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.

Promoting the degradation of organic micropollutants in tertiary moving bed biofilm reactors by controlling growth and redox conditions.

A novel process configuration was designed to increase biofilm growth in tertiary moving bed biofilm reactors (MBBRs) by providing additional substrate from primary treated wastewater in a sidestream reactor under different redox conditions in order to improve micropollutant removal in MBBRs with low substrate availability. This novel recirculating MBBR was operated on pilot scale for 13 months, and a systematic increase was seen in the biomass concentration and the micropollutant degradation rates, compared to a tertiary MBBR without additional substrate. The degradation rates per unit carrier surface area increased in the order of ten times, and for certain micropollutants, such as atenolol, metoprolol, trimethoprim and roxithromycin, the degradation rates increased 20-60 times. Aerobic conditions were critical for maintaining high micropollutant degradation rates. With innovative MBBR configurations it may be possible to improve the biological degradation of organic micropollutants in wastewater. It is suggested that degradation rates be normalized to the carrier surface area, in favor of the biomass concentration, as this reflects the diffusion limitations of oxygen, and will facilitate the comparison of different biofilm systems.

Impact of intermittent feeding on polishing of micropollutants by moving bed biofilm reactors (MBBR).

Moving bed biofilm reactors (MBBRs) were placed at two wastewater treatment plants, where they were constantly fed with effluent and intermittently fed with primary wastewater. Each reactor was subjected to different feast/famine periods and flow rates of primary wastewater, thus the different organic and nutrient loads (chemical oxygen demand(COD), ammonium(NH4-N)) resulted in different feast-famine conditions applied to the biomass. In batch experiments, this study investigated the effects of various feast-famine conditions on the biodegradation of micropollutants by MBBRs applied as an effluent polishing step. Rate constants of micropollutant removals were found to be positively correlated to the load of the total COD and NH4-N, indicating that higher organic loads were favourable for the growth of micropollutant degraders in these MBBRs. Rate constant of atenolol was five times higher when the biomass was fed with the highest COD and NH4-N load than it was fed with the lowest COD and NH4-N load. For diclofenac, mycophenolic acid and iohexol, their maximum rate constants were obtained with feeding of COD and NH4-N of approximately 570 mgCOD/d and 40∼60 mgNH4-N/d respectively. This also supports the concept that co-metabolism (rather competition inhibition or catabolic repression) plays an important role in micropollutants biodegradation in wastewater.

Municipal wastewater treatment targeting pharmaceuticals by a pilot-scale hybrid attached biofilm and activated sludge system (Hybas™).

A hybrid wastewater treatment process with combined attached biofilm (moving bed biofilm reactor) and activated sludge, named as Hybas™, was implemented for the treatment of municipal wastewater. The system consisted of six staged reactors in series including pre-denitrification and nitrification in the Hybas™ line and post-denitrification in a pure MBBR. In addition to the significant removal of nutrients and organic matter from municipal wastewater, Hybas™ also showed removal capacity for pharmaceuticals. Of particular interest was the enhanced removal for pharmaceuticals (i.e. X-ray contrast media) compared to other biological systems. Spiking experiments showed that the maximum removal rate constants (k, h-1) for 10 out of the 21 investigated pharmaceuticals (including diclofenac) were observed to occur within the two aerobic Hybas ™ reactors, operated in a flow-shifting mode that allows even biofilm growth of nitrifying bacteria. In total, 14 out of the 21 pharmaceuticals were removed by more than 50% during continuous flow operation in the all Hybas™ line and post-denitrification MBBR. The calculated and estimated removal contributions of pharmaceuticals by each individual reactor were also assessed.

Coupling electrochemical ammonia extraction and cultivation of methane oxidizing bacteria for production of microbial protein.

Conventional treatment of residual resources relies on nutrient removal to limit pollution. Recently, nutrient recovery technologies have been proposed as more environmentally and energetically efficient strategies. Nevertheless, the upcycling of recovered resources is typically limited by their quality or purity. Specifically, nitrogen extracted from residual streams, such as anaerobic digestion (AD) effluents and wastewaters, could support microbial protein production. In this context, this study was performed as a proof-of-concept to combine nitrogen recovery via electrochemical reactors with the production of high quality microbial protein via cultivation of methanotrophs. Two types of AD effluents, i.e., cattle manure and organic fraction of municipal solid waste, and urine were tested to investigate the nitrogen extraction efficiency. The results showed that 31-51% of the nitrogen could be recovered free of trace chemicals from residual streams depending on the substrate and voltage used. Based on the results achieved, higher nitrogen concentration in the residual streams resulted in higher nitrogen flux between anodic and cathodic chambers. Results showed that the extraction process has an energy demand of 9.97 (±0.7) - 14.44 (±1.19) kWh/kg-N, depending on the substrate and operating conditions. Furthermore, a mixed-culture of methanotrophic bacteria could grow well with the extracted nitrogen producing a total dry weight of 0.49 ± 0.01 g/L. Produced biomass contained a wide range of essential amino acids making it comparable with conventional protein sources.

