Understanding atmospheric intercontinental dispersal of harmful microorganisms.

The atmosphere is a major route for microbial intercontinental dispersal, including harmful microorganisms, antibiotic resistance genes, and allergens, with strong implications in ecosystem functioning and global health. Long-distance dispersal is facilitated by air movement at higher altitudes in the free troposphere and is affected by anthropogenic forcing, climate change, and by the general atmospheric circulation, mainly in the intertropical convergence zone. The survival of microorganisms during atmospheric transport and their remote invasive potential are fundamental questions, but data are scarce. Extreme atmospheric conditions represent a challenge to survival that requires specific adaptive strategies, and recovery of air samples from the high altitudes relevant to study harmful microorganisms can be challenging. In this paper, we highlight the scope of the problem, identify challenges and knowledge gaps, and offer a roadmap for improved understanding of intercontinental microbial dispersal and their outcomes. Greater understanding of long-distance dispersal requires research focus on local factors that affect emissions, coupled with conditions influencing transport and survival at high altitudes, and eventual deposition at sink locations.

Understanding stochastic and deterministic assembly processes in microbial communities along temporal, spatial and environmental scales.

Identifying the main drivers of community assembly remains an open fundamental question in ecology. Dispersal processes introduce randomness in community composition while selection for particular environments creates predictable assemblages. However, the interaction between selection and dispersal processes is still poorly understood. Here, we address this question in bacterial and microeukaryotic communities inhabiting a highly dynamic system of ephemeral (hyper)saline lakes. We show that the combination of beta-diversity decomposition methods and a temporal approach based on colonization and extinction dynamics yields new insights into the relative effect of selection and dispersal along environmental gradients. Selective pressure and dispersal-related processes simultaneously shape each local community with variable strength and effect. The dominance of selection vs. dispersal shifted from stochastic to deterministic assembly as salinity increased along the gradient. This transition also had an impact on the temporal dynamics of the lakes as community turnover decreased at high salinities because both colonization and extinction rates slowed down. Only microeukaryotic richness decreased along the gradient due to lower effective colonization at higher salinities, suggesting that the net effect of selection and dispersal is determined by both environmental conditions and the idiosyncrasy of the different microbial ecologies. Our results emphasize the use of temporal approaches in combination with standard statistical methods for a better understanding of the dynamic processes underlying community assembly.

Global dispersal and potential sources of antibiotic resistance genes in atmospheric remote depositions.

Antibiotic resistance has become a major Global Health concern and a better understanding on the global spread mechanisms of antibiotic resistant bacteria (ARB) and intercontinental ARB exchange is needed. We measured atmospheric depositions of antibiotic resistance genes (ARGs) by quantitative (q)PCR in rain/snow collected fortnightly along 4 y. at a remote high mountain LTER (Long-Term Ecological Research) site located above the atmospheric boundary layer (free troposphere). Bacterial composition was characterized by 16S rRNA gene sequencing, and air mass provenances were determined by modelled back trajectories and rain/snow chemical composition. We hypothesize that the free troposphere may act as permanent reservoir and vector for ARB and ARGs global dispersal. We aimed to i) determine whether ARGs are long-range intercontinental and persistently dispersed through aerosols, ii) assess ARGs long-term atmospheric deposition dynamics in a remote high mountain area, and iii) unveil potential diffuse ARGs pollution sources. We showed that the ARGs sul1 (resistance to sulfonamides), tetO (resistance to tetracyclines), and intI1 (a proxy for horizontal gene transfer and anthropogenic pollution) were long-range and persistently dispersed in free troposphere aerosols. Major depositions of tetracyclines resistance matched with intensification of African dust outbreaks. Potential ARB mostly traced their origin back into agricultural soils. Our study unveils that air masses pathways are shaping ARGs intercontinental dispersal and global spread of antibiotic resistances, with potential predictability for interannual variability and remote deposition rates. Because climate regulates aerosolization and long-range air masses movement patterns, we call for a more careful evaluation of the connections between land use, climate change and ARB long-range intercontinental dispersal.

A long-term atmospheric baseline for intercontinental exchange of airborne pathogens.

