Biofilm colonization and succession in a full-scale partial nitritation-anammox moving bed biofilm reactor.

BACKGROUND: Partial nitritation-anammox (PNA) is a biological nitrogen removal process commonly used in wastewater treatment plants for the treatment of warm and nitrogen-rich sludge liquor from anaerobic digestion, often referred to as sidestream wastewater. In these systems, biofilms are frequently used to retain biomass with aerobic ammonia-oxidizing bacteria (AOB) and anammox bacteria, which together convert ammonium to nitrogen gas. Little is known about how these biofilm communities develop, and whether knowledge about the assembly of biofilms in natural communities can be applied to PNA biofilms. RESULTS: We followed the start-up of a full-scale PNA moving bed biofilm reactor for 175 days using shotgun metagenomics. Environmental filtering likely restricted initial biofilm colonization, resulting in low phylogenetic diversity, with the initial microbial community comprised mainly of Proteobacteria. Facilitative priority effects allowed further biofilm colonization, with the growth of initial aerobic colonizers promoting the arrival and growth of anaerobic taxa like methanogens and anammox bacteria. Among the early colonizers were known 'oligotrophic' ammonia oxidizers including comammox Nitrospira and Nitrosomonas cluster 6a AOB. Increasing the nitrogen load in the bioreactor allowed colonization by 'copiotrophic' Nitrosomonas cluster 7 AOB and resulted in the exclusion of the initial ammonia- and nitrite oxidizers. CONCLUSIONS: We show that complex dynamic processes occur in PNA microbial communities before a stable bioreactor process is achieved. The results of this study not only contribute to our knowledge about biofilm assembly and PNA bioreactor start-up but could also help guide strategies for the successful implementation of PNA bioreactors. Video Abstract.

Inoculation with adapted bacterial communities promotes development of full scale slow sand filters for drinking water production.

Gravity-driven filtration through slow sand filters (SSFs) is one of the oldest methods for producing drinking water. As water passes through a sand bed, undesired microorganisms and chemicals are removed by interactions with SSF biofilm and its resident microbes. Despite their importance, the processes through which these microbial communities form are largely unknown, as are the factors affecting these processes. In this study, two SSFs constructed using different sand sources were compared to an established filter and observed throughout their maturation process. One SSF was inoculated through addition of sand scraped from established filters, while the other was not inoculated. The operational and developing microbial communities of SSFs, as well as their influents and effluents, were studied by sequencing of 16S ribosomal rRNA genes. A functional microbial community resembling that of the established SSF was achieved in the inoculated SSF, but not in the non-inoculated SSF. Notably, the non-inoculated SSF had significantly (p < 0.01) higher abundances of classes Armatimonadia, Elusimicrobia, Fimbriimonadia, OM190 (phylum Planctomycetota), Parcubacteria, Vampirivibrionia and Verrucomicrobiae. Conversely, it had lower abundances of classes Anaerolineae, Bacilli, bacteriap25 (phylum Myxococcota), Blastocatellia, Entotheonellia, Gemmatimonadetes, lineage 11b (phylum Elusimicrobiota), Nitrospiria, Phycisphaerae, subgroup 22 (phylum Acidobacteriota) and subgroup 11 (phylum Acidobacteriota). Poor performance of neutral models showed that the assembly and dispersal of SSF microbial communities was mainly driven by selection. The temporal turnover of microbial species, as estimated through the scaling exponent of the species-time relationship, was twice as high in the non-inoculated filter (0.946 ± 0.164) compared to the inoculated filter (0.422 ± 0.0431). This study shows that the addition of an inoculum changed the assembly processes within SSFs. Specifically, the rate at which new microorganisms were observed in the biofilm was reduced. The reduced temporal turnover may be driven by inoculating taxa inhibiting growth, potentially via secondary metabolite production. This in turn would allow the inoculation community to persist and contribute to SSF function.

Novel and unusual genes for nitrogen and metal cycling in Planctomycetota- and KSB1-affiliated metagenome-assembled genomes reconstructed from a marine subsea tunnel.