Effect of slow biodegradable substrate addition on biofilm structure and reactor performance in two MBBRs filled with different support media.

In this study, two moving-bed biofilm reactors (MBBR1 and MBBR2) filled with different size of carrier media (Kaldnes K1 and Kaldnes K1 micro, respectively) were subjected to soluble (sugar and sodium acetate (Ac)) substrate and mixture of soluble and particulate (particulate potato starch (PS)) substrate in a very high organic loading rate (12 kgCOD/m3·d) at different temperatures (26 and 15°C, in MBBR1 and MBBR2, respectively). The effects of carrier type and substrate on biofilm structure and reactor performance have been studied. Starch was removed by adsorption at the biofilm surface and hydrolyzed which caused substrate gradient in MBBR1, however, hydrolyzed uniformly within biofilm in MBBR2. The biofilm of MBBR1 was irregular due to filamentous structure growth due to the substrate gradient, while, it was regular in MBBR2 due to uniform distribution of substrate. The performance of both MBBRs in ammonium, COD and TN removal decreased significantly when the amount of small particles in the reactor increased owing to feeding by starch, which led to biomass density decline. The type of media affected the quantity and distribution of attached biomass, which in turn influenced the activity of specific microbial functional groups in the biofilm. The biofilm in MBBR2 was thicker and consequently nitrogen removal by denitrification was much higher. The lower temperature did not affect negatively the reactor performance in MBBR2.

Removal of micropollutants during biological phosphorus removal: Impact of redox conditions in MBBR.

Further biological polishing of micropollutants in WWTP effluents is limited by the lack of available carbon for cometabolic degradation. Metabolism of polyhydroxyalkanoates (PHAs) stored intracellularly during enhanced biological phosphorus removal (EBPR) could serve as carbon source for post-denitrification and micropollutant cometabolism. The removal of nine micropollutants (i.e., pharmaceuticals and corrosion inhibitors) was investigated by using Moving Bed Biofilm Reactors (MBBRs), selecting phosphorus (PAO) or glycogen (GAO) accumulating organisms under different redox conditions. Three laboratory-scale MBBRs were operated in sequencing-batch mode under cyclical anaerobic and aerobic/anoxic conditions for phosphorus removal. Batch experiments were performed to evaluate the biodegradation potential of micropollutants along with the utilization of internally stored PHA. Experiments showed that aerobic PAO were able to efficiently remove most of the targeted micropollutants. The removal of benzotriazole, 5­methyl­1H­benzotriazole, carbamazepine, ketoprofen and diclofenac occurred simultaneously to phosphorus uptake and terminated when phosphorus was no longer available. Denitrifying PAO and aerobic GAO exhibited lower removal of micropollutants than aerobic PAO. Degradation profiles of stored PHA suggested a diverse utilization of the different fractions of PHA for phosphorus and micropollutant removal, with PHV (poly 3­hydroxyvalerate) most likely used for the cometabolism of targeted micropollutants.

Reactor staging influences microbial community composition and diversity of denitrifying MBBRs- Implications on pharmaceutical removal.