The atmosphere is a potential pathway for global-scale and long-range dispersal of viable microorganisms, promoting biological interconnections among the total environment. We aimed to provide relevant baseline information for long-range long-term intercontinental exchange of potentially infectious airborne microorganisms of major interest in environmental and health-related disciplines. We used an interannual survey (7-y) with wet depositions fortnightly collected above the boundary layer (free troposphere) at a remote high-elevation LTER (Long-Term-Ecological-Research) site, analyzed by 16S and 18S rRNA genes, and compared to a database of 475 well-known pathogens. We applied a conservative approach on close relatives of pathogenic species (>98% identity) standing their theoretical upper limit for atmospheric baseline relative abundances. We identified c. 2-3% of the total airborne microbiota as potential pathogens. Their most frequent environmental origins were soil, aquatic, and anthropogenic sources. Phytopathogens (mostly fungi) were the potential infectious agents most widely present. We uncovered consistent interannual dynamics with taxa foreseeable over time (i.e., predictable seasonal behavior) and under recurrent environmental scenarios (e.g., Saharan dust intrusions), respectively, being highly valuable microbial forensic environmental indicators. Up to 8 bacterial and 21 fungal genera consistently showed temporal abundances and recurrences unevenly distributed. Incidence of allergenic fungi was lower in summer, and significantly higher in spring. Close relatives to Coccidioides posadasii consistently showed higher signals (i.e., high specificity and high fidelity) in winter, whereas Cryptococcus neoformans had a significant signal in spring. Along Saharan dust intrusions, the bacterial phytopathogens Acidovorax avenae and Agrobacterium tumefaciens and the fungal phytopathogens Pseudozyma hubeiensis and Peniophora sp. consistently showed higher signals. Potential human pathogens showed low proportion, being mostly fungal allergens. Microorganisms related to obligated human, amphibian and fish pathogens were commonly found in winter. More studies in remote field sites above the boundary layer will unveil whether or not a similar trend is found globally.

General decline in the diversity of the airborne microbiota under future climatic scenarios.

Microorganisms attached to aerosols can travel intercontinental distances, survive, and further colonize remote environments. Airborne microbes are influenced by environmental and climatic patterns that are predicted to change in the near future, with unknown consequences. We developed a new predictive method that dynamically addressed the temporal evolution of biodiversity in response to environmental covariates, linked to future climatic scenarios of the IPCC (AR5). We fitted these models against a 7-year monitoring of airborne microbes, collected in wet depositions. We found that Bacteria were more influenced by climatic variables than by aerosols sources, while the opposite was detected for Eukarya. Also, model simulations showed a general decline in bacterial richness, idiosyncratic responses of Eukarya, and changes in seasonality, with higher intensity within the worst-case climatic scenario (RCP 8.5). Additionally, the model predicted lower richness for airborne potential eukaryotic (fungi) pathogens of plants and humans. Our work pioneers on the potential effects of environmental variability on the airborne microbiome under the uncertain context of climate change.

Regional community assembly drivers and microbial environmental sources shaping bacterioplankton in an alpine lacustrine district (Pyrenees, Spain).

Microbial communities in natural ecosystems are subject to strong ecological rules. The study of local communities along a regional metacommunity can reveal patterns of community assembly, and disentangle the underlying ecological processes. In particular, we seek drivers of community assembly at the regional scale using a large lacustrine dataset (>300 lakes) along the geographical, limnological and physico-chemical gradients in the Pyrenees. By using high throughput amplicon sequencing of the 16S rRNA gene, and inferring environmental sources of bacterial immigrants, we showed that surface aquatic bacterial assemblages were strongly influenced by terrestrial populations from soil, biofilms or sediments, and primarily selected by a pH-alkalinity gradient. Indeed, source proportions explained 27% of the community variation, and chemistry 15% of the total variation, half of it shared with the sources. Major taxonomic groups such as Verrucomicrobia, Actinobacteria and Bacteroidetes showed higher aquatic affinities than Parcubacteria, Gammaproteobacteria, Alphaproteobacteria or Betaproteobacteria, which may be recruited and selected through different hydrographic habitats. A regional fingerprint was observed with lower alpha diversity and higher beta diversity in the central Pyrenees than in both ends. We suggest an ecological succession process, likely influenced by complex interactions of environmental source dispersal and environmental filtering along the mountain range geography.

Sponges and Their Microbiomes Show Similar Community Metrics Across Impacted and Well-Preserved Reefs.