The Oslofjord subsea road tunnel is a unique environment in which the typically anoxic marine deep subsurface is exposed to oxygen. Concrete biodeterioration and steel corrosion in the tunnel have been linked to the growth of iron- and manganese-oxidizing biofilms in areas of saline water seepage. Surprisingly, previous 16S rRNA gene surveys of biofilm samples revealed microbial communities dominated by sequences affiliated with nitrogen-cycling microorganisms. This study aimed to identify microbial genomes with metabolic potential for novel nitrogen- and metal-cycling reactions, representing biofilm microorganisms that could link these cycles and play a role in concrete biodeterioration. We reconstructed 33 abundant, novel metagenome-assembled genomes (MAGs) affiliated with the phylum Planctomycetota and the candidate phylum KSB1. We identified novel and unusual genes and gene clusters in these MAGs related to anaerobic ammonium oxidation, nitrite oxidation, and other nitrogen-cycling reactions. Additionally, 26 of 33 MAGs also had the potential for iron, manganese, and arsenite cycling, suggesting that bacteria represented by these genomes might couple these reactions. Our results expand the diversity of microorganisms putatively involved in nitrogen and metal cycling, and contribute to our understanding of potential biofilm impacts on built infrastructure.

Disturbance-based management of ecosystem services and disservices in partial nitritation-anammox biofilms.

The resistance and resilience provided by functional redundancy, a common feature of microbial communities, is not always advantageous. An example is nitrite oxidation in partial nitritation-anammox (PNA) reactors designed for nitrogen removal in wastewater treatment, where suppression of nitrite oxidizers like Nitrospira is sought. In these ecosystems, biofilms provide microhabitats with oxygen gradients, allowing the coexistence of aerobic and anaerobic bacteria. We designed a disturbance experiment where PNA biofilms, treating water from a high-rate activated sludge process, were constantly or intermittently exposed to anaerobic sidestream wastewater, which has been proposed to inhibit nitrite oxidizers. With increasing sidestream exposure we observed decreased abundance, alpha-diversity, functional versatility, and hence functional redundancy, among Nitrospira in the PNA biofilms, while the opposite patterns were observed for anammox bacteria within Brocadia. At the same time, species turnover was observed for aerobic ammonia-oxidizing Nitrosomonas populations. The different exposure regimens were associated with metagenomic assembled genomes of Nitrosomonas, Nitrospira, and Brocadia, encoding genes related to N-cycling, substrate usage, and osmotic stress response, possibly explaining the three different patterns by niche differentiation. These findings imply that disturbances can be used to manage the functional redundancy of biofilm microbiomes in a desirable direction, which should be considered when designing operational strategies for wastewater treatment.

A relationship between phages and organic carbon in wastewater treatment plant effluents.

With stringent effluent requirements and the implementation of new processes for micropollutant removal, it is increasingly important for wastewater treatment plants (WWTPs) to understand the factors affecting effluent quality. Phages (viruses infecting prokaryotes) are abundant in the biological treatment processes. They can contribute to organic carbon in the treated effluent both because they are organic in nature and occur in the effluent and because they cause lysis of microorganisms. Today very little is known about the effects of phages on effluent quality. The goal of this study was, therefore, to determine the relationship between phages and organic carbon in WWTP effluents. We also examined the diversity, taxonomy, and host-association of DNA phages using metagenomics. Effluent samples were collected from four WWTPs treating municipal wastewater. Significant differences in both organic carbon and virus-like particle concentrations were observed between the plants and there was a linear relationship between the two parameters. The phage communities were diverse with many members being taxonomically unclassified. Putative hosts were dominated by bacteria known to be abundant in activated sludge systems such as Comamonadaceae. The composition of phages differed between the WWTPs, suggesting that local conditions shape the communities. Overall, our findings suggest that the abundance and composition of phages are related to effluent quality. Thus, there is a need for further research clarifying the association between phage dynamics and WWTP function.

Metagenomic evidence of a novel family of anammox bacteria in a subsea environment.

