1. Functional Assignment of Metagenomic Data: Insights for the Microbial Nitrogen Cycle

Vikas Sharma, Gaurav Chetal, Todd D. Taylor and Tulika Prakash

Nitrogen is an essential component of basic bio-molecules. Atmospheric nitrogen is inaccessible to living organisms because of its inert nature but it can be fixed into usable forms by nitrogen-fixing microbial communities. The microbial nitrogen cycle is a complex process which occurs through the coordinated functioning of several microbial genes, many of which have been identified primarily from cultivable microbes. However, for unculturable microbes belonging to the community of a given environment, metagenomics is used as an alternative approach to the classical methods of genomics, including polymerase chain reaction based gene identification and restriction fragment length polymorphism. A few metagenomic studies of terrestrial and aquatic environments, including some under moderate and extreme conditions, have been carried out which focus on nitrogen-fixing microbial communities and their functional diversities. These studies highlighted the roles of the resident microbes and their genes in different steps of the nitrogen cycle. Other studies have shown that the use of nitrogen-based fertilizers on agricultural soil has led to alterations in the microbial populations due to increased nitrogen content in the soil. Recently scientists identified novel anammox bacteria which are responsible for the loss of fixed nitrogen from agricultural soil. Presence of anammox along with other non-anammox bacteria indicates a coordinated behaviour of these microbes in the nitrogen-cycling process; however, the complete mechanism of anammox process is not clearly understood. To gain a better understanding of the anammox and other processes of nitrogen-cycling, metagenomic studies should be combined with metatranscriptomic and functional metagenomic approaches which investigate the functional dynamics of a given community.

2. Microbial Metagenomics of Oxygen Minimum Zones

Frank J. Stewart and Osvaldo Ulloa

Marine oxygen minimum zones (OMZs) support complex microbial assemblages with important roles in ocean biogeochemical cycles. The integration of genomic, metagenomic, and metatranscriptomic analyses has significantly enhanced our understanding of OMZ microbial communities, revealing a richness of metabolic processes structured along the OMZ redox gradient and previously unrecognized linkages between community members. Specifically, 'omics studies are clarifying the physiology, in situ activity, and evolutionary history of microbial groups mediating key steps of dissimilatory nitrogen cycling, including OMZ-specific clades of anaerobic ammonium oxidation (anammox) bacteria, aerobic ammonia-oxidizing Thaumarchaeota specialized for high-affinity substrate scavenging, and aerobic nitrite-oxidizing Nitrospina bacteria with adaptations for life under low oxygen conditions. Recent studies have also identified a diverse OMZ community of sulfur-oxidizing autotrophs whose activity appears coupled to reduced sulfur compounds generated by co-occurring sulphate-reducing heterotrophs. We discuss these and other OMZ metabolic processes in relationship to key environmental drivers, including water column nutrient and redox gradients and the microscale partitioning of communities between organic particle-associated and free-living microniches. Coupled ‘omic-biogeochemistry studies are critical for understanding how de-oxygenation structures microbial biogeochemistry in the ocean and for identifying key priorities for future OMZ research.

3. Interactions Between Methane and Nitrogen Cycling; Current Metagenomic Studies and Future Trends

Paul L.E. Bodelier and Anne K. Steenbergh

Wetlands, lakes, soils and sediments are the most important biological sources as well sinks of the greenhouse gas methane. However, the dynamics, variability and uncertainty in methane emission models from these systems is high necessitating better knowledge of the underlying microbial processes. The impact of nitrogenous fertilizers and atmospheric nitrogen deposition on methane production and consumption in freshwater ecosystems (wetlands, lakes, rice paddies) as well as in upland soils has been the subject of intense research the past decades. However, our mechanistic understanding of the observed effects on methane and nitrogen cycling interactions in these ecosystems is poor which is even more so considering the novel microbial groups and pathways discovered. This chapter gives an overview of the main ways the nitrogen cycle interacts with the microbial methane cycling in freshwater wetlands, soils and sediments and summarizes the main current metagenomic studies that carried out on microbial groups involved. It can be concluded that metagenomic techniques developed and applied have the potential to obtain an integrative view of microbial communities and interactions and bear the potential to discover new pathways and organisms. The way forward is to apply these techniques in replicated, manipulative experimental set ups to obtain mechanistic understanding of methane-nitrogen cycle interactions.

