Characterization of the Microbial Communities Within Managed Bioremediation Systems: A Culture- and Genomics-Based Approach

Abstract

Wastewater treatment facilities (WWTF) and biobeds are engineered systems developed to mitigate nutrient and pesticide pollution, respectively, through microbial biodegradation. While understanding the microbiomes of these systems may help inform on optimization and maintenance, genomic characterization of these systems is generally lacking, particularly in reference to biobeds, with many questions regarding which microbes are present, and how these microbial communities change across the treatment process over time. Within this, an additional point of interest relates to the unintended negative effects these systems might pose, specifically as potential environments for the enrichment of antibiotic resistance genes (ARGs). WWTFs have become known hubs for ARG dissemination, with studies reporting increases in both ARGs and antibiotic resistant (ABR) bacteria in effluentreceiving water systems. Here, through culturing, it was identified that the recently upgraded Regina Wastewater Treatment Plant is capable of reducing bacterial populations by 95-99 %, but that resistance to ampicillin, trimethoprim and meropenem is high, and that ABR bacteria are surviving the treatment process. Additionally, through 16S rRNA and cpn60 amplicon sequencing, a significant change in community composition was observed over the treatment continuum, which is most likely driven by significant decreases in diversity, driven through UV disinfection, although results suggest that UV treatment may be selecting for bacteria housing trimethoprim-resistance genes. Finally, multiple multidrug resistant (MDR) Gammaproteobacteria were observed to be surviving the wastewater treatment continuum, and are being released into the Qu’Appelle Valley watershed, indicating that there may be potential for ABR dissemination through this aquatic ecosystem. Lastly, through studying the microbiome of this wastewater system, the sequencing results of the two different amplicon targets – 16S rRNA and cpn60 – were compared. Results suggest a high incidence of primer bias, and indicated that the results provided from these two methods are capable of arriving at different conclusions regarding taxonomy and statistical significance. Biobeds were studied through the context of taxonomy, degradative genes, and also their potential for ARG enrichment through metagenomic and metatranscriptomic sequencing of the Lethbridge Biobed system. Results show that pesticides enrich for bacteria commonly associated with xenobiotic degradation, such as Afipia, Sphingopyxis and Pseudomonas, and enrich for xenobiotic-degrading genes, such as peroxidases, oxygenases, and hydroxylases, among others; the transcription of these genes was able to be directly linked to Pseudomonas, Oligotropha, Mesorhizobium, Rhodopseudomonas, and Stenotrophomonas taxa. Sequencing analyses further demonstrated an absence of ligninolytic fungi, which is contrary to other microbially characterized biobeds, suggesting that biobeds are highly variable in their microbial structure, and that biobeds can function effectively in the absence of fungi. Finally, because ABR selection and enrichment is driven by increased stress, which leads to increased rates of horizontal gene transfer and ARG acquisition, genes that are related to stress response systems, as well as genetic recombination were statistically analyzed alongside ARGs. It was found that the pesticide concentrations being applied to a biobed was high enough to enrich for, and transcribe, genes related to a variety of stress responses, such as repair systems and metabolism modulation, genetic recombination, such as conjugation and plasmids, and ARGs, such as aminoglycoside and MDR genes, showing that biobed systems may need to be monitored for ABR dissemination.