Bacteria in the order 'Candidatus Brocadiales' within the phylum Planctomycetes (Planctomycetota) have the remarkable ability to perform anaerobic ammonium oxidation (anammox). Two families of anammox bacteria with different biogeographical distributions have been reported, marine Ca. Scalinduaceae and freshwater Ca. Brocadiaceae. Here we report evidence of three new species within a novel genus and family of anammox bacteria, which were discovered in biofilms of a subsea road tunnel under a fjord in Norway. In this particular ecosystem, the nitrogen cycle is likely fuelled by ammonia from organic matter degradation in the fjord sediments and the rock mass above the tunnel, resulting in the growth of biofilms where anammox bacteria can thrive under oxygen limitation. We resolved several metagenome-assembled genomes (MAGs) of anammox bacteria, including three Ca. Brocadiales MAGs that could not be classified at the family level. MAGs of this novel family had all the diagnostic genes for a full anaerobic ammonium oxidation pathway in which nitrite was probably reduced by a NirK-like reductase. A survey of published molecular data indicated that this new family of anammox bacteria occurs in many marine sediments, where its members presumably would contribute to nitrogen loss.

Waste surgical masks to fuels via thermochemical co-processing with waste motor oil and biomass.

In this work, the co-processing of waste surgical masks, waste motor oil, and biomass was investigated to reduce the environmental impacts of the increasing medical-derived plastic pollution as well as to elucidate its effect on the production of chemicals . The results showed high yields towards an oily product with an interesting hydrocarbon content in the diesel range. Furthermore, although the initial waste motor oil had a high sulfur content, the oily products showed a low sulfur content, that was logically distributed in the solid and gas phases. In addition, all oily products presented HHVs ​​higher than 44 MJ/Kg, with cetane indices, densities, and viscosities lower than those of petroleum-derived diesel. This work could impact on the management of waste surgical masks and the joint recovery of everyday waste towards high value-added products.

Prediction of potential cysteine synthase inhibitors of Leishmania braziliensis and Leishmania major parasites by computational screening.

Leishmaniasis is a neglected tropical disease considered a public health problem that requires innovative strategies for its chemotherapeutic control. In the present investigation, a molecular docking approach was carried out using the protein cysteine synthase (CS) of Leishmania braziliensis (CSLb) and Leishmania major (CSLm) parasites to identify new compounds as potential candidates for the development of selective leishmaniasis therapy. CS protein sequence similarity, active site, structural modeling, molecular docking, and ADMET properties of compounds were analyzed using bioinformatics tools. Molecular docking analyses identified 1000 ligands with highly promising binding affinity scores for both CS proteins. A total of 182 compounds for CSLb and 173 for CSLm were selected for more detailed characterization based on the binding energy and frequency values and ADMET properties. Based on Principal Component Analysis (PCA) and K-means clusterization for both CS proteins, we classified compounds into 5 clusters for CSLb and 7 for CSLm, thus providing an excellent starting point for verification of enzyme inhibition in in vitro studies. We found the ZINC16524774 compound predicted to have a high affinity and stability for both CSLb and CSLm proteins, which was also evaluated through molecular dynamics simulations. Compounds within each of the five clusters also displayed pharmacological and structural properties that make them attractive drug candidates for the development of selective cutaneous leishmaniasis chemotherapy.

Correction to: Hill-based dissimilarity indices and null models for analysis of microbial community assembly.

An amendment to this paper has been published and can be accessed via the original article.

Hill-based dissimilarity indices and null models for analysis of microbial community assembly.

BACKGROUND: High-throughput amplicon sequencing of marker genes, such as the 16S rRNA gene in Bacteria and Archaea, provides a wealth of information about the composition of microbial communities. To quantify differences between samples and draw conclusions about factors affecting community assembly, dissimilarity indices are typically used. However, results are subject to several biases, and data interpretation can be challenging. The Jaccard and Bray-Curtis indices, which are often used to quantify taxonomic dissimilarity, are not necessarily the most logical choices. Instead, we argue that Hill-based indices, which make it possible to systematically investigate the impact of relative abundance on dissimilarity, should be used for robust analysis of data. In combination with a null model, mechanisms of microbial community assembly can be analyzed. Here, we also introduce a new software, qdiv, which enables rapid calculations of Hill-based dissimilarity indices in combination with null models. RESULTS: Using amplicon sequencing data from two experimental systems, aerobic granular sludge (AGS) reactors and microbial fuel cells (MFC), we show that the choice of dissimilarity index can have considerable impact on results and conclusions. High dissimilarity between replicates because of random sampling effects make incidence-based indices less suited for identifying differences between groups of samples. Determining a consensus table based on count tables generated with different bioinformatic pipelines reduced the number of low-abundant, potentially spurious amplicon sequence variants (ASVs) in the data sets, which led to lower dissimilarity between replicates. Analysis with a combination of Hill-based indices and a null model allowed us to show that different ecological mechanisms acted on different fractions of the microbial communities in the experimental systems. CONCLUSIONS: Hill-based indices provide a rational framework for analysis of dissimilarity between microbial community samples. In combination with a null model, the effects of deterministic and stochastic community assembly factors on taxa of different relative abundances can be systematically investigated. Calculations of Hill-based dissimilarity indices in combination with a null model can be done in qdiv, which is freely available as a Python package ( https://github.com/omvatten/qdiv ). In qdiv, a consensus table can also be determined from several count tables generated with different bioinformatic pipelines. Video Abstract.

