Current production by non-methanotrophic bacteria enriched from an anaerobic methane-oxidizing microbial community.

In recent years, the externalization of electrons as part of respiratory metabolic processes has been discovered in many different bacteria and some archaea. Microbial extracellular electron transfer (EET) plays an important role in many anoxic natural or engineered ecosystems. In this study, an anaerobic methane-converting microbial community was investigated with regard to its potential to perform EET. At this point, it is not well-known if or how EET confers a competitive advantage to certain species in methane-converting communities. EET was investigated in a two-chamber electrochemical system, sparged with methane and with an applied potential of +400 mV versus standard hydrogen electrode. A biofilm developed on the working electrode and stable low-density current was produced, confirming that EET indeed did occur. The appearance and presence of redox centers at -140 to -160 mV and at -230 mV in the biofilm was confirmed by cyclic voltammetry scans. Metagenomic analysis and fluorescence in situ hybridization of the biofilm showed that the anaerobic methanotroph 'Candidatus Methanoperedens BLZ2' was a significant member of the biofilm community, but its relative abundance did not increase compared to the inoculum. On the contrary, the relative abundance of other members of the microbial community significantly increased (up to 720-fold, 7.2% of mapped reads), placing these microorganisms among the dominant species in the bioanode community. This group included Zoogloea sp., Dechloromonas sp., two members of the Bacteroidetes phylum, and the spirochete Leptonema sp. Genes encoding proteins putatively involved in EET were identified in Zoogloea sp., Dechloromonas sp. and one member of the Bacteroidetes phylum. We suggest that instead of methane, alternative carbon sources such as acetate were the substrate for EET. Hence, EET in a methane-driven chemolithoautotrophic microbial community seems a complex process in which interactions within the microbial community are driving extracellular electron transfer to the electrode.

Planctopirus ephydatiae, a novel Planctomycete isolated from a freshwater sponge.

The microbiome of freshwater sponges is rarely studied, and not a single novel bacterial species has been isolated and subsequently characterized from a freshwater sponge to date. A previous study showed that 14.4% of the microbiome from Ephydatia fluviatilis belong to the phylum Planctomycetes. Therefore, we sampled an Ephydatia sponge from a freshwater lake and employed enrichment techniques targeting bacteria from the phylum Planctomycetes. The obtained strain spb1T was subject to genomic and phenomic characterization and found to represent a novel planctomycetal species proposed as Planctopirus ephydatiae sp. nov. (DSM 106606 = CECT 9866). In the process of differentiating spb1T from its next relative Planctopirus limnophila DSM 3776T, we identified and characterized the first phage - Planctopirus phage vB_PlimS_J1 - infecting planctomycetes that was only mentioned anecdotally before. Interestingly, classical chemotaxonomic methods would have failed to distinguish Planctopirus ephydatiae strain spb1T from Planctopirus limnophila DSM 3776T. Our findings demonstrate and underpin the need for whole genome-based taxonomy to detect and differentiate planctomycetal species.

A 192-heme electron transfer network in the hydrazine dehydrogenase complex.

Anaerobic ammonium oxidation (anammox) is a major process in the biogeochemical nitrogen cycle in which nitrite and ammonium are converted to dinitrogen gas and water through the highly reactive intermediate hydrazine. So far, it is unknown how anammox organisms convert the toxic hydrazine into nitrogen and harvest the extremely low potential electrons (-750 mV) released in this process. We report the crystal structure and cryo electron microscopy structures of the responsible enzyme, hydrazine dehydrogenase, which is a 1.7 MDa multiprotein complex containing an extended electron transfer network of 192 heme groups spanning the entire complex. This unique molecular arrangement suggests a way in which the protein stores and releases the electrons obtained from hydrazine conversion, the final step in the globally important anammox process.

Distribution and activity of the anaerobic methanotrophic community in a nitrogen-fertilized Italian paddy soil.

