Correlation of extracellular polymeric substances and microbial community structure in denitrification biofilm exposed to adverse conditions

Microbial community may respond to different adverse conditions and result in the variation of extracellular polymeric substances (EPS) in denitrification biofilm; this study discovered the role of EPS in accordance with the analysis of cyclic diguanylate (c-di-GMP) and electron equilibrium (EE) under low organic loading rate, shock organic loading rate and low temperature conditions. Good nitrate removal performance could be achieved under shock organic loading rate and low temperature conditions; however, owing to the low organic loading rate, the carbon source was preferentially utilized for biomass growth. Tightly bound EPS (TB-EPS) contents progressively increased and facilitated cell adhesion and biofilm formation. The stable TB protein (TB-PN) content in TB-EPS built a cross-linked network to maintain internal biofilm structure and led to the rapid biosynthesis of polysaccharides, which could further enhance microbial adhesion and improve nitrate removal. C-di-GMP played an important role in biomass retention and biofilm formation, based on the correlation analysis of c-di-GMP and EPS. TB polysaccharide (TB-PS) contents presented a significant positive correlation with c-di-GMP content, microbial adhesion and biofilm stabilization was further enhanced through c-di-GMP regulation. In addition, a remarkable negative correlation between electron deletion rate (EDR) and TB-PN and TB-PS was discovered, and TB-PS was required to serve as energy source to enhance denitrification according to EE analysis. Surprisingly, dynamic microbial community was observed due to the drastic community succession under low temperature conditions, and the discrepancy between the dominant species for denitrification was found under shock organic loading rate and low temperature conditions. The notable increase in bacterial strains Simlicispira, Pseudomonas and Chryseobacterium was conducive to biofilm formation and denitrification under shock organic loading rate, while Dechloromonas and Zoogloea dramatically enriched for nitrate removal under low temperature conditions. The high abundance of Dechloromonas improved the secretion of EPS through the downstream signal transduction, and the c-di-GMP conserved in Pseudomonas concurrently facilitated to enhance exopolysaccharide production to shock organic loading rate and low temperature conditions.     

纳米氧化铈作用下活性污泥胞外聚合物及溶解性微生物产物特性

【目的】纳米氧化铈作为一种应用普遍的人工纳米材料,其生物毒性和环境效应得到了越来越多的重视。尝试从微生物代谢产物的角度,解读纳米氧化铈对活性污泥微生物的影响规律和过程。【方法】在实验室活性污泥系统中投加不同质量浓度纳米氧化铈,研究纳米氧化铈短期作用下微生物胞外聚合物(EPS)和溶解性微生物产物(SMP)这两类主要的微生物代谢产物含量和组分的变化规律。【结果】短期作用下,EPS和SMP的总量都随着纳米氧化铈浓度的增加而增加。低浓度纳米CeO_2不会导致活性污泥中松散型胞外聚合物(LB-EPS)、紧密型胞外聚合物(TB-EPS)含量和组分的显著改变。高浓度纳米CeO_2(25 mg/L以上)作用下TB-EPS含量和组分不受影响,而LB-EPS中多糖和蛋白质为抵抗纳米CeO_2毒性而增多。EPS分层组分含量显著提高,且LB-EPS的增幅显著高于TB-EPS增幅。当纳米氧化铈浓度为50 mg/L时,相较于空白对照组,蛋白质和多糖增幅分别达到35.18%和46.57%。当纳米氧化铈超过25 mg/L以上时,SMP不仅出现蛋白质,多糖和腐殖酸的含量也明显增加。【结论】SMP中蛋白质的产生,可能会与纳米材料相结合,以减小纳米材料的毒性。当纳米氧化铈浓度较低时,EPS的吸附作用会抵制其进入细胞内,当纳米氧化铈浓度较高时,刺激细胞产生更多EPS吸附纳米CeO_2,形成更厚的外部屏障层保护细胞。EPS和SMP的共同作用,构成了微生物细胞对纳米CeO_2的毒性抵抗机制。     

Distribution and composition of extracellular polymeric substances in membrane-aerated biofilm

