Nitrification

Nitrogen is an essential element for life, a major component of proteins and nucleic acids. Cultivated ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) provided the basis for investigations into the physiology and biochemistry of nitrification for decades and supported the ecological inferences obtained from field studies. Most of this work was done on AOB, and even now, the NOB are much less studied in the environment. The discovery of novel organisms and novel pathways are the most important findings to be documented in the field of nitrification since the publication of the last monograph in 1986. But just as important, and absolutely critical to these discoveries, are the changes in the study and methodology of nitrification. Both independently and in parallel with these advances in the molecular biology of nitrification, major advances in understanding their biochemistry and regulation have also occurred in the last 25 years. Major insights about ammonia-oxidizing archaea (AOA) genomics and metabolic capabilities are discussed. Microbial ecology has been transformed into molecular ecology, so great has been the impact of molecular biological methods in the study of microbes in natural and managed systems. Ribosomal RNA and functional gene sequence data are now the standard for investigation of microbial diversity, distribution, and activity in the environment. These methods have made it possible to investigate environmental control of nitrification, regulation in response to changing conditions, the discovery of great uncultured diversity, and an understanding of succession and biogeography among functionally similar types.

This chapter covers the current understanding of the biochemical and genetic underpinnings relevant to ammonia oxidation by aerobic bacteria. Ammonia is released into the environment mainly from the decay of organic matter or from the use of NH3-based fertilizers in agriculture and serves as an N supply to plants and microorganisms. Ammonia-oxidizing bacteria (AOB), ammonia oxidizing archaea (AOA), and anaerobic ammonia-oxidizing (anammox) bacteria can derive energy for growth from the oxidation of NH3. Bacterium has the advantages of growing relatively rapid for an AOB and being able to tolerate high concentrations of ammonium (up to 100 mM) and nitrite. The genomes of AOB also show that all encode four specialized proteins perform the oxidation of NH3: ammonia monooxygenase (AMO), hydroxylamine oxidoreductase (HAO), and cytochromes c554 (cyt c554) and cm552 (cyt cm552). The electron transport chain of N. europaea has the same major electron transfer complexes as the electron transport chain of mitochondria.

This chapter discusses archaeal ammonia oxidation and anaerobic ammonia oxidation processes that have important roles in the global nitrogen cycle. It reviews the diversity, distribution, and biogeography of a subset of the ammonia-oxidizing prokaryotes, the aerobic chemolithotrophic ammonia-oxidizing bacteria (AOB). The terrestrial AOB are generally restricted to the Betaproteobacteria, while the marine organisms are found both in the Betaproteobacteria and the Gammaproteobacteria. Phylogeny inference based on the current data set does not support that all nitrosomonads are more closely related to each other than to members of the Nitrosospira lineage; especially problematic is the placement of Nitrosomonas cryotolerans and Nitrosomonas sp. Stable clusters based on 16S rRNA phylogeny are problematic within Nitrosospira because of the overall high levels of identity of the 16S rRNA (>97%). Soils are often dominated by Nitrosospira spp., while marine and freshwater systems often have mixtures of the genera of AOB present. During secondary succession, microbial communities develop on the soils that are often depleted in the number and diversity of microorganisms. In marine habitats, Nitrosococcus, Nitrosomonas, and Nitrosospira coexist with ammonia-oxidizing archaea; their relative contributions and diversity are only beginning to be delineated. The functional cohort of AOB, ammonia-oxidizing archaea, and the nitrite-oxidizing prokaryotes will persist as important model organisms for linking the process of nitrification to microbial diversity and biogeography.

This chapter talks about ammonium-oxidizing bacteria (AOB), addresses the inventory involved in nitrification, attempts metabolic reconstruction of N transformation processes, and provides insights into their evolution. The AOB oxidizes ammonia aerobically as their sole source of energy and reductant belong taxonomically to two monophyletic groups in different proteobacterial classes. amoA-encoding archaea (AEA) is capable of being classified as obligate, ammonia-co-oxidizing mixotrophs, or chemoorganotrophs with nonfunctional amo genes in their genomes. While the anaerobic oxidation of methane by NC10 is coupled to denitrification, it is not yet clear whether the oxidation of ammonia is coupled to nitrite reduction. Ammonification, the production of ammonium from other nitrogen compounds, likely existed within early bacteria and archaea as a consequence of simple fermentations; however, these internal cycles did not likely increase net NH4 +/NH3 availability. The core hydroxylamine ubiquinone redox module (HURM) genes are encoded by a conserved gene cluster, hao-orf2-cycAB, in all AOB. Complete sequences of the genes encoding the HURM proteins had been published from several AOB prior to obtain genome sequences and the protein structures of HAO and c554 have since been resolved.

