recognizable entities. As ever-increasing numbers of unique habitats are examined by use of macromolecular sequence, stable-isotope research, and new cultivation strategies, it is ever more evident that prokaryotic diversity has been regularly underestimated by classical isolation and cultivation approaches. Studies of Buchnera spp., the prokaryotic intracellular symbionts of aphids, are but one reminder that not all organisms of environmental significance are free-living and that some may exist in mutualistic states. With complete and accessible data collection, there is a good chance that even complex systems with multiple species may yield information that is comprehensible, leads to more accurate predictions, and may be used by the greater scientific community.

The challenge for environmental microbiology in the new millennium is to develop a predictive understanding of microbial communities in order to develop effective strategies for important global issues such as greenhouse gas mitigation, clean water generation, pollution remediation, and soil maintenance. Predicting the behavior of complex ecological communities, difficult even when individual elements can be clearly identified such as is the case in plant and animal systems, is further complicated by the large numbers of total individuals and different types of microbes present. The overall section organization and brief summaries of specific chapters are presented in this section. Pure cultures are necessary and an ultimate goal in microbiology; however, it is estimated that 1% or fewer of Earth's microorganisms are currently culturable. In the chapter on cultivation of communities, a community culture is described as one that by design keeps the structure and function of a set of interacting populations intact; this allows for the community to be resilient and adaptive, and if measured at the scale of a biofilm, for example, to be directly observed by using tools available through confocal laser scanning microscopy (CLSM). Advancement in molecular techniques has also enabled the manipulation of the genomes of specific organisms to provide an assayable response to environmental stimuli.

Examination of microorganisms in their natural habitat may be achieved most effectively through the application of a variety of microscope-based techniques. A major goal of the use of microscopic techniques is to achieve minimum disturbance of the system under observation. The major guidelines for all types of laser scanning microscopy (LSM) imaging are to obtain an image using the lowest-intensity laser and the smallest-pinhole aperture to minimize photodamage, optimize image quality (i.e., signal-to-noise ratio), and minimize the thickness of the optical section. Probes useful for fluorescence microscopy may be divided into three different types. Intrinsic probes are already present inside the sample (e.g., pigments such as chlorophyll or phycoerythrins and phycocyanins). Extrinsic probes are those which bind directly to a target (e.g., the general nucleic acid stains, such as 4 ,6 -diamidino-2-phenylindole [DAPI] or the SYTO series). Extrinsic covalently bound probes are usually high-molecular-weight molecules with a high specificity but no fluorescence (e.g., antibodies, lectins, and gene probes). Fluorescence in situ hybridization (FISH) of oligonucleotide probes to specific bacteria has previously been used in conjunction with confocal laser scanning microscopy (CLSM) and epifluorescence microscopy to document microbial diversity in a range of environments, including sewage sludge, river biofilms, and the rhizosphere. A number of emerging microscopy techniques, particularly in fluorescence imaging, may be applied to microbiological samples. These include FLIM, FCS, coherent anti-Stokes Raman scattering (CARS) microscopy, second harmonic imaging microscopy, 4Pi microscopy, stimulated emission depletion microscopy, and near-field scanning optical microscopy (NSOM).

With knowledge of the basic concepts of medium composition and the physical conditions which may limit microbial growth, one can enhance the ability to grow bacteria and fungi in pure culture and to enrich for, isolate, and culture many microorganisms of interest from the environment. The major nutritional requirements for microbial growth that must be considered are sources of carbon; sources of energy; electron acceptors; nitrogen sources; sources of nutrients, such as sulfur, phosphate, magnesium, and calcium; vitamin requirements; and trace metal requirements. Some physiochemical factors affecting growth include temperature, pH, requirement for oxygen, and salinity. This chapter presents a recipe for a basic basal medium for cultivating microorganisms. The selection of the various components and their roles are discussed. The chapter also discusses physicochemical factors for cultivation of bacteria and fungi, the fundamentals of the culture of fungi, larger-scale culture of bacteria, and the progress in culturing the uncultured.

In addition to the algae and protozoa, this chapter also considers the cultivation of one group of prokaryotes, namely, the cyanobacteria. Methods for the isolation and purification of algae and protozoa are broadly similar. The methods employed are comparable to those used for other microorganisms and can be broadly classified as enrichment methods, dilution methods, or physical and chemical methods. Although standardization of methods and media has been achieved for some purposes, particularly ecotoxicity testing, in general the medium composition depends both on the requirements of the algae and on the preferences of the researcher. Some protozoa are algivorous, while others are omnivores and feed on algae facultatively. A variety of algae and protozoa are classified as halophiles, perhaps the best studied being the phototroph Dunaliella, an alga that is commercially cultured for β-carotene. Some organisms obviously change when cultured on artificial media or under axenic conditions. This phenomenon is most commonly observed in cyanobacterial cultures, where loss of the ability to produce colonies, heterocysts, gas vacuoles, or akinetes can occur. To conserve genetic integrity and to minimize the maintenance requirements, long-term preservation methods have been developed for a wide range of biological materials. The methods described in this chapter have the advantage of requiring little routine maintenance of the preserved material and, in the case of cryopreservation, can effectively guarantee genetic stability. Research to improve techniques and to expand the range of organisms that can be maintained by using long-term preservation methods is ongoing.

During the last 50 years, virologists have been able to select cell cultures that are susceptible to many commonly encountered viruses and environmental virologists have developed methods to detect very low numbers of viruses in various environments. This chapter reviews these methods and their practical use and describes basic quality control procedures to maximize their level of sensitivity. The main objective of the cultivation and assay of viruses is to optimize detection methods to a level where even a single infectious unit can be detected with confidence. Several methods for detection of viruses in environmental samples have been described: visualization of the virus by microscopy, detection of viral antigens, detection of viral nucleic acids, and detection of viral infectivity. The detection of infective viruses in environmental samples still relies mainly on cell culture as the method of choice. There are several sources for cell cultures that can be used in virology. Plaque assay is used for a very limited number of viruses and has the lowest sensitivity of the available methods. The main advantages of plaque assay are that each individual virus (or aggregate) forms a single plaque and that each plaque is rarely a mixture of several virus types. Viruses isolated by cell culture can be propagated further in cell culture and identified by a variety of methods, including electron microscopy, serum neutralization, molecular methods, and immunoassays.

Model systems may be used for cultivation of microbial communities, and they offer the advantages of relative simplicity, experimental control, and replication. Some of the criteria used for isolating communities, and other types of cultures, are discussed in this chapter. Cultivation of defined communities requires that community culture methods be used instead of enrichment culture to better define the environment in terms of the concentration and flux of both substrates and products. The chapter emphasizes those culture systems which provide adequate environmental control of substrate flux and concentration throughout the time course of cultivation. Although batch systems may be used to cultivate microbial associations, they should be used and interpreted with caution. Chemostats, nutristats, microstats, and continuous-flow slide cultures (CFSCs) are among the culture methods commonly used to cultivate consortia and communities. Chemostats and other continuous cultures are among the most widely used systems for cultivating microbial communities and microbial consortia. Robbins device is one of the best-known and most widely used systems for studying biofilm communities and pure culture biofilms in applications ranging from medical to industrial and food research. Microstats are used for the cultivation of biofilm communities, as opposed to planktonic communities. Storage of communities under conditions suitable for preserving pure cultures may be inadequate for the preservation of communities.

This chapter discusses how phospholipid fatty acids (PLFA) and other lipids can be used to estimate total viable microbial biomass, community composition, and metabolic status as well as the use of in situ 13C incorporation to determine metabolic pathways in environmental samples. The viable microbial biomass increases with the availability of metabolizable substrates and may decrease after their exhaustion. Gas chromatography-mass spectrometry (GC-MS) of PLFA as their methyl esters provides greater sensitivity and specificity than the LP method and additionally provides detailed information on the microbial community composition and metabolic status. Some PLFA are indicative of particular phylogenic groups, and considered together with the environment from which the sample was retrieved, can be helpful in interpreting results. Biomarkers for the metabolic status of microbial communities include those for starvation and toxicity, unbalanced growth, and aerobic versus anaerobic growth, among others. Ubiquinones (UQ) are biomarkers for oxic respiration that mediate electron transport to oxygen and nitrate, while menaquinones (MK) can carry electrons to any electron acceptor. Besides the major respiratory quinones UQ and MK, Bacteria and Archaea also contain desmethylmenaquinones, methionaquinones, plastoquinones, rhodoquinones, and caldariellaquinones, which can also vary in the length of the side chain and, in the case of MK, in the degree of hydrogenation of the side chain.

While novel methods to discover new types and characterize microbial diversity are valuable, alternative approaches for describing microbial communities in ecologically relevant terms are needed to provide insight into the spatial and temporal patterns in both natural and man-made ecosystems. This chapter concludes with a synthesis of the various community-level physiological profiling (CLPP) approaches, including comparison of their relative strengths and weaknesses, discussion of appropriate applications, and definition of future areas of research. The CLPP assay is a sensitive, simple, rapid, and relatively inexpensive means of discriminating among microbial communities in environmental samples. A number of factors (high substrate concentrations, indirect evaluation of respiration via the redox chemistry, and presence of unknown proprietary ingredients) limit the ability of the Biolog CLPP to examine functionally relevant shifts in microbial communities. Alternative techniques developed to improve functional relevance involve direct measurement of respiration, either based on substrate consumption (O2) or product appearance (CO2), eliminating bias in the Biolog assay as a result of cells that cannot take up or are inhibited by the redox dye. While further incorporation of new technologies to continually improve CLPP is encouraged, the existing techniques represent valuable tools for evaluating factors affecting the metabolic state of heterotrophic microbial communities.

This chapter considers the use of molecular methods for direct measures of abundance, diversity and phylogeny of environmental populations of microorganisms. These molecular methods are mainly based on direct nucleic acid sequence recovery, genomic DNA hybridization, and nucleic acid fingerprinting. There are three basic formats now used to recover DNA sequence information isolated from either pure culture or environmental samples: DNA probe hybridization, restriction enzyme digestion, and chain termination sequencing of cloned (or PCR-amplified) DNA templates. Restriction fragment length polymorphism (RFLP) analysis has been used to characterize extracted total DNA or specific PCR-amplified DNA. Sequence diversity is evaluated by digesting the native or amplfied DNA with a restriction endonuclease(s) followed by size fractionation by electrophoresis on an agarose or a polyacrylamide gel. Genomic DNA hybridization is mainly used in bacterial systematics to determine the degree of genetic similarity between genomic DNA sequences and thus provides limited information of specific sequence content. Molecular fingerprinting methods can be used for rapid surveys using genes that provide for either phylogenetic or functional assessment of populations present in an environmental sample. Terminal RFLP (T-RFLP) of PCR-amplified DNAs is a refined fingerprinting technique based on RFLP. The general steps include PCR amplification of a conserved target sequence (most commonly a region of the small-subunit (SSU) rRNA gene) followed by restriction enzyme digestion and gel fractionation of resulting fragments.

This chapter briefly discusses the different steps in constructing evolutionary trees, including collecting data sets of homologous sequences, generating a multiple-sequence alignment, inferring tree topology, and assessing confidence in the tree. Multiple-sequence alignments are the essential prerequisite for most phylogenetic analyses. There are currently four primary methods for constructing phylogenies from protein and nucleic acid sequence alignments: the distance-based neighbor-joining (NJ) method and the character-based methods, including maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference. The major advantage of bootstrap technique is that it can be applied to basically all tree construction methods, although one has to keep in mind that applying the bootstrap method multiplies the computer time needed by the number of bootstrap samples requested. Until recently, phylogenetic analyses have been routinely based on homologous sequences of a single phylogenetic marker, i.e., the 16S rRNA gene among bacteria. Given the vast number of genome sequences now available, it is possible to compute trees from whole genomes.

This chapter stresses on techniques that are significantly different from other molecular techniques in that they are suitable for complex environments inhabited by a diverse collection of bacteria. While there are several bioreporter genes that might be used, bioreporters that make use of light for bioreporting have significant advantages. The use of either bioluminescent or fluorescent bioreporters is now an established technology, and the uses have expanded greatly over the years. Bioreporters for bioluminescent and fluorescent gene products are described in this chapter. The advantages of bioluminescent bioreporters lie primarily in the relative ease of light measurement. A discussion of the weaknesses of bacterial bioreporters and the means by which these techniques may be improved is provided in the chapter. There are now several fluorescent proteins that can be used as bioreporters in bacterial cells, but the first successful one was green fluorescent protein (GFP). The development of microprobes for the examination of microbial environments has proceeded rapidly thanks to innovative construction techniques. Microprobes have been described for ammonium, nitrate, oxygen, denitrification (by nitrous oxide production), and sulfate reduction. In surface plasmon resonance (SPR), the surface of the waveguide is coated with a thin layer of gold. Multigene analysis will have a substantial impact on the understanding of genetic control. In the area of biosensors, the trend toward miniaturization and commercialization will continue. It is expected that fieldable biosensors will have a great impact on biowarfare monitoring and long-term ecological studies.

Long-term study sites for ecological research exist in a variety of habitats, including forests, deserts, streams, and oceans, providing the observational framework for studying microbial ecological processes over time and space. In this chapter, the authors describe the advantages for microbial ecologists of studies at long-term ecological research sites, the opportunities of various types of research sites and locations available for microbial research and the integration of microbial and ecosystem ecology. The chapter is written from the point of view that studies of the ecology of microbes are a necessary part of gaining a predictive knowledge of ecosystems. There are three types of studies illustrated: correlation between microbial ecology and environmental factors, correlation studies with added data on measurements of a microbial process, and correlation studies that make use of a large-scale and long-term experimental manipulation. Multivariate analysis indicated strong functional differences between meadow and forest soils. Levels of both potential denitrifying enzyme activity (DEA) and potential nitrification were substantially higher in the meadow soils. Denitrifier communities formed distinct groups according to vegetation type and site as evidenced by terminal restriction fragment length polymorphism (TRFLP) data. Nonmetric multidimensional scaling (NMS) analysis of the binary coded TRFLP data was used to assess the effects of fertilization on the composition of the microbial communities.

