Magnus Christensson

Integrated fixed-film activated sludge ANITA™Mox process – a new perspective for advanced nitrogen removal

ANITA™Mox is a Veolia process using moving-bed biofilm reactor (MBBR) technology tested and validated in full-scale for energy- and cost-effective autotrophic N-removal from sidestream effluent using anammox (ANaerobic AMMonium OXidation) bacteria. In order to increase the ANITA™Mox process performances under different operating conditions (e.g. mainstream and sidestream application), substrate transport and accessibility inside the biofilm must be enhanced. In this work, (i) two laboratory scale biofilm ANITA™Mox reactors were operated using different configurations (IFAS - integrated fixed-film activated sludge - and MBBR) and (ii) the distribution of the anammox (AnAOB) and ammonia-oxidizing bacteria (AOB) in the suspended sludge and the biofilm was characterized using molecular tools (qPCR). This study showed that in IFAS configuration, the ANITA™Mox process achieved very high N-removal rate (up to 8 gN/m².d), which was three to four times higher than that achieved in the pure MBBR mode. The high concentration of suspended solids (mixed liquor suspended solids (MLSS)) in the bulk obtained within the IFAS mode induces a very efficient bacterial distribution between the AOB and AnAOB population. AnAOB activity mainly occurs in the biofilm (96% of total AnAOB in the reactor), whereas nitritation by AOB mostly takes place in the suspended phase (93% of total AOB). This spatial distribution observed in the IFAS reactor results from a natural selection due to more easily substrate accessibility for AOB in the bulk (NH4(+), O2) creating higher nitrite concentration in the bulk liquid compare to pure MBBR mode. The efficient control of MLSS level in the IFAS reactor is a key parameter to enhance the nitrite production by AOB and increase the substrate availability in the AnAOB-enriched biofilm leading to higher N-removal rate. These promising results obtained at laboratory scale have been further confirmed in on-going full-scale IFAS ANITA™Mox trials opening new roads for the widespread application of a very compact and robust ANITA™Mox process for sidestream but also mainstream cost-effective N-removal.

Biodegradation of pharmaceuticals in hospital wastewater by a hybrid biofilm and activated sludge system (Hybas)

Hospital wastewater contributes a significant input of pharmaceuticals into municipal wastewater. The combination of suspended activated sludge and biofilm processes, as stand-alone or as hybrid process (hybrid biofilm and activated sludge system (Hybas™)) has been suggested as a possible solution for hospital wastewater treatment. To investigate the potential of such a hybrid system for the removal of pharmaceuticals in hospital wastewater a pilot plant consisting of a series of one activated sludge reactor, two Hybas™ reactors and one moving bed biofilm reactor (MBBR) has been established and adapted during 10 months of continuous operation. After this adaption phase batch and continuous experiments were performed for the determination of degradation of pharmaceuticals. Removal of organic matter and nitrification mainly occurred in the first reactor. Most pharmaceuticals were removed significantly. The removal of pharmaceuticals (including X-ray contrast media, β-blockers, analgesics and antibiotics) was fitted to a single first-order kinetics degradation function, giving degradation rate constants from 0 to 1.49 h(-1), from 0 to 7.78 × 10(-1)h(-1), from 0 to 7.86 × 10(-1)h(-1) and from 0 to 1.07 × 10(-1)h(-1) for first, second, third and fourth reactors respectively. Generally, the highest removal rate constants were found in the first and third reactors while the lowest were found in the second one. When the removal rate constants were normalized to biomass amount, the last reactor (biofilm only) appeared to have the most effective biomass in respect to removing pharmaceuticals. In the batch experiment, out of 26 compounds, 16 were assessed to degrade more than 20% of the respective pharmaceutical within the Hybas™ train. In the continuous flow experiments, the measured removals were similar to those estimated from the batch experiments, but the concentrations of a few pharmaceuticals appeared to increase during the first treatment step. Such increase could be attributed to de-conjugation or formation from other metabolites.

Integration of biopolymer production with process water treatment at a sugar factory.

The present investigation has focused on generating a surplus denitrifying biomass with high polyhydroxyalkanoate (PHA) producing potential while maintaining water treatment performance in biological nitrogen removal. The motivation for the study was to examine integration of PHA production into the water treatment and residuals management needs at the Suiker Unie sugar beet factory in Groningen, the Netherlands. At the factory, process waters are treated in nitrifying-denitrifying sequencing batch reactors (SBRs) to remove nitrogen found in condensate. Organic slippage (COD) in waters coming from beet washing is the substrate used for denitrification. The full-scale SBR was mimicked at laboratory scale. In two parallel laboratory scale SBRs, a mixed-culture biomass selection strategy of anoxic-feast and aerobic-famine was investigated using the condensate and wash water from Suiker Unie. One laboratory SBR was operated as conventional activated sludge with long solids retention time similar to the full-scale (SRT >16 days) while the other SBR was a hybrid biofilm-activated sludge (IFAS) process with short SRT (4-6 days) for the suspended solids. Both SBRs were found to produce biomass with augmented PHA production potential while sustaining process water treatment for carbon, nitrogen and phosphorus for the factory process waters. PHA producing potential in excess of 60 percent g-PHA/g-VSS was achieved with the lab scale surplus biomass. Surplus biomass of low (4-6 days) and high (>16 days) solids retention time yielded similar results in PHA accumulation potential. However, nitrification performance was found to be more robust for the IFAS SBR. Assessment of the SBR microbial ecology based on 16sDNA and selected PHA synthase genes at full-scale in comparison to biomass from the laboratory scale SBRs suggested that the full-scale process was enriched with a PHA storing microbial community. However, structure-function relationships based on RNA levels for the selected PHA synthases could not be established and, towards this ambition, it is speculated that a wider representation of PHA synthesases would need to be monitored. Additionally at the factory, beet tail press waters coming from the factory beet residuals management activities are available as a carbon source for PHA accumulation. At pilot scale, beet tail press waters were shown to provide a suitable carbon source for mixed culture PHA production in spite of otherwise being of relatively low organic strength (≤ 10 g-COD/L). A copolymer of 3-hydroxybutyrate with 3-hydroxyvalerate (PHBV with 15% HV on a molar basis) of high thermal stability and high weight average molecular mass (980 kDa) was produced from the beet tail press water. The mixed culture accumulation process sustained PHA storage with parallel biomass growth of PHA storing bacteria suggesting a strategy to further leverage the utilization of surplus functional biomass from biological treatment systems. Integration of PHA production into the existing factory water management by using surplus biomass from condensate water treatment and press waters from beet residuals processing was found to be a feasible strategy for biopolymer production.

