An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads.

Microbial eukaryotes (protists) are important components of terrestrial and aquatic environments, as well as animal and human microbiomes. Their relationships with metazoa range from mutualistic to parasitic and zoonotic (i.e., transmissible between humans and animals). Despite their ecological importance, our knowledge of protists in urban environments lags behind that of bacteria, largely due to a lack of experimentally validated high-throughput protocols that produce accurate estimates of protist diversity while minimizing non-protist DNA representation. We optimized protocols for detecting zoonotic protists in raw sewage samples, with a focus on trichomonad taxa. First, we investigated the utility of two commonly used variable regions of the 18S rRNA marker gene, V4 and V9, by amplifying and Sanger sequencing 23 different eukaryotic species, including 16 protist species such as Cryptosporidium parvum, Giardia intestinalis, Toxoplasma gondii, and species of trichomonad. Next, we optimized wet-lab methods for sample processing and Illumina sequencing of both regions from raw sewage collected from a private apartment building in New York City. Our results show that both regions are effective at identifying several zoonotic protists that may be present in sewage. A combination of small extractions (1 mL volumes) performed on the same day as sample collection, and the incorporation of a vertebrate blocking primer, is ideal to detect protist taxa of interest and combat the effects of metazoan DNA. We expect that the robust, standardized methods presented in our workflow will be applicable to investigations of protists in other environmental samples, and will help facilitate large-scale investigations of protistan diversity.

Profiling pathogenic protozoan and their functional pathways in wastewater using 18S rRNA and shotgun metagenomics.

Despite extensive research, little is known about the composition of eukaryotic protists in environmental samples. This is due to low parasite concentrations, the complexity of parasite diversity, and a lack of suitable reference databases and standardized protocols. To bridge this knowledge gap, this study used 18S rRNA short amplicon and shotgun metagenomic sequencing approaches to profile protozoan microbial communities as well as their functional pathways in treated and untreated wastewater samples collected from different regions of South Africa. Results demonstrated that protozoan diversity (Shannon index P-value = 0.03) and taxonomic composition (PERMANOVA, P-value = 0.02) was mainly driven by the type of wastewater samples (treated & untreated) and geographic location. However, these WWTPs were also found to contain a core community of protozoan parasites. The untreated wastewater samples revealed a predominant presence of free-living, parasitic, and potentially pathogenic protists typically found in humans and animals, ranging from Alveolata (27 %) phylum (Apicomplexa and Ciliophora) to Excavata (3.88 %) (Discoba and Parasalia) and Amoebozoa (2.84 %) (Entamoeba and Acanthamoeba). Shotgun metagenomics analyses in a subset of the untreated wastewater samples confirmed the presence of public health-importance protozoa, including Cryptosporidium species (3.48 %), Entamoeba hystolitica (6.58 %), Blastocystis hominis (2.91 %), Naegleria gruberi (2.37 %), Toxoplasma gondii (1.98 %), Cyclospora cayetanensis (1.30 %), and Giardia intestinalis (0.31 %). Virulent gene families linked to pathogenic protozoa, such as serine/threonine protein phosphatase and mucin-desulfating sulfatase were identified. Additionally, enriched pathways included thiamine diphosphate biosynthesis III, heme biosynthesis, Methylerythritol 4-Phosphate Pathway, methyl erythritol phosphate (MEP), and pentose phosphate pathways. These findings suggest that protozoan pathogens may possess metabolic and growth potential within WWTPs, posing a severe risk of transmission to humans and animals if inadequately disinfected before release. This study provides a baseline for the future investigation of diverse protozoal communities in wastewater, which are of public health importance.

Biodegradation of PHB/PBAT films and isolation of novel PBAT biodegraders from soil microbiomes.

Polyhydroxyalkanoates (PHAs) are important candidates for replacing petroleum-based plastics. This transition is urgent for the development of a biobased economy and to protect human health and natural ecosystems. PHAs are biobased and biodegradable polyesters that when blended with other polymers, such as poly(butylene adipate-co-terephthalate) (PBAT), acquire remarkable improvements in their properties, which allow them to comply with the requirements of packaging applications. However, the biodegradation of such blends should be tested to evaluate the impact of those polymers in the environment. For instance, PBAT is a compostable aliphatic-aromatic copolyester, and its biodegradation in natural environments, such as soil, is poorly studied. In this work, we evaluated the biodegradation of a bilayer film composed of PHB and PBAT, by a soil microbiome. The bilayer film reached 47 ± 1 % mineralization in 180 days and PHB was no longer detected after this period. The increased crystallinity of the PBAT residue was a clear sign of biodegradation, indicating that the amorphous regions were preferentially biodegraded. Seven microorganisms were isolated, from which 4 were closely related to microorganisms already known as PHB degraders, but the other 3 species, closely related to Streptomyces coelicoflavus, Clonostachys rosea and Aspergillus insuetus, were found for the first time as PHB degraders. Most remarkably, two fungi closely related to Purpureocillium lilacinum and Aspergillus pseudodeflectus (99.83 % and 100 % identity by ITS sequencing) were isolated and identified as PBAT degraders. This is very interesting due to the rarity of isolating PBAT-degrading microorganisms. These results show that the bilayer film can be biodegraded in soil, at mesophilic temperatures, showing its potential to replace synthetic plastics in food packaging.