4. Quantification of Functional Microbial Nitrogen Cycle Genes in Environmental Samples

David Correa-Galeote, Germán Tortosa and Eulogio J. Bedmar

The nitrogen (N) cycle comprises a large number of oxidative and reductive reactions that are catalyzed by wide variety of enzymes. Genes coding for most of the N-cycle enzymes have been shown to be present in a diverse polyphyletic group of microorganisms, including bacteria, archaea and fungi. Therefore, a 16S rRNA phylogeny-based approach to study those microbial populations is not possible. Because cultivation-dependent methods are selective for certain microorganisms, molecular methods have been developed to study the ecology and to assess abundance and diversity composition of nitrogen cycling microorganisms in environmental samples. DNA extraction followed by PCR amplification of genes that encode key functional enzymes of the N-cycle are used to study which genes and/or phylotypes are functionally important in the environment. Methods for DNA isolation and purification from environmental samples will be addressed whilst considering the main functional gene targets used to study the nitrogen fixation, nitrification and denitrification processes within the nitrogen cycle. The fluorescence-based quantitative real-time polymerase chain reaction (qPCR) has proven useful for quantification of nucleic acids in samples obtained from numerous diverse sources. Here, we describe relevant experimental conditions for utilization of qPCR to quantify the 16S rRNA, amoA and nar/nap, nirK/nirS, c-nor/q-nor and nos genes that encode synthesis of key enzymes involved in redox reactions of the N-cycle.

5. Stable Isotope Probing the N Cycle: Current Applications and Future Directions

Boris Wawrik

Much of our understanding of microbial ecophysiology with respect to nitrogen cycling has been derived from cultivation based techniques, which are unable to capture a large proportion of microbial diversity in the environment. ¹⁵N DNA stable isotope probing (SIP) is a technology that allows experimental assessment of nitrogen uptake in systems where requisite organisms cannot be cultivated, by combining evidence for isotopic uptake with phylogenetic inferences via sequence based analysis. The technique has recently been applied in a variety of systems including enrichment cultures, soils, and marine environments. This chapter summarizes some historical perspective on ¹⁵N SIP methodology and reviews the available ¹⁵N SIP literature. Advantages and disadvantages of ¹⁵N SIP are discussed. Much of the potential of ¹⁵N SIP remains unrealized due to important experimental limitation. Recent technological developments that are likely to result in significant improvements in resolution and utility of stable isotope tracer techniques are also described.

6. Application of Metaproteomics to the Exploration of Microbial N-cycling Communities

Cindy Smith and Florence Abram

The recycling of nitrogen is essential for all organisms on earth and microbial communities play crucial roles in the nitrogen biogeochemical cycle. Constant anthropogenic alterations (both positive and negative) and changing environmental conditions (such as climate change and ocean acidification) have profound effects on the nitrogen cycle. In order to fully elucidate the nitrogen cycle, adequately address the consequences of environmental perturbations and mediate nitrogen pollution, nitrogen transformations need to be thoroughly investigated and ideally, modelled. Systems approaches, typically analysing DNA, RNA, proteins and metabolites together with the corresponding metadata prevailing in the ecosystem under study, ultimately aim at developing models to characterise the ecosystem attributes and predict the consequences of changes in environmental conditions in silico. Therefore, systems approaches hold a lot of potential when applied to the nitrogen cycle. In such context, metaproteomics, defined as the analysis of the proteins collectively expressed by all the organisms present in an ecosystem, becomes a crucial requirement. In this chapter we will discuss how metaproteomics, typically combined with other ‘omics' technologies, has advanced our understanding of the nitrogen cycle in different environments. We will also discuss proteomic studies of relevant microbial isolates, as well the application of isotope labelling proteomics to the nitrogen cycle.

