Mike S. M. Jetten

The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms

Abstract Currently available microbiological techniques are not designed to deal with very slowly growing microorganisms. The enrichment and study of such organisms demands a novel experimental approach. In the present investigation, the sequencing batch reactor (SBR) was applied and optimized for the enrichment and quantitative study of a very slowly growing microbial community which oxidizes ammonium anaerobically. The SBR was shown to be a powerful experimental set-up with the following strong points: (1) efficient biomass retention, (2) a homogeneous distribution of substrates, products and biomass aggregates over the reactor, (3) reliable operation for more than 1 year, and (4) stable conditions under substrate-limiting conditions. Together, these points made possible for the first time the determination of several important physiological parameters such as the biomass yield (0.066 ± 0.01 C-mol/mol ammonium), the maximum specific ammonium consumption rate (45 ± 5 nmol/mg protein/min) and the maximum specific growth rate (0.0027 · h−1, doubling time 11 days). In addition, the persisting stable and strongly selective conditions of the SBR led to a high degree of enrichment (74% of the desired microorganism). This study has demonstrated that the SBR is a powerful tool compared to other techniques used in the past. We suggest that the SBR could be used for the enrichment and quantitative study of a large number of slowly growing microorganisms that are currently out of reach for microbiological research.

Missing lithotroph identified as new planctomycete

With the increased use of chemical fertilizers in agriculture, many densely populated countries face environmental problems associated with high ammonia emissions. The process of anaerobic ammonia oxidation (‘anammox’) is one of the most innovative technological advances in the removal of ammonia nitrogen from waste water,. This new process combines ammonia and nitrite directly into dinitrogen gas. Until now, bacteria capable of anaerobically oxidizing ammonia had never been found and were known as “lithotrophs missing from nature”. Here we report the discovery of this missing lithotroph and its identification as a new, autotrophic member of the order Planctomycetales, one of the major distinct divisions of the Bacteria. The new planctomycete grows extremely slowly, dividing only once every two weeks. At present, it cannot be cultivated by conventional microbiological techniques. The identification of this bacterium as the one responsible for anaerobic oxidation of ammonia makes an important contribution to the problem of unculturability.

Anaerobic ammonium oxidation by anammox bacteria in the Black Sea

The availability of fixed inorganic nitrogen (nitrate, nitrite and ammonium) limits primary productivity in many oceanic regions. The conversion of nitrate to N2 by heterotrophic bacteria (denitrification) is believed to be the only important sink for fixed inorganic nitrogen in the ocean. Here we provide evidence for bacteria that anaerobically oxidize ammonium with nitrite to N2 in the worlds largest anoxic basin, the Black Sea. Phylogenetic analysis of 16S ribosomal RNA gene sequences shows that these bacteria are related to members of the order Planctomycetales performing the anammox (anaerobic ammonium oxidation) process in ammonium-removing bioreactors. Nutrient profiles, fluorescently labelled RNA probes, 15N tracer experiments and the distribution of specific ‘ladderane’ membrane lipids indicate that ammonium diffusing upwards from the anoxic deep water is consumed by anammox bacteria below the oxic zone. This is the first time that anammox bacteria have been identified and directly linked to the removal of fixed inorganic nitrogen in the environment. The widespread occurrence of ammonium consumption in suboxic marine settings indicates that anammox might be important in the oceanic nitrogen cycle.

Autotrophic growth of anaerobic ammonium-oxidizing microorganisms in a fluidized bed reactor

An autotrophic, synthetic medium for the enrichment of anaerobic ammonium-oxidizing (Anammox) micro-organisms was developed. This medium contained ammonium and nitrite, as the only electron donor and electron acceptor, respectively, while carbonate was the only carbon source provided. Preliminary studies showed that the presence of nitrite and the absence of organic electron donors were essential for Anammox activity. The conversion rate of the enrichment culture in a fluidized bed reactor was 3 kg NH4 + m-3 d-1 when fed with 30 mM NH4 +. This is equivalent to a specific anaerobic ammonium oxidation rate of 1000-1100 nmol NH4 +h-1 (mg volatile solids)-1. The maximum specific oxidation rate obtained was 1500 nmol NH4 +h-1 (mg volatile solids)-1. Per mol NH4 + oxidized, 0.041mol CO2 were incorporated, resulting in a estimated growth rate of 0.001 h-1. The main product of the Anammox reaction is N2, but about 10% of the N-feed is converted to NO3 -. The overall nitrogen balance gave a ratio of NH4 --conversion to NO2 --conversion and NO3 --production of 1:1-31++0.06:0.22+0.02. During the conversion of NH4 + with NO2 -, no other intermediates or end-products such as hydroxylamine, NO and N2O could be detected. Acetylene, phosphate and oxygen were shown to be strong inhibitors of the Anammox activity. The dominant type of micro-organism in the enrichment culture was an irregularly shaped cell with an unusual morphology. During the enrichment for Anammox micro-organisms on synthetic medium, an increase in ether lipids was observed. The colour of the biomass changed from brownish to red, which was accompanied by an increase in the cytochrome content. Cytochrome spectra showed a peak at 470 nm gradually increasing in intensity during enrichment.

Nitrite-driven anaerobic methane oxidation by oxygenic bacteria

Only three biological pathways are known to produce oxygen: photosynthesis, chlorate respiration and the detoxification of reactive oxygen species. Here we present evidence for a fourth pathway, possibly of considerable geochemical and evolutionary importance. The pathway was discovered after metagenomic sequencing of an enrichment culture that couples anaerobic oxidation of methane with the reduction of nitrite to dinitrogen. The complete genome of the dominant bacterium, named ‘Candidatus Methylomirabilis oxyfera’, was assembled. This apparently anaerobic, denitrifying bacterium encoded, transcribed and expressed the well-established aerobic pathway for methane oxidation, whereas it lacked known genes for dinitrogen production. Subsequent isotopic labelling indicated that ‘M. oxyfera’ bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxide molecules to dinitrogen and oxygen, which was used to oxidize methane. These results extend our understanding of hydrocarbon degradation under anoxic conditions and explain the biochemical mechanism of a poorly understood freshwater methane sink. Because nitrogen oxides were already present on early Earth, our finding opens up the possibility that oxygen was available to microbial metabolism before the evolution of oxygenic photosynthesis.

