Biopharm - the Influence of Macro-substrates & Conditioning on Pharmaceutical Removal Rates by Moving Bed Biofilm Reactors

Organic micropollutants with endocrine disruptive properties are present in the aquatic environment. A major part of their emission is caused by municipal wastewater treatment plants (WWTPs). For this reason, a vast amount of research has been undertaken to remove xenobiotics from municipal wastewater by developing post-treatment technologies with some success. However, these technologies cause considerable environmental costs due their high demand for electrical energy implicating an increase in CO2 emissions. Consequently, existing biological treatments need first to be better understood and subsequently optimized regarding xenobiotic removal before post-treatments are employed.

The study focused on the fate of xenobiotics during biological wastewater treatment. In particular, metabolic strategies of bacteria degrading pharmaceuticals were investigated within moving bed biofilm reactor (MBBR) processes. Two main objectives were tracked. On the one hand, it was to unfold the impact of macro-substrates in terms of type and molecular complexity on the activity of microorganisms and consequently pharmaceutical degradation performance. On the other hand, the study was set out to explore the adaptation of metabolic means regarding exoenzymes and consortia structure during continuous (long-term) exposure to pharmaceuticals. Accordingly, the ability to increase microbial competences during pharmaceutical short-term pulses was the general target of investigation. Both conditions continuous substance flow and short-term peak loads of xenobiotics are believed to occur in urban WWTPs.

A pilot MBBR was set up next to a domestic WWTP. The pilot treated municipal sewage and served as inoculation reservoir for biofilm carriers used for in-depth laboratory experiments. The latter comprised six lab-scale MBBRs featuring flow through operation under controlled conditions regarding temperature, dissolved oxygen, pH, influent flow and influent load. The reactors were conditioned over four weeks with a synthetic sewage providing substrates and micro-nutrients in a similar manner as expected under real conditions. Biofilm was monitored by respirometry and a series of enzyme assays using fluorogenic substrates to capture esterase, phosphatase, alpha- and beta-glucosidase and aminopeptidase activity. All enzymes are essential during organic carbon metabolism. An array of macro-substrates with different molecular complexity was triggering individual enzyme activity profiles. After conditioning, 12 pharmaceuticals being subject to a range of anticipated metabolic pathways and degradation rates were spiked into the MBBRs. Their degradation kinetics were measured by liquid chromatography coupled with tandem mass spectrometry (LC MS/MS). Pseudo first-order kinetics revealed substrate related fingerprints and showed that readily biodegradable substrate leads generally to good pharmaceutical degradation performance compared to synthetic sewage with a mixture of several high molecular organic substrates. The latter was designed to induce the greatest metabolic effort of tested substrates before microbial uptake occurs. However, single substrates triggering exoenzyme activity in a more targeted manner such as maltose and cellobiose showed positive impact on the pseudo first-order rate coefficients of particular pharmaceuticals such as atenolol and diclofenac. Accordingly, alpha-glucosidase activity was found to be directly proportional to atenolol degradation kinetics.

Phylogenetic characterisation of DNA and RNA involving state-of-the-art 16S ribosomal rRNA gene amplification and sequencing techniques was used to explore community structures. Prokaryote diversity in lab-scale MBBRs was in agreement to previous studies which investigated microbial consortia in full-scale systems. Multivariate analysis revealed that bacteria are adapting their active gene pool when the beta-blocker atenolol is continuously present with a concentration in ug/L range. Differential analysis unfolded that the prokaryotic genera Delftia and Lysobacter were thereby exclusively benefiting from the exposure to atenolol. Yet, compared to the influence of macro-substrates, biomass conditioning (training) with atenolol and diclofenac had no notable impact on the degradation performance of pharmaceutical short-term pulses.

The outstanding comprehensive character of the study which encompassed sophisticated experimental design and powerful analytical tools from different scientific domains uncovered some interesting insights in xenobiotic degradation processes. The results finally show how biomass is reacting towards the presence of primary carbon sources and organic micro-pollutants. The outcomes highlight the importance of WWTP influent characterization being indicative of metabolic activity and therefore degradation capacity of xenobiotics. The study further suggests that xenobiotic metabolism and co-metabolism co-exist during biological treatment processes. Co-metabolism is the decisive actor when adaption time is relatively short as it was the case during the lab-scale experiments compared to real conditions. Further, the study indicates some potential of process enhancement of WWTPs ranging from straightforward implementations such as external carbon sources to more elaborated processes of bioaugmentation.