This chapter discusses the principles of quality assurance (QA) and provides a discussion of techniques that can be used to train personnel to QA requirements. Starting with the definition of QA and its counterparts, quality control (QC) and quality improvement, the chapter then moves into a description of general QA program components. Quality assurance is not new to environmental microbiology. However, with the emphasis in environmental sciences being the detection of chemical contaminants and resulting engineered solutions (e.g., Superfund), environmental quality assurance (QA) program requirements have focused on the chemistry and engineering fields. Instructors employed to present the QA program can emphasize problem areas and are present to answer questions. The use of proceduralize scientific notebook, including organization, formatting, entry and attachment techniques, types of reviews, and archival protocols ensure uniform practices. Record-keeping requirements address the types of information to be documented, as well as the techniques to use. Sampling is an integral part of the study design. The selection of sampling sites and sampling strategies is based upon the study objectives and hypotheses. The second and third types of errors may be termed scientific misconduct and can have far-reaching effects, especially in the health fields, resulting in potential harm to humans at the worst and reflecting poorly on the research field at the least. The chapter defines and provides examples, when possible, of typical QA requirements that the environmental microbiologist might implement.

The practice of good science requires a concise description of study objectives and a study design that matches its objectives. A good study design requires (i) definition of the population or factors of interest in the study, (ii) identification of study units, (iii) collection of representative measurements, and (iv) a statistical analysis matched to study objectives and data characteristics. The goal of this chapter is to help one see the statistical issues involved in study design and data analysis. The majority of studies in environmental microbiology involve some form of comparisons since it is through comparisons that we learn where differences exist and/or what factors can influence microbial populations. Experimental designs specify the nature and extent of comparisons that are of interest in a particular study. The goal of data collection, be it in a sampling study or an experimental design, is to obtain a set of measures from a population to gain insight into how values for a particular population characteristic vary from sample unit to sample unit. In this chapter the authors have attempted to raise readers awareness of the role and function of statistics at both the study design and the data analysis steps of environmental microbiological studies.

One of the most important aspects of water microbiology, from a human perspective, is the fact that we acquire numerous diseases from microorganisms found in water. The reservoirs for pathogenic microorganisms found in environmental waters can be humans, animals, or the environment itself, as summarized in this chapter. However, it commonly is presumed that many of those microorganisms that infect humans and are found in our aquatic resources originate from human sources. This anthropogenic contamination can occur during either defecation in water or recreational activities conducted in water. In addition, domestic wastewater is of particular importance as a contributor of the pathogenic contaminants found in aquatic environments; the attendant public health concerns have resulted in the development of methods for studying and reducing the levels of pathogens in wastewater. The treatment of wastewater also is intended to reduce the contamination of crops that may occur when wastewater is eventually discharged onto land surfaces. The goal of this chapter to represent and summarize the current knowledge on public health aspects of water microbiology.

Waterborne transmission is a highly effective means for spreading infectious agents to a large portion of the population. Several water-related modes of transmission of infectious agents are discussed. Infection and development of clinical symptoms depend on a number of specific and nonspecific host factors, such as age, immune status, gastric acidity, nutritional status, vitamin A deficiency, and possibly genetic predisposition. The majority of poliovirus and hepatitis A virus infections in young children are asymptomatic. Most of the information on the risk factors and etiologic agents of waterborne disease comes from investigations of waterborne-disease outbreaks by state and local health departments and the surveillance program maintained by the CDC and the Environmental Protection Agency (EPA). The commonly recognized waterborne pathogens consist of several groups of enteric and aquatic bacteria, enteric viruses, and three enteric protozoa. The most recent list of candidate contaminants was released in February 2005 and includes nine microorganisms: adenoviruses, caliciviruses, coxsackieviruses, echoviruses, Aeromonas hydrophila, Mycobacterium avium and Mycobacterium intracellulare (together referred to as the M. avium complex [MAC]), Helicobacter pylori, cyanobacteria (blue-green algae) and other toxin-producing freshwater algae, and microsporidia (Enterocytozoon and Septata). The section on the classification of water-related diseases, deals with water-related transmission of infectious agents associated not only with microbiological water quality but also with water availability, sanitation, and hygiene. Finally, for infectious agents with multiple transmission routes, it may be difficult to determine the attributable risk associated with waterborne transmission compared to other routes of transmission, especially in areas where waterborne diseases are endemic.

The presence of some microorganisms in waters is used as an indication of possible contamination and as an index of quality deterioration. Gastroenteritis is the most common affliction associated with waterborne pathogens. The ideal manner to determine the microbiological safety of waters is to analyze the waters for the presence of specific enteric pathogens; however, hundreds of different microorganisms have been shown to be involved in waterborne disease outbreaks; thus, it would be impractical to look for every pathogen potentially present in water samples. Bacteroides fragilis is a strict anaerobe found in high concentrations in the human intestinal tract and dies rapidly when discharged into environmental waters. The ratio of fecal coliforms to fecal streptococci was proposed as an indicator of the origin of the contamination because of the different concentrations at which these microorganisms are present in different animals and humans. The detection of thermotolerant coliforms in treated drinking waters should be a cause for concern, since present drinking water treatment processes successfully eliminate indicator microorganisms. Most probable number (MPN) analysis is a statistical method based on the random dispersion of microorganisms in a given sample. Classically, this assay has been performed as a multiple-tube fermentation test. The practice of using groups of bacteria or specific organisms such as Escherichia coli as indicators of the possible presence of fecal contamination or as indicators of the possible presence of pathogenic microorganisms is and has been extremely useful to protect public health.

This chapter deals primarily with human enteric protozoa and more specifically Giardia and Cryptosporidium. Presently, Giardia and Cryptosporidium are of great concern to the water treatment industry, because they are known to be the etiologic agents responsible for a number of episodes of waterborne gastroenteritis. In many cases the only way to accurately identify some free-living protozoans to genus and species is to collaborate with experts in the field. Giardia lamblia is one of the most frequently reported parasitic water-borne pathogens. Detection of G. lamblia and C. parvum in source and finished water has been and continues to be of great interest. It is an immunofluorescence detection procedure performed after concentration of protozoans from large volumes of water. A section assumes use of a microscope, capable of epifluorescence and differential interference contrast (DIC) microscopy or Hoffman modulation optics, with stage and ocular micrometers and 20× (numerical aperture = 0.6) to 100× (numerical aperture = 1.3) oil immersion objectives. Microscopic examination also assumes that the ocular micrometer has been calibrated. The fluorescence in situ hybridization (FISH) assay can also be evaluated with a standard epifluorescence microscope. The major advance in using this approach is that the occurrence of false positives is reduced and that the need for DIC optics is eliminated. Depending upon the source or expert consulted, B. hominis either may or may not be classified as a pathogen.

One concern with the use of Enterococcus as an indicator is that the epidemiology studies on which it was based were conducted at locations where sewage was the primary source of fecal contamination. Present methods to enumerate indicator bacteria in marine coastal waters rely on growth-based assays. Bacterial measurement methods include multiple-tube fermentation (MTF), membrane filtration (MF), and defined substrate (DS) methods. The survival of bacterial indicators in the ambient environment generally differs from that of viral pathogens, and thus, it may be more desirable to measure viral indicators directly. Two types of viral indicators are presently employed in some monitoring programs and epidemiology studies. The first is coliphages, which are viruses that infect coliform bacteria. The second group is enteric viruses, the measurement of which is only recently becoming cost-effective through use of PCR. The most likely coliphage group as a candidate water quality indicator is the F (male)-specific RNA (FRNA) coliphage, because it represents viruses similar in size, shape, and genetic makeup to human enteric viruses; it is more stable than human enteroviruses; and its concentrations in environmental waters have been reported to correlate with sewage contamination.

This chapter describes techniques that can be used for concentrating human enteric viruses from environmental water, drinking water, raw wastewater, wastewater sludges, and wastewater effluents. The chapter presents the use of cartridge filter-based methods for concentrating viruses from environmental water, drinking water, and sewage effluent. The methods presented in this chapter for isolating viruses from raw sewage (raw wastewater) and wastewater sludges effectively utilize a process of directed adsorption and elution but differ in that they rely upon the wastewater solids to serve as an in situ adsorbent. A variety of sampling apparatuses may be needed when detecting viruses in environmental waters. The types of apparatus used for concentrating viruses from large volumes of environmental waters, drinking water, or sewage effluent by means of cartridge filtration are not standard equipment for most environmental microbiology laboratories. All of the cartridge filters described in the chapter can be presterilized within their holders by using ethylene oxide gas treatment before they are transported to the field. Objects less resistant to high heat, such as nonborosilicate glass and some polymer materials, can be surface sterilized by dousing or immersing them in commercial 95% ethanol and then igniting the alcohol with a flame. The infectivity of enteric viruses contained in environmental samples can be examined by inoculating the sample into cultures of either human or animal cells that are prepared in the laboratory as opposed to inoculating them into live animals.

The presence of pathogens and the number of pathogenic bacteria that may be present in wastewater and biosolids are a function of the disease morbidity in the community from which the waste materials are derived and the degree of treatment the waste received. Representative genera of bacterial pathogens that may be found in domestic wastewater and sludge include Salmonella, Shigella, Vibrio, Escherichia, Campylobacter, and Yersinia. Due to the difficulties in the isolation and detection of bacterial pathogens in wastewater and sludges, the use of surrogate (indicator) bacteria has been standard practice in water quality monitoring. Most of the available literature and reported experience in the sphere of wastewater and sludge have been directed towards the detection and enumeration of salmonellae. Environmental water samples may also be centrifuged to concentrate bacteria into a pellet, and the pelleted cells are then lysed in a silica-guanidinium thiocyanate lysis buffer, followed by several washing steps. These crude extracts have been shown to be sufficient to add directly to PCR amplification. Recently, magnetic capture beads have been employed in a wide variety of clinical, food, and environmental applications for the recovery of target nucleic acids that are subsequently amplified by PCR. There are a myriad of difficulties ranging from the huge diversity of microorganisms involved to the considerable variety of methodological approaches one can pursue.

Many types of methodologies have been used for the detection of pathogenic bacteria, viruses, and parasitic protozoa from bivalve molluscs. This chapter presents general processing methodologies, including conventional detection approaches such as microscopic or cultural methods, as well as the molecular approaches that are increasingly being applied to the detection of these pathogens in the bivalve mollusc food matrix. For viruses and parasitic protozoa, the approach used for pathogen extraction and concentration not only affects the efficiency of recovery of targeted pathogens but also influences the sample-associated toxicity or inhibition of the assay. Although efforts to detect protozoan parasites in shellfish have lagged behind those for virus detection, like viruses, it is recommended that parasitic protozoa be concentrated from shellfish tissues prior to the application of detection methods. Most of the molecular approaches applied to the detection of pathogens in shellfish originated from the clinical literature and were first applied to pure cultures (bacteria) or to purified cell culture lysates or fecal suspensions (parasitic protozoa and viruses). The practical application of real-time PCR and reverse transcription-PCR (RT-PCR) for quantification also needs to be investigated further. With consistent developmental effort, much as has been experienced over the last decade, even better methods for the rapid detection of human pathogenic microorganisms in shellfish at naturally occurring levels of contamination should be available in the future.

There are hundreds of different enteric microorganisms that are known to infect humans. In recent years, we have seen an increased reliance on surface water and water recharge or reuse, which are perceived as a more vulnerable source than groundwater in terms of the microbial quality of water. Bacteriophages have been extensively researched as water quality indicators. Antibiotic resistance analysis (ARA) of fecal coliforms and fecal streptococci has been used to determine sources of fecal pollution in natural water. Microbial monitoring is a cornerstone of any watershed quality assessment. Most raw drinking water sources are susceptible to significant water quality changes as stresses are placed on the surrounding environment due to natural, accidental, or intentional contaminations. Groundwater supplies are derived primarily from wells. Water from shallow wells or “hand-dug” wells is not considered groundwater because it is usually under the influence of surface water via runoff and infiltration and hence can have many of the characteristics of surface water. There are several types of physical methods for the treatment and/or disinfection of water, such as reverse osmosis, distillation, and use of UV light. Chlorination is probably the oldest and most widely used form of drinking water disinfection. It has several advantages which make it appealing and have bolstered its popularity. Advanced oxidation processes are processes for producing OH radicals for the oxidation of organic and inorganic impurities in water. Recently, biological treatment has been driven by the concern of increased biodegradable organic matter (BOM) in ozonated waters.

The purpose of wastewater (WW) treatment is to prevent water pollution and thereby protect public health and preserve the value of the receiving water (RW) as a resource. This chapter talks about evaluation of the overall performance of the entire wastewater treatment plant (WWTP) and not with the performance of individual unit processes. The emphasis of the chapter is on an overall evaluation of WWTP treatment efficiency. The objectives of WW treatment, the concepts of efficiency and effectiveness of WW treatment, and the difference between WW treatment processes and sludge treatment processes are reviewed to establish a context for the discussion. Limits for the parameters discussed in the chapter are often conditions of National Pollutant Discharge Elimination System (NPDES) permits. The study was designed to find how new ways of measuring water pollution at bathing beaches can be effectively used to protect swimmer's health. A method for predicting the rate of consumption of dissolved oxygen (DO) by microorganisms in the RW body is essential for assessment of the potential of a final effluent (FE) to cause pollution. Mathematical models are used to extrapolate biochemical oxygen demand (BOD)5 results both to deoxygenation of the receiving water (RW) and to substrate concentration in WW. The chemical oxygen demand (COD) test was developed because the BOD5 test requires 5 days for completion and therefore is not suitable either for operational control of treatment processes or for real-time evaluation of the efficiency of WWTP performance.

This chapter explains some of the quantitative approaches which are applicable to understanding the fate of microorganisms after they are released into water, wastewater and soil. In surface water, during periods when the water velocity is low, the tendency of microorganisms to adsorb onto suspended particulates facilitates the sedimentation of those organisms. Microorganisms released into the environment become susceptible to inactivation by a variety of physical and chemical factors. These include desiccation; thermal or pH related effects upon their biomolecules, which may include denaturation; radiation from sunlight; and effects of inorganic ions. The most important aspect of studies designed to evaluate the fate of microorganisms is to define the study population, which implies controlling or understanding any possible movement of individuals into or out of that population. With these stipulations in mind, a section focuses on the use of regression analysis to develop statistical models that describe temporal changes in microbial titer as a function of experimental parameters and water or soil characteristics. The chapter also talks about mathematically examining the rate at which populations of microorganisms die off following their release into the natural environment. The linear modeling approach requires two major assumptions. The first assumption is that any differences, or distances, between the plotted data points and the graphed equation are due to error. The second assumption is that the use of linear modeling is appropriate for the data being examined.