The subdivision of biofilm reactor in two or more stages (i.e., reactor staging) represents an option for process optimisation of biological treatment. In our previous work, we showed that the gradient of influent organic substrate availability (induced by the staging) can influence the microbial activity (i.e., denitrification and pharmaceutical biotransformation kinetics) of a denitrifying three-stage Moving Bed Biofilm Reactor (MBBR) system. However, it is unclear whether staging and thus the long-term exposure to varying organic carbon type and loading influences the microbial community structure and diversity. In this study, we investigated biofilm structure and diversity in the three-stage MBBR system (S) compared to a single-stage configuration (U) and their relationship with microbial functions. Results from 16S rRNA amplicon libraries revealed a significantly higher microbial richness in the staged MBBR (at 99% sequence similarity) compared to single-stage MBBR. A more even and diverse microbial community was selected in the last stage of S (S3), likely due to exposure to carbon limitation during continuous-flow operation. A core of OTUs was shared in both systems, consisting of Burkholderiales, Xanthomonadales, Flavobacteriales and Sphingobacteriales, while MBBR staging selected for specific taxa (i.e., Candidate division WS6 and Deinococcales). Results from quantitative PCR (qPCR) showed that S3 exhibited the lowest abundance of 16S rRNA but the highest abundance of atypical nosZ, suggesting a selection of microbes with more diverse N-metabolism (i.e., incomplete denitrifiers) in the stage exposed to the lowest carbon availability. A positive correlation (p < 0.05) was observed between removal rate constants of several pharmaceuticals with abundance of relevant denitrifying genes, but not with biodiversity. Despite the previously suggested positive relationship between microbial diversity and functionality in macrobial and microbial ecosystems, this was not observed in the current study, indicating a need to further investigate structure-function relationships for denitrifying systems.

Removal of pharmaceuticals in pre-denitrifying MBBR - Influence of organic substrate availability in single- and three-stage configurations.

Due to the limited efficiency of conventional biological treatment, innovative solutions are being explored to improve the removal of trace organic chemicals in wastewater. Controlling biomass exposure to growth substrate represents an appealing option for process optimization, as substrate availability likely impacts microbial activity, hence organic trace chemical removal. This study investigated the elimination of pharmaceuticals in pre-denitrifying moving bed biofilm reactors (MBBRs), where biofilm exposure to different organic substrate loading and composition was controlled by reactor staging. A three-stage MBBR and a single-stage reference MBBR (with the same operating volume and filling ratio) were operated under continuous-flow conditions (18 months). Two sets of batch experiments (day 100 and 471) were performed to quantify and compare pharmaceutical removal and denitrification kinetics in the different MBBRs. Experimental results revealed the possible influence of retransformation (e.g., from conjugated metabolites) and enantioselectivity on the removal of selected pharmaceuticals. In the second set of experiments, specific trends in denitrification and biotransformation kinetics were observed, with highest and lowest rates/rate constants in the first (S1) and the last (S3) staged sub-reactors, respectively. These observations were confirmed by removal efficiency data obtained during continuous-flow operation, with limited removal (<10%) of recalcitrant pharmaceuticals and highest removal in S1 within the three-stage MBBR. Notably, biotransformation rate constants obtained for non-recalcitrant pharmaceuticals correlated with mean specific denitrification rates, maximum specific growth rates and observed growth yield values. Overall, these findings suggest that: (i) the long-term exposure to tiered substrate accessibility in the three-stage configuration shaped the denitrification and biotransformation capacity of biofilms, with significant reduction under substrate limitation; (ii) biotransformation of pharmaceuticals may have occurred as a result of cometabolism by heterotrophic denitrifying bacteria.

Diffusion and sorption of organic micropollutants in biofilms with varying thicknesses.

Solid-liquid partitioning is one of the main fate processes determining the removal of micropollutants in wastewater. Little is known on the sorption of micropollutants in biofilms, where molecular diffusion may significantly influence partitioning kinetics. In this study, the diffusion and the sorption of 23 micropollutants were investigated in novel moving bed biofilm reactor (MBBR) carriers with controlled biofilm thickness (50, 200 and 500 µm) using targeted batch experiments (initial concentration = 1 µg L-1, for X-ray contrast media 15 µg L-1) and mathematical modelling. We assessed the influence of biofilm thickness and density on the dimensionless effective diffusivity coefficient f (equal to the biofilm-to-aqueous diffusivity ratio) and the distribution coefficient Kd,eq (L g-1). Sorption was significant only for eight positively charged micropollutants (atenolol, metoprolol, propranolol, citalopram, venlafaxine, erythromycin, clarithromycin and roxithromycin), revealing the importance of electrostatic interactions with solids. Sorption equilibria were likely not reached within the duration of batch experiments (4 h), particularly for the thickest biofilm, requiring the calculation of the distribution coefficient Kd,eq based on the approximation of the asymptotic equilibrium concentration (t > 4 h). Kd,eq values increased with increasing biofilm thickness for all sorptive micropollutants (except atenolol), possibly due to higher porosity and accessible surface area in the thickest biofilm. Positive correlations between Kd,eq and micropollutant properties (polarity and molecular size descriptors) were identified but not for all biofilm thicknesses, thus confirming the challenge of improving predictive sorption models for positively charged compounds. A diffusion-sorption model was developed and calibrated against experimental data, and estimated f values also increased with increasing biofilm thickness. This indicates that diffusion in thin biofilms may be strongly limited (f â‰ª 0.1) by the high biomass density (reduced porosity).