In order to mitigate methane emissions from paddy fields, it is important to understand the sources and sinks. Most paddy fields are heavily fertilized with nitrite and nitrate, which can be used as electron acceptors by anaerobic methanotrophs. Here we show that slurry incubations of Italian paddy field soil with nitrate and 13C-labelled methane have the potential for nitrate-dependent anaerobic oxidation of methane (79.9 nmol g-1dw d-1). Community analysis based on 16S rRNA amplicon sequencing and qPCR of the water-logged soil and the rhizosphere showed that anaerobic oxidation of methane-associated archaea (AAA), including Methanoperedens nitroreducens, comprised 9% (bulk soil) and 1% (rhizosphere) of all archaeal reads. The NC10 phylum bacteria made up less than 1% of all bacterial sequences. The phylogenetic analysis was complemented by qPCR showing that AAA ranged from 0.28 × 106 to 3.9 × 106 16S rRNA gene copies g-1dw in bulk soil and 0.27 × 106 to 2.8 × 106 in the rhizosphere. The abundance of NC10 phylum bacteria was an order of magnitude lower. Revisiting published diversity studies, we found that AAA have been detected, but not linked to methane oxidation, in several paddy fields. Our data suggest an important role of AAA in methane cycling in paddy fields.

Simultaneous partial nitritation and anammox at low temperature with granular sludge.

Autotrophic nitrogen removal in the main stream appears as a prerequisite for the implementation of energy autarchic wastewater treatment plants. To investigate autotrophic nitrogen removal a lab-scale gas-lift sequencing batch reactor with granular sludge was operated for more than 500 days. The reactor was operated at temperatures between 20 and 10 °C on autotrophic medium with ammonium (60 and 160 mg-N L(-1)) as only nitrogen compound at an HRT of 0.23-0.3 d. The dissolved oxygen (DO) concentration was shown to be an effective control parameter for the suppression of the undesired nitratation process. DO control guaranteed the effective suppression of the nitratation both at 20 and 15 °C, allowing nitrogen removal rates of 0.4 g-NTot L(-1) d(-1) at nitrogen removal efficiencies of 85-75%. Prolonged operation at 10 °C caused a slow but unrestrainable decrease in anammox activity and process efficiency. This study represents a proof of concept for the application of the autotrophic nitrogen removal in a single reactor with granular sludge at main stream conditions.

Metagenome Analysis of a Complex Community Reveals the Metabolic Blueprint of Anammox Bacterium "Candidatus Jettenia asiatica".

Anaerobic ammonium-oxidizing (anammox) bacteria are key players in the global nitrogen cycle and responsible for significant global nitrogen loss. Moreover, the anammox process is widely implemented for nitrogen removal from wastewaters as a cost-effective and environment-friendly alternative to conventional nitrification-denitrification systems. Currently, five genera of anammox bacteria have been identified, together forming a deep-branching order in the Planctomycetes-Verrucomicrobium-Chlamydiae superphylum. Members of all genera have been detected in wastewater treatment plants and have been enriched in lab-scale bioreactors, but genome information is not yet available for all genera. Here we report the metagenomic analysis of a granular sludge anammox reactor dominated (∼50%) by "Candidatus Jettenia asiatica." The metagenome was sequenced using both Illumina and 454 pyrosequencing. After de novo assembly 37,432 contigs with an average length of 571 nt were obtained. The contigs were then analyzed by BLASTx searches against the protein sequences of "Candidatus Kuenenia stuttgartiensis" and a set of 25 genes essential in anammox metabolism were detected. Additionally all reads were mapped to the genome of an anammox strain KSU-1 and de novo assembly was performed again using the reads that could be mapped on KSU-1. Using this approach, a gene encoding copper-containing nitrite reductase NirK was identified in the genome, instead of cytochrome cd(1)-type nitrite reductase (NirS, present in "Ca. Kuenenia stuttgartiensis" and "Ca. Scalindua profunda"). Finally, the community composition was investigated through MetaCluster analysis, 16S rRNA gene analysis and read mapping, which showed the presence of other important community members such as aerobic ammonia-oxidizing bacteria, methanogens, and the denitrifying methanotroph "Ca. Methylomirabilis oxyfera", indicating a possible active methane and nitrogen cycle in the bioreactor under the prevailing operational conditions.

De novo transcriptome characterization and development of genomic tools for Scabiosa columbaria L. using next-generation sequencing techniques.