This chapter on describes the physiology and biochemical pathways of heterotrophic nitrification and nitrifier denitrification, a description of the genetic and organism diversity involved, and a brief description of techniques to discern one process from another. A final perspective is offered on how anthropogenic input of nitrogen affects microbial transformations of inorganic N with particular emphasis on emissions of gaseous N-oxides to the atmosphere. Ammonia-oxidizing bacteria (AOB) can produce nitrous oxide by two different pathways, hydroxylamine oxidation or nitrifier denitrification. The technical breakthrough to discriminate nitrous oxide production from nitrification, nitrifier denitrification, and denitrification was the detection of individual nitrous oxide isotopomers using isotope ratio mass spectroscopy. The δ15N of nitrous oxide produced from hydroxylamine oxidation was significantly more positive than that from nitrifier denitrification or denitrification. This study found that the site preference of 15N in nitrous oxide was significantly different during nitrifier denitrification by AOB versus denitrification by two species of Pseudomonas. The chapter touches on largely understudied, but highly significant, processes of inorganic nitrogen metabolism that impact the global nitrogen cycle. Many of the studies cited in this chapter suggest that these processes are strongly influenced by the availability of carbon, nitrogen, and oxygen in the environment. It describes microbial populations and processes that make nitrous oxide in response to increased fertilizer use, nitrogen deposition, and hypoxia.

This chapter is divided into two major sections: a brief comparative description of the physiology of ammonia-oxidizing archaea (AOA) in relation to the better-characterized ammonia-oxidizing bacteria (AOB); and a discussion of features that have been gleaned from the genome sequence and its relevance to environmental genomic studies. An understanding of copper homeostasis is fundamental to the understanding of ecological success of the AOA. The genome sequence points to two types of enzyme systems that may be involved in copper handling or response to oxidative stress: genes encoding multicopper oxidases and DsbA-type proteins. Early metagenomics studies and the genome sequence of the sponge symbiont C. symbiosum provided an indication of a capacity for ammonia oxidation. Genomic and metagenomic studies have now shed initial light on the gene inventory of mesophilic AOA. These studies indicate not only that a tremendous diversity of Crenarchaeota may thrive by ammonia oxidation but also that a distinct biochemistry of ammonia oxidation serves energy conservation in these organisms. Broad temperature range, as well as oligotrophy among AOA, shows that apparently unfavorable thermodynamics of ammonia oxidation and the requirement of reverse electron transport for biosynthetic needs do not pose significant limitations on the competitiveness of archaeal nitrifiers for scarce energy resources such as ammonia.

One of the major challenges in studying microorganisms from natural habitats is the inability to cultivate many of them in the laboratory. Recently, the complete genome sequence of C. symbiosum was determined from a metagenomic library providing further insights into the potential physiological properties of uncultured ammonia-oxidizing archaea (AOA). The amo genes of ammonia-oxidizing bacteria (AOB) and AOA are distantly related, and molecular approaches to study the distribution and diversity of ammonia oxidizers rely on specific PCR primers that amplify the archaeal amoA or the bacterial amoA variant. Nitrification is an important process in sedimentary biogeochemistry and particularly in estuarine sediments, which can be exposed to high loads of nutrients from agricultural runoff. There is now unambiguous evidence for the occurrence of AOA in environments of elevated temperature. The mechanisms by which soil pH influences the growth and activity of many microbial functional groups have been determined through a combination of physiological and soil microcosm studies. With their involvement in ammonia oxidation, the majority of mesophilic crenarchaeota (or thaumarchaeota) are most likely major players in biogeochemical cycling. Most studies that attempted to evaluate the actual activity of AOA (and bacteria) seem to reveal that there could, in fact, be an ecological niche differentiation between AOA and AOB.

This chapter presents an overview of the progress that has been made during the last decade with respect to our understanding of the anammox metabolism, focusing on the physiology, cell biology, and information derived from genome sequencing projects. Thereafter, the current concepts on the biochemistry and bioenergetics of the anammox bacteria are discussed, and the chapter concludes with the perspectives and urgent issues in this field of research that need to be addressed. A challenge in culturing of anammox bacteria was their long doubling time. Apparently, anammox bacteria have geared their metabolism toward high affinities for their substrates at the expense of the activity. During growth under autotrophic conditions, anammox bacteria rely on nitrite. In nature, however, the compound is not abundantly present, which raised the question how anammox bacteria obtain nitrite. Over time, when anammox bacteria proliferated, influent concentrations of nitrite and ammonia could be increased from 2.5 mM to 45 mM. Studies during the last decade have revealed many unique structural and metabolic properties of the anammox bacteria. Furthermore, the comparisons between the different genomes may give a clue with respect to niche differentiation. Above all, genome sequencing projects can provide the basic information for future expression studies, both at the gene (genomics) and protein (proteomics) levels. Eventually, such studies will allow an understanding in the way anammox bacteria are able to adapt to changes in the supply of the substrates and other environmental conditions in their highly dynamic habitats.

