This study aimed to analyze the difference between anthropogenic microplastic and natural stone substrates normally present in the environment to examine potential differences in the microbial communities that formed over 90 days in a freshwater riverine system. An additional variable of site impairment was added to evaluate the influence of water body health on microbiome development. Plated coliforms served as the metric to track microbiome adhesion over time and compared to the sequencing data to identify the presence or absence of potentially harmful organisms. Previous testing on this region of the Quinnipiac River used membrane filtration methodology for colony culturing (Mitch et al., 2010) using E.coli as the fecal indicator. With the advent of next-generation sequencing, these indicator species have broadened, and analyses can determine point sources and chronic conditions (McLellan and Eran, 2014). Our study demonstrates that 16S rRNA analysis in freshwater riverine systems provides greater resolution of microplastic microbiome attachment beyond E.coli and that other enteropathogens may be in higher abundance, thus necessitating the inclusion of metagenomic sequencing in water quality monitoring.
The sites selected for this study were purposefully chosen based on their waterbody health status to analyze the impact of site on coliform accumulation. The impaired Quinnipiac River location is in proximity to one of three upriver wastewater treatment plants (Cheshire, CT) along the main river artery. Conversely, the unimpaired Honeypot Brook location is a tributary of the artery fed by surface and groundwater. We found no significant difference in site location for temperature and salinity of the water quality indicators that influence biome development (Oberbeckmann et al., 2018; Marsay et al., 2023). Of the other parameters, dissolved oxygen was lower during the study period, and an elevated D60 pH level was noted at the impaired site. However, with limited data, we cannot speculate on seasonal influence as our study was conducted over the summer months, but future directions could compare winter and summer sampling periods.
Microbiome analysis from 16S rRNA gene sequencing showed that three Proteobacteria classes (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria) were abundant across site and substrate. Wu et al., 2019 found similar percentages (60–77%) when comparing lab-cultured riverine microplastic and stone biofilms. Alphaproteobacteria and Gammaproteobacteria have been shown to establish the microbiome early in the colonization process in marine environments (McCormick et al., 2016; De Tender et al., 2015). Plastic type can also be influential in enriching these classes (Qiang et al., 2021). Similar colonization assemblage has been observed in fresh and waste riverine water where Gammaproteobacteria were more abundant on polyethylene and Betaproteobacteria on polystyrene; however, differences in the representation was theorized to be due to the surrounding environmental flora rather than polymer type (Parrish and Fahrenfeld, 2019).
When looking at the total coliform counts, the impaired site and microplastic substrate showed higher median counts than the unimpaired and stone substrate. However, based on the generalized linear model analyses, both site and substrate appear to additively have the most significant influence on coliform adhesion, suggesting a co-influential action. A laboratory-simulated model using differing concentrations of microplastic substrate and wastewater-treated effluent mixed with fresh riverine water found that the surrounding water heavily influenced the microplastic biofilm community (Eckert et al. (2018). Further, researchers found that higher concentrations of microplastics may drive colonization through the probability of contact with a new particle or through quorum sensing, signaling nearby organisms to attach. As such, a higher microplastic burden in a waterbody would offer more opportunities to colonize these particles. Qiang et al. (2021) found similar evidence of habitat influence in another laboratory-cultured study of the Raritan River in New Jersey, a tidally influenced river similar to the Quinnipiac River Watershed. In this study, microplastic biofilm communities differed in freshwater and estuarine sections of the same river, underscoring the influence of site-specific colonization.
Our alpha diversity indices suggested lower diversity in microplastic substrate over stone, which aligns with similar studies looking at artificial and natural substrates wherein lower diversity can be attributed to critical factors in microplastic microbiome selectivity, including the polymer type and pioneer colonization (Miao et al., 2019; McCormick et al., 2016). However, field-collected microplastics from river estuaries in the Mediterranean Sea and the Atlantic Ocean determined that marine community composition is also primarily geographically influenced; further, these researchers found no common “core” of organisms in the biofilm and concluded that each ‘plastisphere’ should be considered a separate ecosystem, unique from one another (Marsay et al., 2023).