Next-generation sequencing (NGS) technologies are increasingly applied in many organisms, including nonmodel organisms that are important for ecological and conservation purposes. Illumina and 454 sequencing are among the most used NGS technologies and have been shown to produce optimal results at reasonable costs when used together. Here, we describe the combined application of these two NGS technologies to characterize the transcriptome of a plant species of ecological and conservation relevance for which no genomic resource is available, Scabiosa columbaria. We obtained 528,557 reads from a 454 GS-FLX run and a total of 28,993,627 reads from two lanes of an Illumina GAII single run. After read trimming, the de novo assembly of both types of reads produced 109,630 contigs. Both the contigs and the >75 bp remaining singletons were blasted against the Uniprot/Swissprot database, resulting in 29,676 and 10,515 significant hits, respectively. Based on sequence similarity with known gene products, these sequences represent at least 12,516 unique genes, most of which are well covered by contig sequences. In addition, we identified 4320 microsatellite loci, of which 856 had flanking sequences suitable for PCR primer design. We also identified 75,054 putative SNPs. This annotated sequence collection and the relative molecular markers represent a main genomic resource for S. columbaria which should contribute to future research in conservation and population biology studies. Our results demonstrate the utility of NGS technologies as starting point for the development of genomic tools in nonmodel but ecologically important species.

Decreased N2O reduction by low soil pH causes high N2O emissions in a riparian ecosystem.

Quantification of harmful nitrous oxide (N(2)O) emissions from soils is essential for mitigation measures. An important N(2)O producing and reducing process in soils is denitrification, which shows deceased rates at low pH. No clear relationship between N(2)O emissions and soil pH has yet been established because also the relative contribution of N(2)O as the denitrification end product decreases with pH. Our aim was to show the net effect of soil pH on N(2)O production and emission. Therefore, experiments were designed to investigate the effects of pH on NO(3)(-) reduction, N(2)O production and reduction and N(2) production in incubations with pH values set between 4 and 7. Furthermore, field measurements of soil pH and N(2)O emissions were carried out. In incubations, NO(3)(-) reduction and N(2) production rates increased with pH and net N(2)O production rate was highest at pH 5. N(2)O reduction to N(2) was halted until NO(3)(-) was depleted at low pH values, resulting in a built up of N(2)O. As a consequence, N(2)O:N(2) production ratio decreased exponentially with pH. N(2)O reduction appeared therefore more important than N(2)O production in explaining net N(2)O production rates. In the field, a negative exponential relationship for soil pH against N(2)O emissions was observed. Soil pH could therefore be used as a predictive tool for average N(2)O emissions in the studied ecosystem. The occurrence of low pH spots may explain N(2)O emission hotspot occurrence. Future studies should focus on the mechanism behind small scale soil pH variability and the effect of manipulating the pH of soils.

Denitrification at pH 4 by a soil-derived Rhodanobacter-dominated community.

Soil denitrification is a major source of nitrous oxide emission that causes ozone depletion and global warming. Low soil pH influences the relative amount of N2O produced and consumed by denitrification. Furthermore, denitrification is strongly inhibited in pure cultures of denitrifying microorganisms below pH 5. Soils, however, have been shown to denitrify at pH values as low as pH 3. Here we used a continuous bioreactor to investigate the possibility of significant denitrification at low pH under controlled conditions with soil microorganisms and naturally available electron donors. Significant NO3⁻ and N2O reduction were observed for 3 months without the addition of any external electron donor. Batch incubations with the enriched biomass showed that low pH as well as low electron donor availability promoted the relative abundance of N2O as denitrification end-product. Molecular analysis of the enriched biomass revealed that a Rhodanobacter-like bacterium dominated the community in 16S rRNA gene libraries as well as in FISH microscopy during the highest denitrification activity in the reactor. We conclude that denitrification at pH 4 with natural electron donors is possible and that a Rhodanobacter species may be one of the microorganisms involved in acidic denitrification in soils.

Emission of nitrous oxide and nitric oxide from a full-scale single-stage nitritation-anammox reactor.