Sponge diversity has been reported to decrease from well-preserved to polluted environments, but whether diversity and intra-species variation of their associated microbiomes also change as function of environmental quality remains unknown. Our study aimed to assess whether microbiome composition and structure are related to the proliferation of some sponges and not others under degraded conditions. We characterized the most frequent sponges and their associated bacteria in two close areas (impacted and well-preserved) of Nha Trang Bay (Indo-Pacific). Sponge assemblages were richer and more diverse in the well-preserved reefs, but more abundant (individuals/m. transect) in the impacted environments, where two species (Clathria reinwardti and Amphimedon paraviridis) dominated. Sponge microbiomes from the polluted zones had, in general, lower bacterial diversity and core size and consequently, higher intra-species dispersion than microbiomes of sponges from the well-preserved environments. Microbial communities reflect the reduction of diversity and richness shown by their host sponges. In this sense, sponges with less complex and more variable microbiomes proliferate under degraded environmental conditions, following the ecological paradigm that negatively correlates community diversity and environmental degradation. Thereby, the diversity and structure of sponge microbiomes might indirectly determine the presence and proliferation of sponge species in certain habitats.

The DNRA-Denitrification Dichotomy Differentiates Nitrogen Transformation Pathways in Mountain Lake Benthic Habitats.

Effects of nitrogen (N) deposition on microbially-driven processes in oligotrophic freshwater ecosystems are poorly understood. We quantified guilds in the main N-transformation pathways in benthic habitats of 11 mountain lakes along a dissolved inorganic nitrogen gradient. The genes involved in denitrification (nirS, nirK, nosZ), nitrification (archaeal and bacterial amoA), dissimilatory nitrate reduction to ammonium (DNRA, nrfA) and anaerobic ammonium oxidation (anammox, hdh) were quantified, and the bacterial 16S rRNA gene was sequenced. The dominant pathways and associated bacterial communities defined four main N-transforming clusters that differed across habitat types. DNRA dominated in the sediments, except in the upper layers of more productive lakes where nirS denitrifiers prevailed with potential N2O release. Loss as N2 was more likely in lithic biofilms, as indicated by the higher hdh and nosZ abundances. Archaeal ammonia oxidisers predominated in the isoetid rhizosphere and rocky littoral sediments, suggesting nitrifying hotspots. Overall, we observed a change in potential for reactive N recycling via DNRA to N losses via denitrification as lake productivity increases in oligotrophic mountain lakes. Thus, N deposition results in a shift in genetic potential from an internal N accumulation to an atmospheric release in the respective lake systems, with increased risk for N2O emissions from productive lakes.

A long-term survey unveils strong seasonal patterns in the airborne microbiome coupled to general and regional atmospheric circulations.

Airborne microbes (bacteria, archaea, protists, and fungi) were surveyed over a 7-y period via high-throughput massive sequencing of 16S and 18S rRNA genes in rain and snow samples collected fortnightly at a high-elevation mountain Long-Term Ecological Research (LTER) Network site (LTER-Aigüestortes, Central Pyrenees, Spain). This survey constitutes the most comprehensive mountain-top aerobiology study reported to date. The air mass origins were tracked through modeled back-trajectories and analysis of rain water chemical composition. Consistent microbial seasonal patterns were observed with highly divergent summer and winter communities recurrent in time. Indicative microbial taxa were unveiled as a forensic signature, and ubiquitous taxa were observed as common atmosphere inhabitants, highlighting aerosols as a potentially successful mechanism for global microbial dispersal. Source-tracking analyses identified freshwater, cropland, and urban biomes as the most important sources for airborne bacteria in summer, while marine and forest biomes prevailed in winter, in agreement with air mass retrotrajectories and the prevailing general and regional atmospheric circulation.

Showcasing the role of seawater in bacteria recruitment and microbiome stability in sponges.

We studied the core bacterial communities of 19 sponge species from Nha Trang Bay (Central Vietnam), with particular emphasis on the contribution of planktonic seawater bacteria to the sponge core microbiomes. To ensure consistent sponge-microbe associations and accurate identification of planktonic bacteria transmitted from seawater, we were very restrictive with the definition of the sponge core microbiomes (present in all the replicates), and with the identification of valid biological 16S rRNA gene sequences (100% sequence identity) that belonged to potentially different bacterial taxa. We found a high overlap (>50% relative abundance) between the sponge species core microbiome and the seawater bacterial core in ca. a half of the studied species, including representatives of both, HMA and LMA sponges. From our restrictive analysis, we point to horizontal transmission as a relevant way of symbiont acquisition in sponges. Some species-specific recognition mechanisms may act in sponges to enrich specific seawater bacteria in their tissues. These mechanisms would allow the maintenance of bacterial communities in a species across geographical ranges. Moreover, besides contrasting preferences in bacteria selection from seawater, divergent physiological traits may also account for the different microbiomes in species of HMA and LMA sponges.