This chapter provides a synthesis of the broadscale patterns of anammox across a spectrum of aquatic ecosystems and to put forward some hypotheses as to what regulates anammox and the total flux of N in such systems. Research into anammox falls largely into two distinct aquatic ecosystems: (i) its role in the anaerobic oxidation of ammonium in the suboxic layers of aquatic sediments, where the respective reactions and ecophysiologies are compressed into fractions of centimeters; and (ii) the same ecosystem function, but distributed over depths of tens of meters in the oxygen minimum zones (OMZs) of the global ocean. The chapter focuses on the results obtained using slurries of homogenized sediment, and then focuses on the data derived with intact sediment cores, and, finally, draw comparisons between the two at the end, without dwelling too long on the respective complexities of each method. Anammox bacteria appear active in both low-oxygen and suboxic waters, and such conditions are often considered as prerequisites for denitrification, since oxygen represses synthesis and activity of denitrifying enzymes, though the effect may be more subtle.

This chapter presents an overview of physiological parameters relevant for design and operation of the nitritationanammox, based on which different treatment and start-up strategies as well as environmental impact can be evaluated. For a proper evaluation of the one-reactor nitritation-anammox process, such parameters for ammonia oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) are of importance. Ammonium removal with the anammox process always consists of partial nitritation followed by the anammox process. In the denitrification-anammox process, nitrite does not stem from partial oxidation of ammonium but from partial denitrification of nitrate. Reject water treatment can significantly contribute to the overall performance with relatively small reactors. The recently elaborated possibility of removal of nitrogen at very low ammonium concentrations would be a true game-changer: should this become feasible, then the anammox process can be applied in the main line of municipal wastewater treatment plants and treatment of organic matter in wastewater treatment plants (WWTP) can be completely focused on digestion. The chapter concludes with an overview on the state of the art of full-scale implementation of the nitritation-anammox process.

This chapter attempts to place the physiology and biochemistry of nitrite-oxidizing bacteria (NOB) into context with information derived from the annotated Nitrobacter genomes. Daims et al. provide an excellent review of insights gained into the physiology and ecology of Nitrospira that were obtained primarily by using genomics tools and other cultivation-independent methods. The genome of N. hamburgensis is the largest of the Nitrobacter genomes and appears to have maintained a greater level of metabolic flexibility and adaptability than the other sequenced representative. The genes on the largest plasmid, pPB13, appear to be biased toward carbon/energy metabolism. A discussion of the current status of knowledge about carboxysomes is presented in this chapter. The chapter talks about genomic evidence for a potentially different mechanism of CO2 fixation by ''Candidatus Nitrospira defluvii'' presented by Daims et al. The glyoxylate pathway genes were annotated, and several genes encoding for enzymes that facilitate metabolism of pyruvate, acetate, and glycerol were identified. The genome analysis of Nitrobacter has provided some new and confirmatory insights into the biology of this genus.

A comprehensive biological understanding of nitrite-oxidizing bacteria (NOB) will be important to improve the functional stability of wastewater treatment systems and to reduce detrimental effects of nitrification in agriculture. The first part of this chapter provides an overview of the phylogenetic diversity and distribution of NOB in the environment and in engineered systems. Subsequently, the chapter focuses on the ecophysiology and ecological niche differentiation of the genera Nitrobacter and Nitrospira. The third part of this chapter addresses most recent insights into the biology of Nitrospira, which are based on the first sequenced genome from this genus. Future research should clarify whether Nitrobacter strains indeed are ecophysiologically different and how their diversity influences ecosystem functioning. The chapter also provides an overview of selected metabolic characteristics of ‘’Candidatus Nitrospira defluvii’’ as derived from the genomic data. Nevertheless, the microbial genome could be sequenced by using an environmental genomics approach that had been developed for sequencing an uncultured anaerobic ammonium-oxidizing bacterium. Although the phylogeny, might be blurred by past lateral gene transfer events, it is tempting to speculate that the aerobic nitrite-oxidizing system of Nitrospira was derived from an anaerobic protein complex and that the ancestor of Nitrospira was adapted to life under hypoxic conditions. The distant relationship between the NxrA forms of Nitrospira and those of Nitrobacter and Nitrococcus suggests that the use of nitrite as an energy source was invented more than once in the course of bacterial evolution.

