Developing Indicators of Nutrient Pollution in Streams Using 16S rRNA Gene Metabarcoding of Periphyton-Associated Bacteria

Abstract

Indicators based on nutrient-biota relationships in streams can inform water quality restoration and protection programs. Bacterial assemblages could be particularly useful indicators of nutrient effects because they are species-rich, important contributors to ecosystem processes in streams, and responsive to rapidly changing conditions. Here, we sampled 25 streams weekly (12–14 times each) and used 16S rRNA gene metabarcoding of periphyton-associated bacteria to quantify the effects of total phosphorus (TP) and total nitrogen (TN). Threshold indicator taxa analysis identified assemblage-level changes and amplicon sequence variants (ASVs) that increased or decreased with increasing TP and TN concentrations (i.e., low P, high P, low N, and high N ASVs). Boosted regression trees confirmed that relative abundances of gene sequence reads for these four indicator groups were associated with nutrient concentrations. Gradient forest analysis complemented these results by using multiple predictors and random forest models for each ASV to identify portions of TP and TN gradients at which the greatest changes in assemblage structure occurred. Synthesized statistical results showed bacterial assemblage structure began changing at 24 µg TP/L with the greatest changes occurring from 110 to 195 µg/L. Changes in the bacterial assemblages associated with TN gradually occurred from 275 to 855 µg/L. Taxonomic and phylogenetic analyses showed that low nutrient ASVs were commonly Firmicutes, Verrucomicrobiota, Flavobacteriales, and Caulobacterales, Pseudomonadales, and Rhodobacterales of Proteobacteria, whereas other groups, such as Chitinophagales of Bacteroidota, and Burkholderiales, Rhizobiales, Sphingomonadales, and Steroidobacterales of Proteobacteria comprised the high nutrient ASVs. Overall, the responses of bacterial ASV indicators in this study highlight the utility of metabarcoding periphyton-associated bacteria for quantifying biotic responses to nutrient inputs in streams.

Salinization and sedimentation drive contrasting assembly mechanisms of planktonic and sediment‐bound bacterial communities in agricultural streams

“…Genetic approaches like 16S rRNA amplicon sequencing have been used in other systems to better understand planktonic bacterial responses to increasing nutrient concentrations. In other less modified watersheds prone to P enrichment, bacteria begin to respond to increasing TP at 0.052 mg L −1 (upper limit of response range = 0.195 mg L −1 ) (LeBrun et al, 2018a;Pilgrim et al, 2022), which is a considerably lower than our observation of 0.080 mg L −1 (upper limit of response range = 0.220 mg L −1 ). Bacterial responses along TN gradients also occurred at lower, ranges from 0.275-0.9 mg L −1 (LeBrun et al, 2018a;Wagenhoff et al, 2017) in other watersheds compared to our observed range of 0.420-1.020 mg L −1 .…”

“…Lastly, we ranked variables based on response strength (maximum summed z ‐score) to identify which variables were most likely to induce community‐level responses. TITAN has been used recently in other systems to identify bacterial responses to stressor gradients using amplicon sequencing data (Pilgrim et al, 2022; Simonin et al, 2019). Because median D p generally decreased with increasing agricultural land use while sediment nutrients and dissolved ions increased, the negative response for median D p is shown next to the positive responses for other variables to better represent the impacts of increasing agriculture.…”

Periodic Addition of Glucose Suppressed Cyanobacterial Abundance in Additive Lake Water Samples during the Entire Bloom Season

“…Caulobacterales were reported to be more common in low total N and P environments [42]. Caulobacterales and Sphingomonadales populations are promoted in higher temperatures, lower concentrations of oxygen, and increased dissolved organic matter [43].…”

Exploring freshwater stream bacterial communities as indicators of land use intensity

“…To date, relatively few studies have attempted to monitor microbial DNA as a routine tool to monitor freshwater catchments [ 24 , 27 , 43 ], with assessments of fish and macroinvertebrate communities remaining more common [ 15 ]. Capitalising on decades of research facilitating the establishment of macroinvertebrate indices of community health, early efforts used regression analyses to identify microbial community attributes associated with different macroinvertebrate community compositions (i.e., macroinvertebrate community types indicative or poor- to high-quality stream environments, Lau et al [ 27 ]) .…”