Biofilm System Research Articles

Biofilm thickness controls the relative importance of stochastic and deterministic processes in microbial community assembly in moving bed biofilm reactors.

Deterministic and stochastic processes are believed to play a combined role in microbial community assembly, though little is known about the factors determining their relative importance. We investigated the effect of biofilm thickness on community assembly in nitrifying moving bed biofilm reactors using biofilm carriers where maximum biofilm thickness is controlled. We examined the contribution of stochastic and deterministic processes to biofilm assembly in a steady state system using neutral community modelling and community diversity analysis with a null-modelling approach. Our results indicate that the formation of biofilms results in habitat filtration, causing selection for phylogenetically closely related community members, resulting in a substantial enrichment of Nitrospira spp. in the biofilm communities. Stochastic assembly processes were more prevalent in biofilms of 200 µm and thicker, while stronger selection in thinner (50 µm) biofilms could be driven by hydrodynamic and shear forces at the biofilm surface. Thicker biofilms exhibited greater phylogenetic beta-diversity, which may be driven by a variable selection regime caused by variation in environmental conditions between replicate carrier communities, or by drift combined with low migration rates resulting in stochastic historical contingency during community establishment. Our results indicate that assembly processes vary with biofilm thickness, contributing to our understanding of biofilm ecology and potentially paving the way towards strategies for microbial community management in biofilm systems.

Unravelling ciprofloxacin removal in a nitrifying moving bed biofilm reactor: Biodegradation mechanisms and pathways

Although moving bed biofilm reactors (MBBRs) have shown excellent antibiotic removal potentials, the information on underlying mechanisms is yet limited. This work assessed the removal of ciprofloxacin in an enriched nitrifying MBBR by clarifying the contribution of adsorption and microbial-induced biodegradation. Results demonstrated the considerable biomass adsorption (55%) in first 30 min. Limiting nitrite oxidizing bacteria growth or inhibiting nitrification would lead to lower adsorption capacities. The highest ciprofloxacin biodegradation rate constant was 0.082 L g SS−1 h−1 in the presence of ammonium, owing to ammonia oxidizing bacteria (AOB)-induced cometabolism, while heterotrophs played an insignificant role (∼9%) in ciprofloxacin biodegradation. The developed model also suggested the importance of AOB-induced cometabolism and metabolism over heterotrophs-induced biodegradation by analyzing the respective biodegradation coefficients. Cometabolic biodegradation pathways of ciprofloxacin mainly involved the piperazine ring cleavage, probably alleviating antimicrobial activities. It implies the feasibility of nitrifying biofilm systems towards efficient antibiotic removal from wastewater.

Insight into the formation mechanism of algal biofilm in soy sauce wastewater

Microalgal-biofilm-based wastewater treatment refers to a microalgae-dominated biofilm community in non-axenic systems, in which co-existing bacteria and microbial extracellular polymeric substances (EPS) are crucial for the formation of algal biofilm. However, algae-bacteria consortium biofilm-forming mechanism has rarely been studied in wastewaters including soy sauce wastewater (SW). In this study, we conducted algal biofilm cultivation in raw SW (RSW) and sterilized SW (SSW), and determined the characteristics of algal biofilm and its EPS in the two SW treatments. The results demonstrated that the co-existing bacteria in the algal biofilm system significantly enhanced attached biomass and nutrient removal from RSW, compared to SSW without bacteria. The enhancement may be attributed to EPS excreted by dominated Acinetobacter and Comamonas (both Proteobacteria) in the co-existing bacteria community. The algae-bacteria consortium from algal biofilm in RSW excreted the contents of relatively high tightly bound EPS (TB-EPS) and its proteins (TB-PN), which were conducive to forming the algal biofilm. The high TB-EPS content was related to soluble microbial by-product-like substances (mainly proteins), and its high TB-PN content was mainly caused by the increases in hydrophobic glycine and serine contents for stable biofilm formation. Based on these results, we enhanced our understanding of the formation mechanism of algal biofilm in SW remediation, which provided the theoretical basis for biofilm formation by algae-bacteria synergism.

Exploration of the Main Antibiofilm Substance of Lactobacillus plantarum ATCC 14917 and Its Effect against Streptococcus mutans.