This chapter addresses organisms which are infectious for humans, and while some of these represent aquatic contaminants that come from either human or animal reservoirs, others represent organisms which are naturally present in the environment. Five main variables must be considered when the risk of acquiring infectious disease by ingestion of water is estimated. These are the concentration of pathogenic organisms in water which are infectious by the route of ingestion, the amount of water ingested by an individual per unit of time, and the probabilities of infection, illness, and death associated with ingesting those pathogenic organisms. A recent estimate had found that the raw water entering that area’s municipal drinking water treatment plants contained an average level of 3.3 viruses per liter (viruses capable of infecting cultured mammalian cells and thus presumably capable of infecting humans). Mycobacterium tuberculosis, which causes tuberculosis, is one of those microbes particularly adept at avoiding immunological defenses. While the mycobacterium is not waterborne, sadly other species of pathogenic mycobacteria are waterborne. Several important issues must be recognized and their implications must be understood when risk assessment estimations for infectious diseases are made. The first three of these issues specifically apply to estimations for drinking water. The last three apply not only to drinking water but also to any infectious disease risks regardless of the source of the pathogenic organisms.

The biological process by which toxic photosynthetic microbes can be harmful is also nontoxic to humans. There are six significant types of marine poisonings which regularly occur in different parts of the world. The classes of algae responsible for producing the majority of toxin poisonings in the marine environment are dinoflagellates and diatoms. These types of poisoning events and the organisms that produce them are discussed. Saxitoxin (and its derivatives) is one of the most potent toxins known. This toxin is also produced by certain species of freshwater toxic photosynthetic algae. Marine cyanobacteria generally produce toxins classified as lipopolysaccharides (LPS), contact irritants, and neurotoxins. Lyngbya is one of the most diverse genera of toxic photosynthetic microbes. For the sake of brevity, only the most potent of the toxins produced by Lyngbya in marine environments are discussed. In 1963, large blooms of Trichodesmium spp. plagued the northeastern coast of Brazil, causing Tamandaré fever. Secondly, the facts concerning toxic photosynthetic microbes must be passed to the people of the world in a morally responsible manner. Lastly, continuing research studies must be performed to ensure that the safety of water is maintained. One of the most interesting findings discussed here is that some toxins have very different chemical structures but have the same toxic effects. Toxic photosynthetic microbes are a few significant species of algae that can cause major damage to our individual, societal, and economic health.

This chapter reflects the integrative nature of aquatic microbial ecology. It has been structured around areas that influence the role of the microbial community in ecosystem function such as diversity and community structure of primary and secondary producers, growth and grazing of primary and secondary producers, the role of bacteria in geochemical cycling, and specialized environments. The field of aquatic microbial ecology is being revolutionized by genetic and genomic approaches. Pioneering work in the late 1980s, which started with community analysis based on the PCR amplification of the 16S rRNA gene from many different types of bacteria, has expanded to encompass the analysis of multiple genomes from many individuals in many populations in the community (metagenome). Tringe et al. used environmental gene tags (EGTs), or short sequences that contain fragments of functional genes from whole genome shotgun libraries, to compare the microbial communities from three different ecosystems. In general, there are two approaches to measuring bacterial community activity in aquatic systems: (i) the direct tracer approach, which measures the conversion of a labeled substrate to a product of interest, and (ii) isotope dilution methods, in which the product pool is labeled and the dilution of the labeled product pool by new unlabeled product formation gives an estimate of the rate of production. Direct tracer methods have been employed for organic carbon, inorganic carbon, sulfur, nitrogen, phosphorus, and metal cycling. The future challenge is going to be to integrate activity and community structure measurements with ecosystem measurements of physicochemical fluxes.

In 1993, Don Button and colleagues broke new ground with their theoretical description and subsequent application of an extinction culturing method for aquatic microbial cells. Their approach was specifically tailored toward the isolation of oligotrophic cells by taking advantage of the unique ability those cells have to effectively compete at very low nutrient concentrations. The volume of water required for medium preparation and inoculation depends on the concentration of microbial cells in the raw sample and the design and goals of the particular cultivation experiment. As with any technique involving the cultivation or manipulation of live microorganisms, measures taken to minimize and eliminate the risk of contamination are crucial for a successful experiment. In seawater processing the entire collection container is acid and heat sterilized. The tangential flow filtration (TFF) unit and peristaltic pump are set up as directed by the manufacturer. Sterile seawater is stored at 4°C to minimize potential contamination and should be inspected for continued sterility via microscopy immediately prior to each use by following either the DAPI (4',6'-diamidino-2-phenylindole) staining protocol or a similar procedure. Coupled with rapid, sensitive, and specific screening methods such as fluorescence in situ hybridization (FISH), T-RFLP, or screening for the presence of specific functional genes via PCR, the high-throughput culturing (HTC) method allows scientists to tailor extensive cultivation efforts that target maximum overall diversity or focus their efforts on narrowly defined target microorganisms.

Analyses of long-term data sets have demonstrated the utility of primary productivity measurements as indicators of either natural or anthropogenic ecosystem alterations. A suite of microscopic identification and enumeration techniques including various cytological stains, immuno-and autofluorescence, and microautoradiography coupled to fluorescence in situ hybridization are available. These techniques add to the sensitivity, specificity, relevance, and utility of productivity measurements as indicators of ecosystem structure, function, and change. The general use and application of these ancillary techniques are discussed in this chapter. The chapter discusses methods for measuring primary pathways in aquatic habitats. Studies on diverse marine and freshwater systems have shown that the highest rates of primary productivity occur during mid to late morning, prior to the period of maximum irradiance. Oxygen can be measured electrochemically by using several types of electrodes. The construction and general application of cathode-style microelectrodes in microbial O2 production and consumption studies are discussed in detail by Revsbech and Jørgensen. Standard measurements of primary productivity can be complemented by modifications, ancillary techniques, and procedures that enhance physiological, ecological, and taxonomic interpretations of productivity measurements. Microautoradiography and radiolabeling of diagnostic microalgal pigments can enhance the specificity and dimensionality of primary productivity measurements. Both natural perturbations and human activities can strongly impact aquatic primary production by modifying nutrient, sediment, toxin, and other xenobiotic inputs. Evolving remote-sensing techniques will help clarify and evaluate the nutrient-production interactions on scales appropriate for a broad range of aquatic ecosystems.

Planktonic heterotrophic bacteria (bacterioplankton) are now recognized to be a large and metabolically active group that contributes significantly to the biomass and to the flow of carbon in aquatic systems. Various methods available to determine bacterial biomass production (BBP), those that employ radiolabeled precursors to estimate the rate of synthesis of nucleic acids and proteins have become the most widely used and are the focus of this chapter. In this chapter, the rationale, advantages, and disadvantages of the most commonly used methods based on the incorporation of thymidine (TdR) and leucine (Leu) methods are discussed. In addition, methods to determine empirically a conversion factor from thymidine or leucine incorporation to cells produced is presented as well as several procedures designed to test various assumptions of these methods. Alternative methods to determine BBP that do not rely on the uptake of radiolabeled compounds are also discussed in the chapter. The BBP measurement methods presented in this chapter estimate the total BBP and do not provide information on the relationship between bacterial diversity and metabolism. The chapter concludes with a review of novel methods that combine measurements of BBP with microscopy and molecular techniques to determine the proportion of total bacteria that are active, and the contribution to total BBP of specific phylogenetic groups.

This chapter outlines the various approaches that are used to determine community structure and some guidelines regarding which methods may be most appropriate for specific aquatic environments and specific scientific questions. It describes new molecular biological and other highly technical approaches in detail, even though they are still under development, because they are likely to become particularly important in the future. The oldest method for obtaining information on microbial community structure is to examine the sample with a microscope and characterize the microbes by their morphology. The most common traditional method of determining community structure involves culturing the organisms from the habitat in question and identifying the cultures by standard techniques. Immunological approaches have been used primarily to characterize and count nitrifying bacteria and cyanobacteria. The molecular size distribution pattern of low-molecular weight RNA (including tRNAs and 5S rRNA) is thought to be unique within narrow phylogenetic groups of microorganisms. The PCR methods have been used with samples from sediments and microbial mats, as well as deep-sea holothurian guts, often showing unexpected bacterial and archaeal diversity. Some studies have used information about the 16S rRNA clones short of partial or full sequences. There is still a question about possible biases and errors in the molecular methods. There is also the question of quantitative biases. In the quantitative analysis of community structure, oligonucleotide probes are powerful tools that avoid possible biases in cloning and yield a more direct measure of the target groups of interest.

This chapter brings together methods that can be used to count and examine the genetic diversity of communities and populations of aquatic viral communities, although many of the methods can be adapted for other environments. The first studies on viral abundance in aquatic systems used transmission electron microscopy (TEM) to count virus-like particles (VLPs). Interestingly, although TEM images of natural communities suggest that viruses with small noncontractile tails are most abundant, the most frequently isolated viruses are tailed sipho-and myoviruses. This discrepancy may be due to the method of sample preparation used in many TEM studies of natural virus communities or due to the fact that viral isolates are not representative of native viral communities. Breitbart et al. constructed a metagenomic library of two coastal DNA phage communities, using the linkeramplified shotgun library (LASL) method. In this study, 200 liters of seawater was prefiltered and concentrated by tangential flow filtration. Community approaches are useful for studies of the total abundance of viral particles or for documentation of the dominant morphotypes in viral communities. Viruses are an abundant, dynamic, and ecologically important component of aquatic ecosystems, and there is now strong evidence that they are the most genetically diverse biological entities on the planet. Targeting the viral DNA polymerase, this research revealed a vast amount of genetic variation that was not represented in cultures and showed that very similar sequences were distributed on a global scale.

Protistan assemblages of aquatic ecosystems are the focus of extensive research in aquatic ecology. One stimulus for this work has been the long-standing recognition that phototrophic protists (the unicellular algae) constitute a major fraction of the primary productivity within aquatic ecosystems. We have learned a great deal about the taxonomic composition and trophic structure of aquatic protistan communities through the application of traditional approaches of morphological analysis and culture. Nevertheless, the tremendous diversity of protistan assemblages and the varied methods required for identifying protistan species and their abundances, biomass, and trophic activity continue to hamper in-depth understanding of the structure and function of these communities. The success of using molecular (genetic/immunological) signatures for assessing the community structure of natural protistan assemblages will ultimately depend on linking these signatures to classical (morphological) species descriptions and to the physiological abilities of protistan phylotypes. Ultimately, molecular approaches, in combination with classical methods, will provide new tools for studying the emergent physiological, ecological, and biogeochemical processes that are created and/or affected by protistan community structure. Probably the most distinct difference between freshwater and marine protistan communities is the restriction of the larger sarcodines (acantharia, radiolaria, and foraminifera) to brackish and marine ecosystems. Modern molecular biological approaches have revealed unexpected, and as yet largely uncharacterized, protistan diversity in a wide variety of ecosystems.

This chapter focuses on leaf decomposition in streams, which is dominated by aquatic hyphomycetes. It emphasizes on methods used to analyze the decomposition of various substrates and on methods to characterize fungal community structure. The procedures used to collect and expose leaves in streams will affect several interdependent aspects of decomposition. The use of dried material in litter bags has been the method of choice to study decay in submerged or emergent macrophytes. Their decomposition is therefore best studied by marking individual plants or leaves with electrical cable ties and measuring decay and microbial colonization in situ. Application of this technique has profoundly altered our view of the fungal involvement in macrophyte decomposition in marshes. Many of the considerations for choosing and collecting samples apply regardless of subsequent analysis. The great advantage of molecular methods is that they are based on a much greater number of unique traits than traditional approaches (primarily nucleotide sequences rather than morphological data), resulting in much higher resolution. In addition, molecular traits are essentially independent of life stage; there is no need to induce a reproductive stage to allow unequivocal identification and phylogenetic assessment. Currently, neither molecular nor traditional methods lead to comprehensive species lists; therefore statistical evaluations are needed to extrapolate from our samples to ecosystems.

This chapter introduces some of the important aspects of bacterial organic carbon cycling and to provides an update on the molecular biological approaches currently in use for examining carbon cycling by bacteria without cultivation. Such investigations by microbial ecologists are motivated by the desire to understand how the composition of microbial communities dictates the way that food webs and carbon cycling function in the oceans and other aquatic environments. The goal of one study by Cottrell and Kirchman was to determine whether the relative contributions of various types of bacteria to DOM consumption depend solely on the relative abundance of these types of bacteria in the community. This study used microautoradiography and fluorescent in situ hybridization (FISH) to test the hypothesis that low-molecular-weight compounds are used by all bacteria and high-molecular-weight compounds are used by a smaller, less diverse group of bacteria. In another study, Brennan et al. examined microbial xylanases in insect guts. Although the study did not include aquatic bacteria, the approach used in this study should be applicable to aquatic microbes with appropriate modification by using a DNA extraction method for aquatic microbial DNA. The great phylogenetic distance between the novel xylanases and the known xylanases suggests that microbes in the insect gut have evolved in isolation from other microbes that have been successfully cultivated.

Facultative methylotrophs are capable of growth on multicarbon compounds, while obligate methylotrophs are not. Knowledge of the content and structure of the genomes of methylotrophic bacteria is instrumental for detecting C1 metabolism genes in the environment. Comparative analysis of methylotroph genomes, as well as the proteomic analyses, provides knowledge on the complement of the genes essential for C1 metabolism in the environment. The current knowledge of the methylotrophy modules, however, remains incomplete. While originally developed for detection of copper-containing membrane-bound methane monooxygenase (pMMO), this protocol may be adapted to detect other genes encoding key enzymes of C1 metabolism. Detection of specific groups is based on light scattering and/or autofluorescence. In the absence of natural autofluorescence, microbial cells can be differentiated by various types of fluorescence staining, using either immunofluorescent labeling or fluorescent in situ hybridization (FISH). The first is the prohibitive cost, and the second is the computational difficulty of assembling the large databases of random DNA sequences . The progress in environmental genomics will bring about new clues as to the possible means of cultivating microbes that have so far resisted cultivation. The field of environmental genomics is still in its infancy, and it is easy to predict that the future will be full of surprises.