Reviews of opportunistic infections by water-borne or water-based organisms underscore the global threat of acquiring these diseases from impaired freshwater systems (Stec et al., 2022; Borque and Vinetz, 2018). This particular section of the river and others within the watershed are unsuitable for recreation, fishing, or sustaining aquatic life. Based on plated results, we did confirm the presence of fecal coliforms in higher abundance on the microplastic substrate at the impaired (QR) site. However, these data only present a generalized picture of waterbody health. Here, through sequence analysis of D90 genera from the Enterobacteriaceae class, we confirmed that the genera Escherichia were not a significant driver of biomass accumulation (Fig. S3). Interestingly, non-Escherichia genera were more abundant, suggesting that expanding analyses to all species in the Enterobacteriaceae genera is warranted. Additionally, the results showed significantly increased relative abundance for several other pathogenic genera. Wastewater-enriched systems host a wide diversity of organisms, many of which can survive the treatment processes (Varela and Manaia, 2013); the presence of these non-fecal genera further supports high-resolution sequence methodologies.
Research into freshwater riverine systems is significant due to their proximity to urban areas and the services provided through transport, wastewater discharge, and maritime or recreational activity. The socio-economic disparities in regions of pollution burden tend to favor wealthier communities at the expense of the poor: the so-called “luxury effect” (Schell et al., 2020). These affluence gaps are linked to uneven distributions of abiotic stressors, floral and faunal diversity, and pressure from pollutant sources. A review of 122 US urban Green Infrastructure plans showed that 80% omitted or poorly defined terms concerning ‘equity’ and ‘justice’. A meager 6% contained best practices in procedures that ensure equitable distribution of environmental services. By comparing biofilm assemblage in an impacted riverine section with an unimpacted section, we demonstrated the potential disparate effect of water quality and the influence of microplastic burden that could be present in a freshwater urban and non-urban riverine system (Grabowski et al., 2023).
At the D60 harvest date, it was noted that the sample cage at the unimpaired Honeypot Brook location was removed from the brook. A previous visit a week prior confirmed the position, and it was unclear how long the cage had been out of the water. The cage was replaced in the brook and allowed to soak for several minutes to rehydrate potentially desiccated biomass. The extent of biofilm disruption due to remaining out of the brook is unknown, though sequencing data did not indicate any significant reduction in feature counts.
It is also suspected that by D90, the accumulated detritus on the exterior of the infuser may have reduced flow-through to the substrate itself; this would have affected both substrates similarly. Alternatives for future studies include employing a less fine mesh containing the 3 mm substrate size, allowing for uninhibited water flow.
Future directions for this study include additional exploration of community richness and evenness over time, which could be an interesting focus. As Qiang et al. (2021) described, the dominant taxon was distinct from primary colonizers in the first 18 days of lab incubation, with a leveling off from days 18 to 31. Though outside of the scope of our analyses, a similar observation using Pielou’s evenness suggested significant change within groups between D30 and D60 (p = 6.13e07) (Fig. S4). Examination of successional growth over an extended study period may provide more information into early and later biofilm development and the influence of seasonal variability. Comparing environmental communities from water samples (Marsay et al., 2023; Qiang et al., 2021) with that of the adhered community to the microplastic substrate would be another approach to analyze the incident of preferential selection.
The limitation of selective media for coliform monitoring is that the methodology indicates the presence or absence of E.coli and non-E.coli organisms. While we did not see a significant abundance of E.coli in the substrate microbiomes, it was evident that the latter was more problematic and too general to determine what fraction of these organisms may be pathogens. Although more costly, using 16S rRNA gene sequencing elucidated the specific genera included in this category. The microbiomes of the microplastic and stone substrate were not overwhelmingly distinct at the class level as first hypothesized; however, there appears to be support that an impaired waterbody with microplastics may suffer from an additive impact of their presence. That is, the discharge of these particles in riverine systems alone does not address the issue's totality; the system's condition must also be considered.
Microplastic biomes present a complicated environmental issue that has the potential to be more impactful in impaired urban riverine ecosystems. The results of this study demonstrated a paired interaction of site and microplastic substrate concerning coliform attachment and adhesion of known pathogenic organisms. Given the importance of these waterways for communities, particularly those near polluting sources, improving water quality should be a top priority.