Dental plaque, a complex biofilm system established by cariogenic bacteria such as Streptococcus mutans (S. mutans), is the initiator of dental caries. Studies have found that the cell-free supernatant (CFS) of Lactobacilli could inhibit S. mutans biofilm formation. However, the main antibiofilm substance of the Lactobacilli CFS that acts against S. mutans is unclear. The present study found that the CFS of Lactobacillus plantarum (L. plantarum) ATCC 14917 had the strongest antibiofilm effect among the five tested oral Lactobacilli. Further bioassay-guided isolation was performed to identify the main antibiofilm substance. The antibiofilm effect of the end product, named 1-1-4-3, was observed and the structure of it was elucidated by using Q-TOF MS, 2D NMR and HPLC. The results showed that several components in the CFS had an antibiofilm effect; however, the effect of 1-1-4-3 was the strongest, as it could reduce the generation of exopolysaccharides and make the biofilm looser and thinner. After structure elucidation and validation, 1-1-4-3 was identified as a mixture of lactic acid (LA) and valine. Additionally, LA was shown to be the main antibiofilm substance in 1-1-4-3. In summary, this study found that the antibiofilm effect of the L. plantarum CFS against S. mutans was attributable to the comprehensive effect of multiple components, among which LA played a dominant role.

Identification and Regulatory Roles of a New Csr Small RNA from Arctic Pseudoalteromonas fuliginea BSW20308 in Temperature Responses.

Small RNAs (sRNAs) play a very important role in gene regulation at the posttranscriptional level. However, sRNAs from nonmodel microorganisms, extremophiles in particular, have been rarely explored. We discovered a putative sRNA, termed Pf1 sRNA, in Pseudoalteromonas fuliginea BSW20308 isolated from the polar regions in our previous work. In this study, we identified the sRNA and investigated its regulatory role in gene expression under different temperatures. Pf1 sRNA was confirmed to be a new member of the CsrB family but has little sequence similarity with Escherichia coli CsrB. However, Pf1 sRNA was able to bind to CsrA from E. coli and P. fuliginea BSW20308 to regulate glycogen synthesis. The Pf1 sRNA knockout strain (ΔPf1) affected motility, fitness, and global gene expression in transcriptomes and proteomes at 4°C and 32°C. Genes related to carbon metabolism, amino acid metabolism, salinity tolerance, antibiotic resistance, oxidative stress, motility, chemotaxis, biofilm, and secretion systems were differentially expressed in the wild-type strain and the ΔPf1 mutant. Our study suggested that Pf1 sRNA might play an important role in response to environmental changes by regulating global gene expression. Specific targets of the Pf1 sRNA-CsrA system were tentatively proposed, such as genes involved in the type VI secretion system, TonB-dependent receptors, and response regulators, but most of them have an unknown function. Since this is the first study of CsrB family sRNA in Pseudoalteromonas and microbes from the polar regions, it provides a novel insight at the posttranscriptional level into the responses and adaptation to temperature changes in bacteria from extreme environments. This study also sheds light on the evolution of sRNA in extreme environments and expands the bacterial sRNA database. IMPORTANCE Previous research on microbial temperature adaptation has focused primarily on functional genes, with little attention paid to posttranscriptional regulation. Small RNAs, the major posttranscriptional modulators of gene expression, are greatly underexplored, especially in nonpathogenic and nonmodel microorganisms. In this study, we verified the first Csr sRNA, named Pf1 sRNA, from Pseudoalteromonas, a model genus for studying cold adaptation. We revealed that Pf1 sRNA played an important role in global regulation and was indispensable in improving fitness. This study provided us a comprehensive view of sRNAs from Pseudoalteromonas and expanded our understanding of bacterial sRNAs from extreme environments.

Compartmentalization of a synergistic fungal-bacterial consortium to boost lactic acid conversion from lignocellulose via consolidated bioprocessing

A simplified aerobic fungal and facultative bacterial consortium can co-exist owing to the different oxygen spatial niches created by the biofilm system, achieving efficient lactic acid production from cellulose via CBP.