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.

Three-dimensional stratification of bacterial biofilm populations in a moving bed biofilm reactor for nitritation-anammox.

Moving bed biofilm reactors (MBBRs) are increasingly used for nitrogen removal with nitritation-anaerobic ammonium oxidation (anammox) processes in wastewater treatment. Carriers provide protected surfaces where ammonia oxidizing bacteria (AOB) and anammox bacteria form complex biofilms. However, the knowledge about the organization of microbial communities in MBBR biofilms is sparse. We used new cryosectioning and imaging methods for fluorescence in situ hybridization (FISH) to study the structure of biofilms retrieved from carriers in a nitritation-anammox MBBR. The dimensions of the carrier compartments and the biofilm cryosections after FISH showed good correlation, indicating little disturbance of biofilm samples by the treatment. FISH showed that Nitrosomonas europaea/eutropha-related cells dominated the AOB and Candidatus Brocadia fulgida-related cells dominated the anammox guild. New carriers were initially colonized by AOB, followed by anammox bacteria proliferating in the deeper biofilm layers, probably in anaerobic microhabitats created by AOB activity. Mature biofilms showed a pronounced three-dimensional stratification where AOB dominated closer to the biofilm-water interface, whereas anammox were dominant deeper into the carrier space and towards the walls. Our results suggest that current mathematical models may be oversimplifying these three-dimensional systems and unless the multidimensionality of these systems is considered, models may result in suboptimal design of MBBR carriers.

Removal of pharmaceuticals in conventionally treated wastewater by a polishing moving bed biofilm reactor (MBBR) with intermittent feeding

Previous studies have demonstrated that aerobic moving bed biofilm reactors (MBBRs) remove pharmaceuticals better than activated sludge. Thus we used a MBBR system to polish the effluent of an activated sludge wastewater treatment plant. To overcome that effluent contains insufficient organic matter to sustain enough biomass, the biofilm was intermittently fed with raw wastewater. The capacity of pharmaceutical degradation was investigated by spiking pharmaceuticals. Actual removal during treatment was assessed by sampling the inlets and outlets of reactors. The removal of the majority of pharmaceuticals was enhanced through the intermittent feeding of the MBBR. First-order rate constants for pharmaceutical removal, normalised to biomass, were significantly higher compared to other studies on activated sludge and suspended biofilms, especially for diclofenac, metoprolol and atenolol. Due to the intermittently feeding, degradation of diclofenac occurred with a half-life of only 2.1h and was thus much faster than any hitherto described wastewater bioreactor treatment.

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.

Partial nitrification in MBBRs for mainstream deammonification with thin biofilms and alternating feed supply

A new principle for mainstream nitrogen removal through nitritation followed by anammox was studied in a two-stage moving bed biofilm reactor (MBBR) configuration. The first stage was optimized for nitritation by using thin biofilms and a feed alternating between synthetic mainstream wastewater at 15°C and, for shorter periods, synthetic reject water at 30 °C. The exposure of the biofilm to reject water conditions aimed to improve the growth conditions for ammonia oxidizing bacteria, while inhibiting nitrite oxidizing bacteria. The biofilm thickness was maintained below 200 μm to ensure high exposure of the total biomass to the bulk reactor conditions. Nitritation was successfully achieved in the configuration, with a nitrite accumulation ratio above 75% during the majority of the study, and ammonia removal rates between 0.25 and 0.50 g NH4-N/L,d. The anoxic second stage, optimized for anammox, was fed with the effluent from the nitritation reactor, reaching nitrogen removal rates above 0.20 g TN/L,d.

Mainstream Deammonification with ANITA™Mox Process

ANITATMMox process was successfully applied to treat mainstream effluent with low COD content at both pilotand prototype-scale using the Integrated Fixed-Film Activated Sludge (IFAS) configuration. The IFAS ANITA Mox unique feature of easy and robust separation system (i.e. media sieves) to retain anammox biomass in the reactor is a clear advantage for mainstream application. Continuous bioaugmentation strategies based on carriers recirculation and alternating feed between sidestream and mainstream have been developed to boost anammox activity and strongly repress the NOB. Preliminary pilot trials on low COD effluent from C-stage MBBR achieved NH4-removal rate of 0.8-1.3 gN/m 2 .d for operating temperature between 17-23°C. Results from a 50m 3 prototype where bioaugmentation strategy was implemented show efficient suppression of the NOB activity while achieving a NH4-removal rate up to 0.2-0.3 kgN/m 3 .d at 16-18°C. Additional studies are underway to further demonstrate the advantage of the IFAS ANITA Mox process for mainstream deammonification application and how this process can be integrated in a general WWTP scheme to deliver all the foresee benefits on energy recovery and OPEX savings.

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.