At a full-scale single-stage nitritation-anammox reactor, off-gas measurement for nitric oxide (NO) and nitrous oxide (N(2)O) was performed. NO and N(2)O are environmental hazards, imposing the risk of improving water quality at the cost of deteriorating air quality. The emission of NO during normal operation of a single-stage nitritation-anammox process was 0.005% of the nitrogen load while the N(2)O emission was 1.2% of the nitrogen load to the reactor, which is in the same range as reported emission from other full-scale wastewater treatment plants. The emission of both compounds was strongly coupled. The concentration of NO and N(2)O in the off-gas of the single-stage nitritation-anammox reactor was rather dynamic and clearly responded to operational variations. This exemplifies the need for time-dependent measurement of NO and N(2)O emission from bioreactors for reliable emission estimates. Nitrite accumulation clearly resulted in increased NO and N(2)O concentrations in the off-gas, yielding higher emission levels. Oxygen limitation resulted in a decrease in NO and N(2)O emission, which was unexpected as oxygen limitation is generally assumed to cause increased emissions in nitrogen converting systems. Higher aeration flow dramatically increased the NO emission load and also seemed to increase the N(2)O emission, which stresses the importance of efficient aeration control to limit NO and N(2)O emissions.

N2O emission hotspots at different spatial scales and governing factors for small scale hotspots.

Chronically nitrate-loaded riparian buffer zones show high N(2)O emissions. Often, a large part of the N(2)O is emitted from small surface areas, resulting in high spatial variability in these buffer zones. These small surface areas with high N(2)O emissions (hotspots) need to be investigated to generate knowledge on the factors governing N(2)O emissions. In this study the N(2)O emission variability was investigated at different spatial scales. Therefore N(2)O emissions from three 32 m(2) grids were determined in summer and winter. Spatial variation and total emission were determined on three different scales (0.3 m(2), 0.018 m(2) and 0.0013 m(2)) at plots with different levels of N(2)O emissions. Spatial variation was high at all scales determined and highest at the smallest scale. To test possible factors inducing small scale hotspots, soil samples were collected for slurry incubation to determine responses to increased electron donor/acceptor availability. Acetate addition did increase N(2)O production, but nitrate addition failed to increase total denitrification or net N(2)O production. N(2)O production was similar in all soil slurries, independent of their origin from high or low emission soils, indicating that environmental conditions (including physical factors like gas diffusion) rather than microbial community composition governed N(2)O emission rates.

Global impact and application of the anaerobic ammonium-oxidizing (anammox) bacteria.

In the anaerobic ammonium oxidation (anammox) process, ammonia is oxidized with nitrite as primary electron acceptor under strictly anoxic conditions. The reaction is catalysed by a specialized group of planctomycete-like bacteria. These anammox bacteria use a complex reaction mechanism involving hydrazine as an intermediate. The reactions are assumed to be carried out in a unique prokaryotic organelle, the anammoxosome. This organelle is surrounded by ladderane lipids, which make the organelle nearly impermeable to hydrazine and protons. The localization of the major anammox protein, hydrazine oxidoreductase, was determined via immunogold labelling to be inside the anammoxosome. The anammox bacteria have been detected in many marine and freshwater ecosystems and were estimated to contribute up to 50% of oceanic nitrogen loss. Furthermore, the anammox process is currently implemented in water treatment for the low-cost removal of ammonia from high-strength waste streams. Recent findings suggested that the anammox bacteria may also use organic acids to convert nitrate and nitrite into dinitrogen gas when ammonia is in short supply.

Role of nitrogen oxides in the metabolism of ammonia-oxidizing bacteria.

Ammonia-oxidizing bacteria (AOB) can use oxygen and nitrite as electron acceptors. Nitrite reduction by Nitrosomonas is observed under three conditions: (i) hydrogen-dependent denitrification, (ii) anoxic ammonia oxidation with nitrogen dioxide (NO(2)) and (iii) NO(x)-induced aerobic ammonia oxidation. NO(x) molecules play an important role in the conversion of ammonia and nitrite by AOB. Absence of nitric oxide (NO), which is generally detectable during ammonia oxidation, severely impairs ammonia oxidation by AOB. The lag phase of recovery of aerobic ammonia oxidation was significantly reduced by NO(2) addition. Acetylene inhibition tests showed that NO(2)-dependent and oxygen-dependent ammonia oxidation can be distinguished. Addition of NO(x) increased specific activity of ammonia oxidation, growth rate and denitrification capacity. Together, these findings resulted in a hypothetical model on the role of NO(x) in ammonia oxidation: the NO(x) cycle.