Effect of Different Operational Conditions on the Treatment Performance of Milk Processing Wastewater (MPW) Using a Single Stage Flexible Fibre Biofilm Reactor (SS-FFBR)

The performance of a biofilm system, single-stage flexible fibre biofilm reactor (SS-FFBR) treating milk processing wastewater (MPW) is evaluated under various process and operational conditions. The system behavior is analyzed with different biological and physical parameters. Results show that the high COD removal efficiency of 95% is obtained at a low CODin concentration of 809 mg/L. However, the COD removal is slightly decreased to 91.7% once the CODin concentration incremented to nearly 4000 mg/L. The effect of organic loading rate (OLR) on the SS-FFBR performance is examined as total suspended solids removal efficiency, dissolved oxygen, and turbidity. The SS-FFBR showed considerable performance, so that 89.9% and 89.7% removal efficiencies in terms of COD and TSS removals, respectively, obtained at the highest OLR of 11.7 kg COD/m3d. TSS removal efficiency of 96.7% is obtained at a low OLR of 1.145 kg COD/m3d. A linear relationship between the OLR and COD removal rate was revealed. The COD removal rate was incremented from 1.08 to 10.68 kg COD/m3d as the OLR increased from 1.145 to 11.7 kg COD/m3d. Finally, the operating system is a promising technique recommended to treat various industrial wastewaters with high OLR.

Perspectives on the Monod model in biological wastewater treatment

AbstractThe Monod model has been widely used in activated sludge and biofilm models to mathematically describe the kinetics of microbial growth. Mathematical models of biological systems can be distinguished according to their dimensionality as zero‐dimensional (0D), one‐dimensional (1D), two‐dimensional (2D) and three‐dimensional (3D). The Monod model is a 0D model, which does not take cell (aggregate) structure into account, yet has been used in different ways in activated sludge and biofilm modelling: In activated sludge modelling, it is used in its original sense (i.e. 0D), whilst in dimensional biofilm modelling, it is used to model intrinsic kinetics in the biofilm and thus in 1D, 2D and 3D. We believe that the latter is not the correct use of the Monod model, because both activated sludge and biofilm systems consist of similar cell aggregates that both feature concentration gradients. Diffusional mass transfer limitations are manifested by the values of kinetic parameters in the Monod model and should not be described explicitly by Fick's laws of diffusion. The 0D concept has been adopted in state‐of‐the‐art activated sludge models and more recently in 0D biofilm models. Measured kinetic parameters can be directly transferred to 0D models. The application of Fick's second law of diffusion in combination with the Monod model (i.e. the reaction–diffusion equation) therefore becomes irrelevant. In this manner, the Monod equation becomes a simple, universal approach to modelling that contains variable parameters. © 2022 Society of Chemical Industry (SCI).

The application of post-denitrification fixed biofilm reactor for polishing secondary effluent: Nitrate removal, soluble microbial products and micropollutants biotransformation

This study was conducted to comprehensively evaluate the role of hydraulic retention time (HRT) on simultaneous removal of nitrate and micropollutants (MPs) in secondary effluent from wastewater treatment plants in post-denitrification fixed biofilm reactor (PDFBR). Results showed that PDFBR was favorable for nitrate removal (above 94%). Prolonged HRT promoted the bio-utilization of nonaromatic soluble microbial products with low molecular weight and reduced biomass production. MPs was partially removed in PDFBRs (below 48%). Microbial diversity increased along the extending of HRT and thus partially enhanced MPs removal. Batch experiments showed that changing HRT had no direct impact on the biodegradation rates of the selected MPs. Correlation analysis revealed that Dechloromonas, Terrimonas, and Phreatobacter were reasonable for simultaneous removal of MPs and nitrate. The abundance of nosZ gene had a rapid decrease under extreme HRT. This study provides insights into polishing nitrate and MPs from secondary effluent in a denitrifying biofilm system.