7. Functional Molecular Analysis of Microbial Nitrogen Cycle by Microarray-based GeoChip: Insights for Climate Change, Agriculture and Other Ecological Studies

Kai Xue, Joy D. Van Nostrand, Zhili He and Jizhong Zhou

Nitrogen (N) is a common constraining factor for biological communities, e.g. plants and microbes, in terrestrial ecosystems. Its cycling is mainly mediated by soil microbial communities. Environmental changes may affect functional genes of soil microbial communities involved in N cycling and hence cause substantial changes in related geochemical processes. A microarray-based metagenomics technique, GeoChip, includes a comprehensive set of functional genes involved in almost all processes of N transformation, e.g. N fixation, nitrification, denitrification, etc. GeoChip analysis allows us to monitor the entire N ecological network by a single hybridization to detect changes of all these genes. In this chapter, we will focus on the development and application of GeoChip methodology for analyzing functional genes of microbial communities involved in N processes under different treatments in various ecosystems. This chapter will reveal how information obtained from GeoChip enhances our understanding of ecological consequences of climatic changes like rising temperature and CO2 or fertilization treatments. Moreover, the challenges of this technique will also be discussed.

8. Functional and Taxonomic Diversity of the Nitrogen Cycling Guild in the Sargasso Sea Metagenomes

Germán Bonilla-Rosso, Luis Eguiarte and Valeria Souza

The number and quality of available metagenomes from the oligotrophic Sargasso Sea provides an unprecedented opportunity to study taxonomic and functional diversity in its microbial communities. Here, we dissect the diversity within seven functional guilds involved in nitrogen cycling, and contextualize them with overall taxonomic diversity. Our results reveal a large preference for fixed nitrogen regeneration and reutilization, as opposed to novel incorporation of inorganic nitrogen. An overall relationship was found between taxonomic and functional richness, but not evenness. All the metabolic pathways analized were present in all sampling sites and all size fractions, suggesting a similarity in the ecological niches available for these guilds. Nevertheless, we found significant differences in the phylogenetic composition and community structure within these guilds. The numerical dominance of versatile oligotrophs that can use a wide variety of simple molecules is evident, while the guild inspection revealed the presence of a relatively abundant, uncharacterised or uncultured group of organisms specialized in the degradation of complex organic matter. The presence of proteorhodopsins in many of these organisms suggests the existence of an unexplored link between nitrogen cycling and phototrophy.

9. Microbial Nitrogen Cycle: Determination of Microbial Functional Activities and Related N-compounds in Environmental Samples

D. Correa-Galeote, G. Tortosa and E.J. Bedmar

Nitrogen (N) is part of essential compounds such as proteins, nucleic acids, hormones, etc. Although N makes up to about 80 per cent of the Earth's atmosphere, it is not readily available for plant and animal consumption. Free-living and symbiotic microbes contain the enzyme nitrogenase which initiates the N-cycle in the biosphere by reducing dinitrogen gas to bio-available ammonia, a process called nitrogen fixation. Ammonia is subsequently oxidized to nitrate by nitrification, a two-step aerobic pathway during which ammonia is oxidized to nitrate and nitrite by the enzymes ammonia monooxygenase and nitrite oxidoreductase, rexpectively. Finally, nitrate is reduced to dinitrogen gas by denitrifying microorganisms, thereby closing the N cycle. Denitrification is carried out by the sequential activity of the enzymes nitrate-, nitrite, nitric oxide and nitrous oxide-reductase, respectively. Ammonia can also be incorporated into cellular biomass via the glutamine synthetase-glutamate synthase and glutamate dehydrogenase pathways to form amino acids and other nitrogen compounds. After cellular death, organic nitrogen compounds are released to the environment to be mineralized by microbial activities. Widely-used procedures for determination of microbial functional activities of the nitrogen cycling microorganisms and of N-compounds produced during the redox reactions of the cycle will be addressed. In addition, we will consider new methodologies being developed for further understanding of the N-cycle.

