Elena Torresi

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.

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.

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.