Unveiling the effects of soluble starch, ethanol, and sodium acetate on the interactions of functional microorganisms and nitrogen removal in a partial nitritation and anammox biofilm system

Different organic matters have different effects on the nitrogen removal performance of partial nitritation and anammox (PN/A) biofilm system. In this study, the effects of adding soluble starch, ethanol, and sodium acetate on the functional microorganisms and nitrogen removal in the PN/A biofilm system are investigated. The results show that ethanol had the strongest effects on the ammonium-oxidizing bacteria (AOB) growth, enrichment, and ammonium nitrogen removal rate, followed by soluble starch and sodium acetate. However, the suppression of anaerobic ammonium oxidation bacteria (AnAOB) and decrease in total nitrogen removal rate were affected the most by ethanol, followed by soluble starch and sodium acetate. In addition, the enrichment of nitrifying oxidizing bacteria (NOB) was the most strongly influenced by ethanol, followed by soluble starch and sodium acetate. Furthermore, the enrichment of phylum Proteobacteria was facilitated by ethanol > soluble starch > sodium acetate. The enrichment of phylum Nitrospirae and genus Nitrospira were influenced by ethanol > soluble starch > sodium acetate. However, the suppression of the phylum Planctomycetes and the genus Candidatus Brocadia were affected by ethanol > soluble starch > sodium acetate. These results show that the PN/A biofilm system is suitable for the treatment of ammonium nitrogen wastewaters containing organic salt (sodium acetate) but not for treating wastewaters containing organic alcohol (ethanol) and macromolecular organic matter (soluble starch).

Robust nitrogen removal from municipal wastewater by partial nitrification anammox at ultra-low dissolved oxygen in a pure biofilm system

Efficient nitrogen removal from municipal wastewater applying a pure biofilm system has promise. In this study, a partial nitrification anammox (PNA) pure biofilm system was established in a sequencing batch reactor with anaerobic/oxic/anoxic operation; using this reactor, robust nitrogen removal from municipal wastewater at ambient temperature was achieved with a nitrogen removal efficiency (NRE) of 93.3 %. Partial nitrification with anammox could be coupled at dominant nitrite-oxidizing bacteria (NOB) abundance by controlling ultra-low dissolved oxygen (<0.1 mg/L) in the aerobic section where the contribution to nitrogen removal was 79.4 %. Microorganisms with different oxygen affinity spatially distributed on the carrier. Ammonia-oxidizing bacteria (AOB) dominated on the surface of the carrier, while anammox bacteria dominated on the interior of the carrier, with their relative abundance increasing from 0.26 % to 1.78 %. The intercalary NOB were inhibited by the restricted oxygen transfer. Overall, this study provides a new approach to realize PNA in biofilm system.

Ammonium concentration determines oxygen penetration depth to impact the suppression of nitrite-oxidizing bacteria inside partial nitritation and anammox biofilms

Biofilm-based Partial Nitritation and Anammox (PN/A) process has gained wide popularity in treating high ammonium (∼1000 mg NH4+-N/L) sidestream wastewater while facing a significant challenge in terms of nitrite-oxidizing bacteria (NOB) suppression when applied for low-strength municipal wastewater treatment with low ammonium concentration (∼50 mg N/L). Here, the significance of residual ammonium (NH4+) concentration to the mainstream PN/A biofilm system was systematically investigated through long-term and short-term experiments, in conjunction with analysis of the stratification of functional microorganisms and measurement of oxygen penetration. The experimental results collectively showed that a residual NH4+ concentration of ∼ 50 mg N/L was essential to stable NOB suppression, while a lower concentration of ∼ 5 mg N/L undesirably caused NOB proliferation. This result suggests that low NH4+ concentration in mainstream wastewater would lead to the intrinsic difficulty of NOB suppression in a biofilm PN/A process. Moreover, this study reveals that high NH4+ concentration promotes the activity of ammonia oxidizing bacteria (AOB) occupying the biofilm surface, thereby reducing oxygen penetration depth. Accordingly, NOB that inhabit a deep region of biofilm are suppressed by the limiting oxygen, while anammox bacteria are favoured instead. These findings provide important knowledge for the process control and expand the understanding of microbial interaction in mainstream PN/A biofilm systems.

