Thermophilic biological nitrogen removal in industrial wastewater treatment.

Nitrification is an integral part of biological nitrogen removal processes and usually the limiting step in wastewater treatment systems. Since nitrification is often considered not feasible at temperatures higher than 40 °C, warm industrial effluents (with operating temperatures higher than 40 °C) need to be cooled down prior to biological treatment, which increases the energy and operating costs of the plants for cooling purposes. This study describes the occurrence of thermophilic biological nitrogen removal activity (nitritation, nitratation, and denitrification) at a temperature as high as 50 °C in an activated sludge wastewater treatment plant treating wastewater from an oil refinery. Using a modified two-step nitrification-two-step denitrification mathematical model extended with the incorporation of double Arrhenius equations, the nitrification (nitrititation and nitratation) and denitrification activities were described including the cease in biomass activity at 55 °C. Fluorescence in situ hybridization (FISH) analyses revealed that Nitrosomonas halotolerant and obligatehalophilic and Nitrosomonas oligotropha (known ammonia-oxidizing organisms) and Nitrospira sublineage II (nitrite-oxidizing organism (NOB)) were observed using the FISH probes applied in this study. In particular, this is the first time that Nitrospira sublineage II, a moderatedly thermophilic NOB, is observed in an engineered full-scale (industrial) wastewater treatment system at temperatures as high as 50 °C. These observations suggest that thermophilic biological nitrogen removal can be attained in wastewater treatment systems, which may further contribute to the optimization of the biological nitrogen removal processes in wastewater treatment systems that treat warm wastewater streams.

NH4+ ad-/desorption in sequencing batch reactors: simulation, laboratory and full-scale studies.

Significant NH4-N balance deficits were found during the measurement campaigns for the data collection for dynamic simulation studies at five full-scale sequencing batch reactor (SBR) waste water treatment plants (WWTPs), as well as during subsequent calibrations at the investigated plants. Subsequent lab scale investigations showed high evidence for dynamic, cycle-specific NH4+ ad-/desorption to the activated flocs as one reason for this balance deficit. This specific dynamic was investigated at five full-scale SBR plants for the search of the general causing mechanisms. The general mechanism found was a NH4+ desorption from the activated flocs at the end of the nitrification phase with subsequent nitrification and a chemical NH4+ adsorption at the flocs in the course of the filling phases. This NH4+ ad-/desorption corresponds to an antiparallel K+ ad/-desorption.One reasonable full-scale application was investigated at three SBR plants, a controlled filling phase at the beginning of the sedimentation phase. The results indicate that this kind of filling event must be specifically hydraulic controlled and optimised in order to prevent too high waste water break through into the clear water phase, which will subsequently be discarded.

Physiological and phylogenetic characterization of a novel lithoautotrophic nitrite-oxidizing bacterium, 'Candidatus Nitrospira bockiana'.

A new isolate of a lithoautotrophic nitrite-oxidizing bacterium was obtained from internal corrosion deposits from a steel pipeline of the Moscow heating system. The organism oxidized nitrite as the sole energy source and fixed carbon dioxide as the only carbon source. The cells were extremely pleomorphic: loosely wound spirals, slightly curved and even straight rods were detected, as well as coccoid cells. The highest rate of nitrite consumption (1.5 mM nitrite as substrate) was measured at 42 degrees C, with a temperature range of 28-44 degrees C. In enrichment cultures with Nocardioides sp. as an accompanying organism, optimal oxidation of 5.8 mM nitrite occurred at 45 degrees C, with a range of 28-48 degrees C. Neither pyruvate nor yeast extract stimulated nitrification. Organotrophic growth was not observed. Phylogenetic analysis of 16S rRNA gene sequences revealed that the novel isolate represents a new sublineage of the genus Nitrospira. On the basis of physiological, chemotaxonomic and molecular characteristics, the name 'Candidatus Nitrospira bockiana' is proposed.

Ecophysiology and niche differentiation of Nitrospira-like bacteria, the key nitrite oxidizers in wastewater treatment plants.

Nitrite-oxidizing bacteria of the genus Nitrospira are key nitrifiers in wastewater treatment plants. Pure cultures of these organisms are unavailable, but cultivation-independent molecular methods make it possible to detect Nitrospira-like bacteria in environmental samples and to investigate their ecophysiology. Comprehensive screening of natural and engineered habitats and of public databases for 16S rRNA sequences of Nitrospira-like bacteria revealed a surprisingly high biodiversity in the genus Nitrospira, which comprises at least four phylogenetic sublineages. All Nitrospira-like bacteria detected in wastewater treatment plants belonged to the sublineages I and II. Subsequently, the population dynamics of different Nitrospira-like bacteria were monitored, by quantitative fluorescence in situ hybridization with rRNA-targeted probes, confocal laser scanning microscopy and digital image analysis, during incubation of nitrifying activated sludge in media containing different nitrite concentrations. These experiments showed that Nitrospira-like bacteria, which were affiliated with the phylogenetic sublineages I or II of the genus Nitrospira, responded differently to nitrite concentration shifts. Previously unknown properties of Nitrospira-like bacteria were discovered in the course of an environmental genomics project. Implications of the obtained results for fundamental understanding of the microbial ecology of nitrite oxidizers as well as for future improvement of nutrient removal in wastewater treatment plants are discussed.