Deciphering the evolution and metabolism of an anammox bacterium from a community genome

Anaerobic ammonium oxidation (anammox) has become a main focus in oceanography and wastewater treatment. It is also the nitrogen cycles major remaining biochemical enigma. Among its features, the occurrence of hydrazine as a free intermediate of catabolism, the biosynthesis of ladderane lipids and the role of cytoplasm differentiation are unique in biology. Here we use environmental genomics—the reconstruction of genomic data directly from the environment—to assemble the genome of the uncultured anammox bacterium Kuenenia stuttgartiensis from a complex bioreactor community. The genome data illuminate the evolutionary history of the Planctomycetes and allow us to expose the genetic blueprint of the organisms special properties. Most significantly, we identified candidate genes responsible for ladderane biosynthesis and biological hydrazine metabolism, and discovered unexpected metabolic versatility.

A microbial consortium couples anaerobic methane oxidation to denitrification

Modern agriculture has accelerated biological methane and nitrogen cycling on a global scale. Freshwater sediments often receive increased downward fluxes of nitrate from agricultural runoff and upward fluxes of methane generated by anaerobic decomposition. In theory, prokaryotes should be capable of using nitrate to oxidize methane anaerobically, but such organisms have neither been observed in nature nor isolated in the laboratory. Microbial oxidation of methane is thus believed to proceed only with oxygen or sulphate. Here we show that the direct, anaerobic oxidation of methane coupled to denitrification of nitrate is possible. A microbial consortium, enriched from anoxic sediments, oxidized methane to carbon dioxide coupled to denitrification in the complete absence of oxygen. This consortium consisted of two microorganisms, a bacterium representing a phylum without any cultured species and an archaeon distantly related to marine methanotrophic Archaea. The detection of relatives of these prokaryotes in different freshwater ecosystems worldwide indicates that the reaction presented here may make a substantial contribution to biological methane and nitrogen cycles.

Nitrous oxide emission during wastewater treatment

Nitrous oxide (N(2)O), a potent greenhouse gas, can be emitted during wastewater treatment, significantly contributing to the greenhouse gas footprint. Measurements at lab-scale and full-scale wastewater treatment plants (WWTPs) have demonstrated that N(2)O can be emitted in substantial amounts during nitrogen removal in WWTPs, however, a large variation in reported emission values exists. Analysis of literature data enabled the identification of the most important operational parameters leading to N(2)O emission in WWTPs: (i) low dissolved oxygen concentration in the nitrification and denitrification stages, (ii) increased nitrite concentrations in both nitrification and denitrification stages, and (iii) low COD/N ratio in the denitrification stage. From the literature it remains unclear whether nitrifying or denitrifying microorganisms are the main source of N(2)O emissions. Operational strategies to prevent N(2)O emission from WWTPs are discussed and areas in which further research is urgently required are identified.

Revising the nitrogen cycle in the Peruvian oxygen minimum zone

The oxygen minimum zone (OMZ) of the Eastern Tropical South Pacific (ETSP) is 1 of the 3 major regions in the world where oceanic nitrogen is lost in the pelagic realm. The recent identification of anammox, instead of denitrification, as the likely prevalent pathway for nitrogen loss in this OMZ raises strong questions about our understanding of nitrogen cycling and organic matter remineralization in these waters. Without detectable denitrification, it is unclear how NH4+ is remineralized from organic matter and sustains anammox or how secondary NO2− maxima arise within the OMZ. Here we show that in the ETSP-OMZ, anammox obtains 67% or more of NO2− from nitrate reduction, and 33% or less from aerobic ammonia oxidation, based on stable-isotope pairing experiments corroborated by functional gene expression analyses. Dissimilatory nitrate reduction to ammonium was detected in an open-ocean setting. It occurred throughout the OMZ and could satisfy a substantial part of the NH4+ requirement for anammox. The remaining NH4+ came from remineralization via nitrate reduction and probably from microaerobic respiration. Altogether, deep-sea NO3− accounted for only ≈50% of the nitrogen loss in the ETSP, rather than 100% as commonly assumed. Because oceanic OMZs seem to be expanding because of global climate change, it is increasingly imperative to incorporate the correct nitrogen-loss pathways in global biogeochemical models to predict more accurately how the nitrogen cycle in our future ocean may respond.

Completely autotrophic nitrogen removal over nitrite in one single reactor

The microbiology and the feasibility of a new, single-stage, reactor for completely autotrophic ammonia removal were investigated. The reactor was started anoxically after inoculation with biomass from a reactor performing anaerobic ammonia oxidation (Anammox). Subsequently, oxygen was supplied to the reactor and a nitrifying population developed. Oxygen was kept as the limiting factor. The development of a nitrifying population was monitored by Fluorescence In Situ Hybridization and off-line activity measurements. These methods also showed that during steady state, anaerobic ammonium-oxidizing bacteria remained present and active. In the reactor, no aerobic nitrite-oxidizers were detected. The denitrifying potential of the biomass was below the detection limit. Ammonia was mainly converted to N2 (85%) and the remainder (15%) was recovered as NO3-. N2O production was negligible (less than 0.1%). Addition of an external carbon source was not needed to realize the autotrophic denitrification to N2.