Benthic diatoms modify riverine silicon export to a marine zone in a hypertidal estuarine environment

Riverine dissolved silicon (DSi) and biogenic silica (BSi) are modulated along the estuarine gradient by several biotic and abiotic processes governed by physical forcings. An important area controlling silicon transport in alluvial estuaries with large intertidal mudflats is the benthic diatom-dominated biofilm system. Here, the hypertidal Severn Estuary, UK, has been used as a case study to improve our understanding of silicon transport in these benthic-dominated systems. We present the first time-series dataset of Si concentrations in the Severn. River and tidal hydrodynamics drove spatio-temporal changes in DSi. The longitudinal profile of DSi followed the classical view of dilution with downstream transport. Despite low riverine supply of BSi and low siliceous-phytoplankton production, relatively high BSi concentrations were measured in the Severn Estuary (maximum of 14.9 mg/L), which accounted for over 70% of the total bioavailable silicon present and were characterised by isotopically heavy waters (δ30Si of + 0.9 to + 1.1‰). Benthic biofilms (microphytobenthos) on the intertidal mudflats contained significant biomass (measured as chlorophyll a concentration with a maximum of 116.8 ± 16.2 µg/g dw. sed) with high productivity, driven by their photoprotective adaptions to these harsh intertidal environments, contributing to isotopically heavy mudflat water (δ30Si of + 1.19 to + 2.03‰), and resulting in high benthic BSi content in the spring (0.74 ± 0.03%) and summer (0.76 ± 0.05%). The fast-flowing tidal currents resulted in high bottom shear stress which likely exceeded the erosion thresholds of the biofilms, transporting the sediment-BSi matrix into the water column. Suspended particulate matter (SPM) and BSi remained tightly coupled in the estuarine water column (bioflocculation), and experienced the series of erosion–deposition events, burial/dissolution and export out of the estuary. Our novel observations improve understanding of the complex processes governing Si transport in hypertidal, benthic-dominated estuaries, and highlights the importance of tightly coupled benthic-pelagic systems in influencing the terrestrial silicon export to a marine zone.

Flocs enhance nitrous oxide reduction capacity in a denitrifying biofilm-based system: Mechanism of electron competition

Nitrous oxide (N2O) accumulation induced by insufficient electron donors and substrate diffusion restriction is inevitably occurred in denitrifying biofilm-based systems, yet rare mechanism is intensively explored. This study discovered the mechanism of electron competition affecting N2O reduction capacity in two denitrifying biofilm-based systems, and uncovered the role of flocs for mitigating N2O production with varying exogenous organic carbon levels. The results clearly delineated that substrate diffusion restriction of biofilms deteriorated electron donors availability, leading to fiercer electron competition (ratio of 1.95) in the single denitrifying biofilm system. Such competition also occurred with the decreasing carbon loading rates (from 180 to 30 mg-COD/g VSS/h), further resulting in a higher N2O accumulation of 0.95 mg-N/L. By contrast, flocs from the denitrifying biofilm/flocs system can efficiently reduce overall N2O accumulation by 32 %. Also, flocs promoted the higher proportion of the electrons distributed to nitrous oxide reductase (Nos), indicating a strong N2O-reducing capacity. In addition, electrons from Nos inclinedly distributed to nitrate reductase (Nar) with an increase of 21.4 % under co-occurrence of NO3−, NO2− and N2O in the single denitrifying biofilm system, suggesting the mechanism of electron competition was the competition in upstream electron pool. In contrast, electrons from Nos tended to nitrite reductase (Nir) (5.9 %) instead of Nar (2.1 %) in the denitrifying biofilm/flocs system implied the competition in downstream electron pool. This study unraveled that flocs can be applied in denitrifying biofilm-based systems to alleviate N2O emissions and total carbon footprints at wastewater treatment plants (WWTPs).

Microbial activity along the depth of biofilm in the simultaneous partial nitrification, anammox and denitrification (SNAD) system

ABSTRACT Simultaneous partial nitrification, anammox and denitrification (SNAD) is a sustainable and cost-effective technology for nitrogen removal from low-strength wastewater. However, knowledge of the biofilm microenvironment of the SNAD system is currently unsatisfactory. The purpose of this study was to evaluate organic carbon effects on the microenvironment and microbial growth in the SNAD biofilm system. Microelectrodes were used to investigate microbial activity in-depth within biofilms. ORP distribution of the SNAD system was positively related to anammox activity(R 2 = 0.9), and had some influence on microbial community structure. The synergistic effect of anammox bacteria and denitrifiers could be achieved when the abundance ratio of anammox bacteria to denitrifying bacteria is greater than 1.2.