Cultivation-independent, semiautomatic determination of absolute bacterial cell numbers in environmental samples by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has found widespread application for analyzing the composition of microbial communities in complex environmental samples. Although bacteria can quickly be detected by FISH, a reliable method to determine absolute numbers of FISH-stained cells in aggregates or biofilms has, to our knowledge, never been published. In this study we developed a semiautomated protocol to measure the concentration of bacteria (in cells per volume) in environmental samples by a combination of FISH, confocal laser scanning microscopy, and digital image analysis. The quantification is based on an internal standard, which is introduced by spiking the samples with known amounts of Escherichia coli cells. This method was initially tested with artificial mixtures of bacterial cultures and subsequently used to determine the concentration of ammonia-oxidizing bacteria in a municipal nitrifying activated sludge. The total number of ammonia oxidizers was found to be 9.8 x 10(7) +/- 1.9 x 10(7) cells ml(-1). Based on this value, the average in situ activity was calculated to be 2.3 fmol of ammonia converted to nitrite per ammonia oxidizer cell per h. This activity is within the previously determined range of activities measured with ammonia oxidizer pure cultures, demonstrating the utility of this quantification method for enumerating bacteria in samples in which cells are not homogeneously distributed.

In situ characterization of Nitrospira-like nitrite-oxidizing bacteria active in wastewater treatment plants.

Uncultivated Nitrospira-like bacteria in different biofilm and activated-sludge samples were investigated by cultivation-independent molecular approaches. Initially, the phylogenetic affiliation of Nitrospira-like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Subsequently, a phylogenetic consensus tree of the Nitrospira phylum including all publicly available sequences was constructed. This analysis revealed that the genus Nitrospira consists of at least four distinct sublineages. Based on these data, two 16S rRNA-directed oligonucleotide probes specific for the phylum and genus Nitrospira, respectively, were developed and evaluated for suitability for fluorescence in situ hybridization (FISH). The probes were used to investigate the in situ architecture of cell aggregates of Nitrospira-like nitrite oxidizers in wastewater treatment plants by FISH, confocal laser scanning microscopy, and computer-aided three-dimensional visualization. Cavities and a network of cell-free channels inside the Nitrospira microcolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources by Nitrospira-like bacteria within their natural habitat under different incubation conditions was studied by combined FISH and microautoradiography. Under aerobic conditions, the Nitrospira-like bacteria in bioreactor samples took up inorganic carbon (as HCO(3)(-) or as CO(2)) and pyruvate but not acetate, butyrate, and propionate, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by the Nitrospira-like bacteria of any of the carbon sources tested was observed under anoxic or anaerobic conditions.

Nitrification in sequencing biofilm batch reactors: lessons from molecular approaches.

The nitrifying microbial diversity and population structure of a sequencing biofilm batch reactor receiving sewage with high ammonia and salt concentrations (SBBR 1) was analyzed by the full-cycle rRNA approach. The diversity of ammonia-oxidizers in this reactor was additionally investigated using comparative sequence analysis of a gene fragment of the ammonia monooxygenase (amoA), which represents a key enzyme of all ammonia-oxidizers. Despite the "extreme" conditions in the reactor, a surprisingly high diversity of ammonia- and nitrite-oxidizers was observed to occur within the biofilm. In addition, molecular evidence for the existence of novel ammonia-oxidizers is presented. Quantification of ammonia- and nitrite-oxidizers in the biofilm by Fluorescent In situ Hybridization (FISH) and digital image analysis revealed that ammonia-oxidizers occurred in higher cell numbers and occupied a considerably larger share of the total biovolume than nitrite-oxidizing bacteria. In addition, ammonia oxidation rates per cell were calculated for different WWTPs following the quantification of ammonia-oxidizers by competitive PCR of an amoA gene fragment. The morphology of nitrite-oxidizing, unculturable Nitrospira-like bacteria was studied using FISH, confocal laser scanning microscopy (CLSM) and three-dimensional visualization. Thereby, a complex network of microchannels and cavities was detected within microcolonies of Nitrospira-like bacteria. Microautoradiography combined with FISH was applied to investigate the ability of these organisms to use CO2 as carbon source and to take up other organic substrates under varying conditions. Implications of the obtained results for fundamental understanding of the microbial ecology of nitrifiers as well as for future improvement of nutrient removal in wastewater treatment plants (WWTPs) are discussed.

The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set.

In situ hybridization with rRNA-targeted oligonucleotide probes has become a widely applied tool for direct analysis of microbial population structures of complex natural and engineered systems. In such studies probe EUB338 (AMANN et al., 1990) is routinely used to quantify members of the domain Bacteria with a sufficiently high cellular ribosome content. Recent reevaluations of probe EUB338 coverage based on all publicly available 16S rRNA sequences, however, indicated that important bacterial phyla, most notably the Planctomycetales and Verrucomicrobia, are missed by this probe. We therefore designed and evaluated two supplementary versions (EUB338-II and EUB338-III) of probe EUB338 for in situ detection of most of those phyla not detected with probe EUB338. In situ dissociation curves with target and non-target organisms were recorded under increasing stringency to optimize hybridization conditions. For that purpose a digital image software routine was developed. In situ hybridization of a complex biofilm community with the three EUB338 probes demonstrated the presence of significant numbers of probe EUB338-II and EUB338-III target organisms. The application of EUB338, EUB338-II and EUB338-III should allow a more accurate quantification of members of the domain Bacteria in future molecular ecological studies.