Extracellular polymeric substances (EPS) are one of the main components of the biofilm and perform important functions in the biofilm system. In this study, two membrane-aerated biofilms (MABs) were developed for the thin and thick biofilms under different surface loading rates (SLRs). Supplies of oxygen and substrates in the MAB were from two opposite directions. This counter diffusion of nutrients resulted in a different growth environment, in contrast to conventional biofilms receiving both oxygen and substrates from the same side. The compositions, distributions and physicochemical properties (solubility and bindability) of EPS in the MABs of different thicknesses under different SLRs were studied. The effect of dissolved oxygen (DO) concentration within the MAB on EPS properties and distribution was investigated. Experimental results showed the different biofilm thicknesses produced substantially different profiles of EPS composition and distribution. Soluble proteins were more dominant than soluble polysaccharides in the inner aerobic layer of the biofilms; in contrast, bound proteins were greater than bound polysaccharides in the outer anoxic or anaerobic layer of the biofilms. The biofilm-EPS matrix consisted mainly of bound EPS. Bound EPS exhibited a hump-shaped profile with the highest content occurring in an intermediate region in the thin MAB and relatively more uniformly in the one half of the biofilm close to the membrane side and then declined towards the biofilm-liquid interface in the thick MAB. The profiles of soluble EPS presented a similar declining trend from the membrane towards the outer region in both thin and thick MABs. The study suggested that not only EPS composition but also EPS distribution and properties (solubility and bindability) played a crucial role in controlling the cohesiveness and maintaining the structural stability and stratification of the MABs.     

Distinct roles of extracellular polymeric substances in Pseudomonas aeruginosa biofilm development

Bacteria form surface attached biofilm communities as one of the most important survival strategies in nature. Biofilms consist of water, bacterial cells and a wide range of self-generated extracellular polymeric substances (EPS). Biofilm formation is a dynamic self-assembly process and several distinguishable stages are observed during bacterial biofilm development. Biofilm formation is shown to be coordinated by EPS production, cell migration, subpopulation differentiation and interactions. However, the ways these different factors affect each other and contribute to community structural differentiation remain largely unknown. The distinct roles of different EPS have been addressed in the present report. Both Pel and Psl polysaccharides are required for type IV pilus-independent microcolony formation in the initial stages of biofilm formation by Pseudomonas aeruginosa PAO1. Both Pel and Psl polysaccharides are also essential for subpopulation interactions and macrocolony formation in the later stages of P. aeruginosa PAO1 biofilm formation. Pel and Psl polysaccharides have different impacts on Pseudomonas quinolone signal-mediated extracellular DNA release in P. aeruginosa PAO1 biofilms. Psl polysaccharide is more important than Pel polysaccharide in P. aeruginosa PAO1 biofilm formation and antibiotic resistance. Our study thus suggests that different EPS materials play distinct roles during bacterial biofilm formation.     

Volumetric measurements of bacterial cells and extracellular polymeric substance glycoconjugates in biofilms

In this study an enrichment culture developed from activated sludge was used to investigate the architecture of fully hydrated multispecies biofilms. The assessment of biofilm structure and volume was carried out using confocal laser scanning microscopy (CLSM). Bacterial cell distribution was determined with the nucleic acid-specific stain SYTO 60, whereas glycoconjugates of extracellular polymeric substances (EPS) were stained with the Alexa-488-labeled lectin of Aleuria aurantia. Digital image analysis was employed for visualization and quantification of three-dimensional CLSM data sets. The specific volumes of the polymeric and cellular biofilm constituents were quantified. In addition, gravimetric measurements were done to determine dry mass and thickness of the biofilms. The data recorded by the CLSM technique and the gravimetric data were then compared. It was shown that the biofilm thicknesses determined with both methods agree well for slow-growing heterotrophic and chemoautotrophic biofilms. In addition, for slow-growing biofilms, the volumes and masses calculated from CLSM and the biomass calculated from gravimetric measurements were also comparable. For fast-growing heterotrophic biofilms cultivated with high glucose concentrations the data sets fit to a lesser degree, but still showed the same common trend. Compared with traditional gravimetric measurements, CLSM allowed differential recording of multiple biofilm parameters with subsequent three-dimensional visualization and quantification. The quantitative three-dimensional results recorded by CLSM are an important basis for understanding, controlling, exploiting, and modeling of biofilms.     