Biomineralization and AHLs-guided quorum sensing enhanced phosphorus recovery in the alternating aerobic/anaerobic biofilm system under metal ion stress

The alternating aerobic/anaerobic biofilm system had been applied for phosphorus (P) enrichment and recovery because of the advantage of low energy consumption and high efficiency. The metal ions and N-acyl-L-homoserine lactones (AHLs) in system were studied to better clarify the mechanism of P uptake/release under metal ion stress. The results indicated that the increase of metal ions stimulated the release of AHLs, and AHLs-guided quorum sensing (QS) enhanced P uptake. Moreover, biomineralization could stimulate the increase of P content in biofilm (Pbiofilm). Meanwhile, some ortho-p was converted to short-chain poly-p in extracellular polymer substance (EPS), and others were transferred into cell through EPS to synthesize poly-p. With the Pbiofilm increased, more P could be absorbed/released due to the shift in the metabolic model of polyphosphate accumulating organisms (PAOs). The release of AHLs between microorganisms was also inhibited when PAOs reached the state of P saturation (75.6 ± 2.5 mg/g SS), which meant that the effect of signaling function would tend to stabilize, and the 169.2 ± 2.6 mg/L P concentration in the enriched solution was obtained due to the P release was inhibited. Moreover, P was rapidly transferred to the new enriched solution after the P was recovered, and PAOs restored its capability of P uptake/release. In addition, 31P-NMR analysis demonstrated that EPS played a major role in PAOs compared to cell, and inorganic phosphorus (IP) played an essential role in the uptake/release of P compared to organic phosphorus (OP). Furthermore, the microbiological analysis showed that Candidatus Accumulibacter was positively correlated with AHLs (P < 0.05). This study provided essential support for clarifying the P metabolism mechanism of PAOs.

Recovering phosphorus as vivianite using an alternating aerobic/anaerobic biofilm and fluidized bed crystallization coupled system

An alternating aerobic/anaerobic biofilm (AABF) and fluidized bed crystallization (FBC) coupled system was constructed to examine the feasibility of using phosphorus (P) metabolic mechanism to enrich P and the availability of large size and high-purity vivianite crystals in this study. The results demonstrated that the stable operation of AABF system could be maintained by operating the biofilm system through P metabolic mechanism of polyphosphate accumulating organisms (PAOs), and the P concentration of 204.5 ± 3.2 mg/L could be obtained. The metabolic mode of PAOs shifted from GAM to PAM as the Pbiofilm increased, which led to PAOs being able to absorb/release more P, wherein extracellular polymer substance (EPS) and cell both played an indispensable role in the metabolism of P uptake/release. In the P recovery stage, the maximum P recovery efficiency reached 94.6 ± 3.1 %, and the main crystals formed were vivianite crystals. Moreover, the percentage of FeP and CaP was 80–93 % and 3–9 % in precipitate, and the percentage of phosphate used to synthesize vivianite was 83.2 %. Furthermore, the size of vivianite crystal increased with the generated vivianite as seed crystal. What's more, the LCA revealed that carbon dioxide equivalent (CO2 eq.) emissions, sulfur dioxide equivalent (SO2 eq.) emissions and phosphate equivalent (PO4− eq.) emissions were 13.3–15.1 kg CO2 eq./kg vivianite, 0.10–0.11 kg SO2 eq./kg vivianite, 0.031–0.037 kg PO4− eq./kg vivianite, respectively. The pH = 7 and Fe: P = 1.5:1 was the optimum condition to ensure high P recovery efficiency and reduced potential environmental emissions. This process system laid the groundwork for recovering P from wastewater.

Degradation efficiency of the azo dye acid orange 2 and microbial community characteristics in a rotating algal biofilm reactor

Under the traditional suspended culture system for algae, processing high concentrations of azo dyes is difficult because of poor light transmittance in wastewater. The rotating algal biofilm (RAB) system enables algal biofilms to receive adequate light, even in the presence of high concentrations of dye. The degradation efficiency of acid orange 2 (AO-2) at concentrations of 50–600 mg/L exceeded 95 % within 12–24 h, and the highest removal rate was 592.35 mg/(L·d). Fourier transform infrared spectroscopy showed that the azo bond was broken in AO-2, and the main decomposition products were amines and olefins. The sulfate concentration did not influence the AO-2 removal, whereas the pH had a significant effect (P < 0.001). The dye degradation efficiencies under acidic and neutral conditions were higher than those under alkaline conditions. Decolorization experiments indicated that the extracellular and intracellular enzymes synergistically degraded AO-2. High-throughput sequencing revealed that biofilms in RAB reactors contained a variety of taxa, including cyanobacteria, bacteria, Rotifera, and Cercozoa. The potential cause of AO-2 biodegradation by algae-bacterial systems is that the biofilm can use AO-2 as a nitrogen source for its own growth.

Model-based identification and testing of appropriate strategies to minimize N2O emissions from biofilm deammonification.

Based on a one-year pilot plant operation of a two-step biofilm nitritation-anammox pilot plant, N2O mitigation strategies were identified by applying a newly developed biofilm modeling approach. Due to adapted plant operation, the N2O emission could be diminished by 75% (8.8% → 2.3% of NH4-Noxidized_AOB). The results (measurement and simulation) confirm the huge importance of denitrification as an N2O source or N2O sink, depending on the boundary conditions. A significant reduction of N2O emissions could only be achieved with a one-step deammonification system, which is related to low nitrite and HNO2 concentrations. Increased oxygen concentrations in the bulk phase are not related to decreased emissions. N2O formation by ammonium-oxidizing bacteria (AOB) just shifts deeper into the biofilm; zones with low oxygen concentrations are not avoidable in biofilm systems. Low oxygen concentrations in the bulk phase, however, result in a reduction of the total net N2O formation due to increased activity of heterotrophic bacteria directly at the source of N2O formation (outer biofilm layer). For the model-based identification of mitigation strategies, the standard modeling approaches for biofilms were expanded by including the factor-based N2O formation and emission approach. The new model 'Biofilm/N2OISAH' was successfully validated using data from pilot-scale measurement campaigns. Altogether, the investigation confirms that the employed digital model can strongly support the development of N2O mitigation strategies without the need for specialized measurement inside the biofilm.

Discovery of a new genus of anaerobic ammonium oxidizing bacteria with a mechanism for oxygen tolerance

In the past 20 years, there has been a major stride in understanding the core mechanism of anaerobic ammonium-oxidizing (anammox) bacteria, but there are still several discussion points on their survival strategies. Here, we discovered a new genus of anammox bacteria in a full-scale wastewater-treating biofilm system, tentatively named “Candidatus Loosdrechtia aerotolerans”. Next to genes of all core anammox metabolisms, it encoded and transcribed genes involved in the dissimilatory nitrate reduction to ammonium (DNRA), which coupled to oxidation of small organic acids, could be used to replenish ammonium and sustain their metabolism. Surprisingly, it uniquely harbored a new ferredoxin-dependent nitrate reductase, which has not yet been found in any other anammox genome and might confer a selective advantage to it in nitrate assimilation. Similar to many other microorganisms, superoxide dismutase and catalase related to oxidative stress resistance were encoded and transcribed by “Ca. Loosdrechtia aerotolerans”. Interestingly, bilirubin oxidase (BOD), likely involved in oxygen resistance of anammox bacteria under fluctuating oxygen concentrations, was identified in “Ca. Loosdrechtia aerotolerans” and four Ca. Brocadia genomes, and its activity was demonstrated using purified heterologously expressed proteins. A following survey of oxygen-active proteins in anammox bacteria revealed the presence of other previously undetected oxygen defense systems. The novel cbb3-type cytochrome c oxidase and bifunctional catalase-peroxidase may confer a selective advantage to Ca. Kuenenia and Ca. Scalindua that face frequent changes in oxygen concentrations. The discovery of this new genus significantly broadens our understanding of the ecophysiology of anammox bacteria. Furthermore, the diverse oxygen tolerance strategies employed by distinct anammox bacteria advance our understanding of their niche adaptability and provide valuable insight for the operation of anammox-based wastewater treatment systems.