10. Functional Metagenomics of the Nitrogen Cycle in Freshwater Lakes with Focus on Methylotrophic Bacteria

Ludmila Chistoserdova

In this chapter, insights from metagenomic sequencing of samples originating from a freshwater lake, Lake Washington in Seattle, are presented, including data for communities responding to the stimuli of methane and nitrate at varying concentrations of oxygen. Data are also presented on the presence and the diversity of genes for nitrate metabolism in the genomes of methylotrophs isolated from Lake Washington, along with observations on the physiology of some of the major species active in methane and nitrate metabolism. A potential for partnerships between the Methylococcaceae and other organisms is discussed in terms of the connection between methanotrophy and nitrate metabolism.

11. The Fungal Contribution to the Nitrogen Cycle in Agricultural Soils

Markus Gorfer, Sylvia Klaubauf, Harald Berger and Joseph Strauss

Nitrate assimilation and its regulation is well studied for several model fungi but little is known about the fungal contribution to microbial nitrogen cycles in agricultural soils, although fungal dominated systems are less prone to nitrogen losses via leaching and denitrification. Tools were recently developed to characterise gene pools and expression levels of fungal assimilatory nitrate reductases in environmental samples and carbon source availability was identified as one of the major parameter determining nitrate assimilation capacities. Even though the majority of the environmental sequences could be classified to family, genus or species level, a considerably high number could only be assigned to higher taxonomic levels. Ongoing addition of new fungal nitrate reductase genes from whole genome sequencing efforts and from PCR amplification from pure culture isolates already led to improved affiliation of environmental sequences. Precaution is however necessary in the interpretation of higher order affiliations: Horizontal gene transfer events seem to be rather common in the evolution of fungal nitrate assimilation. No fungal nitrate reductases are found in publicly accessible soil metagenomic and metatranscriptomic libraries. Future studies will focus on integration of data from metagenomics, metatranscriptomics, stable isotope probing, isotope pool dilution assays and modelling approaches. The goal of these studies is to obtain a comprehensive picture of the fungal contribution to the soil microbial nitrogen cycle.

12. Biofilms in Nitrogen Removal: Population Dynamics and Spatial Distribution of Nitrifying- and Anammox Bacteria

Robert Almstrand, Frank Persson and Malte Hermansson

Efficient nitrogen removal at wastewater treatment plants (WWTPs) is necessary to avoid eutrophication of recipient waters. The most commonly used approach consists of aerobic nitrification and subsequent anaerobic denitrification resulting in the release of dinitrogen gas into the atmosphere. Nitrification is a two-step process performed by ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) often grown in biofilms at WWTPs. Alternatively, anaerobic ammonium oxidation (anammox) where anammox bacteria convert ammonium and nitrite directly into dinitrogen gas may be utilized. These groups of recalcitrant bacteria grow very slowly and are sensitive to perturbations, which may result in decreased efficiency or even breakdown of the respective process. Thus, their ecology, activity and the structure of the biofilms in which they grow require detailed investigation to improve our understanding of the nitrification and anammox processes. This in turn will facilitate the design of more efficient nitrogen-removal strategies. To assess the population dynamics and spatial distribution of nitrifying and anammox bacteria, culture-independent methods are essential. Therefore, the application of methods such as quantitative PCR (qPCR), terminal restriction fragment length polymorphism (T-RFLP) and Fluorescence In Situ Hybridization (FISH) in combination with advanced microscopy techniques and digital image analysis for the study of nitrifying and anammox biofilms will be reviewed in this chapter.