An intracellular pH gradient in the anammox bacterium Kuenenia stuttgartiensis as evaluated by 31P NMR.

The cytoplasm of anaerobic ammonium oxidizing (anammox) bacteria consists of three compartments separated by membranes. It has been suggested that a proton motive force may be generated over the membrane of the innermost compartment, the "anammoxosome". 31P nuclear magnetic resonance (NMR) spectroscopy was employed to investigate intracellular pH differences in the anammox bacterium Kuenenia stuttgartiensis. With in vivo NMR, spectra were recorded of active, highly concentrated suspensions of K. stuttgartiensis in a wide-bore NMR tube. At different external pH values, two stable and distinct phosphate peaks were apparent in the recorded spectra. These peaks were equivalent with pH values of 7.3 and 6.3 and suggested the presence of a proton motive force over an intracytoplasmic membrane in K. stuttgartiensis. This study provides for the second time--after discovery of acidocalcisome-like compartments in Agrobacterium tumefaciens--evidence for an intracytoplasmic pH gradient in a chemotrophic prokaryotic cell.

Response of anaerobic ammonium-oxidizing bacteria to hydroxylamine.

Anaerobic ammonium oxidation is a recent addition to the microbial nitrogen cycle, and its metabolic pathway, including the production and conversion of its intermediate hydrazine, is not well understood. Therefore, the effect of hydroxylamine addition on the hydrazine metabolism of anaerobic ammonium-oxidizing (anammox) bacteria was studied both experimentally and by mathematical modeling. It was observed that hydroxylamine was disproportionated biologically in the absence of nitrite into dinitrogen gas and ammonium. Little hydrazine accumulated during this process; however, rapid hydrazine production was observed when nearly all hydroxylamine was consumed. A mechanistic model is proposed in which hydrazine is suggested to be continuously produced from ammonium and hydroxylamine (possibly via nitric oxide) and subsequently oxidized to N(2). The electron acceptor for hydrazine oxidation is hydroxylamine, which is reduced to ammonium. A decrease in the hydroxylamine reduction rate, therefore, leads to a decrease in the hydrazine oxidation rate, resulting in the observed hydrazine accumulation. The proposed mechanism was verified by a mathematical model which could explain and predict most of the experimental data.

The membrane bioreactor: a novel tool to grow anammox bacteria as free cells.

In a membrane bioreactor (MBR), fast growth of anammox bacteria was achieved with a sludge residence time (SRT) of 12 days. This relatively short SRT resulted in a--for anammox bacteria--unprecedented purity of the enrichment of 97.6%. The absence of a selective pressure for settling, and dedicated cultivation conditions led to growth in suspension as free cells and the complete absence of flocs or granules. Fast growth, low levels of calcium and magnesium, and possibly the presence of yeast extract and a low shear stress are critical for the obtainment of a completely suspended culture consisting of free anammox cells. During cultivation, a population shift was observed from Candidatus "Brocadia" to Candidatus "Kuenenia stuttgartiensis." It is hypothesized that the reason for this shift is the higher affinity for nitrite of "Kuenenia." The production of anammox bacteria in suspension with high purity and productivity makes the MBR a promising tool for the cultivation and study of anammox bacteria.

Dynamics of nitric oxide and nitrous oxide emission during full-scale reject water treatment.

Emission of NO and N2O from a full-scale two-reactor nitritation-anammox process was determined during a measurement campaign at the Dokhaven-Sluisjesdijk municipal WWTP (Rotterdam, NL). The NO and N2O levels in the off-gas responded to the aeration cycles and the aeration rate of the nitritation reactor, and to the nitrite and dissolved oxygen concentration. Due to the strong fluctuations in the NO and N2O levels in both the nitritation and the anammox reactor, only time-dependent measurements could yield a reliable estimate of the overall NO and N2O emissions. The NO emission from the nitritation reactor was 0.2% of the nitrogen load and the N2O emission was 1.7%. The NO emission from the anammox reactor was determined to be 0.003% of the nitrogen load and the N2O emission was 0.6%. Emission of NO2 could not be detected from the nitritation-anammox system. Denitrification by ammonia-oxidizing bacteria was considered to be the most probable cause of NO and N2O emission from the nitritation reactor. Since anammox bacteria have not been shown to produce N2O under physiological conditions, it is also suspected that ammonia-oxidizing bacteria contribute most to N2O production in the anammox reactor. The source of NO production in the anammox reactor can be either anammox bacteria or denitrification by heterotrophs or ammonia-oxidizing bacteria. Based on the results and previous work, it seems that a low dissolved oxygen or a high nitrite concentration are the most likely cause of elevated NO and N2O emission by ammonia-oxidizing bacteria. The emission was compared with measurements at other reject water technologies and with the main line of the Dokhaven-Sluisjesdijk WWTP. The N2O emission levels in the reject water treatment seem to be in the same range as for the main stream of activated sludge processes. Preliminary measurements of the N2O emission from a one-reactor nitritation-anammox system indicate that the emission is lower than in two-reactor systems.

Startup of reactors for anoxic ammonium oxidation: experiences from the first full-scale anammox reactor in Rotterdam.

The first full-scale anammox reactor in the world was started in Rotterdam (NL). The reactor was scaled-up directly from laboratory-scale to full-scale and treats up to 750 kg-N/d. In the initial phase of the startup, anammox conversions could not be identified by traditional methods, but quantitative PCR proved to be a reliable indicator for growth of the anammox population, indicating an anammox doubling time of 10-12 days. The experience gained during this first startup in combination with the availability of seed sludge from this reactor, will lead to a faster startup of anammox reactors in the future. The anammox reactor type employed in Rotterdam was compared to other reactor types for the anammox process. Reactors with a high specific surface area like the granular sludge reactor employed in Rotterdam provide the highest volumetric loading rates. Mass transfer of nitrite into the biofilm is limiting the conversion of those reactor types that have a lower specific surface area. Now the first full-scale commercial anammox reactor is in operation, a consistent and descriptive nomenclature is suggested for reactors in which the anammox process is employed.