Synthesis of ordered microporous/macroporous MOF-808 through modulator-induced defect-formation, and surfactant self-assembly strategies.

Ordered materials with interconnected porosity allow the diffusion of molecules within their inner porous structure to access the active sites located in the microporous core. As a follow-up of our work on engineering of MOF-808, in this contribution, we study the synthesis of defective MOF-808 using two different strategies: the use of modulators and the surfactant-assisted synthesis to obtain materials with ordered and interconnected pores. The results of the study indicated that (i) the use of modulators of different chain length led to the formation of microporous/mesoporous MOFs through the formation of missing linker defects. However, the use of the acetic acid contributes to the formation of MOFs with larger mesoporous size distributions compared to materials synthesized with formic and propionic acids as modulators, and (ii) the self-assembly of CTAB surfactant produced an ordered microporous/macroporous network which enhanced crystallinity. However, the surface properties of the materials seem to be unaffected by the use of surfactants during synthesis. These results contribute to the development of ordered materials with a broad range of pore size distributions and give rise to new opportunities to extend the applications of MOF-808.

Long-term stability of partial nitritation-anammox for treatment of municipal wastewater in a moving bed biofilm reactor pilot system.

Nitrogen removal from the mainstream of municipal wastewater with partial nitritation-anammox (PNA) would be highly beneficial with regard to the uses of energy and organic carbon. However, the challenges of process instability, low nitrogen removal rates (NRR) and unwanted aerobic nitrite oxidation need to be solved to reach large-scale implementation. Here, we have operated pilot-scale moving bed biofilm reactors (MBBRs) for mainstream treatment, together with sidestream treatment of sludge liquor from anaerobic digestors, for over 900 days to investigate process stability, reactor performance and microbial community structure at realistic conditions. The MBBR biofilm contained stable and high relative abundances of anammox bacteria (10-32%) consisting of two major Brocadia sp. populations, and several populations of aerobic ammonia-oxidising bacteria (AOB) within Nitrosomonas sp. (0.2-3.1%), as assessed by 16S rDNA amplicon sequencing. In addition, nitrite-oxidising bacteria (NOB) consisting of Nitrospira sp. (0.4-0.8%) and Nitrotoga sp. (up to 0.4%) were present. Nitrogen was removed at a peak rate of 0.66 g N m-2 d-1 (0.13 kg N m-3 d-1) with a nitrate production over ammonium consumption of 15% by the NOB, at operation with continuous aeration at 15 °C. However, during most periods with continuous aeration, the NRR was lower (≈ 0.45 g N m-2 d-1), with larger relative nitrate production (≈40%), presumably due to problems to maintain stable residual ammonium concentrations during wet-weather mainstream flows. Changing reactor operation to intermittent aeration decreased the NRR but did not help in suppressing the NOB. The study shows that with MBBRs, stable mainstream PNA can be attained at realistic NRR, but with need for post-treatment of nitrate, since effective NOB suppression was hard to achieve.

Thickness determines microbial community structure and function in nitrifying biofilms via deterministic assembly.