Extraction of extracellular polymeric substances from extreme acidic microbial biofilms

The efficiency of five extraction methods for extracellular polymeric substances (EPS) was compared on three benthic eukaryotic biofilms isolated from an extreme acidic river, Río Tinto (SW, Spain). Three chemical methods (MilliQ water, NaCl, and ethylenediamine tetraacetic acid [EDTA]) and two physical methods (Dowex 50.8 and Crown Ether cation exchange resins) were tested. The quality and quantity of the EPS extracted from acidic biofilms varied according to which EPS extraction protocol was used. Higher amounts were obtained when NaCl and Crown Ether resins were used as extractant agents, followed by EDTA, Dowex, and MilliQ. EPS amounts varied from approximately 155 to 478 mg g⁻¹ of dry weight depending on the extraction method and biofilm analyzed. EPS were primarily composed of carbohydrate, heavy metals, and humic acid, plus small quantities of proteins and DNA. Neutral hexose concentrations corresponded to more than 90% of the total EPS dry weight. The proportions of each metals in the EPS extracted with EDTA are similar to the proportions present in the water from each locality where the biofilms were collected except for Al, Cu, Zn, and Pb. In this study, the extracellular matrix heavy metal sorption efficiencies of five methods for extracting EPS from eukaryotic acidic biofilms were compared.     

Biofouling of reverse osmosis membranes: effects of cleaning on biofilm microbial communities,membrane performance,and adherence of extracellular polymeric substances

Laboratory-scale reverse osmosis (RO) flat-sheet systems were used with two parallel flow cells, one treated with cleaning agents and a control (ie undisturbed). The cleaning efforts increased the affinity of extracellular polymeric substances (EPS) to the RO membrane and altered the biofilm surface structure. Analysis of the membrane biofilm community composition revealed the dominance of Proteobacteria. However, within the phylum Proteobacteria, γ-Proteobacteria dominated the cleaned membrane biofilm, while β-Proteobacteria dominated the control biofilm. The composition of the fungal phyla was also altered by cleaning, with enhancement of Ascomycota and suppression of Basidiomycota. The results suggest that repeated cleaning cycles select for microbial groups that strongly attach to the RO membrane surface by producing rigid and adhesive EPS that hampers membrane performance.     

A methodological review on the characterization of microalgal biofilm and its extracellular polymeric substances

Biofilm secreted by microalgae are extracellular polymeric substances (EPSs) composed mainly of polysaccharides, proteins, nucleic acids and lipids. These EPSs immobilize the cells and stabilize biofilm, mediating adhesion towards solid surfaces. The EPSs valorization through industrial exploitations and scientific works is becoming more popular, but the bottleneck of such studies is the lack of consensus among researchers on the selection of detection techniques to be used, especially for novice researchers. It is a daunting task for any inexperienced researcher when they fail to identify the right tools needed for microalgal biofilm studies. In this review, a well-refined analysis protocol about microalgal biofilm and EPSs were prepared including its extraction and characterization. Pros and cons of various detection techniques were addressed and cutting-edge methods to study biofilm EPSs were highlighted. Future perspectives were also presented at the end of this review to bridge research gaps in studying biofilm adhesion via EPSs production. Ultimately, this review aims to assist novice researchers in making the right choices in their research studies on microalgal biofilms in accordance to the available technologies and needs.     

Spatial and successional dynamics of microbial biofilm communities in a grassland stream ecosystem

Biofilms represent a metabolically active and structurally complex component of freshwater ecosystems. Ephemeral prairie streams are hydrologically harsh and prone to frequent perturbation. Elucidating both functional and structural community changes over time within prairie streams provides a general understanding of microbial responses to environmental disturbance. We examined microbial succession of biofilm communities at three sites in a third‐order stream at Konza Prairie over a 2‐ to 64‐day period. Microbial abundance (bacterial abundance, chlorophyll a concentrations) increased and never plateaued during the experiment. Net primary productivity (net balance of oxygen consumption and production) of the developing biofilms did not differ statistically from zero until 64 days suggesting a balance of the use of autochthonous and allochthonous energy sources until late succession. Bacterial communities (MiSeq analyses of the V4 region of 16S rRNA) established quickly. Bacterial richness, diversity and evenness were high after 2 days and increased over time. Several dominant bacterial phyla (Beta‐, Alphaproteobacteria, Bacteroidetes, Gemmatimonadetes, Acidobacteria, Chloroflexi) and genera (Luteolibacter, Flavobacterium, Gemmatimonas, Hydrogenophaga) differed in relative abundance over space and time. Bacterial community composition differed across both space and successional time. Pairwise comparisons of phylogenetic turnover in bacterial community composition indicated that early‐stage succession (≤16 days) was driven by stochastic processes, whereas later stages were driven by deterministic selection regardless of site. Our data suggest that microbial biofilms predictably develop both functionally and structurally indicating distinct successional trajectories of bacterial communities in this ecosystem.     