Microorganisms of the S cycle are extremely diverse. The anaerobic sulfate-reducing bacteria (SRB), which are unique physiologically and genetically, are represented by several genera, most of which were discovered in the last 20 years. Chemolithotrophic S oxidation is mediated aerobically by colorless S bacteria, some purple S bacteria, and SRB. For enumeration, isolation, and rate measurements, it is important to process samples quickly after they are obtained and to maintain samples near ambient temperatures. Rates of SO4 2- reduction have become routine measurements in studies of the biogeochemistry of anoxic aquatic environments. SRB are readily isolated from many environments, including freshwater and salt water, soils, oil-bearing shales and strata, intestinal contents, sewage, and paper mill effluent. In recent years, molecular methods based on PCR, gene cloning, and hybridization probes have been developed to examine natural populations of bacteria directly without cultivation. Fluorescence in situ hybridization (FISH) is widely used for detection, identification, and quantification of microorganisms in the environment; the analysis of sulfur-oxidizing bacteria gives particularly interesting examples for FISH applications. Sulfide is produced from degradation of sulfur-containing organic matter and by dissimilatory sulfate reduction. Microbial metabolism of sulfide competes with chemical oxidation, either by O2 or Fe2+. Cultivation and enumeration of sulfur oxidizers are most successful in CO3 2- buffered media. High-performance liquid chromatography with fluorometric detection can also be used to measure oxidized and reduced S species.

This chapter focuses on the three core nitrogen cycling processes that are performed wholly or predominantly by prokaryotes: nitrogen fixation, nitrification, and denitrification. A phylogenetically and physiologically diverse array of bacteria and archaea carry out the process, using a highly conserved and complex suite of enzymes, in a variety of aquatic environments. Major advances in the study of nitrogen-fixing microbial populations in recent years have also occurred in the direction of assessing the potential importance of nitrogen fixers other than Trichodesmium. The ammoniaoxidizing bacteria (AOB) are considered to be strict autotrophs, while the nitrite-oxidizing bacteria (NOB) have a limited heterotrophic potential; growth of some strains is enhanced by metabolism of small organic acids. The most widely used approach for assessment of denitrifier diversity and community structure is based on the genes that encode nitrite reductase. Direct measurements of in situ denitrification rates in the open ocean are much rarer than those of nitrogen fixation. Recent advances in the study of the anammox process have highlighted how poorly conventional denitrification is understood, especially in the open ocean, and have focused attention on the need to improve methods for the direct in situ measurement of denitrification rates. Metagenomic approaches will allow us to link function, regulation, and population assessments by allowing us to identify multiple genes and operons and to link them to the identity of uncultivated organisms.

Field investigation of the marine phosphorus (P) cycle requires the use of a variety of methods to measure the ambient concentrations of total dissolved and particulate (both organic and inorganic) matter to assess local inventories of P and to estimate P fluxes. The latter include the delivery to and losses from the ecosystem in question and the rates of microbial P uptake and microbial decomposition of P-containing organic matter. In seawater, the most commonly measured P pools are (i) soluble reactive P (SRP), (ii) total dissolved P (TDP), and (iii) particulate P (PP). The structure and function of the marine P cycle are both time and space (vertical and horizontal) variable; a comprehensive understanding will require a four-dimensional resolution of the key P inventories and fluxes. Measurements of P-ATP in marine ecosystems date back to the classic work of Holm-Hansen and Booth, who developed the theoretical basis for its use in total microbial biomass estimation. Marine microorganisms assimilate D-ATP for P and for purine salvage. The central portions of all major ocean basins are characterized as “marine deserts” because of the small standing stocks of photosynthetic organisms and small nutrient inventories.

This chapter focuses on microbial metal redox metabolism, with an emphasis on iron (Fe) and manganese (Mn) cycling in the water column and surface sediments. It deals exclusively with metal cycling in circumneutral pH environments. The majority of the Fe and Mn that enters aquatic systems comes in the form of insoluble oxides (Fe and Mn) and silicate phases (Fe only; Mn-rich silicates are uncommon), which are produced during weathering of rock-forming silicate minerals (e.g., olivines, pyroxenes, and amphiboles) in the terrestrial environment and transported to coastal marine environments and lakes by rivers and streams. Although insoluble oxide phases are by far the most abundant forms of Fe(III) and Mn(IV) in neutral-pH aquatic environments, several recent studies suggested that small but significant quantities of soluble Fe(III) exist in circumneutral sediment pore fluids. A review by Emerson provides an overview of the history of research on circum-neutral bacterial Fe(II) oxidation as well as the physiology and systematics of Fe(II)-oxidizing bacteria (FeOB). In light of the foregoing analysis of the role of microbes in the oxidative side of the Fe and Mn cycles in aquatic environments, it is clear that Fe and Mn redox cycling is an example of microbial syntrophy analogous to the well-known syntrophic relationships among N- and S-oxidizing and -reducing microorganisms in natural systems. Both the water column and sediment chemical profiles illustrate several key aspects of the Fe-Mn redox cycling systems in aquatic environments.

Recent progress in studies of the microbiology of the surfaces of marine algae and sponges has provided new insights into a series of ecological processes. This chapter presents contemporary approaches to the study of several such processes, including the establishment of community composition and diversity, gene function in complex microbial consortia, chemically mediated interactions between prokaryotes and eukaryotes, key events in bacterial interspecies competition and colonization of surfaces, and factors facilitating persistence and survival of pathogens in the environment. While several aspects of modern technologies and methods are discussed, the intent of this chapter is to familiarize the reader with such approaches in the context of the biological processes displayed by marine microbial epiphytic and epibiotic communities on living surfaces. Biofilms occur on practically all surfaces, and most bacteria can make biofilms. The formation of biofilms on living surfaces has consequences for the individual (plant or animal), and hence, a number of defenses have evolved to either prevent such colonization or recruit specific bacteria to their surface that thwart subsequent colonization by other bacteria. Bacteria are the primary colonizing organisms on surfaces and serve as a focus for the attachment and growth of other organisms within the marine environment, including complex fouling communities of invertebrates, sessile plants, and animals. Similar to other model biofilms, Pseudoalteromonas tunicata biofilms develop a complex architecture consisting of matrix-enclosed structures called microcolonies separated by a network of open water channels. The chapter focuses on biofilm formation by vibrio spp. on living surfaces.

The observation that the vast majority of Earth’s microorganisms reside in extreme aquatic environments, given the immense volume of the deep ocean, is a simple fact. Some combination of extreme temperature, pressure, food supply, acidity, redox conditions, or even water activity describes the norm, not the rarity, for aquatic microbial habitats. This chapter provides some history, current trends, and specific examples of strategies and experimental protocols for studying microorganisms (Bacteria and Archaea) that inhabit the largest volume of extreme (or any inhabited) environment on the planet, the pressurized deep ocean and its sub-seafloor realm. Although few scientists have ready access to submersible operations in the deep sea or even to standard sampling expeditions by surface ships, the new investigator can find established researchers forthcoming with expertise, field samples, or cultured strains. Yet another variant on the theme of end-point experiments at high temperatures and pressures is the recent development in the Jørgensen laboratory of a high-pressure thermal gradient block, generally patterned after the system described for low-temperature, high-pressure research. The problem for extreme deep-sea environments, as elsewhere, lies with the available approaches to measuring activity, not with limitations to deep-sea sampling or seafloor experimentation. The latter are limited only by resources (and perhaps motivation), since a wide variety of sampling gear and instrumentation, including pressure retaining devices, are available for in situ study of deep-sea extremophiles.

To the uninitiated, it might seem almost paradoxical that such an outwardly humble substance as soil should bear such advanced technical treatments as those presented here: microarrays, reporter genes, molecular probes, fluorescence microscopy, metagenomics, proteomics, and others. However, soil microbial communities are arguably among the most complex biological entities, dwelling in extremely heterogeneous and complex physical environments. The study of plant-associated microbial communities presents challenges similar to those encountered in soils. Environmental microbiology is a dynamic field that has produced many innovations in recent decades. A section presents both classical and cutting-edge techniques in an attempt to summarize the current state of the field.

This chapter provides a first step into the analysis of the complex world of surface soil and a selection of conceptual approaches and resources for sampling surface soils so that the components of interest can be properly acquired and handled for subsequent microbial ecological studies. The focus on surface soils is one that distinguishes the chapter from others dealing with subsurface or aquifer sampling. The chapter also focuses on approaches that enable the scientist to acquire surface soil materials that are of appropriate quality and quantity for subsequent studies and provides information and resources for ensuring that soils are appropriately transported, stored, and distributed prior to use in experiments. The methods and approaches discussed are general ones that are provided to enable scientists to address their questions. The intact systems are generally more difficult to implement and maintain than those soil microbiological studies for which no such maintenance is required. Even in cases where the rhizosphere relationship is maintained, sampling from such chambers is problematic due to the immediate disturbance and destruction of the rhizosphere zone that takes place upon sample removal. Air drying is preferably done under a stream of sterile air flow to decrease the likelihood of introduction of laboratory microbial strains or other airborne propagules into the soil samples. The purpose of sieving is both to remove rocks and larger plant debris and invertebrates and to make the soils easier to distribute to experimental chambers.

The author’s goal in writing this chapter is twofold: (i) to persuade the researcher that investigating soil microbial communities by using DNA sequence data is the most appropriate method for making community comparisons or for inferring ecological processes based on community membership, and (ii) to present the best available analytic methods, the steps needed to use those methods, and how to interpret the results. The advent of molecular techniques and cloning allowed microbiology to escape the petri dish and radically changed our understanding of microbial diversity. A section of the chapter presents a brief summary of how to construct phylogenies followed by descriptions of techniques to compare microbial communities and to infer ecological processes. The results suggested that successional changes associated with marked shifts from the wet, cool conditions of winter and spring to the drier and warmer conditions of summer involve the turnover of highly divergent groups rather than shifts in the relative abundance of closely related species. Lineage-per-time plots were introduced by Nee and coworkers, and others as a means of inferring rates of speciation and extinction from phylogenies. Webb developed methods for testing whether sampled phylogenetic lineages comprising ecological communities were more closely related or more distantly related to each other than expected by chance based on the available species pool. Both methods have been applied to the analysis of microbial communities with intriguing results. The best methods require understanding models of DNA sequence evolution, phylogenetic inference, and character evolution, challenging subjects and areas of active research.

This chapter focuses on the methodology used to detect animal viruses in samples of soil. This methodology generally relies on elution and subsequent concentration of viruses from the soil, after which either cytopathogenicity or plaque formation assays are used to detect the viruses. These assays are based on the use of cultured animal cells as hosts for viral replication. The chapter also describes the use of plaque formation methodology to detect bacteriophages, viruses which infect bacteria. Other types of assay procedures, such as those based on the PCR, have also been developed to detect viruses in soil samples. Many different types of apparatus can be used to collect soil samples. These range from spoons and spatulas to shovels and powered augers. The available options for suitable sample containers include wide-mouthed screw-cap plastic jars and zipper closure plastic bags. Soil samples should be kept chilled to reduce thermal inactivation of the viruses. Bacteriophages can be detected by direct assay of soil suspensions using a plaque formation technique. If the presence of soil particles in the assay causes a problem, either because the resulting turbidity obscures assessment of the results or the number of contaminating soil bacteria and fungi carried along with the soil particles complicates plaque enumeration, then the bacteriophages can be eluted from the soil particles and the eluate can be assayed.

This chapter summarizes methods to (i) isolate and estimate numbers of soilborne propagules of arbuscular mycorrhizal (AM) fungi, (ii) propagate AM fungi by traditional and innovative methods, and (iii) detect and assess properties of these fungi by using recent biochemical and molecular technology. Soilborne propagules of AM fungi may include chlamydospores or azygospores, colonized roots, and hyphae. Isolates of special interest should be given a unique isolate code and then classified to the species level at a later date. To observe AM structures within the root, it is necessary to clear cortical cells of cytoplasm and phenolic compounds and then to differentially stain the fungal tissue. The most commonly used methods to obtain an estimate of the total number of propagules are the most probable number (MPN) and infectivity assays. The culture of AM fungi on plants in disinfested soil, using spores, roots, or infested soil as inocula, has been the most frequently used technique for increasing propagule numbers. Conducive environmental conditions for cultures of AM fungi are a balance of high light intensity, adequate moisture, and moderate soil temperature without detrimental additions of fertilizers or pesticides. Biochemical methods have been used to improve the means of detection and quantification of AM fungi in the environment. Although current molecular methods improve our ability to detect AM fungi in the field, monitoring of the abundance and distribution of individual fungal species remains laborious and expensive.

This chapter reviews endophytic bacteria and outlines procedures useful in establishing their presence, culture, and physiological and metabolic interactions with the host and their biotechnological uses. Alternatively, full-strength bleach may be used as described for endophytic fungal isolation. Isolation techniques may be modified to include techniques for isolating xylem-inhabiting endophytic bacteria from woody stems, although such bacteria make poor endophytes for biotechnological uses. The roots are plated on one or more media for bacterial isolations. It may be necessary to cut off and discard the end of each root when it is removed from the chloramine-T solution. Two general methods are used to isolate bacterial endophytes from surface-disinfected plant material: plant tissue direct and physical extraction isolation techniques. McInroy and Kloepper recommend the use of three media as a routine screening procedure during the isolation of endophytic bacteria: medium R2A (Difco) for oligotrophic bacteria, TSA (Difco) for culturable heterotrophic bacteria, and medium SC for the growth of fastidious organisms. The major use of endophytic bacteria has been for the control of plant diseases, and the manner in which this is accomplished is the subject of current research. The uses for phytoremediation with endophytic bacteria range from reducing petroleum hydrocarbon contamination in soils to reducing soil heavy-metal concentrations, including water-soluble and volatile organic xenobiotics, such as benzene, toluene, ethylbenzene, high-ammonia waste and animal manure, chloroform, dichloromethane, xylene, and other hydrophobic pollutants.