Microbial biofilms are ubiquitous in aquatic environments where they provide important ecosystem functions. A key property believed to influence the community structure and function of biofilms is thickness. However, since biofilm thickness is inextricably linked to external factors such as water flow, temperature, development age and nutrient conditions, its importance is difficult to quantify. Here, we designed an experimental system in a wastewater treatment plant whereby nitrifying biofilms with different thicknesses (50 or 400 µm) were grown in a single reactor, and thus subjected to identical external conditions. The 50 and 400 µm biofilm communities were significantly different. This beta-diversity between biofilms of different thickness was primarily caused by deterministic factors. Turnover (species replacement) contributed more than nestedness (species loss) to the beta-diversity, i.e. the 50 µm communities were not simply a subset of the 400 µm communities. Moreover, the two communities differed in the composition of nitrogen-transforming bacteria and in nitrogen transformation rates. The study illustrates that biofilm thickness alone is a key driver for community composition and ecosystem function, which has implications for biotechnological applications and for our general understanding of biofilm ecology.

Subsea tunnel reinforced sprayed concrete subjected to deterioration harbours distinct microbial communities.

Deterioration of concrete is a large societal cost. In the Oslofjord subsea tunnel (Norway), deterioration of sprayed concrete and corrosion of reinforcing steel fibres occur under biofilm formed at sites with intrusion of saline groundwater. In this study, the microbial community structure, in situ environmental gradients and chemical composition of the biofilms were examined at three tunnel sites. Ammonia- and nitrite-oxidising microorganisms, in particular Nitrosopumilus sp., and iron-oxidising bacteria within Mariprofundus sp., were omnipresent, together with a diversity of presumably heterotrophic bacteria. Alpha- and beta diversity measures showed significant differences in richness and community structure between the sites as well as over time and null-models suggested that deterministic factors were important for the community assembly. The superficial flow of water over the biofilm had a strong effect on oxygen penetration in the biofilm and was identified as one major environmental gradient that varied between the sites, likely being important for shaping the microbial communities.

Enfermedad de Kawasaki / Kawasaki disease

Resumen La enfermedad de Kawasaki es un estado febril agudo, cuya incidencia es de 175 por cada 100.000 niños menores de 5 años. Es una vasculitis con predilección por los vasos de pequeño y mediano calibre, especialmente por las arterias coronarias; sin tratamiento pueden desarrollarse aneurismas coronarios que conllevan riesgo de infarto agudo de miocardio y muerte súbita. Se describe el caso de un niño de 4 años a quien se diagnosticó enfermedad de Kawasaki y aneurismas fusiformes moderados en las arterias coronarias derecha e izquierda. En control a los dos meses desarrolló nuevo cuadro febril con hipercolesterolemia, trombocitosis y alteración de las pruebas hepáticas. En ecocardiograma de control se evidenció aneurismagigante en la coronaria izquierda y mediante angiografía coronaria se detectaron dos aneurismas en la coronaria derecha. En vista de que se consideró un caso de evolución atípica, se realizó revisión de la literatura mundial en enfermedad de Kawasaki y se recomendó diagnóstico y tratamiento precoz para disminuir la morbilidad con compromiso cardiovascular severo y/o la mortalidad.

Abstract Kawasaki disease is an acute febrile state with an incidence of 175 per 100,000 children under the age of 5. It is a vasculitis with a preference for small and mediumsized vessels, especially for coronary arteries. If untreated, coronary aneurysms may pose the risk of acute myocardial infarction and sudden death. The case of a 4 year-old child who was diagnosed of Kawasaki disease and moderate fusiform aneurysms in the right and left coronary arteries is described. Follow-up visit after two months revealed fever again with hypercholesterolemia, thrombocytosis and abnormal liver testing. Control echocardiogram evidenced a giant aneurysm in the left coronary and a coronary angiography detected two additional aneurysms in the right coronary. As this was considered an atypical progress of the condition, review of global Kawasaki disease literature was carried out and early diagnosis and treatment were recommended to reduce morbidity with severe cardiovascular involvement and/or mortality.

¿Son pobres las familias que viven en condiciones de pobreza? / Are families living in poverty conditions poor?

