Freshwater plastisphere: a review on biodiversity, risks, and biodegradation potential with implications for the aquatic ecosystem health.

The plastisphere, a unique microbial biofilm community colonizing plastic debris and microplastics (MPs) in aquatic environments, has attracted increasing attention owing to its ecological and public health implications. This review consolidates current state of knowledge on freshwater plastisphere, focussing on its biodiversity, community assembly, and interactions with environmental factors. Current biomolecular approaches revealed a variety of prokaryotic and eukaryotic taxa associated with plastic surfaces. Despite their ecological importance, the presence of potentially pathogenic bacteria and mobile genetic elements (i.e., antibiotic resistance genes) raises concerns for ecosystem and human health. However, the extent of these risks and their implications remain unclear. Advanced sequencing technologies are promising for elucidating the functions of plastisphere, particularly in plastic biodegradation processes. Overall, this review emphasizes the need for comprehensive studies to understand plastisphere dynamics in freshwater and to support effective management strategies to mitigate the impact of plastic pollution on freshwater resources.

Microbial colonization patterns and biodegradation of petrochemical and biodegradable plastics in lake waters: insights from a field experiment.

Introduction: Once dispersed in water, plastic materials become promptly colonized by biofilm-forming microorganisms, commonly known as plastisphere. Methods: By combining DNA sequencing and Confocal Laser Scanning Microscopy (CLSM), we investigated the plastisphere colonization patterns following exposure to natural lake waters (up to 77 days) of either petrochemical or biodegradable plastic materials (low density polyethylene - LDPE, polyethylene terephthalate - PET, polylactic acid - PLA, and the starch-based MaterBi® - Mb) in comparison to planktonic community composition. Chemical composition, water wettability, and morphology of plastic surfaces were evaluated, through Transform Infrared Spectroscopy (ATR-FTIR), Scanning Electron Microscopy (SEM), and static contact angle analysis, to assess the possible effects of microbial colonization and biodegradation activity. Results and Discussion: The phylogenetic composition of plastisphere and planktonic communities was notably different. Pioneering microbial colonisers, likely selected from lake waters, were found associated with all plastic materials, along with a core of more than 30 abundant bacterial families associated with all polymers. The different plastic materials, either derived from petrochemical hydrocarbons (i.e., LDPE and PET) or biodegradable (PLA and Mb), were used by opportunistic aquatic microorganisms as adhesion surfaces rather than carbon sources. The Mb-associated microorganisms (i.e. mostly members of the family Burkholderiaceae) were likely able to degrade the starch residues on the polymer surfaces, although the Mb matrix maintained its original chemical structure and morphology. Overall, our findings provide insights into the complex interactions between aquatic microorganisms and plastic materials found in lake waters, highlighting the importance of understanding the plastisphere dynamics to better manage the fate of plastic debris in the environment.

Interplay between abiotic and microbial biofilm-mediated processes for travertine formation: Insights from a thermal spring (Piscine Carletti, Viterbo, Italy).

Active hydrothermal travertine systems are ideal environments to investigate how abiotic and biotic processes affect mineralization mechanisms and mineral fabric formation. In this study, a biogeochemical characterization of waters, dissolved gases, and microbial mats was performed together with a mineralogical investigation on travertine encrustations occurring at the outflow channel of a thermal spring. The comprehensive model, compiled by means of TOUGHREACT computational tool from measured parameters, revealed that mineral phases were differently influenced by either abiotic conditions or microbially driven processes. Microbial mats are shaped by light availability and temperature gradient of waters flowing along the channel. Mineralogical features were homogeneous throughout the system, with euhedral calcite crystals, related to inorganic precipitation induced by CO2 degassing, and calcite shrubs associated with organomineralization processes, thus indicating an indirect microbial participation to the mineral deposition (microbially influenced calcite). The microbial activity played a role in driving calcite redissolution processes, resulting in circular pits on calcite crystal surfaces possibly related to the metabolic activity of sulfur-oxidizing bacteria found at a high relative abundance within the biofilm community. Sulfur oxidation might also explain the occurrence of gypsum crystals embedded in microbial mats, since gypsum precipitation could be induced by a local increase in sulfate concentration mediated by S-oxidizing bacteria, regardless of the overall undersaturated environmental conditions. Moreover, the absence of gypsum dissolution suggested the capability of microbial biofilm in modulating the mobility of chemical species by providing a protective envelope on gypsum crystals.