Viscosity dynamics and the production of extracellular polymeric substances and soluble microbial products during anaerobic digestion of pulp and paper mill wastewater sludges

Bioprocess and Biosystems Engineering - The production processes of the pulp and paper industry often run in campaigns, leading to large variations in the composition of wastewaters and waste...     

Effect of biofilm in irrigation pipes on microbial quality of irrigation water

Aims: The focus of this work was to investigate the contribution of native Escherichia coli to the microbial quality of irrigation water and to determine the potential for contamination by E. coli associated with heterotrophic biofilms in pipe‐based irrigation water delivery systems. Methods and Results: The aluminium pipes in the sprinkler irrigation system were outfitted with coupons that were extracted before each of the 2‐h long irrigations carried out with weekly intervals. Water from the creek water and sprinklers, residual water from the previous irrigation and biofilms on the coupons were analysed for E. coli. High E. coli concentrations in water remaining in irrigation pipes between irrigation events were indicative of E. coli growth. In two of the four irrigations, the probability of the sample source, (creek vs sprinkler), being a noninfluential factor, was only 0·14, that is, source was an important factor. The population of bacteria associated with the biofilm on pipe walls was estimated to be larger than that in water in pipes in the first three irrigation events and comparable to one in the fourth event. Conclusion: Biofilm‐associated E. coli can affect microbial quality of irrigation water and, therefore, should not be neglected when estimating bacterial mass balances for irrigation systems. Significance and Impact of the Study: This work is the first peer‐reviewed report on the impact of biofilms on microbial quality of irrigation waters. Flushing of the irrigation system may be a useful management practice to decrease the risk of microbial contamination of produce. Because microbial water quality can be substantially modified while water is transported in an irrigation system, it becomes imperative to monitor water quality at fields, rather than just at the intake.     

Confocal imaging of in situ natural microbial communities and their extracellular polymeric secretions using Nanoplast resin

A novel method using excision and fixation in Nanoplast, a hydrophilic embedding resin, allows confocal imaging of natural microbial communities and their extracellular polymeric secretions (EPS) while in situ. Prestaining with fluorescent probes permits the observation of specific cellular and extracellular components. Marine stromatolite sediments were examined using this method. Optical sectioning using confocal laser scanning microscopy (CLSM) permitted high-resolution imaging through sediments. Delicate arrangements of the EPS that are associated with sedimentary microbial biofilms were imaged using a fluorescein isothiocyanate (FITC)-labeled lectin (concanavalin-A) probe. Close microspatial associations of heterotrophic bacteria cells and autotrophic cyanobacteria cells were also observed. The nanoplast resin produces no detectable autofluorescence. Further coupling of multi-photon scanning laser microscopy (2P-LSM) with a conventional single photon CLSM allowed concurrent imaging of DAPI-labeled microbial cells, FITC-labeled EPS and autofluorescent carbonate sand grains. The multi-photon infrared laser permits deep (approximately 1 mm) penetration of samples and the excitation of DAPI, which normally requires UV-excitation with minimal disturbance to samples. The unique combination of Nanoplast with fluorescent probes, CLSM and 2P-LSM allows for the preservation and imaging of natural microbial communities in their in situ state, a method easily adapted for examinations of other microbial systems.     

微生物生物膜研究的新进展

微生物在生长过程中为适应生存环境而形成了生物膜,Dr.Costerton JW在生物膜方面的研究为我们开拓了微生物学的新领域。微生物生物膜是由微生物群体及其包被的细胞外多聚物和基质网组成,它们彼此黏附或者黏附到组织或物体的表面。微生物生物膜与微生物的耐药性形成、基因的转移以及引起机体的持续性感染等都密切相关。目前对生物膜的研究重点已经深入到微生物相互间的信号传递、致病基因的转移以及如何干预微生物生物膜的形成等方面。此外,在治理污水和环境保护工程、生物材料工程和食品工业等方面,微生物生物膜技术已经得到了应用。     