Introducción: la familia es el núcleo primario de referencia de las personas, que determina que la forma en que interpreta la realidad se constituye como un modelo que guía y da sentido a las acciones de sus integrantes, por lo que puede ser un factor protector en condiciones de pobreza. Objetivo: describir la relación entre las concepciones teóricas de familia y pobreza con la experiencia familiar de 15 mujeres que viven en esta condición. Métodos: estudio cualitativo en una comuna de Santiago. Se utilizaron entrevistas semi-estructuradas y la Escala de Resiliencia SV-RES. Los datos se recolectaron entre noviembre 2012 y enero 2013. El análisis se realizó según los procedimientos de la Grounded Theory. Resultados: la familia se define por tres aspectos centrales e irrenunciables: a) comunicación, b) afecto y c) proyecto de vida. La pobreza es definida y asociada a la falta de recursos y a relaciones de dependencia. Las metas compartidas surgen como elementos diferenciadores en las familias que seguirán trayectorias de pobreza, no así respecto de los recorridos individuales. Conclusiones: la familia en situación de pobreza debe ser descubierta en su realidad, para ayudarla a enfrentar la tarea de emerger con sus propias fuerzas y potencialidades, fortaleciendo su capacidad formadora de capital humano, potenciando la integración real de todos quienes forman la sociedad y permitiendo la construcción de sistemas de representación inclusivos(AU)

Introduction: The family is the people's primary reference center, which determines that the way in which they interpret reality is constituted as a model that guides and gives meaning to the actions of its members, so it can be a protective factor under poverty conditions. Objective: To describe the relationship between the theoretical conceptions about family and poverty with the family experience of 15 women living under this condition Methods: Qualitative study in a commune of Santiago. Semi-structured interviews and the SV-RES Resilience Scale were used. Data were collected between November 2012 and January 2013. The analysis was performed according to the Grounded Theory procedures. Results: The family is defined by three central and indispensable aspects: a) communication, b) affection and c) life prospects. Poverty is defined and associated with the lack of resources and by the dependence relationships. The shared goals appear as differentiating elements in the families that will follow the trajectories of poverty, not in relationship with individual routes. Conclusions: The family in a poverty situation must be discovered in their reality, in order to help them face the task of emerging with their own strengths and potentialities, strengthening their capacity to form human capital, enhancing the real integration of everyone who forms the society and allowing the construction of inclusive representation systems(AU)

¿Son pobres las familias que viven en condiciones de pobreza? / Are families living in poverty conditions poor?

Introducción: la familia es el núcleo primario de referencia de las personas, que determina que la forma en que interpreta la realidad se constituye como un modelo que guía y da sentido a las acciones de sus integrantes, por lo que puede ser un factor protector en condiciones de pobreza. Objetivo: describir la relación entre las concepciones teóricas de familia y pobreza con la experiencia familiar de 15 mujeres que viven en esta condición. Métodos: estudio cualitativo en una comuna de Santiago. Se utilizaron entrevistas semi-estructuradas y la Escala de Resiliencia SV-RES. Los datos se recolectaron entre noviembre 2012 y enero 2013. El análisis se realizó según los procedimientos de la Grounded Theory. Resultados: la familia se define por tres aspectos centrales e irrenunciables: a) comunicación, b) afecto y c) proyecto de vida. La pobreza es definida y asociada a la falta de recursos y a relaciones de dependencia. Las metas compartidas surgen como elementos diferenciadores en las familias que seguirán trayectorias de pobreza, no así respecto de los recorridos individuales. Conclusiones: la familia en situación de pobreza debe ser descubierta en su realidad, para ayudarla a enfrentar la tarea de emerger con sus propias fuerzas y potencialidades, fortaleciendo su capacidad formadora de capital humano, potenciando la integración real de todos quienes forman la sociedad y permitiendo la construcción de sistemas de representación inclusivos(AU)

Introduction: The family is the people's primary reference center, which determines that the way in which they interpret reality is constituted as a model that guides and gives meaning to the actions of its members, so it can be a protective factor under poverty conditions. Objective: To describe the relationship between the theoretical conceptions about family and poverty with the family experience of 15 women living under this condition Methods: Qualitative study in a commune of Santiago. Semi-structured interviews and the SV-RES Resilience Scale were used. Data were collected between November 2012 and January 2013. The analysis was performed according to the Grounded Theory procedures. Results: The family is defined by three central and indispensable aspects: a) communication, b) affection and c) life prospects. Poverty is defined and associated with the lack of resources and by the dependence relationships. The shared goals appear as differentiating elements in the families that will follow the trajectories of poverty, not in relationship with individual routes. Conclusions: The family in a poverty situation must be discovered in their reality, in order to help them face the task of emerging with their own strengths and potentialities, strengthening their capacity to form human capital, enhancing the real integration of everyone who forms the society and allowing the construction of inclusive representation systems(AU)

Community structure of partial nitritation-anammox biofilms at decreasing substrate concentrations and low temperature.