Treatment of nitrogen-rich wastewater using partial nitrification and anammox in the CANON process.

Partial nitrification combined with Anammox in a single reactor (the CANON process) is an energy-efficient N-removal technology that could substantially lower the N-load of a WWTP by separate treatment of nitrogen-rich side streams, preventing the need for extensive expansion and reducing the total energy requirement. This study looks at the enrichment of Anammox from activated sludge and its application in the CANON process on lab-scale. The aim was to identify the critical process control parameters necessary for successful operation of CANON. An Anammox culture capable of removing 0.6 kg N/m3/d was enriched in 14 weeks in a sequencing batch reactor. Nitrifying biomass was inoculated into the Anammox reactor (10% v/v) together with limited oxygen supply (< 8 mL/min) to initiate the CANON process in continuous culture. The small flocs formed by the biomass (< 1000 microm) were sensitive to low O2 concentrations (< 0.1 mg/L) which prevented simultaneous nitrification and Anammox. Operation with 20 min aerobiosis and 30 min anaerobiosis was necessary to achieve sustained, completely autotrophic N-removal for an extended period at a rate of 0.08 kg N/m3/d. Essential process control parameters for stable CANON operation were the nitrite concentration, oxygen concentration, pH and the temperature.

Enrichment of anammox from activated sludge and its application in the CANON process.

A microbial culture capable of actively oxidizing ammonium to dinitrogen gas in the absence of oxygen, using nitrite as the electron acceptor, was enriched from local activated sludge (Western Australia) in <14 weeks. The maximum anaerobic ammonium oxidation (i.e., anammox) activity achieved by the anaerobic culture was 0.26 mmol NH (4) (+) (g biomass)(-1) h(-1) (0.58 kg total-N m(-3) day(-1)). Qualitative FISH analysis (fluorescence in situ hybridization) confirmed the phylogenetic position of the enriched microorganism as belonging to the order Planctomycetales, in which all currently identified anammox strains fall. Preliminary FISH analysis suggests the anammox strain belongs to the same phylogenetic group as the Candidatus 'Brocadia anammoxidans' strain discovered in the Netherlands. However, there are quite a few differences in the target sites for the more specific probes of these organisms and it is therefore likely to represent a new species of anammox bacteria. A small amount of aerobic ammonium-oxidizing biomass was inoculated into the anammox reactor (10% v/v) to initiate completely autotrophic nitrogen removal over nitrite (the CANON process) in chemostat culture. The culture was always under oxygen limitation and no organic carbon was added. The CANON reactor was operated as an intermittently aerated system with 20 min aerobiosis and 30 min anaerobiosis, during which aerobic and anaerobic ammonium oxidation were performed in sequential fashion, respectively. Anammox was not inhibited by repeated intermittent exposure to oxygen, allowing sustained, completely autotrophic ammonium removal (0.08 kg N m(-3) day(-1)) for an extended period of time.

1994-2004: 10 years of research on the anaerobic oxidation of ammonium.

The obligately anaerobic ammonium oxidation (anammox) reaction with nitrite as primary electron acceptor is catalysed by the planctomycete-like bacteria Brocadia anammoxidans, Kuenenia stuttgartiensis and Scalindua sorokinii. The anammox bacteria use a complex reaction mechanism involving hydrazine as an intermediate. They have a unique prokaryotic organelle, the anammoxosome, surrounded by ladderane lipids, which exclusively contains the hydrazine oxidoreductase as the major protein to combine nitrite and ammonia in a one-to-one fashion. In addition to the peculiar microbiology, anammox was shown to be very important in the oceanic nitrogen cycle, and proved to be a very good alternative for treatment of high-strength nitrogenous waste streams. With the assembly of the K. stuttgartiensis genome at Genoscope, Evry, France, the anammox reaction has entered the genomic and proteomic era, enabling the elucidation of many intriguing aspects of this fascinating microbial process.

Complete conversion of nitrate into dinitrogen gas in co-cultures of denitrifying bacteria.