Soil microbial communities probably are the most complex of natural communities, and one study estimated that there may be as many as 1,000,000 “distinct genomes” per g of soil. This chapter is divided into two sections, the first covering approaches that require laboratory cultivation or incubation and the second addressing those based on analysis of indicator molecules that have been extracted directly from soil communities. A commonly used approach for identification of environmental isolates is based on growth on specific substrates and fermentative abilities. A more precise method of determining the phylogenetic affiliation of an isolate is by characterization of its rRNA. The technique is based on the concept that rRNA molecules, particularly the 16S and 23S rRNA molecules of prokaryotes, are highly conserved throughout evolution and are therefore useful as specific indicators of the phylogenetic affiliations of environmental isolates. A variety of DNA fingerprinting techniques can be used to rapidly differentiate closely related environmental strains. Regardless of the specific technique, the data obtained are DNA fragments that, when separated on agarose or polyacrylamide gels, yield a banding pattern specific to the genome under investigation. Rapid screening methods are of three categories that include: denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), and terminal restriction fragment length polymorphism (T-RFLP) analysis. The chapter talks about automated ribosomal intergenic spacer analysis (ARISA). An alternative approach to soil microbial community analysis is cloning of the soil “metagenome,” defined as the genomes of the total microbiota found in soils.

There are two primary types of microarrays, cDNA-based and oligonucleotide-based microarrays. This chapter focuses primarily on oligonucleotide-based technology because of the specialized problems encountered in agricultural and environmental microbiology. In general, oligonucleotide microarrays are designed by using some of the basic principles of primer and probe design for PCR and Southern hybridization. Microarrays targeting mRNA enable researchers to relate community structure to community function. The design of these types of microarrays allows investigators to evaluate gene expression and therefore to expose important metabolic activities of specific microbial communities. There are several issues related to the use of microarrays that limit their use in environmental studies. At the front end of this potentially powerful tool is the need for sensitive nucleic acid extraction from complicated sample matrices. Direct extraction of nucleic acids from environmental samples may coextract humic acids or other organic materials that may affect nucleic acid hybridization with microarrays. PCR amplification has been used by several groups to amplify nucleic acids prior to microarray analysis. In situations where uncharacterized organisms are present, exhaustive gene or clone libraries for the target gene need to be gathered in order to design group- or clade-specific probes for detection and distinction among all possible targets within the studied ecosystem.

This chapter introduces the primary concepts behind PCR, describes different flavors or specialized derivatives of PCR that are used to overcome various problems, provides an overview of ways in which the use of PCR can enhance soil microbiology research, and also provides information on equipment and other products designed for PCR. Ultimately, the PCR copies and amplifies the target sequence from the nucleic acid template, doubling the number of copies during each cycle of temperature. The different PCR methodologies are Nested and Seminested PCRs, Touchdown PCR, hot-start PCR, Booster PCR, Two-Step PCR, Multiplex PCR and Reverse transcriptase (RT) PCR. In recent years there has been increased interest in the use of DNA fingerprinting methodologies to identify bacteria at the isolate level and to subtype pathogenic bacteria. A more recent method, terminal-restriction fragment length polymorphism (T-RFLP) analysis, has also been used to study microbial community structures. T-RFLP analysis is based on the restriction endonuclease digestion of PCR products that have been fluorescently end labeled through the use of labeled primers. It is probably already apparent that the choice of the primer sequences is critical for the successful amplification of a specific DNA sequence. The degree of specificity can be varied by primer design and also by changing the annealing temperature. Sensitivity can be evaluated in terms of whole-cell lysates or pure genomic DNA preparations. Overall, PCR adds a useful new technology to aid in the study of plant and soil microbes.

This chapter describes some of the most useful methods for measuring the most important soil nitrogen transformations (N2 fixation, nitrogen mineralization, nitrification, and denitrification) and includes briefer descriptions of less important soil nitrogen transformations (NO3 - immobilization, heterotrophic nitrification, dissimilatory NO3 - reduction to NH4 +, and anammox) and newer functional gene detection methods. The most recently developed method for estimating N2 fixation is the use of acetylene as a surrogate substrate for the nitrogenase enzyme. In most soils, autotrophic nitrification is the dominant NO3 - producing transformation; however, NO3 - production directly from organic N by heterotrophic microorganisms can be important in some soils. The simplest and least expensive is the so-called acetylene block method, which takes advantage of the fact that acetylene inhibits the last step in the denitrification process, the reduction of N2O to N2. Two applications of denitrification method are presented here, namely, an assay for denitrifier enzyme activity and field measurements using soil cores. Most commonly, the composition or diversity of a given functional gene has been assayed either by using fingerprinting techniques, such as denaturing gradient gel electrophoresis (DGGE) or terminal restriction fragment length polymorphism analysis, or by cloning and sequencing. More recently, functional gene copy numbers have been quantified by the use of approaches such as competitive or quantitative PCR, which should serve as indexes of the population size of the organisms harboring specific genes.

The seemingly infinite variety of metabolic processes and products suggests potential for a correspondingly vast array of detection methodologies, and the scope of available technology for monitoring microbial metabolic activity is increasing so rapidly that it is difficult to catalog the available methods. Soil, the rhizosphere, and the phyllosphere are populated by complex communities of organisms, including microbes (bacteria, archaea, fungi, and protists) but also typically including plant and animal components. The rhizosphere and phyllosphere are defined by the presence of a plant, so it is inherently impossible to characterize microbial activity in these habitats through the study of genetically uniform microbial strains in isolation. Respirometry is increasingly being used for determination of biodegradation kinetics, and microcosm screening studies often are performed under controlled conditions to evaluate biodegradability potential and options for bioremediation. Several methods for estimating the metabolic activity levels of microbial populations involve quantification of cellular pools and rates of synthesis of specific biochemical components including RNA, DNA, ATP, and total adenine nucleotide. A number of methods for the quantification of metabolic activity and/or biomass of individuals, populations, or microbial communities involve direct microscopic observation of cells. This chapter has attempted to provide an overview of a number of these new methodologies, but it only presents a snapshot in time, as the number of new techniques and the opportunities they present for environmental microbiology continue to rapidly expand.

Extracellular enzymes are the proximate agents of organic matter transformation in soils. Data on microbial distributions and diversity are accumulating rapidly, and advances in molecular biology are providing new tools that are applicable to extracellular enzyme studies. In particular, proteomic approaches can be used to identify the extracellular enzymes that link genomic information with ecological processes. This chapter presents brief overviews of recent advances in extracellular enzyme research. Root activity may be supplemented by enzyme production from mycorrhizal fungi. Arbuscular mycorrhizal fungi are primarily involved in phosphorus (P) capture via production of phosphatases, accounting for 48 to 59% of total P uptake when P is supplied in organic form but only 22 to 33% when supplied in inorganic form. There is good evidence that invasive plant species alter nutrient cycling processes and that these changes are sometimes mediated by extracellular enzyme activity (EEA). Microplate technology also enables well-known colorimetric assays to be scaled down for high-throughput analyses. Two main challenges must be overcome to reduce variability to acceptable levels in these assays. First, there must be adequate homogenization of the environmental sample to ensure that the slurry in the microplate wells is representative of the initial material. The second challenge is that particles from the sample homogenate scatter the light beam of the microplate reader and make absorbance readings highly variable. Innovative approaches based on molecular biology are resolving long-standing questions about the mechanisms of biogeochemical processes and the controls on microbial diversity.

This chapter describes the application of fluorescent molecular probes used with immunofluorescence microscopy (IFM) and fluorescence in situ hybridization (FISH) techniques for studies of microbial autecology, with an emphasis on soil and root-associated microbes. Proper filter sets must be used to match the specific optical requirements for excitation and emission of fluorescent light by different fluorochromes. Fluorescein isothiocyanate (FITC), and to a lesser extent tetramethyl rhodamine isothiocyanate (TRITC), are common fluorochromes for single labeling experiments of IFM. There are two general approaches in immunofluorescence staining: direct and indirect. Both approaches involve the production, in an immunologically competent animal (e.g., rabbit), of a primary specific antibody against the antigen of interest. The exciting innovations in image analysis technology featured in Center for Microbial Ecology Image Analysis System (CMEIAS) v. 3.0 software will undoubtedly enhance the ecological analysis of in situ bacterial colonization using immunofluorescence and other discriminating microscopy techniques operating at single cell resolution. A large online probeBase database provides an overview of more than 700 published oligonucleotide probes and their characteristics for prokaryotic rRNAs suitable for FISH. The potential ability of FISH-MAR techniques to target the ecological niche for physiological groups of microorganisms in environmental samples may help to close the gap to the general enzymatic measurements, which are also very much increased in sensitivity. CMEIAS can extract an abundance of quantitative information on microbial community structure from the multiprobe FISH image.

This chapter focuses on those reporter gene systems that are useful in assessing the in situ transcriptional activities of promoters in bacterial cells in soil or associated with plant tissues or surfaces. More recently, reporter gene systems that are useful in assessing in situ gene expression by bacteria in natural habitats have been described. Due to the ease and sensitivity of its detection and the large number of plasmid vectors and transposons available for making transcriptional and translational fusions, lacZ is probably the most common reporter gene used in studies of gene regulation by bacteria in culture. gusA has also been useful in assessing gene expression by bacterial pathogens and symbionts of plants, especially in resolving spatial patterns of in situ transcriptional activity of bacterial cells. The xylE reporter was used successfully to study in situ gene expression by Pseudomonas putida inhabiting the rhizosphere. Reporter gene systems provide an excellent opportunity for microbiologists to gain new perspectives on the activities of bacteria inhabiting natural substrates, including their expression of specific genes, their recognition of environmental signals, their metabolic activities, and the chemical nature of the habitats that they occupy. A number of innovative and complementary reporter gene systems that are useful in environmental microbiology are now available. The discussion in this chapter focuses on the ice nucleation and green fluorescent protein (GFP) reporter genes, which have several unique attributes that make them extremely useful in studies evaluating in situ gene expression by bacteria inhabiting natural environments.

This chapter examines experimental design considerations for a population-based approach for identifying microorganisms involved in specific in situ functions. Although this chapter focuses on a particular population-based approach, many of the experimental design considerations discussed here apply to a wide range of rRNA gene-based population studies and sequence selective PCR assays. This chapter examines an experimental approach that uses the population-based strategy. The approach has the following three phases: (i) identifying populations of rRNA genes whose abundances correlate with the functional parameter, (ii) validating the rRNA gene correlates identified in phase I by using an independent quantitative assay, and (iii) isolating the microorganisms identified by the rRNA gene correlates and reintroducing them into the environment to assess their functions in situ. This approach was recently used to identify microorganisms that suppress the population development of the plant parasitic nematode Heterodera schachtii in southern California soil. Functional gradients are created by manipulating the microbial community with methods such as differential heat treatments, targeted antimicrobial agents, and nutritional amendments. Microbial community composition is examined by rRNA gene analysis. Nucleotide sequence analysis of rRNA gene clone libraries can be used to generate detailed depictions of microbial community composition. PCR assays can be validated by using them to amplify DNAs extracted from different environmental samples and then cloning and sequencing several randomly selected clones from each sample. The assays can be considered selective if they exclusively amplify the target sequence.

This chapter concentrates on characteristics of the different types of mobile elements that facilitate horizontal gene transfer (HGT) in bacteria. It begins with a discussion of the HGT; then describes the tremendous variation both within and between different classes of mobile gene transfer elements; and ends with a brief description of the techniques for studying HGT in environmental microbial communities. HGT is facilitated through some combination of the activity of mobile gene elements (MGEs) and/or host and recipient cellular enzyme systems. The chapter primarily focuses on the characteristics of the MGEs themselves. There are five basic classes of MGEs: phages, plasmids, transposons, integrons, and integrative conjugative elements (ICEs). Metagenomic techniques have revealed support for long-distance HGT in a number of bacteria and archaea. Metagenomic approaches have also been used to obtain phage genomes from environmental microbial communities. Many researchers have also taken a prospective approach to examine the frequency and factors influencing gene transfer in environmental microbial communities. Traditionally, prospective approaches have included the seeding of microcosms or environments with bacteria containing marker genes or naked DNA or plasmids and the selection of transformants.

The majority of methods that have been developed for studying microorganisms associated with arthropods in soil focus on arthropod pathogens, and these methods are emphasized in this chapter. Soil-dwelling microorganisms for which research methods are discussed in the chapter include viruses, bacteria, fungi, protists, and nematodes. All groups of arthropods are included in this chapter, but pestiferous species are the focus of the development of many methods for studying interactions with microorganisms. The chapter describes in more detail some of the major methods used for detecting, isolating, quantifying, and studying microorganisms, arthropods, and the interactions between these groups in the soil environment. Protocols for different types of microorganisms are varied based on characteristics of the specific groups of microorganisms. An effective technique often used for isolating some types of microorganisms is use of selective media. Microscopy has also been used to count arthropod-associated viruses, bacteria, and protists in soil. Soil-dwelling arthropods also exhibit behaviors to prevent infection, such as actively dislodging microorganisms from the body surface or applying glandular secretions inhibitory to entomopathogens. For ecological studies of communities of microorganisms, such techniques have principally been used to date for bacteria, but community profiling of other types of microorganisms is now also possible by using universal primers. Molecular techniques for profiling microbial communities have been used for studies of both bacterial and fungal communities.

This chapter provides an overview of lipid-based methods used to characterize microbial communities, specifically targeting the soil environment. The authors summarize the methods commonly used in investigations of soil communities, consider analytical and technical challenges of soil, describe statistical approaches for analyzing fingerprint data, and present applications to illustrate the types of information generated and questions that can be addressed with these methods. Types of lipid-based methods applied to soil include analyses of phospholipid fatty acids (PLFA), whole-cell fatty acid methyl esters (FAME) (also called EL-MIDI, MIDI method, and TS-FAME) in soil, sterols, and respiratory quinones. Fatty acid-based methods such as PLFA generally produce chromatographs (gas chromatography (GC) or gas chromatography-mass spectrophotometer (GC-MS)) consisting of multiple fatty acids. Some of the fatty acids have masses that are close to detection. PLFA analysis has provided insights into how soil microbial communities respond, in agricultural soils, to different management practices and, in natural ecosystems, to invasion by exotic plant species. In a comparison of the effects of different hay and fertilizer inputs, microbial community composition, based on PLFA fingerprinting, was significantly affected by the different treatments yet microbial C and N pools, as well as respiration, did not specifically respond to inputs. PLFA analysis is a direct wet-chemistry method (unlike, e.g., PCR-based methods) and generates quantitative information (e.g., nanomoles of different fatty acids).