Impact of external carbon dose on the removal of micropollutants using methanol and ethanol in post-denitrifying Moving Bed Biofilm Reactors.

Addition of external carbon sources to post-denitrification systems is frequently used in wastewater treatment plants to enhance nitrate removal. However, little is known about the fate of micropollutants in post-denitrification systems and the influence of external carbon dosing on their removal. In this study, we assessed the effects of two different types and availability of commonly used carbon sources -methanol and ethanol- on the removal of micropollutants in biofilm systems. Two laboratory-scale moving bed biofilm reactors (MBBRs), containing AnoxKaldnes K1 carriers with acclimated biofilm from full-scale systems, were operated in continuous-flow using wastewater dosed with methanol and ethanol, respectively. Batch experiments with 22 spiked pharmaceuticals were performed to assess removal kinetics. Acetyl-sulfadiazine, atenolol, citalopram, propranolol and trimethoprim were easily biotransformed in both MBBRs (biotransformations rate constants kbio between 1.2 and 12.9 L gbiomass-1 d-1), 13 compounds were moderately biotransformed (rate constants between 0.2 and 2 L gbiomass-1 d-1) and 4 compounds were recalcitrant. The methanol-dosed MBBR showed higher kbio (e.g., 1.5-2.5-fold) than in the ethanol-dosed MBBR for 9 out of the 22 studied compounds, equal kbio for 10 compounds, while 3 compounds (i.e., targeted sulfonamides) were biotransformed faster in the ethanol-dosed MBBR. While biotransformation of most of the targeted compounds followed first-order kinetics, removal of venlafaxine, carbamazepine, sulfamethoxazole and sulfamethizole could be described with a cometabolic model. Analyses of the microbial composition in the biofilms using 16S rRNA amplicon sequencing revealed that the methanol-dosed MBBR contained higher microbial richness than the one dosed with ethanol, suggesting that improved biotransformation of targeted compounds could be associated with higher microbial richness. During continuous-flow operation, at conditions representative of full-scale denitrification systems (hydraulic residence time = 2 h), the removal efficiencies of micropollutants were below 35% in both MBBRs, with the exception of atenolol and trimethoprim (>80%). Overall, this study demonstrated that MBBRs used for post-denitrification could be optimized to enhance the biotransformation of a number of micropollutants by accounting for optimal carbon sources and extended residence time.

Biofilm Thickness Influences Biodiversity in Nitrifying MBBRs-Implications on Micropollutant Removal.

In biofilm systems for wastewater treatment (e.g., moving bed biofilms reactors-MBBRs) biofilm thickness is typically not under direct control. Nevertheless, biofilm thickness is likely to have a profound effect on the microbial diversity and activity, as a result of diffusion limitation and thus substrate penetration in the biofilm. In this study, we investigated the impact of biofilm thickness on nitrification and on the removal of more than 20 organic micropollutants in laboratory-scale nitrifying MBBRs. We used novel carriers (Z-carriers, AnoxKaldnes) that allowed controlling biofilm thickness at 50, 200, 300, 400, and 500 µm. The impact of biofilm thickness on microbial community was assessed via 16S rRNA gene amplicon sequencing and ammonia monooxygenase (amoA) abundance quantification through quantitative PCR (qPCR). Results from batch experiments and microbial analysis showed that (i) the thickest biofilm (500 µm) presented the highest specific biotransformation rate constants (kbio, L g(-1) d(-1)) for 14 out of 22 micropollutants; (ii) biofilm thickness positively associated with biodiversity, which was suggested as the main factor for the observed enhancement of kbio; (iii) the thinnest biofilm (50 µm) exhibited the highest nitrification rate (gN d(-1) g(-1)), amoA gene abundance and kbio values for some of the most recalcitrant micropollutants (i.e., diclofenac and targeted sulfonamides). Although thin biofilms favored nitrification activity and the removal of some micropollutants, treatment systems based on thicker biofilms should be considered to enhance the elimination of a broad spectrum of micropollutants.