活性污泥微生物胞外多聚物生物合成途径与菌胶团形成的调控机制

活性污泥法是借助活性污泥微生物菌胶团形成来实现泥水重力分离和部分污泥回用,辅以曝气供氧,在曝气池中高密度的微生物细胞可将溶解性有机污染物迅速降解、转化后为己所用,外排的剩余污泥带走大量有机质和氮磷,水质得以净化。活性污泥微生物所合成的胶质状胞外多聚物(Extracellular polymeric substances,EPS)是污泥菌胶团形成必不可少的"黏合剂",吸水性极高,这也造成剩余污泥难以处置和利用。我们初步总结了活性污泥微生物宏基因组研究概况,利用分子遗传学和基因组学手段,对活性污泥优势种动胶菌(Zoogloea)和其他菌胶团形成菌的EPS生物合成途径和菌胶团形成与调控机制加以研究,鉴定出一个约40 kb的胞外多糖生物合成大型基因簇和一个由7个基因组成的小型基因簇,该基因簇中除胞外多糖合成相关基因外,还编码组氨酸激酶Prs K和反应调节蛋白Prs R双组分系统,可激活RpoNσ因子共同调控一类称之为PEP-CTERM的新型胞外蛋白质的表达,参与菌胶团的形成。PEP-CTERM富含天冬酰胺(缩写为Asn或者N)残基,可能与胞外多糖通过N-连锁的糖基化形成复合物,包裹微生物细胞群体来介导菌胶团的形成。类似的PEP-CTERM基因和胞外多糖合成基因簇在许多重要的活性污泥细菌如聚磷菌和全程氨氧化菌中存在,说明这些细菌也是菌胶团形成菌,可通过污泥沉淀和回用在活性污泥中得以富集。这些研究结果可供活性污泥膨胀控制、污泥减量和剩余污泥资源和能源回收利用参考。     

Atomic force microscopy characterization of silver nanoparticles interactions with marine diatom cells and extracellular polymeric substance

This study highlights the capacity of atomic force microscopy (AFM) for investigating nanoparticle (NP) algal cell interaction with a subnanometer resolution. We designed a set of AFM experiments to characterize NP size, shape, and structure to visualize changes in the cell morphology induced by NPs and to characterize NP interaction with the extracellular polymeric substance (EPS). Samples for AFM imaging were prepared using the same protocol-drop deposition on mica and imaged in air. Here we address the interactions of Ag NPs with ubiquitous, lightly silicified marine diatoms Cylindrotheca fusiformis and Cylindrotheca closterium and their EPS. In natural seawater used throughout this study, the single Ag NPs adopted truncated tetrahedron morphology with particle heights of 10, 20, 30, and 40 nm. This size class Ag NPs penetrates the cell wall through the valve region built of silica NPs embedded in organic matrix. The Ag NPs cause a local damage inside the cell without disintegration of the cell wall. The EPS production has been shown to increase as a feedback response to Ag NP exposure and may contribute to detoxification mechanisms. Imaging EPS at high resolution revealed the incorporation of Ag NPs and their aggregates into the EPS-gel matrix, proving their detoxifying capacity.     

Optically transparent porous medium for nondestructive studies of microbial biofilm architecture and transport dynamics

We describe a novel and noninvasive, microscopy-based method for visualizing the structure and dynamics of microbial biofilms, individual fluorescent microbial cells, and inorganic colloids within a model porous medium. Biofilms growing in flow cells packed with granules of an amorphous fluoropolymer could be visualized as a consequence of refractive index matching between the solid fluoropolymer grains and the aqueous immersion medium. In conjunction with the capabilities of confocal microscopy for nondestructive optical sectioning, the use of amorphous fluoropolymers as a solid matrix permits observation of organisms and dynamic processes to a depth of 2 to 3 mm, whereas sediment biofilms growing in sand-filled flow cells can only be visualized in the region adjacent to the flow cell wall. This method differs fundamentally from other refractive index-matching applications in that optical transparency was achieved by matching a solid phase to water (and not vice versa), thereby permitting real-time microscopic studies of particulate-containing, low-refractive-index media such as biological and chromatographic systems.     

Membrane biofouling by extracellular polymeric substances or soluble microbial products from membrane bioreactor sludge

This study extracted the soluble microbial products and loosely bound and tightly bound extracellular polymeric substances (EPS) from suspended sludge from a membrane bioreactor, original and aerobically/anaerobically digested, and compared their fouling potentials on a microfiltration membrane. The resistance of cake layer accounts for 95–98% of the total filtration resistances when filtering the whole sludges, with anaerobically digested sludge presenting the highest resistance among the three tested sludges. The tightly bound EPS has the highest potential to foul the membrane; however, the loosely bound EPS contribute most of the filtration resistances of the whole sludges. The foulants corresponding to the irreversible fouling have chemical fingerprints similar to those from loosely bound EPS, which have a greater predilection to proteins and humic substances than to polysaccharides.     

