Metagenomics and Metatranscriptomics Suggest Pathways of 3-Chloroaniline Degradation in Wastewater Reactors

Abstract

Biological wastewater treatment systems are often affected by major shifts in influent quality, including the input of various toxic chemicals. Yet the mechanisms underlying adaptation of activated sludge process performance when challenged with a sustained toxin input have not been studied in a controlled and replicated experiment. Three replicate bench-scale bioreactors were subjected to a chemical disturbance in the form of 3-chloroaniline (3-CA) input over 132 days, following an acclimation period of 58 days, while three control reactors received no 3-CA input. The nitrification process was initially affected by 3-CA input; yet all three treatment reactors evolved to biologically degrade 3-CA within three weeks of the experiment, resulting in partial nitrification recovery. 16S rRNA amplicon sequencing revealed that ammonia oxidizers were initially impacted by 3-CA, and a new ammonia oxidizing community emerged with the onset of 3-CA degradation. Combining process and microbial community data from amplicon sequencing with potential functions gleaned from assembled metagenomics and metatranscriptomics data, 3-CA degradation was shown to likely occur via a phenol monooxygenase followed by ortho-cleavage of the aromatic ring. The relative abundance of genera Gemmatimonas, Saprospiraceae OLB8 and Taibaiella correlated significantly with 3-CA degradation. This study demonstrated the impact of a sustained stress on the activated sludge community and wastewater treatment process and its subsequent recovery. Using a combination of techniques in a controlled and replicated experiment, we showed that microbial communities can evolve degradative capacity following a sustained xenobiotic input, specific functions like nitrification can fully or partially recover and targeted culture-independent approaches can be used to elucidate the mechanisms of adaptation.

Diversity and Ecophysiology of the Genus OLB8 and Other Abundant Uncultured Saprospiraceae Genera in Global Wastewater Treatment Systems

“…Interestingly, filamentous Saprospiraceae such as Haliscomenobacter can occasionally be involved in bulking problems in WWTPs (Xu et al, 2018). However, the most abundant genera, such as OLB8, are widely found in different environments such as activated sludge (Al Ali et al, 2020;Aqeel and Liss, 2020;Seshan et al, 2021) and anammox reactors (Speth et al, 2016). The first metagenome-assembled genome (MAG) of the genus OLB8 was recovered from partial nitrification anammox bioreactors, where the bacterium was known to function as an aerobe heterotroph (Speth et al, 2016).…”

“…The first metagenome-assembled genome (MAG) of the genus OLB8 was recovered from partial nitrification anammox bioreactors, where the bacterium was known to function as an aerobe heterotroph (Speth et al, 2016). Members of the genus OLB8 were also found in high abundance in reactors with polyphosphate-accumulating organism (PAO)enriched biomass (Arumugam et al, 2019), bioreactors with a high ratio of food to microorganisms (Roy et al, 2020), reactors with increased concentration of ammonia (Aqeel and Liss, 2020), deammonification reactors (Chini et al, 2020), and also in reactors related to degradation of aromatic compounds (Seshan et al, 2021). Over the years, OLB8 bacteria were speculated to be involved in phosphorus and nitrogen removal in addition to the degradation of different carbon compounds in WWTPs, but their function was never fully resolved.…”

Press Xenobiotic 3-Chloroaniline Disturbance Favors Deterministic Assembly with a Shift in Function and Structure of Bacterial Communities in Sludge Bioreactors

“…However, the general rise in percent removal toward the end of the experiment, which coincided with the rise in effluent nitrite concentration, indicates the recovery of ammonia oxidation. A more detailed analysis of changes in process performance under the 3-CA disturbance in this experiment was the focus of an additional study . In that work, the analysis of process variables are explored in full SBR cycles during the course of the experiment, together with an assessment of the metabolic mechanisms behind 3-CA removal, for a deeper understanding of the effect of the 3-CA disturbance on activated sludge performance.…”

“…In control reactors, complete nitrification was achieved by the end of an SBR cycle, meaning that after discharging half the reactor volume there was enough nitrate present to support denitrifying organisms like Paracoccus and Alicycliphilus , which could use it as a terminal electron acceptor during the anoxic phase of the cycle. The prevailing nitrifying genera, Nitrosomonas and Nitrospira , were relatively less abundant in treatment reactors than in control reactors, coincident with a reduction in ammonia and nitrite oxidation activity (details in Seshan et al). On the other hand, organisms like Candidatus Competibacter (a glycogen accumulating organism) and Gemmatimonas (a PAO) prevailed in treatment reactors, as was also reported for microcosm sludge bioreactors under a similar 3-CA pollutant disturbance .…”

“…Composition-aware correlation network analysis on day 164 via SparCC also displayed separate clusters of bacterial ASVs based on node modularity (Figure 4B). See Seshan et al 31 for a detailed analysis of the effect of a 3-CA press disturbance on nitrifying genera and the putative microorganisms involved in 3-CA degradation.…”