Partial nitritation-anammox (PNA) permits energy effective nitrogen removal. Today PNA is used for treatment of concentrated and warm side streams at wastewater treatment plants, but not the more diluted and colder main stream. To implement PNA in the main stream, better knowledge about microbial communities at the typical environmental conditions is necessary. In order to investigate the response of PNA microbial communities to decreasing substrate availability, we have operated a moving bed biofilm reactor (MBBR) at decreasing reactor concentrations (311-27 mg-N l-1 of ammonium) and low temperature (13°C) for 302 days and investigated the biofilm community using high throughput amplicon sequencing; quantitative PCR; and fluorescence in situ hybridization. The anammox bacteria (Ca. Brocadia) constituted a large fraction of the biomass with fewer aerobic ammonia oxidizing bacteria (AOB) and even less nitrite oxidizing bacteria (NOB; Nitrotoga, Nitrospira and Nitrobacter). Still, NOB had considerable impact on the process performance. The anammox bacteria, AOB and NOB all harboured more than one population, indicating some diversity, and the heterotrophic bacterial community was diverse (seven phyla). Despite the downshifts in substrate availability, changes in the relative abundance and composition of anammox bacteria, AOB and NOB were small and also the heterotrophic community showed little changes in composition. This indicates stability of PNA MBBR communities towards decreasing substrate availability and suggests that even heterotrophic bacteria are integral components of these communities.

The inhibitory effects of reject water on nitrifying populations grown at different biofilm thickness.

Suppression of nitrite oxidizing bacteria (NOB) is of vital importance to achieve successful, energy efficient, mainstream anammox processes for wastewater treatment. In this study, biofilm carriers from a fully nitrifying MBBR system, fed with mainstream wastewater, were temporarily exposed to reject water from sludge dewatering, to evaluate this as a possible strategy to inhibit NOB and achieve nitrite production under realistic conditions. Two different carrier types were compared, in which biofilm thickness was maintained at approximately 400 and 50 µm, respectively, and reject treatment was tested at different exposure time and loading rates. Reject exposure almost always resulted in an increased nitrite production in the thinner biofilm, and overall, nitrifiers growing in the thin biofilm were more sensitive than those grown in the thicker biofilm. The effect from reject exposure remained in the systems for four days after returning to mainstream operation, with nitrite production gradually increasing for three days. Increased concentrations of free ammonia correlated with reject exposure and may be the cause of inhibition, although other factors cannot be excluded.

Predation of nitritation-anammox biofilms used for nitrogen removal from wastewater.

Predation is assumed to be a major cause of bacterial mortality in wastewater treatment plants (WWTP). Grazing on the slowly growing autotrophic ammonia oxidizing bacteria (AOB) and anaerobic ammonium oxidizing bacteria (AMX) may result in loss of biomass, which could compromise nitrogen removal by the nitritation-anammox process. However, predation, particularly of anaerobic AMX, is unknown. We investigated the presence of protozoa, AOB and AMX and the possible predation in nitritation-anammox biofilms from several WWTPs. By fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM), predator and prey were localized in intact biofilm cryosections. Different broad morphological types of protozoa were found at different biofilm depths. Large variations in abundance of protozoa were seen. One reactor showed a predation event of amoeba-like protozoa, forming grazing fronts reaching deep biofilm regions that were dominated by the anaerobic AMX. Both AOB and AMX were grazed by the amoeba, as revealed by FISH-CLSM. Hence, even AMX, living in the deeper layers of stratified biofilms, are subjected to predation. Interestingly, different colocalization was observed between the amoeba-like protozoa and two different Ca. Brocadia AMX sublineages, indicating different grazing patterns. The findings indicate that predation pressure can be an important factor regulating the abundance of AOB and AMX, with implications for nitrogen removal from wastewater.