To date, the majority of evidence indicates that most subsurface environments possess climax ecological communities that are well adapted to the environment in which they live. Like their counterparts on the surface, subsurface ecosystems are characterized by a high degree of microbiological diversity, they possess trophic structure, and they exhibit material cycling and energy transfer. Members of such ecosystems typically possess structural, physiological, or reproductive adaptations that allow them to disperse and survive in such habitats. Current studies argue that microbial activities influence the geochemical processes in both landfills and subsurface environments and that the altered geochemistry, in turn, influences the selection of microorganisms proliferating in the habitat. However, while microbial activity may often be limited by the availability of electron donors in uncontaminated aquifer systems, this is rarely the case with landfills. Sections in this chapter provide investigators with a greater understanding of the experimental approaches needed to study the microbiology of the terrestrial subsurface and an appreciation of interpretational limits imposed by the existing methodologies. The hope is that further study of subsurface microorganisms will provide insights into the process of microbial evolution and possibly into the origins of life itself.

Sampling of subsurface solids requires specialized techniques for drilling (advancing the borehole) and coring (collecting samples of subsurface materials). The goal of this chapter is to provide background information and general guidelines necessary for obtaining representative samples of subsurface solids and groundwater for microbiological and geochemical analyses. To this end, approaches for collecting representative solid and groundwater samples, processing of subsurface solids to reduce and assess microbiological and geochemical alterations during coring, and evaluating the quality of subsurface samples are described. The major advantages of coring through hollow-stem augers are that it is relatively simple, commonly available, and economical to use for obtaining nearly undisturbed unsaturated or saturated subsurface samples. A major source of microbial and solute contamination during hollow-stem auger coring beneath the water table is from sediment and water in the borehole. Selection of tracer type and deployment method is dependent upon the goals of the sampling, the types of lithologies being sampled, the type and source(s) of contaminants being traced, and the drilling and coring methods being used. The chapter provides background information and general guidelines for obtaining representative samples from subsurface environments for microbiological and geochemical analyses.

This chapter discusses techniques for the study of anaerobic biological reactions and microorganisms involved in refuse decomposition as it occurs in a landfill as well as the aerobic biological reactions that occur in the landfill cover. The chapter begins with a brief description of the major components of a sanitary landfill followed by a discussion of municipal solid waste (MSW) composition. Next, the manner in which cellulosic substrates are converted to CH4 and CO2 is described, followed by a discussion of CH4 oxidation in landfill cover soils. Factors that influence both anaerobic decomposition rates in landfills and aerobic CH4 oxidation in landfill covers are discussed. This is followed by a section on systems that can be used to simulate refuse decomposition and techniques that can be used to measure refuse biodegradation and microbial activity in landfills. Cellulose and hemicellulose are the principal biodegradable components of MSW. Carboxylic acids and H2 will accumulate and the pH of the system will fall, thus inhibiting methanogenesis. After placement of refuse in a landfill, several months or longer is necessary for the proper growth conditions and the required microbiological system to become established for biological decomposition. The presence of anaerobic protozoa in refuse excavated from landfills has been documented, and many of the protozoa contained symbiotic methanogenic bacteria that utilize H2 released by the host’s hydrogenosomes. Common contaminants in older landfills and leachate plumes include alkylbenzenes, ketones, and chlorinated aliphatic hydrocarbons.

Many studies have examined the differences in bacterial numbers, composition, and activity between groundwater and sediment samples. The majority of the literature has suggested higher percentages of attached bacteria than of unattached bacteria in aquifer systems, including in pristine aquifers and in aquifers contaminated with petroleum, creosote, sewage, and landfill leachate. In studies of aquifer biogeochemistry, much useful information regarding the microbial ecology of the system can be obtained by looking at organic compound and electron acceptor concentrations. An overview of approaches for identifying the redox characteristics of sediment is given in Christensen et al., and methods specific for determining reactive iron species in aquifers are reviewed by Heron et al. and Tuccillo et al. Other solid-phase electron acceptors that are important in aquifer systems include Mn(IV) oxides and barite. Important biogeochemical reactions catalyzed by indigenous microorganisms also are studied using a variety of experimental approaches including laboratory batch and column experiments as well as field-based in situ microcosms, tracer tests, and push-pull tests. The advantage of using a radiolabeled tracer in a study was that the reaction rates could be determined for the different steps in the denitrification pathway. Historically, researchers trained in geochemistry and hydrology created and tested hypotheses about aquifer biogeochemistry through laboratory assays and field-based geo-chemical measurements and experiments. Jeon et al. extended this research by using push-pull tests combined with stable-isotope probing to identify the specific members of the microbial community actively degrading naphthalene and rates of naphthalene degradation.

Microorganisms obtain their energy for metabolism by catalyzing a variety of oxidation-reduction reactions. The distribution of the terminal electron-accepting reactions in an aquifer is dictated by several factors. The sequence of terminal electron-accepting reactions occurs in the order shown in a table, but very long periods can be necessary before the supply of a given electron acceptor is depleted. Determining whether aerobic respiration is the dominant terminal process is relatively straightforward. When oxygen is present, it is the electron acceptor, not only for the thermodynamic reasons but also because it is toxic to the obligately anaerobic processes (iron reduction, sulphate reduction, and methanogenesis) and inhibits the expression and the function of the denitrification enzymes. In many aspects, iron reduction differs from the other terminal electron-accepting reactions. The substrate, Fe(III), is present in many forms, all crystalline solids, and often in large quantities in the saturated subsurface. Concentrations of dissolved hydrogen can serve as an additional indicator of the predominant terminal electron-accepting reaction. An integral part of future characterizations and insights regarding microbial communities in the subsurface must necessarily involve the context of the terminal electron-accepting reactions.

This chapter describes and discusses laboratory and field techniques for studying microbial transport behavior in aquifer materials and model porous media. Changes in ionic strength (I) during transport studies may occur inadvertently as a result of using halides as conservative tracers and may lead to density-induced sinking of the tracer cloud. Substantive increases in I as a result of injection of high concentrations of halide tracers can also result in overestimations of microbial attachment. In order to differentiate "test" microorganisms from indigenous subsurface populations and/or from other inadvertently introduced populations, microorganisms used in laboratory or in situ transport tests are typically labeled a priori with a stable tag. Other methods of labeling microorganisms for use in in situ and column transport studies have involved the use of stable isotopes ratio mass spectrometry (IRMS). The characteristics of the conservative tracer breakthrough curve can then be used comparatively to determine some of the major transport parameters exhibited by the introduced microorganisms. Most controlled field investigations of subsurface microbial transport are conducted on limited spatial scales relative to the scales of interest to those concerned with pathogen transport to water supply wells, with microbially enhanced oil recovery from petroleum reservoirs, and with the feasibility of using introduced bacteria for aquifer restoration.

This chapter discusses the methods used to characterize the microbial populations in oil reservoirs, to detect and control their detrimental activities such as souring and corrosion, and to stimulate their beneficial activities such as improved oil recovery. To be effective for enhanced oil recovery (EOR), the biosurfactant must reduce the interfacial tension between oil and brine by several orders of magnitude. Nitrite-reducing bacteria (NRB) can be placed in two functional groupings, the heterotrophic NRB (hNRB), and the nitrate- or nitrite-reducing, sulfide-oxidizing bacteria (NR-SOB). Samples to enumerate sulfate-reducing bacteria (SRB), general aerobic bacteria (GAB), or acid-producing bacteria (APB) should be taken at various points in the system, and the same sample should be used for the enumeration of each group of bacteria. The presence of SRB indicates a potential for corrosion and souring. The design of a microbially enhanced oil recovery (MEOR) process varies depending on the reservoir and problems that limits oil production. Oil reservoirs contain diverse and metabolically active microbial communities. Cultivation-dependent and cultivation-independent approaches can be used to characterize these communities and determine how their activities may affect operations. Knowledge of the potential microbial activities present in the surface facilities and the reservoir is critical to avoid the stimulation of unwanted microbial activities such as corrosion, souring, and biofouling during petroleum exploitation activities. This knowledge can also be used to stimulate the beneficial activities of microorganisms to improve oil recovery or alter the terminal electron-accepting process by nitrate addition.

Source Water Assessment and Protection Programs (SWAPPs) require states to delineate and assess the areas of land that contribute to public water systems using both surface and groundwaters. An integral part of these programs is an analysis of the susceptibilities of these systems to chemical and microbial contamination. There are several methods that can be used to establish placement of drinking water wells to minimize microbial contamination. The six most common delineation methods, listed in order of increasing technical complexity, are as follows: arbitrary-fixed-radius method; calculated-fixed-radius method; simplified variable shape method; analytical method; hydrogeologic mapping; and numerical transport models. An analysis of hydrogeology, an understanding of the contaminants and the factors that control their fate and transport in specific environments, and an analysis of the effectiveness of existing prevention and mitigation measures are essential so that states can apply the assessment results to source water protection. There are several components of the proposed groundwater rule (GWR) that require similar assessments of groundwater vulnerability or sensitivity to microbial contamination to those performed by the SWAPP. There are many different methods that can be used to delineate zones around drinking water wells to protect the water supply from microbial contamination. Finally, many of the wells that are used for drinking water are owned and operated by small communities and individual businesses.

This chapter introduces the study of airborne microorganisms and their by-products, discusses indoor and anthropogenic outdoor sources of airborne microorganisms that affect human health and the environment, reviews the association of bioaerosols and indoor environmental quality, and briefly presents background information on airborne microorganisms as potential bioterrorism agents. The transport and ultimate settling of a bioaerosol are affected by its physical properties and by the environmental conditions that it encounters while airborne. The most important physical characteristics are the size, density, and shape of the droplets or particles, while the most significant environmental conditions are the temperature, relative humidity, and magnitude of air currents. Numerous anthropogenic activities serve as the origin of bioaerosols in outdoor environments, especially agricultural practices and wastewater treatment processes. In summary, interest in the populations of airborne microorganisms in agricultural and industrial settings, health care facilities, residences, offices, and classroom environments has increased in recent years. The threat of purposeful release of microorganisms as bioterrorism agents has prompted renewed interest in aerobiology, and research activity in this area of environmental microbiology has rapidly expanded.

This chapter presents various bioaerosol sampling and analysis methods to facilitate the selection of instrumentation and techniques. The principles of bioaerosol sampling are presented, followed by a review of sampling methods and techniques currently available, 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. Measurement of airborne microorganisms with a bioaerosol sampler often aims at documenting the presence of specific sources. The impaction method separates particles from the air stream by utilizing the inertia of the particles to force their deposition onto a solid or semisolid collection surface. Liquid impingement is similar to impaction in that the inertial force of the particle is the principal force removing it from the air. Filtration achieves the separation of particles from the air stream by passage of the air through a porous medium, usually a membrane filter. The collection of airborne microorganisms onto a filter material is used in bioaerosol monitoring due to its simplicity, low cost, and versatility. Validation of surface sampling methods and the development of standardized sampling and analysis protocols are vital for the detection of biothreat agents, the determination of their concentration and distribution, and the evaluation of the effectiveness of remediation procedures.

Respiratory exposure to certain pathogenic or toxigenic microorganisms and/or elevated concentrations of environmental organisms could result in health effects, such as allergic reactions, irritant responses, toxicosis, and respiratory illness. This chapter presents an overview of available methods for the analysis of bioaerosols. In addition, the potential use of enhanced monitoring of bioaerosols with PCR, biochemical, and immunological assays is discussed. Several conditions, such as pH, temperature, water activity, nutrients, antibiotics, light, and aeration, can be manipulated to favor the growth of a selected group of organisms. In contrast with culture techniques, microscopic analysis allows enumeration of both culturable and nonculturable microorganisms. However, identification of microorganisms to the species level is usually not possible without the aid of a taxon-specific technique, such as immunospecific fluorescence staining. Flow cytometry offers an alternative to microscopic enumeration of total cells. In a different application of flow cytometry, researchers coupled sandwich immunoassay utilizing microsphere beads with flow cytometry for the detection of biothreat agents. Fluorescence immunoassay consists of staining samples with a fluorescently labeled antibody that binds specifically to the antigens on the surfaces of the target organisms and enumeration by epifluorescence microscopy. Hybrid technologies are being developed that combine more than one analysis method to maximize the accuracy of the results obtained. One such system utilizes immunoassay, flow cytometry, and PCR analysis in various combinations for the continuous monitoring of airborne biological threat agents.

The fate and transport of microorganisms in the atmosphere are complicated issues involving many physical and biochemical factors. The transport of bioaerosols is primarily governed by hydrodynamic and kinetic factors, while their fate is dependent upon the specific biological composition, chemical makeup, and the meteorological parameters to which they are exposed. Local atmospheric conditions such as wind speed, temperature, and relative humidity (RH) are strongly influenced by the features of large-scale flow fields, geographical locations, and local topography. The most significant environmental factors influencing viability are RH, solar irradiance, temperature, and oxygen concentration. Additional influences are exerted through air ions and open-air factors (OAF). The state of water and the water content associated with bioaerosols are fundamental factors influencing the fate or viability of these microorganisms. Studies to determine the effect of temperature on aerosol stability have generally shown that increases in temperature tend to decrease the viability of airborne microorganisms. RH, water activity, oxygen concentration, aerosol age, and the presence of other gases all influence the effect radiation exerts on airborne microorganisms. Data are presented in a section relating aerosol stability or infectivity studies of bacteria, animal viruses, bacterial viruses (phages), and other microorganisms. Before a bioaerosol project is initiated, a substantial amount of study must be completed to determine methods to maximize the recovery of aerosolized microorganisms. These studies should include effects concerning sampling media, counting media, generation media, and the makeup of test atmospheres.