Influence of edaphic, climatic, and agronomic factors on the composition and abundance of nitrifying microorganisms in the rhizosphere of commercial olive crops.

The microbial ecology of the nitrogen cycle in agricultural soils is an issue of major interest. We hypothesized a major effect by farm management systems (mineral versus organic fertilizers) and a minor influence of soil texture and plant variety on the composition and abundance of microbial nitrifiers. We explored changes in composition (16S rRNA gene) of ammonia-oxidizing archaea (AOA), bacteria (AOB), and nitrite-oxidizing bacteria (NOB), and in abundance of AOA and AOB (qPCR of amoA genes) in the rhizosphere of 96 olive orchards differing in climatic conditions, agricultural practices, soil properties, and olive variety. Majority of archaea were 1.1b thaumarchaeota (soil crenarchaeotic group, SCG) closely related to the AOA genus Nitrososphaera. Most AOB (97%) were identical to Nitrosospira tenuis and most NOB (76%) were closely related to Nitrospira sp. Common factors shaping nitrifiers assemblage composition were pH, soil texture, and olive variety. AOB abundance was positively correlated with altitude, pH, and clay content, whereas AOA abundances showed significant relationships with organic nitrogen content and exchangeable K. The abundances of AOA differed significantly among soil textures and olive varieties, and those of AOB among soil management systems and olive varieties. Overall, we observed minor effects by orchard management system, soil cover crop practices, plantation age, or soil organic matter content, and major influence of soil texture, pH, and olive tree variety.

Environmental controls and composition of anoxygenic photoheterotrophs in ultraoligotrophic high-altitude lakes (Central Pyrenees).

The phylogenetic composition of freshwater anoxygenic photoheterotrophs (APs) has been poorly investigated as compared with their marine counterparts. In this study, we explored a set of ultraoligotrophic cold high mountain lakes (Central Pyrenees, Spain) by both pufM gene denaturing gradient gel electrophoresis fingerprinting, and cloning and sequencing of selected lakes samples. Different ranges of limnological and physico-chemical values were explored as environmental drivers of APs richness and composition. We did not observe significant relationships between richness/diversity of pufM and any of the limnological characteristics measured or trophic status, but a negative correlation with ammonia concentration. Conductivity, pH and nitrate concentration were significantly related to changes in APs community composition, whereas lake area, altitude, temperature and trophic status did not. Most of the sequences (> 85%) had the pufM sequences of Limnohabitans (Betaproteobacteria) as the closest relative in databases, whereas less abundant clones were more closely related to Rhodobacter, Sulfitobacter and Brevundimonas (Alphaproteobacteria), in agreement with 16S rRNA gene sequences previously found in the area. Congregibacter-like Gammaproteobacteria were not detected. Comparison with available studies in inland waters showed taxonomic partitioning along salinity gradients, and Congregibacter-like sequences restricted to high saline conditions in continental water bodies.

Ecotoxicity of Cr, Cd, and Pb on two Mediterranean soils.

Three potentially toxic elements [chromium (Cr), lead (Pb), and cadmium (Cd)] were tested to assess their effects on two soils of different properties and origin. The soils were a granitic soil (Haplic Arenosol), which meets the requirements of OECD ecotoxicity testing, and a calcareous soil (Calcaric Regosol) with properties often found in the Mediterranean areas. The metal concentrations used ranged from 0.001 to 5,000 mg kg(-1) soil. The effects on soil microbial activity and community composition (respirometry and polymerase chain reaction-denaturing gradient gel electrophoresis analysis), as well as the effects on plant germination and elongation (Lactuca sativa), were assessed. The toxicity of the soil water extracts was also evaluated by the growth inhibition of algal populations (Pseudokirschneriella subcapitata). Cr showed the highest level of toxicity to soil organisms in the assays performed because this element remains in soil as anionic form and is less retained by the soil solid matrix than Cd and Pb. The lowest observed-adverse effect level for Cr ranged from approximately 0.1 mg kg(-1) [substrate induced respiration (SIR) test for the granitic soil] to 10 mg kg(-1) (basal respiration and SIR tests for the calcareous soil). For Pb (SIR) and Cd (SIR and alga tests), these levels were approximately 100 mg kg(-1). Germination and algal tests showed higher sensitivity in Regosol soil than in Arenosol soil for Cr due to differences in the bioavailability between the soils. In the cases or areas where alkaline soils are abundant, these should also be considered in laboratory ecotoxicity testing to avoid underestimation of ecotoxicological risks.