Plastisphere in lake waters: Microbial diversity, biofilm structure, and potential implications for freshwater ecosystems.

Once dispersed in water, microplastic (MP) particles are rapidly colonised by aquatic microbes, which can adhere and grow onto solid surfaces in the form of biofilms. This study provides new insights on microbial diversity and biofilm structure of plastisphere in lake waters. By combining Fourier Confocal Laser Scanning Microscopy (CLSM), Transform Infrared Spectroscopy (FT-IR) and high-throughput DNA sequencing, we investigated the microbial colonization patterns on floating MPs and, for the first time, the occurrence of eukaryotic core members and their possible relations with biofilm-forming bacterial taxa within the plastisphere of four different lakes. Through PCR-based methods (qPCR, LAMP-PCR), we also evaluated the role of lake plastisphere as long-term dispersal vectors of potentially harmful organisms (including pathogens) and antibiotic resistance genes (ARGs) in freshwater ecosystems. Consistent variation patterns of the microbial community composition occurred between water and among the plastisphere samples of the different lakes. The eukaryotic core microbiome was mainly composed by typical freshwater biofilm colonizers, such as diatoms (Pennales, Bacillariophyceaea) and green algae (Chlorophyceae), which interact with eukaryotic and prokaryotic microbes of different trophic levels. Results also showed that MPs are suitable vectors of biofilm-forming opportunistic pathogens and a hotspot for horizontal gene transfer, likely facilitating antibiotic resistance spread in the environments.

Benthic Microbial Communities in a Seasonally Ice-Covered Sub-Arctic River (Pasvik River, Norway) Are Shaped by Site-Specific Environmental Conditions.

The Pasvik River experiences chemical, physical, and biological stressors due to the direct discharges of domestic sewage from settlements located within the catchment and runoff from smelter and mine wastes. Sediments, as a natural repository of organic matter and associated contaminants, are of global concern for the possible release of pollutants in the water column, with detrimental effects on aquatic organisms. The present study was aimed at characterizing the riverine benthic microbial community and evaluating its ecological role in relation to the contamination level. Sediments were sampled along the river during two contrasting environmental periods (i.e., beginning and ongoing phases of ice melting). Microbial enzymatic activities, cell abundance, and morphological traits were evaluated, along with the phylogenetic community composition. Amplified 16S rRNA genes from bacteria were sequenced using a next-generation approach. Sediments were also analyzed for a variety of chemical features, namely particulate material characteristics and concentration of polychlorobiphenyls, polycyclic aromatic hydrocarbons, and pesticides. Riverine and brackish sites did not affect the microbial community in terms of main phylogenetic diversity (at phylum level), morphometry, enzymatic activities, and abundance. Instead, bacterial diversity in the river sediments appeared to be influenced by the micro-niche conditions, with differences in the relative abundance of selected taxa. In particular, our results highlighted the occurrence of bacterial taxa directly involved in the C, Fe, and N cycles, as well as in the degradation of organic pollutants and toxic compounds.

High concentrations of dissolved biogenic methane associated with cyanobacterial blooms in East African lake surface water.

The contribution of oxic methane production to greenhouse gas emissions from lakes is globally relevant, yet uncertainties remain about the levels up to which methanogenesis can counterbalance methanotrophy by leading to CH4 oversaturation in productive surface waters. Here, we explored the biogeochemical and microbial community variation patterns in a meromictic soda lake, in the East African Rift Valley (Kenya), showing an extraordinarily high concentration of methane in oxic waters (up to 156 µmol L-1). Vertical profiles of dissolved gases and their isotopic signature indicated a biogenic origin of CH4. A bloom of Oxyphotobacteria co-occurred with abundant hydrogenotrophic and acetoclastic methanogens, mostly found within suspended aggregates promoting the interactions between Bacteria, Cyanobacteria, and Archaea. Moreover, aggregate sedimentation appeared critical in connecting the lake compartments through biomass and organic matter transfer. Our findings provide insights into understanding how hydrogeochemical features of a meromictic soda lake, the origin of carbon sources, and the microbial community profiles, could promote methane oversaturation and production up to exceptionally high rates.

Uncovering the release of micro/nanoplastics from disposable face masks at times of COVID-19.