In the past 10 years many molecular aspects of microbial nitrate reduction have been elucidated, but the ecophysiology of this process is hardly understood. In this contribution, our efforts to study the complex microbial communities and interactions involved in the reduction of nitrate to dinitrogen gas are summarized. The initial work concentrated on emission of the greenhouse gas nitrous oxide during incomplete denitrification by Alcaligenes faecalis. As more research methods became available, the fitness of A. faecalis could be tested in mixed cultures with other denitrifying bacteria, most notably with the nitrate-reducing bacterium Pseudomonas G9. Finally, the advancement of molecular diagnostic tools made it possible to survey complex microbial communities using specific primer sets for/and antibodies raised against the various NO(x) reductases. Given the enormous complexity of substrates and environmental conditions, it is evident that mixed cultures rather than single species are responsible for denitrification in man-made and natural ecosystems. However, it is surprising that even for the breakdown of a single compound, such as acetate, mixed cultures are responsible, and that the consecutive denitrification steps are commonly performed by mutualistic co-operating species. Our observations also indicate that we seldom know the identity of the major key players in the nitrogen cycle of these ecosystems.

Stability of the ANAMMOX process in a gas-lift reactor and a SBR.

In the last years, the ANAerobic AMMonium OXidation (ANAMMOX) process has been put forward as a promising alternative to treat ammonium rich wastewaters. An ANAMMOX gas-lift reactor and a sequential batch reactor (SBR) were operated during around 200 days in this study, reaching nitrogen loading rates (NLRs) of 2.0 and 0.75 g l(-1) per day, respectively. The efficiency in the nitrite (limiting substrate) removal was 99%. The ammonium and nitrite influent concentrations were increased stepwise until biomass in the reactors started to float. These flotation events coincided with periods when the NLR exceeded the maximum specific ANAMMOX activity (MSAA) of the sludge. The MSAA, determined in batch experiments, was 0.9 and 0.44 g g(-1) per day for biomasses from the gas-lift reactor and the SBR, respectively. Flotation of the biomass occurred most likely due to a granule density decrease caused by dinitrogen gas accumulation inside the granules and an apparent breakage of the granules. Further research is needed to understand this phenomenon and to optimise the corresponding strategies to counteract the flotation.

Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria.

Recently, two fresh water species, " Candidatus Brocadia anammoxidans" and " Candidatus Kuenenia stuttgartiensis", and one marine species, " Candidatus Scalindua sorokinii", of planctomycete anammox bacteria have been identified. " Candidatus Scalindua sorokinii" was discovered in the Black Sea, and contributed substantially to the loss of fixed nitrogen. All three species contain a unique organelle--the anammoxosome--in their cytoplasm. The anammoxosome contains the hydrazine/hydroxylamine oxidoreductase enzyme, and is thus the site of anammox catabolism. The anammoxosome is surrounded by a very dense membrane composed almost exclusively of linearly concatenated cyclobutane-containing lipids. These so-called 'ladderanes' are connected to the glycerol moiety via both ester and ether bonds. In natural and man-made ecosystems, anammox bacteria can cooperate with aerobic ammonium-oxidising bacteria, which protect them from harmful oxygen, and provide the necessary nitrite. The cooperation of these two groups of ammonium-oxidising bacteria is the microbial basis for a sustainable one reactor system, CANON (completely autotrophic nitrogen-removal over nitrite) to remove ammonia from high strength wastewater.

CANON and Anammox in a gas-lift reactor.

Anoxic ammonium oxidation (Anammox) and Completely Autotrophic Nitrogen removal Over Nitrite (CANON) are new and promising microbial processes to remove ammonia from wastewaters characterized by a low content of organic materials. These two processes were investigated on their feasibility and performance in a gas-lift reactor. The Anammox as well as the CANON process could be maintained easily in a gas-lift reactor, and very high N-conversion rates were achieved. An N-removal rate of 8.9 kg N (m(3) reactor)(-1) day(-1) was achieved for the Anammox process in a gas-lift reactor. N-removal rates of up to 1.5 kg N (m(3) reactor)(-1) day(-1) were achieved when the CANON process was operated. This removal rate was 20 times higher compared to the removal rates achieved in the laboratory previously. Fluorescence in situ hybridization showed that the biomass consisted of bacteria reacting to NEU, a 16S rRNA targeted probe specific for halotolerant and halophilic Nitrosomonads, and of bacteria reacting to Amx820, specific for planctomycetes capable of Anammox.