The exposition of a calcareous Mediterranean soil to toxic concentrations of Cr, Cd and Pb produces changes in the microbiota mainly related to differential metal bioavailability.

The involvement of the bacterial community of an agricultural Mediterranean calcareous soil in relation to several heavy metals has been studied in microcosms under controlled laboratory conditions. Soil samples were artificially polluted with Cr(VI), Cd(II) and Pb(II) at concentrations ranging from 0.1 to 5000 mg kg(-1) and incubated along 28 d. The lowest concentrations with significant effects in soil respirometry were 10 mg kg(-1) Cr and 1000 mg kg(-1) Cd and Pb. However, only treatments showing more than 40% inhibition of respirometric activity led to significant changes in bacterial composition, as indicated by PCR-DGGE analyses. Presumable Cr- and Cd-resistant bacteria were detected in polluted microcosms, but development of the microbiota was severely impaired at the highest amendments of both metals. Results also showed that bioavailability is an important factor determining the impact of the heavy metals assayed, and even an inverted potential toxicity ranking could be achieved if their soluble fraction is considered instead of the total concentration. Moreover, multiresistant bacteria were isolated from Cr-polluted soil microcosms, some of them showing the capacity to reduce Cr(VI) concentrations between 26% and 84% of the initial value. Potentially useful strains for bioremediation were related to Arthrobacter crystallopoietes, Stenotrophomonas maltophilia and several species of Bacillus.

Ecotoxicity of chlorophenolic compounds depending on soil characteristics.

Three chlorophenolic compounds (2-chlorophenol, 2,4,6-trichlorophenol, and pentachlorophenol) were tested to assess their effects on two soils with different properties: a granitic soil (Haplic Arenosol) and a calcareous one (Calcaric Regosol). Different concentrations of the pollutants (ranging from 0.001 to 10,000 mg kg(-1) soil, d.w.) were assayed for their effects on soil microbial activity and composition, using manometric respirometry and PCR-DGGE analysis, respectively. Other ecotoxicity tests such as Lactuca sativa seedling growth in the contaminated soils and algal growth inhibition (Pseudokirschneriella subcapitata) in their water extracts were done. The behaviour of the pollutants in the soils with respect to biodegradability and volatilization was also investigated. In the Haplic Arenosol, volatilization is the main process affecting 2-chlorophenol. Degradation and fixation of this compound in the soil matrix are favored in the Calcaric Regosol. This is the least toxic pollutant assayed. For 2,4,6-trichlorophenol, the soil pH is a critical parameter in the toxicity assays due to the neutral pKa of the compound. It is toxic in the soil microbial activity assay, but some recovery of the biotic processes can be observed, particularly in the Calcaric Regosol. This compound is more toxic in the Haplic Arenosol than in the Calcaric Regosol. Pentachlorophenol is ionized in both soils due to its low pKa, increasing its water solubility. It is highly toxic to the soil microbiota, thus inhibiting respiration, biodegradation and other biotic dissipation processes. Plant and alga tests, were more sensitive than soil microbial tests, except for PCP. The microbial populations tend to show changes at lower concentrations than the microbial activity. Some soil types (abundant in the Mediterranean area), with alkaline pH and fine textures could show higher level of ecotoxicity for ionizable organic pollutants than the soil type recommended by the OECD in ecotoxicity testing.

Impact of chlorophenols on microbiota of an unpolluted acidic soil: microbial resistance and biodegradation.

