Microbiome response in an urban river system is dominated by seasonality over wastewater treatment upgrades

Microorganisms such as coliform-forming bacteria are commonly used to assess freshwater quality for drinking and recreational use. However, such organisms do not exist in isolation; they exist within the context of dynamic, interactive microbial communities which vary through space and time. Elucidating spatiotemporal microbial dynamics is imperative for discriminating robust community changes from ephemeral ecological trends, and for improving our overall understanding of the relationship between microbial communities and ecosystem health. We conducted a seven-year (2013-2019) microbial time-series investigation in the Chicago Area Waterways (CAWS): an urban river system which, in 2016, experienced substantial upgrades to disinfection processes at two wastewater reclamation plants (WRPs) that discharge into the CAWS and improved stormwater capture, to improve river water quality and reduce flooding. Using culture-independent and culture-dependent approaches, we compared CAWS microbial ecology before and after the intervention. Examinations of time-resolved beta distances between WRP-adjacent sites showed that community similarity measures were often consistent with the spatial orientation of site locations to one another and to the WRP outfalls. Fecal coliform results suggested that upgrades reduced coliform-associated bacteria in the effluent and the downstream river community. However, examinations of whole community changes through time suggest that the upgrades did little to affect overall riverine community dynamics, which instead were overwhelmingly driven by yearly patterns consistent with seasonality. Such results emphasize the dynamic nature of microbiomes in open environmental systems such as the CAWS, but also suggest that the seasonal oscillations remain consistent even when perturbed. Importance This study presents a systematic effort to combine 16S rRNA gene amplicon sequencing with traditional culture-based methods to evaluate the influence of treatment innovations and systems upgrades on the microbiome of the Chicago Area Waterway System, representing the longest and most comprehensive characterization of the microbiome of an urban waterway yet attempted. We found that the systems upgrades were successful in improving specific water quality measures immediately downstream of wastewater outflows. Additionally, we found that the implementation of the water quality improvement measures to the river system did not disrupt the overall dynamics of the downstream microbial community, which remained heavily influenced by seasonal trends.


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High quality fresh water is a critical natural asset that is under increasing risk of overuse  Without this intervention, Combined Sewer Overflow (CSO) directly discharges into the CAWS 116 during rainy weather and high flow conditions. The implementation of these initiatives during 117 our longitudinal study provided a unique opportunity to examine how microbial community 118 dynamics in a wastewater-impacted water system may be affected when presented with 119 substantial wastewater management upgrades.

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The primary goals of this study were to a) characterize the microbial communities of the 121 CAWS and its spatio-temporal dynamics across 12 riverine sites and 2 wastewater treatment 122 plants for 63 timepoints over 7 years, b) compare such characterizations to the trends found in a 123 traditional, culture-dependent approach to measuring water quality, and c) examine the impact of 124 water quality improvement interventions on the microbial ecology of the CAWS. The 125 combination of sequencing data, fecal coliform data, and physicochemical data thus enabled us 126 to parameterize the microbial ecology of the CAWS to gain nuanced insights into how 127 environmental disturbance impacts the ecology of this already dynamic microbial system.  Sample collection 139 All CAWS locations were grab-sampled monthly for water (i.e. river, effluent, sewage) 140 and sediment by MWRD personnel. A total of 500 mL of surface water and 100 g of sediment 141 were collected in sterile containers for microbiome analysis. Temperature, pH, conductivity, and 142 turbidity of water samples were measured using a handheld YSI multiparameter digital water 143 quality meter. NO2 -/NO3ratios, NH3, and PO4 3values were measured using a Lachat      Alpha and beta diversity analysis 188 Alpha and beta diversity were calculated between different sample types as well as by year.

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Alpha diversity for all sample types was measured using Shannon's index. Beta diversity was    March relative to August, and the 10 most differentially abundant ASVs in August relative to 228 March, separately with both water and effluent samples. Once March-associated and August-229 associated ASVs were identified, we examined changes in their log-ratios across the entire rarefied 230 dataset of water and effluent samples to test whether the identified ASVs follow a predictable 231 seasonal gradient through time. We used the following formula: with a pseudo count of one applied to the table before taking the ratio to avoid undefined values. and effluent types were highly significant (p < 0.001).

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Beta diversity analyses using both weighted and unweighted UniFrac distance metrics 286 indicated significant differences in community composition across sample types (weighted 287 UniFrac PERMANOVA p < 0.001, unweighted UniFrac PERMANOVA p < 0.001, Figure 1B).  -Supplemental S3). When aggregating beta distance results across sites, we found evidence for 308 seasonality in effluent and water but not sewage and sediment ( Figure 1C). Water and effluent samples contained strong seasonal signals in community composition, 318 therefore we performed compositionally-aware differential abundance analyses (Morton et al. 3B, see Supplemental Table S2 for PERMANOVA results).

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Examinations of time-resolved beta distances between WRP-adjacent sites showed that 344 community similarity measures were often consistent with the spatial orientation of site locations 345 to one another and to the WRP outfalls, particularly with water samples ( Figure 3C) Within sediment communities, we found that each site also contained compositionally 357 distinct microbial communities whose differences remained robust through time (Supplementary   358   Table S2, Figure 3C). Additionally, we note that unlike in the water communities, sediment 359 community similarities did not follow a spatial orientation consistent with site location; microbial 360 communities from all sites had mostly the same beta diversity distance to the effluent, for both the 361 Calumet and O'Brien regions ( Figure 3C). Similar results were found when measuring beta 362 distances with weighted UniFrac distances (Supplemental figure S5). 363 We found minimal evidence that the WRP upgrades caused any significant changes in site-  Figure 3C). Re-analysis with weighted UniFrac distances also did not identify meaningful 368 community changes as a result of the intervention (Supplemental figure S5). Unsurprisingly, 369 differential abundance models attempting to identify key microbial ASVs that significantly  (Table 1). Overall, we found no 404 statistical support that the WRP upgrades caused significant shifts in physicochemical to the natural seasonal variability captured at upstream sites ( Figure S3).  coliform-associated bacteria to the waterway besides the WRP effluent and pre-TARP CSO events. 477 We additionally note that this described spatio-temporal pattern was nearly identical between the  Notably, we found that sewage and sediment did not appear to be affected by seasonal sewage to treated effluent, although confirmation would require a more nuanced study. As for the 511 sediment, communities were found to be relatively stable through time and instead were most 512 definable by site, suggesting that despite their close proximity to water, their dynamics are distinct 513 from those of water. 514 River systems are unique environments for examining microbial community dynamics, in 515 that they offer spatial complexity that is integrated with temporal patterns. In this seven-year study, 516 we characterized the microbial community of the CAWS across a broad spatio-temporal gradient 517 and described its response to a prominent environmental change. Our results from this seven-year 518 long microbiome study are nuanced; on one hand, we provide evidence that wastewater Competing interests 535 The authors declare that they have no competing interests.