Effects of anthropogenic stress on hosts and their microbiomes: Treated wastewater alters performance and gut microbiome of a key detritivore (Asellus aquaticus).

Human activity is a major driver of ecological and evolutionary change in wild populations and can have diverse effects on eukaryotic organisms as well as on environmental and host-associated microbial communities. Although host-microbiome interactions can be a major determinant of host fitness, few studies consider the joint responses of hosts and their microbiomes to anthropogenic changes. In freshwater ecosystems, wastewater is a widespread anthropogenic stressor that represents a multifarious environmental perturbation. Here, we experimentally tested the impact of treated wastewater on a keystone host (the freshwater isopod Asellus aquaticus) and its gut microbiome. We used a semi-natural flume experiment, in combination with 16S rRNA amplicon sequencing, to assess how different concentrations (0%, 30%, and 80%) of nonfiltered wastewater (i.e. with chemical toxicants, nutrients, organic particles, and microbes) versus ultrafiltered wastewater (i.e. only dissolved pollutants and nutrients) affected host survival, growth, and food consumption as well as mid- and hindgut bacterial community composition and diversity. Our results show that while host survival was not affected by the treatments, host growth increased and host feeding rate decreased with nonfiltered wastewater - potentially indicating that A. aquaticus fed on organic matter and microbes available in nonfiltered wastewater. Furthermore, even though the midgut microbiome (diversity and composition) was not affected by any of our treatments, nonfiltered wastewater influenced bacterial composition (but not diversity) in the hindgut. Ultrafiltered wastewater, on the other hand, affected both community composition and bacterial diversity in the hindgut, an effect that in our system differed between sexes. While the functional consequences of microbiome changes and their sex specificity are yet to be tested, our results indicate that different components of multifactorial stressors (i.e. different constituents of wastewater) can affect hosts and their microbiome in distinct (even opposing) manners and have a substantial impact on eco-evolutionary responses to anthropogenic stressors.

Wastewater microorganisms impact microbial diversity and important ecological functions of stream periphyton.

Effluents of wastewater treatment plants can impact microbial communities in the receiving streams. However, little is known about the role of microorganisms in wastewater as opposed to other wastewater constituents, such as nutrients and micropollutants. We aimed therefore at determining the impact of wastewater microorganisms on the microbial diversity and function of periphyton, key microbial communities in streams. We used a flow-through channel system to grow periphyton upon exposure to a mixture of stream water and unfiltered or ultra-filtered wastewater. Impacts were assessed on periphyton biomass, activities and tolerance to micropollutants, as well as on microbial diversity. Our results showed that wastewater microorganisms colonized periphyton and modified its community composition, resulting for instance in an increased abundance of Chloroflexi and a decreased abundance of diatoms and green algae. This led to shifts towards heterotrophy, as suggested by the changes in nutrient stoichiometry and the increased mineralization potential of carbon substrates. An increased tolerance towards micropollutants was only found for periphyton exposed to unfiltered wastewater but not to ultra-filtered wastewater, suggesting that wastewater microorganisms were responsible for this increased tolerance. Overall, our results highlight the need to consider the role of wastewater microorganisms when studying potential impacts of wastewater on the receiving water body.

Wastewater microorganisms impact the micropollutant biotransformation potential of natural stream biofilms.

Biotransformation is the most important process removing manmade chemicals from the environment, yet mechanisms governing this essential ecosystem function are underexplored. To understand these mechanisms, we conducted experiments in flow-through systems, by colonizing stream biofilms under different conditions of mixing river water with treated (and ultrafiltered) wastewater. We performed biotransformation experiments with those biofilms, using a set of 75 micropollutants, and could disentangle potential mechanisms determining the biotransformation potential of stream biofilms. We showed that the increased biotransformation potential downstream of wastewater treatment plants that we observed for specific micropollutants contained in household wastewaters (downstream effect) is caused by microorganisms released with the treated effluent, rather than by the in-stream exposure to those micropollutants. Complementary data from 16S rRNA amplicon-sequencing revealed 146 amplicon sequence variants (ASVs) that followed the observed biotransformation patterns. Our results align with findings for community tolerance, and provide clear experimental evidence that microorganisms released with treated wastewater integrate into downstream biofilms and impact crucial ecosystem functions.

Impact of wastewater on the microbial diversity of periphyton and its tolerance to micropollutants in an engineered flow-through channel system.

Wastewater treatment plants (WWTPs) play an important role in retaining organic matter and nutrients but to a lesser extent micropollutants. Therefore, treated wastewater is recognized as a major source of multiple stressors, including complex mixtures of micropollutants. These can potentially affect microbial communities in the receiving water bodies and the ecological functions they provide. In this study, we evaluated in flow-through channels the consequences of an exposure to a mixture of stream water and different percentages of urban WWTP effluent, ranging from 0% to 80%, on the microbial diversity and function of periphyton communities. Assuming that micropollutants exert a selective pressure for tolerant microorganisms within communities, we further examined the periphyton sensitivity to a micropollutant mixture extracted from passive samplers that were immersed in the wastewater effluent. As well, micropollutants in water and in periphyton were comprehensively quantified. Our results show that micropollutants detected in periphyton differed from those found in water, both in term of concentration and composition. Especially photosystem II inhibitors accumulated in periphyton more than other pesticides. Although effects of other substances cannot be excluded, this accumulation may have contributed to the observed higher tolerance of phototrophic communities to micropollutants upon exposure to 30% and 80% of wastewater. On the contrary, no difference in tolerance was observed for heterotrophic communities. Exposure to the gradient of wastewater led to structural differences in both prokaryotic and eukaryotic communities. For instance, the relative abundance of cyanobacteria was higher with increasing percentage of wastewater effluent, whereas the opposite was observed for diatoms. Such results could indicate that differences in community structure do not necessarily lead to higher tolerance. This highlights the need to consider other wastewater constituents such as nutrients and wastewater-derived microorganisms that can modulate community structure and tolerance. By using engineered flow-through channels that mimic to some extent the required field conditions for the development of tolerance in periphyton, our study constitutes a base to investigate the mechanisms underlying the increased tolerance, such as the potential role of microorganisms originating from wastewater effluents, and different treatment options to reduce the micropollutant load in effluents.