This chapter reviews existing literature on airborne fungi, with emphasis on indoor fungal growth and contamination as well as the health effects of mycotoxins and fungal volatile organic compounds (VOCs). A wealth of literature on outdoor airborne fungi can also be found in reviews by various researchers. The majority of airborne fungi collected on samplers and grown on agar media are Deuteromycotina and Zygomycetes. The detection of airborne fungi does not necessarily suggest growth and amplification of fungi indoors. However, it is believed that actively growing fungi in the indoor environment are the primary cause of the adverse health effects due to exposure to indoor fungal allergens, mycotoxins, and fungal VOCs. Fungi are commonly known to cause infections of the skin and other body organs, as well as allergies and respiratory problems. These conditions are briefly discussed. Fungal by-products (i.e., mycotoxins) have ciliostatic effects in the respiratory tract, which can be one of the important pathological mechanisms causing diminished mucociliary clearing and local inflammatory effects in the airways and sinuses. Organic dust toxic syndrome (ODTS), also called toxic pneumonitis, is a nonallergic, noninfectious form of an acute inflammatory lung reaction to high-level fungal dust exposure. An overview of clinically important health disorders based on various case reports and results of disease cluster investigations is presented for the most important mycotoxin producers.

Livestock production and concentrated animal feeding operations (CAFOs) are highly contaminated environments. These include swine, poultry, dairy, and beef cattle facilities, and Staphylococcus, Pseudomonas, Bacillus, Listeria, Enterococcus, Nocardia, and Lactobacillus spp. are commonly present. Bacteria, endotoxin, and organic dust are the major airborne contaminants in swine CAFOs, and culturable airborne bacterial levels as high as 105 CFU/m3 have been reported. The cotton textile, vegetable- and seed-processing, machining, and forest products industries and slaughterhouse facilities are environments with moderate concentrations of bacteria and endotoxin. Outdoor environments also may contain high airborne concentrations of bacterial contaminants resulting from their proximity to bacterium-laden industries or activities. Endotoxins are regularly found in indoor and outdoor air, water, soil, and food. Endotoxins exist as lipopolysaccharides (LPS) or lipooligosaccharides depending on the genus. Acute inhalation exposure to purified endotoxin or to particulate matter containing endotoxin can induce airflow obstruction, airway hyperreactivity, inflammation, and systemic symptoms including fever, chills, myalgia, and malaise. The inflammation is marked by the appearance of neutrophils and proinflammatory cytokines in lung lavage fluid and includes clinical conditions such as asthma, asthma-like syndrome, organic dust toxic syndrome, and byssinosis. The biological activity of endotoxin from various organisms varies depending on their chemical structure and bioavailability. Susceptibilities differ between individuals dependent on genetic differences in the recognition and response pathways.

Legionellae are now associated with two forms of respiratory illness, collectively referred to as legionellosis. Inhalation of legionellae in aerosolized droplets is the primary means of transmission for legionellosis. The number and types of sites that should be tested to detect legionellae must be determined on an individual basis. Two primary sample types should be collected when sampling for legionellae: water samples and swabs of point-of-use devices or system surfaces. Examination of water samples is the most efficient microbiologic method for identifying sources of legionellae. Most investigations of epidemic legionellosis have used culture to detect legionellae in the environment. Practical information concerning treatment processes that effectively control legionellae is limited. Practices to control legionellae in amplifying reservoirs can be divided into two categories, routine maintenance and emergency decontamination procedures. Some control strategies are intended to prevent exposure of susceptible individuals to aerosols which may contain legionellae. Early investigations of Legionnaires’ disease associated with cooling towers resulted in a recommendation for relocation of cooling towers or air intake vents so that cooling tower exhaust would not be carried directly into the heating, ventilation, and air-conditioning systems of buildings. Effective prevention strategies require more-effective decontamination techniques and approaches to prevent amplification of legionellae in reservoirs.

Viruses can become airborne through the release of contaminated liquids or dried material and can then be carried by air currents indoors, and outdoors. The methodology for generating, storing, and collecting viral aerosols has already been reviewed. Therefore, this chapter is focused on a critical review of the information on the role of air in the spread of vertebrate viruses. Work with airborne viruses also requires stringent safety precautions. In spite of the limitations, ultrafine threads are the best means to study the influence of atmospheric chemicals and light and irradiation on airborne viruses. According to Pike, 27% of the cases of laboratory-acquired infections were due to airborne viruses; cases in research settings accounted for more than 67% of such infections. Increasing use of recycled air will further enhance the risk of exposure of susceptible individuals to airborne viruses. Recently, alteration of the lung airway surface properties by spraying normal saline into the respiratory tract has been found to diminish the number of exhaled bioaerosols by human subjects by over 70%. Immunization of humans and animals by exposure to artificially aerosolized virus is a very attractive alternative to current practices of parenteral or oral vaccination. Many human pathogenic viruses and animal pathogenic viruses, some of which are relatively obscure and outside the mainstream, continue to be considered potentially useful bioweapons, and recent accounts of their production and stockpiling attest this fact.

Although propagules of plant pathogens are dispersed by wind, rain, soil water, insects, and even humans, this chapter focuses on the airborne spread of those pathogens. In general, spores of most fungal pathogens are adapted for airborne transport; however, much of the aerobiological research on agricultural pathogens has focused on a limited number of fungi that cause economically important diseases. The chapter examines a few of the significant fungal pathogens that are dispersed by the aerobiological pathway. Today, rust fungi still remain among the most serious agricultural pathogens. The major vehicles for disease spread are the uredospores, which are easily carried by wind from one plant to another, giving rise to epidemics. The major dispersal agents of smut fungi are the asexual teliospores. The chapter talks about many plant pathogens that can propagate on hosts at various distances and can cause extensive damage and economic loss; thus, the focus on plant pathogens as anticrop weapons is understandable. The ultimate goal of forecasting is to reduce costly and environmentally hazardous pesticide applications during periods when disease occurrence is not likely. Aerobiological studies must be part of any effort to understand the distribution and epidemiology of agricultural pathogens that rely on air currents for dispersal.

Biotransformation and biodegradation have been active areas of research since the very beginnings of microbiology and particularly since the beginning of the industrial revolution in the late 18th and early 19th centuries, when the first of many thousands of anthropogenic chemicals began to be introduced into the environment. Much of the historical work in these areas has involved the study of pure cultures of microorganisms isolated by enrichment culture for their abilities to degrade or transform a particular chemical. Many elegant methods employing biochemical, physiological, and/or genetic approaches have been developed over the decades to elucidate the intricate and evolutionarily beautiful pathways employed by such pure cultures to degrade or transform natural and anthropogenic chemicals.

The major advantage of microarrays is that attachment to the solid surface confers the ability to print an enormous number of different probes (thousands to hundreds of thousands per square centimeter) on an individual array. The authors proposed that microarrays of potential use for environmental samples can be divided into three or more major groups based on the type of nucleic acid probes arrayed including (i) phylogenetic oligonucleotide arrays (POAs), (ii) community genome arrays (CGAs), and (iii) functional gene arrays (FGAs). Due to the analytical versatility of FGAs, this chapter focuses on their development, application, and use in characterizing functional genes involved in biogeochemical processes and bioremediation. Along with the size and sequence of the FGA probes, the temperature and composition of the hybridization solution control the stringency of hybridization. It is preferable to use highly specific hybridization conditions for FGAs because of the potential for many similar sequences to be present. Researchers have successfully used FGAs to examine microbial involvement in several environmental processes including nitrogen fixation, nitrification, denitrification, and sulfate reduction in freshwater and marine systems; degradation of organic contaminants including polychlorinated biphenyls and polycyclic aromatic hydrocarbons in soils and sediments; and methane-oxidizing capacity and diversity in landfill-simulating soil. Development of prokaryotic mRNA amplification methods will likely broaden the range of samples for which FGA analysis of microbial activity is possible.

Metagenomics (synonymous with environmental or community genomics) is the construction and analysis of libraries containing random DNA fragments cloned from naturally occurring microbial communities. The goals are to (i) describe the genomic structure of microbial communities, (ii) decipher the physiology and ecology of uncultured prokaryotes, and (iii) identify novel genes, enzymes, and molecules for biotechnology. Most methods for extracting DNA from soil are intended for PCR-based applications such as the amplification of 16S rRNA or other genes rather than direct cloning, which is especially challenging because of humic acids in soil that coextract with DNA and inhibit restriction enzymes. This chapter provides detailed methods to obtain DNA of sufficient purity for cloning into plasmid, cosmid, fosmid, or bacterial artificial chromosome (BAC) vectors. The most common techniques for soil metagenomic library construction entail partial digestion of soil DNA with various restriction enzymes. Chemical and enzymatic lysis methods are normally employed, which may bias the extraction towards easily lysed cells. There are two basic approaches to high-molecular- weight (HMW) DNA extraction from soil: (i) direct lysis of cells in the soil matrix and (ii) separation of cells from soil followed by lysis. Cell separation is more time-consuming than direct lysis, but larger DNA can be extracted when the cells are embedded and lysed within an agarose plug.

Genetic algorithms (GAs) have a number of specific advantages over other optimization techniques that make them especially attractive for such use in microbial ecology. This chapter provides a general outline of the GA approach to optimization and lists a number of specific considerations for microbial ecological applications. For the microbial ecological applications discussed here in which algorithm speed is not a concern, this can be implemented by going over every gene on the chromosome of every new individual and tossing a weighted coin. From a fundamental ecological point of view, using GAs to optimize functions in microbial ecosystems offers great promise. Genetic algorithms belong to the larger field of evolutionary computation, which contains other population-based optimization methods that are similarly inspired by the biological principle of natural evolution. Evolutionary programming and evolution strategies are similar to genetic algorithms but typically do not include a recombination or crossover step. These two types of methods may provide an alternative to genetic algorithms for the optimization of functions of microbial ecosystems, especially when the optimization task is to find the appropriate level of various ecosystem factors, rather than more simply to find the right combination of such factors. The genetic programming approach essentially uses genetic algorithms to evolve computer programs, typically represented as tree structures. This approach seems less suitable for the optimization of functions of microbial ecosystems because there is no obvious way to tie an evolving computer program to the properties of an ecosystem.

Isolated microorganisms constitute only a minor fraction of the global microbial diversity, which may comprise millions of species. This chapter discusses some cautionary guidelines that should be followed in the attempts to link phylogeny with function. The simplest and most commonly used approach to identify organisms involved in biodegradation is to isolate microbial strains capable of utilizing the target substrate, xenobiotics, or pollutant as a sole C/N source. Sequence-dependent approaches to identify genes are largely limited by an a priori knowledge of gene(s) mediating the biodegradation processes but have proven invaluable for an understanding of the key enzymes in these metabolic pathways. The majority of control in prokaryotes is thought to occur at the transcriptional level, so the presence of an mRNA is strong evidence for the expression of that gene product. Methods for amplifying genes requiring only one gene-specific primer impose less sequence-dependent bias than standard two-primer PCR amplification procedures. These PCR-based strategies have been used for the recovery of the up- or downstream regions flanking a single PCR primer for the recovery of full-length genes. The minimum number of clones that need to be screened in order to find a gene of interest in the metagenomic library increases with the presence of eukaryotic DNA (a eukaryotic genome is 3 to 140,000 Mbp compared to prokaryotic organisms of 0.6 to 9.5 Mbp). In summary, metagenomic techniques have begun to address both the identity of active organisms and their gene products that mediate biodegradation.

This chapter reviews methods for proteomic analyses of white-rot fungi, which can degrade both the carbohydrate and the lignin components of wood to CO2. Microbial degradation of lignocellulosic material, due to the heterogeneity of the substrate, involves an ensemble of extracellular enzymes. Woody biomass has three major components: cellulose, hemicellulose, and lignin. Lignin is formed by free radical coupling of the phenylpropanoid units and thus can be linked by more than 12 types of linkages. Another group of wood-degrading Basidiomycetes, the brown-rot fungi, can also degrade fully lignified tissue but without substantial depletion of the lignin. Ascomycetes and Fungi Imperfecti cause the soft-rot type of wood decay which was categorized only within the past 30 years. The enzymes that utilize H2O2 and molecular oxygen to oxidize organic and inorganic substrates are the peroxidases and the laccases, respectively. Phenol extraction proved to be very effective in extracting plant proteins from interfering substances to obtain good resolution in 2-D gels. Methods are available for processing of the complete extracellular protein mixture by protease digestion and separation and identification by liquid chromatography and tandem mass spectrometry (MS-MS). For heavily glycoslyated proteins, CapLC–nanoelectrospray ionization–MS-MS can be used instead to analyze in-gel tryptic digests.

Mycoremediation, or fungus-based remediation, is an ex situ form of bioaugmentation, in which hazardous organics are degraded or detoxified by fungi that are introduced into the contaminated soil via a fungal inoculum. Due to the ability of white-rot fungi (WRF) to degrade extremely recalcitrant contaminants (e.g., high-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs), work on fungus-based remediation has focused on the treatment of soils contaminated with these types of chemicals. In addition, work has progressed on the soil microbiology of mycoremediation and on the development of techniques to monitor the fate of fungi inoculated into soils. Fallout of PCDDs and PCDFs from combustion at incineration facilities has resulted in serious pollution of the surrounding soil environment because of their extremely high persistence and toxicity. Importantly, the extent of the enhancement varied with soil and fungal species. While degradation of fluorene was complete in all three treatments, the extents of degradation of the low-molecular-weight (LMW) PAHs phenanthrene and pyrene were less in the soil inoculated with Pleurotus ostreatus than in soil inoculated with Antrodia vaillantii or noninoculated soil. Several techniques have been adapted or developed for tracking of inoculated fungi in soils treated with mycoremediation. The phospholipid fatty acid (PLFA) method has the added benefit of revealing interactions between inoculated fungi and indigenous soil bacteria by simultaneous extraction of other marker PLFAs.