Characteristics of extracellular polymeric substances from sludge and biofilm in a simultaneous nitrification and denitrification system under high salinity stress

The composition and distribution of extracellular polymeric substance (EPS) both from suspended sludge and attached biofilm were investigated in a simultaneous nitrification and denitrification (SND) system with the increase of the salinity from 1.0 to 3.0 %. Fourier-transform infrared (FTIR) spectroscopy and three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy were used to examine proteins (PN), polysaccharides (PS) and humic substances (HS) present in EPS. High total nitrogen removal (above 83.9 %) via SND was obtained in the salinity range of 1.0–2.5 %. Total EPS in the sludge increased from 150.2 to 200.6 mg/gVSS with the increase of salinity from 1.0 to 3.0 %, whereas the corresponding values in the biofilm achieved the maximum of 288.6 mg/g VSS at 2.0 % salinity. Dominant composition of EPS was detected as HS in both sludge and biofilm, having the percentages of 50.6–68.6 and 41.1–69.9 % in total EPS, respectively. Both PN and PS contents in soluble EPS (S-EPS), loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) of sludge and biofilm increased with the increased salinity. The FTIR spectrum and 3D-EEM fluorescence spectroscopy of S-EPS, LB-EPS and TB-EPS in the sludge and biofilm showed the changes of functional groups and conformations of the compositions in EPS with the increase of salinity. The results demonstrated that the characteristics of EPS varied from sludge to biofilm. The obtained results could provide a better understanding of the salinity effect on the EPS characteristics in a SND system.     

Persistence of microbial extracellular enzymes in soils under different temperatures and water availabilities

Microbial extracellular enzyme activity (EEA) is critical for the decomposition of organic matter in soils. Generally, EEA represents the limiting step governing soil organic matter mineralization. The high complexity of soil microbial communities and the heterogeneity of soils suggest potentially complex interactions between microorganisms (and their extracellular enzymes), organic matter, and physicochemical factors. Previous studies have reported the existence of maximum soil EEA at high temperatures although microorganisms thriving at high temperature represent a minority of soil microbial communities. To solve this paradox, we attempt to evaluate if soil extracellular enzymes from thermophiles could accumulate in soils. Methodology at this respect is scarce and an adapted protocol is proposed. Herein, the approach is to analyze the persistence of soil microbial extracellular enzymes at different temperatures and under a broad range of water availability. Results suggest that soil high‐temperature EEA presented longer persistence than enzymes with optimum activity at moderate temperature. Water availability influenced enzyme persistence, generally preserving for longer time the extracellular enzymes. These results suggest that high‐temperature extracellular enzymes could be naturally accumulated in soils. Thus, soils could contain a reservoir of enzymes allowing a quick response by soil microorganisms to changing conditions. This study suggests the existence of novel mechanisms of interaction among microorganisms, their enzymes and the soil environment with relevance at local and global levels.     

Sequentially aerated membrane biofilm reactors for autotrophic nitrogen removal: microbial community composition and dynamics

Membrane‐aerated biofilm reactors performing autotrophic nitrogen removal can be successfully applied to treat concentrated nitrogen streams. However, their process performance is seriously hampered by the growth of nitrite oxidizing bacteria (NOB). In this work we document how sequential aeration can bring the rapid and long‐term suppression of NOB and the onset of the activity of anaerobic ammonium oxidizing bacteria (AnAOB). Real‐time quantitative polymerase chain reaction analyses confirmed that such shift in performance was mirrored by a change in population densities, with a very drastic reduction of the NOB Nitrospira and Nitrobacter and a 10‐fold increase in AnAOB numbers. The study of biofilm sections with relevant 16S rRNA fluorescent probes revealed strongly stratified biofilm structures fostering aerobic ammonium oxidizing bacteria (AOB) in biofilm areas close to the membrane surface (rich in oxygen) and AnAOB in regions neighbouring the liquid phase. Both communities were separated by a transition region potentially populated by denitrifying heterotrophic bacteria. AOB and AnAOB bacterial groups were more abundant and diverse than NOB, and dominated by the r‐strategists Nitrosomonas europaea and Ca. Brocadia anammoxidans, respectively. Taken together, the present work presents tools to better engineer, monitor and control the microbial communities that support robust, sustainable and efficient nitrogen removal.