The impact of 2-monochlorophenol (MCP), 2,4,6-trichlorophenol (TCP) and pentachlorophenol (PCP) on the microbial community of an acidic forest soil was studied under controlled laboratory conditions by spiking microcosms with the pollutants at concentrations ranging from 0.1 to 5000 mg kg(-1). A decrease in the cumulative respirometric values and changes in the bacterial and fungal community composition were detected at 1000 mg MCP kg(-1), 100 mg TCP kg(-1) and 100 and 1000 mg PCP kg(-1). However, drastic effects on the microbial community were revealed only at higher concentrations of MCP and TCP, although the toxicity of PCP was expected to be stronger. The acidic condition of the soil presumably reduces bioavailability of PCP, leading to less pronounced effects than the other pollutants. This finding highlights the consideration of pollutant bioavailability in each environment to adequately assess contamination effects. Twenty-two different chlorophenol-resistant and potentially degrading microorganisms were isolated from highly polluted microcosms. The most resistant isolates were related to Burkholderia arboris, Bacillus circulans, Paenibacillus taichungensis, Luteibacter rhizovicina and Janibacter melonis. These isolates also showed the capacity to reduce the concentration of TCP or PCP between 15% and 35% after 5 days of incubation (initial concentration of 50 mg L(-1)). The isolate related to B. circulans is an atypical case of a member of the Firmicutes group for which chlorophenol-degrading capacities have been described.

The microbiota of an unpolluted calcareous soil faces up chlorophenols: Evidences of resistant strains with potential for bioremediation.

To highlight the effects of a variety of chlorophenols (CP) in relation to the response of an indigenous bacterial community, an agricultural Mediterranean calcareous soil has been studied in microcosms incubated under controlled laboratory conditions. Soil samples were artificially polluted with 2-monochlorophenol (MCP), 2,4,6-trichlorophenol (TCP) and pentachlorophenol (PCP), at concentrations ranging from 0.1 up to 5000 mg kg(-1). Both activity and composition of the microbial community were assessed during several weeks, respectively, by respirometric methods and PCR-DGGE analysis of extracted DNA and RNA. Significant decreases in soil respirometric values and changes in the bacterial community composition were observed at concentrations above 1000 mg kg(-1) MCP and TCP, and above 100 mg kg(-1) PCP. However, the persistence of several active bacterial populations in soil microcosms contaminated with high concentration of CP, as indicated by DGGE fingerprints, suggested the capacity of these native bacteria to survive in the presence of the pollutants, even without a previous adaptation or contact with them. The isolation of potential CP degraders was attempted by culture plating from microcosms incubated with high CP concentrations. Twenty-three different isolates were screened for their resistance to TCP and PCP. The most resistant isolates were identified as Kocuria palustris, Lysobacter gummosus, Bacillus sp. and Pseudomonas putida, according to 16S rRNA gene homology. In addition, these four isolates also showed the capacity to reduce the concentration of TCP and PCP from 15% to 30% after 5d of incubation in laboratory assays (initial pollutant concentration of 50 mg L(-1)). Isolate ITP29, which could be a novel species of Bacillus, has been revealed as the first known member in this bacterial group with potential for CP bioremediation applications, usually wide-spread in the soil natural communities, which has not been reported to date as a CP degrader.

Development of batch-culture enrichment coupled to molecular detection for screening of natural and man-made environments in search of anammox bacteria for N-removal bioreactors systems.

This investigation probed for anammox bacterial populations that would be suitable to start-up a biological reactor for N-removal. Samples of sludge and sediments from different environments were screened and used as inoculum for enrichment of anammox bacteria in batch cultures. Enrichments were monitored in order to detect anammox bacteria or their potential activity. Candidatus "Brocadia anammoxidans" was successfully enriched, detected, and identified in five of the twelve batch cultures. Furthermore, this organism was retrieved for the first time from a brackish environment. Wide-range primers used in several Polymerase Chain Reaction (PCR) attempts were unable to successfully amplify the 16S rRNA sequence from anammox populations, but were used to search for hypothetical ecological partners of anammox. A nested PCR approach with specific primers was also employed since conventional PCR was unable to amplify anammox DNA from the inocula of the successful enrichments. However, it was impossible to obtain optimal results with the different strategies utilized to improve PCR performance (higher annealing temperature or more specific primers), and only the primer set Amx368F-Amx820R resulted in an acceptable balance between both specificity and sensitivity. Although the enrichment process is relatively slow and requires lengthy incubation periods, it proved to be useful as the molecular analyses were not sensitive enough to detect anammox in the original samples or even after short enrichment periods. Therefore, a batch enrichment procedure coupled with molecular techniques was an appropriate approach to achieve successful inocula for starting-up anammox biological reactor.