Wearing face masks is a fundamental prevention and control measure to limit the spread of COVID-19. The universal use and improper disposal of single-use face masks are raising serious concerns for their environmental impact, owing to the foregone contribution to plastic water pollution during and beyond the pandemic. This study aims to uncover the release of micro/nanoplastics generated from face mask nonwoven textiles once discarded in the aquatic environment. As assessed by microscopy and flow cytometry, the exposure to different levels of mechanical stress forces (from low to high shear stress intensities) was proved effective in breaking and fragmenting face mask fabrics into smaller debris, including macro-, micro-, and nano-plastics. Even at the low level of fabric deterioration following the first second of treatment, a single mask could release in water thousands of microplastic fibers and up to 108 submicrometric particles, mostly comprised in the nano-sized domain. By contributing to the current lack of knowledge regarding the potential environmental hazards posed by universal face masking, we provided novel quantitative data, through a suitable technological approach, on the release of micro/nanoplastics from single-use face masks that can threaten the aquatic ecosystems to which they finally end-up.

Adaptation of Microbial Communities to Environmental Arsenic and Selection of Arsenite-Oxidizing Bacteria From Contaminated Groundwaters.

Arsenic mobilization in groundwater systems is driven by a variety of functionally diverse microorganisms and complex interconnections between different physicochemical factors. In order to unravel this great ecosystem complexity, groundwaters with varying background concentrations and speciation of arsenic were considered in the Po Plain (Northern Italy), one of the most populated areas in Europe affected by metalloid contamination. High-throughput Illumina 16S rRNA gene sequencing, CARD-FISH and enrichment of arsenic-transforming consortia showed that among the analyzed groundwaters, diverse microbial communities were present, both in terms of diversity and functionality. Oxidized inorganic arsenic [arsenite, As(III)] was the main driver that shaped each community. Several uncharacterized members of the genus Pseudomonas, putatively involved in metalloid transformation, were revealed in situ in the most contaminated samples. With a cultivation approach, arsenic metabolisms potentially active at the site were evidenced. In chemolithoautotrophic conditions, As(III) oxidation rate linearly correlated to As(III) concentration measured at the parental sites, suggesting that local As(III) concentration was a relevant factor that selected for As(III)-oxidizing bacterial populations. In view of the exploitation of these As(III)-oxidizing consortia in biotechnology-based arsenic bioremediation actions, these results suggest that contaminated aquifers in Northern Italy host unexplored microbial populations that provide essential ecosystem services.

Water and microbial monitoring technologies towards the near future space exploration.

Space exploration is demanding longer lasting human missions and water resupply from Earth will become increasingly unrealistic. In a near future, the spacecraft water monitoring systems will require technological advances to promptly identify and counteract contingent events of waterborne microbial contamination, posing health risks to astronauts with lowered immune responsiveness. The search for bio-analytical approaches, alternative to those applied on Earth by cultivation-dependent methods, is pushed by the compelling need to limit waste disposal and avoid microbial regrowth from analytical carryovers. Prospective technologies will be selected only if first validated in a flight-like environment, by following basic principles, advantages, and limitations beyond their current applications on Earth. Starting from the water monitoring activities applied on the International Space Station, we provide a critical overview of the nucleic acid amplification-based approaches (i.e., loop-mediated isothermal amplification, quantitative PCR, and high-throughput sequencing) and early-warning methods for total microbial load assessments (i.e., ATP-metry, flow cytometry), already used at a high readiness level aboard crewed space vehicles. Our findings suggest that the forthcoming space applications of mature technologies will be necessarily bounded by a compromise between analytical performances (e.g., speed to results, identification depth, reproducibility, multiparametricity) and detrimental technical requirements (e.g., reagent usage, waste production, operator skills, crew time). As space exploration progresses toward extended missions to Moon and Mars, miniaturized systems that also minimize crew involvement in their end-to-end operation are likely applicable on the long-term and suitable for the in-flight water and microbiological research.

Reuse of ultrafiltered effluents for crop irrigation: On-site flow cytometry unveiled microbial removal patterns across a full-scale tertiary treatment.

Reuse of treated wastewater for crop irrigation has been widely adopted to mitigate the effects of water scarcity on agricultural yields and to help preserving the integrity of aquatic ecosystems. This paper presents the outcomes of one-year monitoring of a full-scale agro-industrial wastewater treatment plant designed for water reuse, with a multistage tertiary treatment based on sand filtration, membrane ultrafiltration, storage and on-demand UV disinfection. We aimed to test flow cytometry as a monitoring tool to provide on-site indications on tertiary treatment performances and on the quality of treated wastewater along the treatment scheme. Membrane ultrafiltration retained prokaryotic cells and E. coli (>3 log). During storage of treated effluents, a significant decay of E. coli was observed together with the growth of prokaryotic and eukaryotic cells, and the UV disinfection was effective only against fecal indicators. The microbial quality of the treated effluent was comparable to the control groundwater locally used for irrigation. On-site rapid assessments by flow cytometry allowed unveiling crucial aspects affecting the microbiological quality of ultrafiltration permeate and treated effluent immediately after sampling, including plant operating performances and microbial removal patterns across the treatment train.