Tracking microeukaryotic footprint in a peri-urban watershed, China through machine-learning approaches.

Microeukaryotes play a significant role in biogeochemical cycling and can serve as bioindicators of water quality in freshwater ecosystems. However, there is a knowledge gap on how freshwater microeukaryotic communities are assembled, especially that how terrestrial microeukaryotes influence freshwater microeukaryotic assemblages. Here, we used a combination of 18S rRNA gene amplicon sequencing and community-based microbial source tracking (MST) approaches (i.e., SourceTracker and FEAST) to assess the contribution of microeukaryotes from surrounding environments (i.e., soils, river sediments, swine wastewater, influents and effluents of decentralized wastewater treatment plants) to planktonic microeukaryotes in the main channel, tributaries and reservoir of a peri-urban watershed, China in wet and dry seasons. The results indicated that SAR (~ 49% of the total communities), Opithokonta (~ 34%), Archaeplastida (~ 9%), and Amoebozoa (~ 2%) were dominant taxa in the watershed. The community-based MST analysis revealed that sewage effluents (7.96 - 21.84%), influents (2.23 - 13.97%), and river sediments (2.56 - 11.71%) were the major exogenous sources of riverine microeukaryotes. At the spatial scale, the downstream of the watershed (i.e., main channel and tributaries) received higher proportions of exogenous microeukaryotic OTUs compared to the upstream reservoirs, while at the seasonal scale, the sewage effluents and influents contributed higher exogenous microeukaryotes to river water in wet season than in dry season. Moreover, the swine and domestic wastewater led to the presence of Apicomplexa in wet season only, implying rainfall runoff may enhance the spread of parasitic microeukaryotes. Taken together, our study provides novel insights into the immigration patterns of microeukaryotes and their dominant supergroups between terrestrial and riverine habitats.

Water Column Microbial Communities Vary along Salinity Gradients in the Florida Coastal Everglades Wetlands.

Planktonic microbial communities mediate many vital biogeochemical processes in wetland ecosystems, yet compared to other aquatic ecosystems, like oceans, lakes, rivers or estuaries, they remain relatively underexplored. Our study site, the Florida Everglades (USA)-a vast iconic wetland consisting of a slow-moving system of shallow rivers connecting freshwater marshes with coastal mangrove forests and seagrass meadows-is a highly threatened model ecosystem for studying salinity and nutrient gradients, as well as the effects of sea level rise and saltwater intrusion. This study provides the first high-resolution phylogenetic profiles of planktonic bacterial and eukaryotic microbial communities (using 16S and 18S rRNA gene amplicons) together with nutrient concentrations and environmental parameters at 14 sites along two transects covering two distinctly different drainages: the peat-based Shark River Slough (SRS) and marl-based Taylor Slough/Panhandle (TS/Ph). Both bacterial as well as eukaryotic community structures varied significantly along the salinity gradient. Although freshwater communities were relatively similar in both transects, bacterioplankton community composition at the ecotone (where freshwater and marine water mix) differed significantly. The most abundant taxa in the freshwater marshes include heterotrophic Polynucleobacter sp. and potentially phagotrophic cryptomonads of the genus Chilomonas, both of which could be key players in the transfer of detritus-based biomass to higher trophic levels.

Multitrophic interactions in the rhizosphere microbiome of wheat: from bacteria and fungi to protists.

Plants modulate the soil microbiota by root exudation assembling a complex rhizosphere microbiome with organisms spanning different trophic levels. Here, we assessed the diversity of bacterial, fungal and cercozoan communities in landraces and modern varieties of wheat. The dominant taxa within each group were the bacterial phyla Proteobacteria, Actinobacteria and Acidobacteria; the fungi phyla Ascomycota, Chytridiomycota and Basidiomycota; and the Cercozoa classes Sarcomonadea, Thecofilosea and Imbricatea. We showed that microbial networks of the wheat landraces formed a more intricate network topology than that of modern wheat cultivars, suggesting that breeding selection resulted in a reduced ability to recruit specific microbes in the rhizosphere. The high connectedness of certain cercozoan taxa to bacteria and fungi indicated trophic network hierarchies where certain predators gain predominance over others. Positive correlations between protists and bacteria in landraces were preserved as a subset in cultivars as was the case for the Sarcomonadea class with Actinobacteria. The correlations between the microbiome structure and plant genotype observed in our results suggest the importance of top-down control by organisms of higher trophic levels as a key factor for understanding the drivers of microbiome community assembly in the rhizosphere.