Advances in methods for detection of anaerobic ammonium oxidizing (anammox) bacteria

Anaerobic ammonium oxidation (anammox), the biochemical process oxidizing ammonium into dinitrogen gas using nitrite as an electron acceptor, has only been recognized for its significant role in the global nitrogen cycle not long ago, and its ubiquitous distribution in a wide range of environments has changed our knowledge about the contributors to the global nitrogen cycle. Currently, several groups of methods are used in detection of anammox bacteria based on their physiological and biochemical characteristics, cellular chemical composition, and both 16S rRNA gene and selective functional genes as biomarkers, including hydrazine oxidoreductase and nitrite reductase encoding genes hzo and nirS, respectively. Results from these methods coupling with advances in quantitative PCR, reverse transcription of mRNA genes and stable isotope labeling have improved our understanding on the distribution, diversity, and activity of anammox bacteria in different environments both natural and engineered ones. In this review, we summarize these methods used in detection of anammox bacteria from various environments, highlight the strengths and weakness of these methods, and also discuss the new development potentials on the existing and new techniques in the future

Phylogenetic and functional marker genes to study ammonia-oxidizing microorganisms (AOM) in the environment

The oxidation of ammonia plays a significant role in the transformation of fixed nitrogen in the global nitrogen cycle. Autotrophic ammonia oxidation is known in three groups of microorganisms. Aerobic ammonia-oxidizing bacteria and archaea convert ammonia into nitrite during nitrification. Anaerobic ammonia-oxidizing bacteria (anammox) oxidize ammonia using nitrite as electron acceptor and producing atmospheric dinitrogen. The isolation and cultivation of all three groups in the laboratory are quite problematic due to their slow growth rates, poor growth yields, unpredictable lag phases, and sensitivity to certain organic compounds. Culture-independent approaches have contributed importantly to our understanding of the diversity and distribution of these microorganisms in the environment. In this review, we present an overview of approaches that have been used for the molecular study of ammonia oxidizers and discuss their application in different environments

Importance and controls of anaerobic ammonium oxidation influenced by riverbed geology

Rivers are an important global sink for excess bioavailable nitrogen: they convert approximately 40% of terrestrial N runoff per year (∼47 Tg) to biologically unavailable N 2 gas and return it to the atmosphere. At present, riverine N 2 production is conceptualized and modelled as denitrification. Anaerobic ammonium oxidation, known as anammox, is an alternative pathway of N 2 production important in marine environments, but its contribution to riverine N 2 production is not well understood. Here we use in situ and laboratory measurements of anammox activity using 15 N tracers and molecular analyses of microbial communities to evaluate anammox in clay-, sand-and chalk-dominated river beds in the Hampshire Avon catchment, UK during summer 2013. Abundance of the hzo gene, which encodes an enzyme central to anammox metabolism, varied across the contrasting geologies. Anammox rates were similar across geologies but contributed different proportions of N 2 production because of variation in denitrification rates. In spite of requiring anoxic conditions, anammox, most likely coupled to partial nitrification, contributed up to 58% of in situ N 2 production in oxic, permeable riverbeds. In contrast, denitrification dominated in low-permeability clay-bed rivers, where anammox contributes roughly 7% to the production of N 2 gas. We conclude that anammox can represent an important nitrogen loss pathway in permeable river sediments

Ammonium oxidation at the oxic/anoxic interface

Applied Science

The CANON system (completely autotrophic nitrogen-removal over nitrite) under ammonium limitation: Interaction and competition between three groups of bacteria

The CANON system (Completely Autotrophic Nitrogen Removal Over Nitrite) can potentially remove ammonium from wastewater in a single, oxygen-limited treatment step. The usefulness of CANON as an industrial process will be determined by the ability of the system to recover from major disturbances in feed composition. The CANON process relies on the stable interaction between only two bacterial populations: Nitrosomonas-like aerobic and Planctomycete-like anaerobic ammonium oxidising bacteria. The effect of extended periods of ammonium limitation was investigated at the laboratory scale in two different reactor types (sequencing batch reactor and chemostat). The lower limit of effective and stable nitrogen removal to dinitrogen gas in the CANON system was 0.1 kg N m-3 day-1. At this loading rate, 92% of the total nitrogen was removed. After prolonged exposure (>1 month) to influxes lower than this critical NH4+-influx, a third population of bacteria developed in the system and affected the CANON reaction stoichiometry, resulting in a temporary decrease in nitrogen removal from 92% to 57%. The third group of bacteria were identified by activity tests and qualititative FISH (Fluorescence In Situ Hybridisation) analysis to be nitrite-oxidising Nitrobacter and Nitrospira species. The changes caused by the NH4+-limitation were completely reversible, and the system re-established itself as soon as the ammonium limitation was removed. This study showed that CANON is a robust system for ammonium removal, enduring periods of up to one month of ammonium limitation without irreversible damage

13C-contents of bacterial lipids in a shallow sulfidic monomictic lake (Lake Ciso, Spain)

Stable carbon isotopic analysis was performed on sedimentary biomarkers of a shallow sulfide-rich monomictic lake, Lake Cisó (NE Spain). Specific biomarkers derived from phototrophic sulfur bacteria in Lake Cisó were considerably depleted in 13C, most likely due to the depleted 13C-content of the dissolved inorganic carbon that was photosynthetically fixed. Recycling of respired CO2, a well-known phenomenon in monomictic lakes, probably caused this depletion. The stable carbon isotopic composition of terrestrial markers, such as C25 33 odd-carbon-numbered n-alkanes and C22 30 even-carbon-numbered n-alkan-1-ols and fatty acids, were rather similar to each other and their 13C depleted values (c. −31 to −35 ) indicated that they were derived from the surrounding vegetation. Phytol was predominantly derived from the bacteriochlorophylls of phototrophic purple sulfur bacteria as were specific fatty acids

Characterization of 3-chlorobenzoate degrading aerobic bacteria isolated under various environmental conditions

The rates of bacterial growth in nature are often restricted by low concentrations of oxygen or carbon substrates. In the present study the metabolic properties of 24 isolates that had been isolated using various concentrations of 3-chlorobenzoate, benzoate and oxygen as well as using continuous culture at high and low growth rates were determined to investigate the effects of these parameters on the metabolism of monoaromatic compounds. Bacteria were enriched from different sampling sites and subsequently isolated. In batch culture this was done both under low oxygen (2% O-2) and air-saturated concentrations. Chemostat enrichments were performed under either oxygen or 3-chlorobenzoate limiting conditions. Bacteria metabolizing aromatics with gentisate or protocatechuate as intermediates (gp bacteria) as well as bacteria metabolizing aromatic compounds via catechols (cat bacteria) were isolated from batch cultures when either benzoate or 3CBA were used as C sources, regardless of the enrichment conditions applied. In contrast, enrichments performed in chemostats at low dilution rates resulted in gp-type organisms only, whereas at high dilution rates cat-type organisms were enriched, irrespective of the oxygen and 3-chlorobenzoate concentration used during enrichment. It is noteworthy that the gp-type of bacteria possessed relatively low Irm, values on 3CBA and benzoate along with relatively high substrate and oxygen affinities for these compounds. This is in contrast with cat-type of bacteria, which seemed to be characterized by high maximum specific growth rates on the aromatic substrates and relatively high apparent half saturation constants. In contrast, bacteria degrading chlorobenzoate via gentisate or protocatechuate may possibly be better adapted to conditions leading to growth at reduced rates such as low oxygen and low substrate concentrations. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved