SupplementaryInfo_Fowleretal_MBBR.docx from 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 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

Modeling Nitrous Oxide Production during Biological Nitrogen Removal via Nitrification and Denitrification: Extensions to the General ASM Models

Nitrous oxide (N2O) can be formed during biological nitrogen (N) removal processes. In this work, a mathematical model is developed that describes N2O production and consumption during activated sludge nitrification and denitrification. The well-known ASM process models are extended to capture N2O dynamics during both nitrification and denitrification in biological N removal. Six additional processes and three additional reactants, all involved in known biochemical reactions, have been added. The validity and applicability of the model is demonstrated by comparing simulations with experimental data on N2O production from four different mixed culture nitrification and denitrification reactor study reports. Modeling results confirm that hydroxylamine oxidation by ammonium oxidizers (AOB) occurs 10 times slower when NO2– participates as final electron acceptor compared to the oxic pathway. Among the four denitrification steps, the last one (N2O reduction to N2) seems to be inhibited first when O2 is present. Overall, N2O production can account for 0.1–25% of the consumed N in different nitrification and denitrification systems, which can be well simulated by the proposed model. In conclusion, we provide a modeling structure, which adequately captures N2O dynamics in autotrophic nitrification and heterotrophic denitrification driven biological N removal processes and which can form the basis for ongoing refinements

Aeration Strategies To Mitigate Nitrous Oxide Emissions from Single-Stage Nitritation/Anammox Reactors

Autotrophic nitrogen removal is regarded as a resource efficient process to manage nitrogen-rich residual streams. However, nitrous oxide emissions of these processes are poorly documented and strategies to mitigate emissions unknown. In this study, two sequencing batch reactors performing single-stage nitritation/anammox were operated under different aeration strategies, gradually adjusted over six months. At constant but limiting oxygen loading, synthetic reject water was fed (0.75g-N/L·d) and high nitrogen removal efficiencies (83 ± 5 and 88 ± 2%) obtained. Dynamics of liquid phase nitrous (N₂O) and nitric oxide (NO) concentrations were monitored and N₂O emissions calculated. Significant decreases in N₂O emissions were obtained when the frequency of aeration was increased while maintaining a constant air flow rate (from >6 to 1.7% ΔN₂O/ΔTN). However, no significant effect on the emissions was noted when the duration of aeration was increased while decreasing air flow rate (10.9 ± 3.2% ΔN₂O/ΔTN). The extant ammonium oxidation activity (mgNH₄⁺-N/gVSS·min) positively correlated with the specific N₂O production rate (mgN₂O-N/gVSS·min) of the systems. Operating under conditions where anaerobic exceeds aerobic ammonium oxidation activity is proposed to minimize N₂O emissions from single-stage nitritation/anammox reactors; increasing the frequency of aeration cycling is an efficient way of obtaining those conditions

Biodegradation in a Partially Saturated Sand Matrix: Compounding Effects of Water Content, Bacterial Spatial Distribution, and Motility

Bacterial pesticide degraders are generally heterogeneously distributed in soils, leaving soil volumes devoid of degradation potential. This is expected to have an impact on degradation rates because the degradation of pollutant molecules in such zones will be contingent either on degraders colonizing these zones or on pollutant mass transfer to neighboring zones containing degraders. In a model system, we quantified the role exerted by water on mineralization rate in the context of a heterogeneously distributed degradation potential. Alginate beads colonized by Pseudomonas putida KT2440 were inserted at prescribed locations in sand microcosms so that the initial spatial distribution of the mineralization potential was controlled. The mineralization rate was strongly affected by the matric potential (decreasing rate with decreasing matric potential) and by the initial distribution of the degraders (more aggregated distributions being associated with lower rates). The mineralization was diffusion-limited, as confirmed with a mathematical model. In wet conditions, extensive cell dispersal was observed for the flagellated wild type and, albeit to a lesser extent, for a nonflagellated mutant, partially relieving the diffusion limitation. Dry conditions, however, sustained low mineralization rates through the combined effects of low pollutant diffusivity and limited degrader dispersal

Evaluation of Bioaugmentation with Entrapped Degrading Cells as a Soil Remediation Technology

Soil augmentation with microbial degraders immobilized on carriers is evaluated as a potential remediation technology using a mathematical model that includes degradation within spatially distributed carriers and diffusion or advection-dispersion as contaminant mass transfer mechanisms. The total volume of carriers is a critical parameter affecting biodegradation performance. In the absence of advection, 320 and 20 000 days are required to mineralize 90% of the herbicide linuron by Variovorax sp. SRS16 encapsulated in 2 mm beads with 5 and 20 mm spacings, respectively. Given that many pesticide degraders have low intrinsic degradation rates and that only limited carrier to soil volume ratios are practically feasible, bioaugmented soils are characterized by low effective degradation rates and can be considered fully mixed. A simple exponential model is then sufficient to predict biodegradation as verified by comparisons with published experimental data. By contrast, the full spatially distributed model is needed to adequately model the degradation of faster degrading contaminants such as naphthalene and benzene which can be mass-transfer limited. Dimensionless Damköhler numbers are proposed to determine whether the spatially distributed model is required. Results show that field scale applications of immobilized degraders will be limited by the amount of carriers required to reach acceptable degradation rates

Antecedent Growth Conditions Alter Retention of Environmental <i>Escherichia coli</i> Isolates in Transiently Wetted Porous Media

The physical transport of Escherichia coli in terrestrial environments may require control to prevent its dissemination from potential high-density sources, such as confined animal feedlot operations. Biobarriers, wherein convective flows carrying pathogens pass through a porous matrix with high retentive capacity, may present one such approach. Eight environmental E. coli isolates were selected to conduct operational retention tests (ORT) with potential biobarrier materials Pyrax or dolomite, or silica glass as control. The conditions in the ORT were chosen to simulate conditioning by manure solutes, a pulse application of a bacterial load followed by rainfall infiltration, and natural drainage.Removal was limited, and likely caused by the relatively high velocities during drainage, and the conditioning of otherwise favorable adhesion sites. Flagella-mediated motility showed the strongest correlation to biobarrier retention. Significant variability was observed across the E. coli isolates, but consistently higher retention was observed for cells with external versus intestinal pregrowth histories. E. coli O157:H7 was retained the least with all examined matrices, while E. coli K-12 displayed moderate retention and may not serve as representative model strain. Pyrax is a good candidate biobarrier material given its superior removal ability across the tested E. coli strains

Sequential Aeration of Membrane-Aerated Biofilm Reactors for High-Rate Autotrophic Nitrogen Removal: Experimental Demonstration

One-stage autotrophic nitrogen (N) removal, requiring the simultaneous activity of aerobic and anaerobic ammonium oxidizing bacteria (AOB and AnAOB), can be obtained in spatially redox-stratified biofilms. However, previous experience with Membrane-Aerated Biofilm Reactors (MABRs) has revealed a difficulty in reducing the abundance and activity of nitrite oxidizing bacteria (NOB), which drastically lowers process efficiency. Here we show how sequential aeration is an effective strategy to attain autotrophic N removal in MABRs: Two separate MABRs, which displayed limited or no N removal under continuous aeration, could remove more than 5.5 g N/m2/day (at loads up to 8 g N/m2/day) by controlled variation of sequential aeration regimes. Daily averaged ratios of the surficial loads of O2 (oxygen) to NH4+ (ammonium) (LO2/LNH4) were close to 1.73 at this optimum. Real-time quantitative PCR based on 16S rRNA gene confirmed that sequential aeration, even at elevated average O2 loads, stimulated the abundance of AnAOB and AOB and prevented the increase in NOB. Nitrous oxide (N2O) emissions were 100-fold lower compared to other anaerobic ammonium oxidation (Anammox)-nitritation systems. Hence, by applying periodic aeration to MABRs, one-stage autotrophic N removal biofilm reactors can be easily obtained, displaying very competitive removal rates, and negligible N2O emissions

Estimating the Transfer Range of Plasmids Encoding Antimicrobial Resistance in a Wastewater Treatment Plant Microbial Community

Wastewater treatment plants (WWTPs) have been suggested as reservoirs and sources of antibiotic resistance genes (ARGs) in the environment. In a WWTP ecosystem, human enteric and environmental bacteria are mixed and exposed to pharmaceutical residues, potentially favoring genetic exchange and thus ARG transmission. However, the contribution of microbial communities in WWTPs to ARG dissemination remains poorly understood. Here, we examined for the first time plasmid permissiveness of an activated sludge microbial community by utilizing an established fluorescent bioreporter system. The activated sludge microbial community was challenged in standardized filter matings with one of three multidrug resistance plasmids (pKJK5, pB10, and RP4) harbored by <i>Escherichia coli</i> or <i>Pseudomonas putida</i>. Different donor–plasmid combinations had distinct transfer frequencies, ranging from 3 to 50 conjugation events per 100000 cells of the WWTP microbial community. In addition, transfer was observed to a broad phylogenetic range of 13 bacterial phyla with several taxa containing potentially pathogenic species. Preferential transfer to taxa belonging to the predicted evolutionary host range of the plasmids was not observed. Overall, the ARG dissemination potential uncovered in WWTP communities calls for a thorough risk assessment of ARG transmission across the wastewater system, before identification of possible mitigation strategies