This chapter reviews the methods for fungal solid-state fermentation (SSF) for bioconversion of lignocellulosic biomass. The major chemical components of lignocellulosic biomass include cellulose, hemicelluloses, and lignin. The SSF process has great potential for the bioconversion of lignocellulosic biomass. The most efficient degraders of lignocellulosic biomass are reported to be higher fungi, including white rot fungi and some mushrooms. SSF has great potential in microbial conversion of lignocellulosic biomass into biofuel, enzymes, animal feed, and biofertilizer. Major fungi involved in lignocellulosic biomass conversion belong to the groups Ascomycetes, Deuteromycetes, and Basidiomycetes. The major disadvantage of spores is that they are metabolically dormant, and hence metabolic activities must be induced and the appropriate enzyme systems must be synthesized before the fungus begins to utilize the substrate and grow. The solid substrate should be continuously mixed for proper aeration at selected intervals to prevent lumping and to obtain uniform mold growth and bioconversion. The lignin-degrading system of the white rot fungi has potential applications in the area of lignocellulose bioconversion by SSF. The selection of a leaching method such as percolation, pulsed plug flow extraction, countercurrent extraction, or hydraulic pressing is probably the most important step in SSF downstream processing, since the economics of the process will be dictated by the leached product concentration. After the leaching step, the extract may be clarified by either filtration or centrifugation to remove suspended cells, spores, or solid residues.

Microeukaryotes, including fungi and protists, are important members of communities within natural environments. This chapter focuses exclusively on available molecular approaches for investigating microeukaryote assemblages within various natural environments. Oligotrophic environments generally require more sampling effort to obtain adequate biomass for molecular characterization than do more nutrient-rich environments. Sediment sampling achieved by the implementation of sterile mud samplers and coring devices has been described for hydrothermal sediments and anoxic sediments. The 18S rRNA gene contains both conserved and variable regions that can be exploited for characterization of the microeukaryote assemblages within an environment. Multiple PCRs containing amplicons of expected size can be pooled to minimize bias from single reactions. Immunological approaches have proven useful in ecological studies for the detection and enumeration of microeukaryotes. Quantitative real-time PCR uses the rate of accumulation of amplified target DNA during the PCR to estimate the copy number of the target in the original sample. Denaturing gradient gel electrophoresis is a PCR-based method in which the amplified 18S rRNA gene fragments are separated by polyacrylamide gel electrophoresis containing a linearly increasing gradient of denaturant.

This chapter provides an overview of selected techniques that are used to detect, quantify, and evaluate the activity of low-molecular-weight metabolites produced by the brown rot fungi. The role of low-molecular-weight fungal metabolites in the brown rot decay process and their potential use in bioremediation and industrial processes are also briefly considered. The chapter also talks about the techniques used in the purification, quantification, and characterization of selected types of low-molecular-weight metabolites produced by brown rot fungi. Detection of hydroxyl radicals is limited by their extremely short half-life and high level of chemical activity. The chapter talks about selected methods that have been used in the characterization of wood or lignocellulose colonized by brown rot fungi or treated with isolated fungal metabolites. These include cellulose chain length determination, X-ray analysis, molecular beam mass spectroscopy (MBMS) and near infrared spectroscopy (NIR) evaluation of complex substrates, and 13C thermochemolysis characterization of lignin modification. Bioremediation applications are particularly intriguing because of the demonstrated ability of brown rot fungi to ramify through soil and colonize wood and other substrates in the natural environment. The ability to characterize the underlying nonenzymatic microbial processes utilized by the brown rot fungi will contribute to our ability to both control and utilize these unique degradative organisms in a better manner.

Uncultivated magnetotactic bacteria (MTB) can be studied most easily in microcosms set up in the laboratory. In their simplest form, microcosms consist of about one-third sediment together with about two-thirds overlying water and are placed in a flask or aquarium (0.1 to 5 liters) and incubated in dim light at room temperature. Many MTB can easily be detected and collected by taking advantage of their active, directed migration along magnetic field lines. Cell morphology and flagellar patterns of specific MTB can be determined by using negatively stained preparations of cells and transmission electron microscopy. Magnetosomes are electron-dense structures that can be visualized in cells without staining. The high abundance, conspicuous morphology, and magnetic response of the MTB allow for their easy microscopic identification and have made them an attractive subject for the early application of cultivation-independent, rRNA based techniques. Isolated cultures of MTB need to be properly preserved, as both the magnetic polarity and the capability of biomineralizing magnetosomes can be irreversibly lost upon prolonged subcultivation and storage in the lab due to genetic instability. For the establishment of a genetic system for the MTB, the ability to grow cells as colonies on agar plates is a necessity, since colony formation on the surfaces of agar plates is required for clonal selection in genetic analysis.

The availability of bacteria that are able to interact with electrodes presents opportunities for conversion of organic compounds into electricity. While the obvious application of this phenomenon involves power generation, it also implies new routes to biological sensing and control of oxidative or reductive biocatalytic reactions. A combination of the standard fuel cell and poised-electrode approaches is the use of potentiostats to perform analyses such as cyclic voltammetry and electrochemical impedance spectroscopy on mature microbial biofilms, which can demonstrate the presence of redox-active proteins or mediator pools on or near electrodes. While devices used for the study of electrode-reducing bacteria are typically custom-constructed to meet the needs of the experiment or organism being studied and procedures are continually evolving to obtain higher current densities or allow voltammetry measurements, some sample experimental protocols that illustrate issues of sterility and anaerobic technique are provided in this chapter. By combining microbial physiology with an understanding of electrical and chemical engineering, an equally rich spectrum of devices that are able to study and control the growth of electrode-reducing bacteria can be imagined and should be explored.

Microbial communities are recognized to mediate iron geochemical cycling in aquatic, terrestrial, and subsurface ecosystems. This chapter discusses the role that anaerobic, Fe(II)-oxidizing microorganisms (FOM) play in iron biogeochemical cycling, identification of the metabolism, and isolation of anaerobic FOM. In the anoxic zone, Fe(III) oxides provide an electron sink and are chemically or biologically reduced. The significance of the phototrophic Fe(II) oxidation processes to contemporary iron biogeochemical cycling is limited to the photic zone as light penetration of soil and particulate matter is only between 8 to 200 μm. At circumneutral pH, light-independent microbially mediated oxidation of Fe(II)aq and Fe(II)s coupled to nitrate reduction has been demonstrated in a variety of freshwater and saline environmental systems. To date the presence of anaerobic, Fe(II)-oxidizing bacteria in paddy soil, pond, wastewater, stream, ditch, brackish lagoon, lake, wetland, aquifer, hydrothermal, subsurface, and deep-sea sediments has been identified using traditional microbiological techniques. Due to the broad phylogenetic diversity of these organisms and a lack of knowledge of the functional genes involved, molecular approaches to population studies and community dynamics of this metabolism are limited.

The study of extreme environments and the microorganisms that inhabit these environments, the so-called extremophiles, has become increasingly popular in recent years. One important class of extreme environments is those of low pH, which are inhabited by prokaryotic and eukaryotic microorganisms referred to as acidophiles. The ability of microbes to grow at low pH is a seemingly ancient trait, as acidophiles are widely distributed throughout the two prokaryotic domains. Heterotrophic acidophiles can be enriched for, and cultivated in, liquid media containing a variety of single or complex carbon sources. Iron-oxidizing acidophiles were particularly problematic, with some (e.g., Leptospirillum ferrooxidans) being categorized as being incapable of growing on solid media. Recent advances in this area have led to the development of techniques that allow all categorized species of acidophilic prokaryotes to be grown on solid media. Most probable number (MPN) microbial counts of cultures in specified liquid media (e.g., acidic ferrous sulfate medium) and incubated at an appropriate temperature continue to be used to enumerate acidophiles on a physiological basis. More recently, 16S rRNA gene libraries have been prepared from DNA samples obtained at an abandoned pyrite mine at the Iron Mountain site and acidic geothermal sites on the volcanic island of Montserrat.

Many toxic metals appear to be able to enter cells by import systems for essential elements or molecules, and although chromosomally encoded homeostasis systems for essential metals are widespread, in bacteria many toxic metal resistance genes are found on mobile genetic elements such as plasmids and transposons. Highthroughput methods in DNA sequencing, metagenomics, and the postgenomic technologies are going to revolutionize our understanding of toxic metal-bacterial cell interactions and the diversity of toxic metal(loid) resistances. With this in mind, this chapter talks about the options and potential workflows open to researchers wishing to use molecular techniques for the study of these resistance genes. Most of the techniques discussed in the chapter are equally applicable to the study of other nonmetal resistance phenotypes of environmental bacteria, because the techniques are generic. The chapter, however, focuses on and discusses the merits of molecular biology techniques that can be used to investigate bacterial resistance mechanisms. The outline of the experimental procedure and primary purpose of this technique is to culture toxic-metal-resistant bacteria, so that the genetic and biochemical basis for their resistance mechanisms can be further investigated. Although we are now entering a new phase in molecular research into toxic metal resistances, the techniques described in this chapter still have a valuable place in describing new metal-resistant bacteria.

X rays are a powerful probe for investigating metal and radionuclide transformations in soils, sediments, and groundwaters. In particular, synchrotron-based X-ray investigations can identify the changes in an element’s valence state and chemical speciation that often result from microbially mediated electron transfer. This chapter describes some of the synchrotron-based X-ray techniques (X-ray absorption spectroscopy, X-ray fluorescence, and X-ray microscopy) that can be used to improve understanding of metal transformations. The X-ray absorption near-edge structure (XANES) technique provides an in situ probe of an element’s oxidation state and clearly can contribute significantly to an understanding of the fate of elements in the environment, in both solid and solution phases. In addition to its utility, XANES is relatively easy to implement and has been perhaps the most commonly used synchrotron-based X-ray technique for monitoring metal transformations in environmental studies. XANES spectroscopy focuses on the energy range near an element's absorption edge, which is related to the element's valence state. Extended X-ray absorption fine-structure (EXAFS) focuses on the energy region well above the absorption edge and yields information on the local chemical environment of the absorbing element. Investigators can initiate the use of synchrotron radiation in their research in a number of ways.

This chapter provides an overview of the range of techniques that are available to help characterize the critical microbiological and geological factors underpinning metal-microbe interactions. These include techniques to study microbial cells, the mineral substrates that they live on, and the surrounding geochemical environment that together control the rate and extent of metal biotransformations. The chapter focuses on examining current techniques used in understanding the transformations of long-lived, redox-active radionuclides, such as uranium (U), technetium (Tc), neptunium (Np), and plutonium (Pu), in pure culture and laboratory simulation experiments and highlights the safe handling and measurement of these radionuclides. It highlights how the real need to understand the transformation behavior of radionuclides is balanced against the safe handling requirements for working with radiation. In summary, specialist radiochemistry laboratories are needed for handling all of the radionuclides mentioned above, with the possible exception of uranium. The long-lived radionuclides U, Tc, Np, and Pu are redox active, and all of these radionuclides have the potential to form less soluble species as reducing conditions develop. The chapter discusses two forms of microscopy based on conventional light and electron beams to study the fate of metals in geomicrobiological experiments. The focus here is on visualizing the interactions between “microbiological structures” and metals, while in a later section more general methods to assess mineralogical substrates, using a range of techniques, including microscopy are discussed.

This chapter highlights the various approaches and techniques that others have used to investigate the geomicrobiology of arsenate respiration in subsurface and aqueous environments. This includes the discussion of classical microbiological techniques (e.g, enrichment culture and pure strain studies), environmental DNA methods, and culture-dependent microcosm studies. The overall goal is to provide investigators with a concise overview of the tools used to better understand the biological mechanisms influencing the arsenic geochemical cycle. To date most arsenate-respiring microbes have been isolated as heterotrophs. To investigate how iron influences the arsenic geochemical cycle, many studies have used synthetic hydrous ferric oxide (HFO) as an adsorbant for either As(V) or As(III). This HFO-As mineral is then used in batch or flowthrough experiments with iron and/or arsenate reducers to characterize how microbes affect the mineralogy and mobilization of arsenic. The results of any sediment microcosm study should be interpreted with caution, especially when extrapolating to generalizations about what occurs in the environment. Manipulation of oxygenation may also provide useful information regarding the types of respiration that contribute to arsenic transformations. The substrate in the column can be sampled at the end of an experiment and analyzed for mineralogical transformations, changes in cell densities and spatial variations, and also for molecular markers such as 16S rRNA gene if natural sediments are used.

The redox properties of manganese (Mn), make it central to a variety of biological processes and result in significant and often rapid biogeochemical cycling that is mediated by abiotic and biotic oxidation and reduction, biological uptake, and mineral formation. In nature Mn occurs in three different oxidation states, +II, +III, and +IV. Mn(III) and Mn(IV) are found in environmentally prevalent ferromanganese (oxyhydr)oxide minerals, which often occur as layer-type or tunnel-structure minerals. As biotic and abiotic processes both play important roles in the oxidation and reduction of Mn, a major challenge to understanding Mn biotransformations is the differentiation and subsequent quantification of the biotic and abiotic components of the processes. Total manganese concentrations are facilely measured by using the formaldoxime colorimetric technique. In general, two considerations are important for the assays. First, the Mn(II) concentration utilized must not inhibit biological activity. Second, effective abiotic controls must be employed to distinguish biological Mn(II) removal and/or oxidation from the adsorption and autocatalytic oxidation of Mn(II) on Mn oxide surfaces. The assay proceeds with few modifications to the leucoberbelin blue (LBB) and formaldoxime methods and can be scaled to different volumes, Mn concentrations.

The dissimilatory Fe(III)-reducing bacteria (FeRB) are anaerobic prokaryotes that couple the oxidation of an electron donor such as hydrogen or short-chain fatty acids such as acetate to the reduction of Fe(III) as an energy-conserving metabolic process. The researchers pumped an anaerobic medium mixed with a green fluorescent protein-expressing strain of Shewanella oneidensis through the aerobic flow cell (AFC) and across the mineral surface. Surface pitting from mineral dissolution was heterogeneous, as was the precipitation of secondary Fe(II) minerals. These studies highlight the importance of considering the effects of open systems on the physiology and biogeochemistry of iron-microbe interactions. The geochemistry of the environment under study can provide the first indications of microbial Fe(III) reduction. Most 16S rRNA-based molecular ecology tools can be, and have been, applied to the FeRB. The volume of literature focusing on the biochemistry and physiology of FeRB has increased dramatically in the last 3 years. Based on numerous studies, it is clear that with all FeRB studied, there are two common themes shared by all of these organisms. The first is that cell attachment to mineral surfaces is common in all FeRB studied and is probably essential to Fe(III) reduction. The second theme is the abundance of c-type cytochromes in almost every organism studied.