This chapter focuses on very recent developments and their implications for nitrogen cycling in the marine environment. Nitrification does not influence the net N inventory of the ocean directly except by small losses to the gaseous pool of nitrous oxide, but it does determine the distribution of N among important dissolved inorganic nitrogen pools. The ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) oxidize ammonium to nitrite, and nitrite oxidizers, convert the nitrite to nitrate, which can be a very important N source for many kinds of phytoplankton. The only cultivated ammonia oxidizers were bacteria. These cultures have provided the basis of physiological inferences about ecological niches and environmental regulation of nitrification in the ocean. The most important development in the study of nitrification in the ocean in the last decade is the discovery of AOA. The implications of this discovery may not result in big changes in our understanding of the rates and distribution of nitrification in the ocean. Despite their somewhat restricted phylogenetic range, the bacterial nitrifiers are polyphyletic, and the phenotype has apparently arisen independently numerous times. Recent discoveries in the marine nitrogen cycle and in nitrification, in particular, point out the important gaps in our understanding, both at the level of microorganisms and at the ecosystem level.

This chapter provides an overview of soil nitrification, paying particular attention to these recent advances. The focus is on the specific factors associated with soil that influence the nitrification process and nitrifier communities. Thus, the chapter considers how soil nitrification differs from nitrification in pure cultures growing in liquid medium or in oceans, estuaries, and wastewater treatment plants. The links between phylogenetic diversity and physiological or functional diversity are discussed in relation to soil characteristics and environmental factors. Three important aspects of surface growth are discussed: effects on growth and inhibition, survival and recovery from starvation, and protection from effects of low pH. Cell activities, and other kinetic parameters, are being reassessed using cultivation-independent quantitative PCR (qPCR) techniques to assess cell abundance used this approach to determine the influence of ammonia concentration on growth characteristics of soil communities in microcosms and in the field. Although low pH inhibits ammonia oxidizers in laboratory culture, a metastudy of nitrification in almost 300 soils provided no evidence for a significant effect of soil pH on nitrification. The chapter deals with more general aspects of the impact of heterogeneity. The focus is on a single process within the nitrogen cycle, but soil nitrification is intimately linked to mineralization, soil organic matter decomposition, and denitrification.

This chapter presents the latest information on nitrification in inland waters, and deals mostly with ammonium-oxidizing bacteria (AOB) as they are primarily important for the onset of the process of nitrification, although their activity might be influenced by the presence of active nitrite-oxidizing bacteria, especially after starvation for ammonium. Nitrification in lakes takes place in the sediment as well as in the water column. As in lakes, nitrification in streams and rivers occurs primarily in the oxic surface layers of the sediment. The use of the amoA gene led to quite different results: no amoA gene fragments related to the Nitrosospira lineage were found in any of the compartments; whereas the pelagic and the epiphytic compartments had only amoA gene fragments of the N. oligotropha lineage, and the benthic compartments contained a mixture of fragments of the N. oligotropha, N. europaea, and Nitrosomonas sp. Nm143 lineages. In summary, nitrification in streams and rivers appears to be associated with particles, and the nature of the particles may determine the size of the nitrification rate. Overall, members of the N. oligotrophalineage are most numerous among the ammonia-oxidizing betaproteobacteria, both in rivers and lakes. After being detected in large numbers in soils and marine environments, the first observations of crenarchaea containing the amoA gene are published, but their role in nitrification in inland waters still has to be demonstrated.

Microbial nitrification is a necessary step in removing nitrogen from wastewaters via biological denitrification and is becoming more important due to strict regulations on nitrogen discharge. However, microbial nitrification is recognized as being difficult to maintain in practical wastewater treatment plants (WWTPs) owing to the lower kinetics, yields, and sensitivity of nitrifying bacteria to physical, chemical, and environmental disturbances as mentioned, even though nitrification has been studied more than any other specific biochemical reactions occurring in wastewater treatment to date. Influent NO2 -, chromium, and nickel influenced the AOB community structure, while correlations between other metals analyzed in this study and the AOB community structure were insignificant. As an oxidation process, nitrification significantly consumes oxygen, and dissolved oxygen (DO) concentration is a key factor for maintaining nitrification stably as well as pH. In an activated sludge process, 3% salt inhibited both the maximum utilization rate and the saturation constant, suggesting uncompetitive inhibition. Nitrification in wastewater treatment systems has been studied extensively. Despite their importance, knowledge about the identity and ecology of nitrifying bacteria carrying out nitrification in WWTPs has been scarce. Thus, biological nitrogen removal processes have been regarded as “a black box” in practice because the lack of fundamental microbiological understanding hampers knowledge-driven process design and operation.