Investigating the influences of quorum quenching and nutrient conditions on activated sludge flocs at a short-time scale.

Quorum sensing signals regulate various functions within activated sludge processes such as formation of microbial aggregates. Disturbance of this signaling system, known as quorum quenching (QQ), provides opportunities for eliminating some problems related to biological wastewater treatment (e.g., biofouling and excess sludge production). However, it is poorly understood how and to what extent QQ systems can affect the microbial aggregation processes and the following floc formation. In particular, an in-depth structural characterization at the scale of microbial aggregate while considering nutrient conditions in the reactor is still largely disregarded. Here, we evaluated the QQ effects at the short-term time scale (i.e., after 4 h for the exogenous period and 19 h for exogenous/endogenous period), by combining advanced techniques for microbial characterization (flow cytometry, CARD-FISH, and confocal laser scanning microscopy) and conventional physical-chemical assessments. The results indicated that by implementing QQ agents (immobilized Acylase I enzyme in porous alginate beads) the abundance of single cells and suspended microbial aggregates in the supernatant did not show significant changes during the exogenous period. Conversely, at the end of the exogenous/endogenous period a significant increase of single prokaryotic cells, small and large microbial aggregates favored the growth of grazers, including free-living nanoflagellates and ciliates. Flocs became looser and thinner than those in the control reactor, thus affecting the sludge settling behavior. Inability of microbial community in degradation of soluble protein during the endogenous period confirmed that the QQ agents are likely to inhibit the secretion of protease enzyme within microbial communities of activated sludge.

Six artificial recharge pilot replicates to gain insight into water quality enhancement processes.

The processes that control water quality improvement during artificial recharge (filtering, degradation, and adsorption) can be enhanced by adding a reactive barrier containing different types of sorption sites and promoting diverse redox states along the flow path, which increases the range of pollutants degraded. While this option looks attractive for renaturazing reclaimed water, three issues have to be analyzed prior to broad scale application: (1) a fair comparison between the system with and without reactive barrier; (2) the role of plants in prevention of clogging and addition of organic carbon; and (3) the removal of pathogens. Here, we describe a pilot installation built to address these issues within a waste water treatment plant that feeds on water reclaimed from the secondary outflow. The installation consists of six systems of recharge basin and aquifer with some variations in the design of the reactive barrier and the heterogeneity of the aquifer. We report preliminary results after one year of operation. We find that (1) the systems are efficient in obtaining a broad range of redox conditions (at least iron and manganese reducing), (2) contaminants of emerging concern are significantly removed (around 80% removal, but very sensitive to the compound), (3) pathogen indicators (E. coli and Enterococci) drop by some 3-5 log units, and (4) the recharge systems maintained infiltration capacity after one year of operation (only the system without plants and the one without reactive barrier displayed some clogging). Overall, the reactive barrier enhances somewhat the performance of the system, but the gain is not dramatic, which suggests that barrier composition needs to be improved.

Effects of a Simulated Acute Oil Spillage on Bacterial Communities from Arctic and Antarctic Marine Sediments.

BACKGROUND: The bacterial community responses to oil spill events are key elements to predict the fate of hydrocarbon pollution in receiving aquatic environments. In polar systems, cold temperatures and low irradiance levels can limit the effectiveness of contamination removal processes. In this study, the effects of a simulated acute oil spillage on bacterial communities from polar sediments were investigated, by assessing the role of hydrocarbon mixture, incubation time and source bacterial community in selecting oil-degrading bacterial phylotypes. METHODS: The bacterial hydrocarbon degradation was evaluated by gas chromatography. Flow cytometric and fingerprinting profiles were used to assess the bacterial community dynamics over the experimental incubation time. RESULTS: Direct responses to the simulated oil spill event were found from both Arctic and Antarctic settings, with recurrent bacterial community traits and diversity profiles, especially in crude oil enrichment. Along with the dominance of Pseudomonas spp., members of the well-known hydrocarbon degraders Granulosicoccus spp. and Cycloclasticus spp. were retrieved from both sediments. CONCLUSIONS: Our findings indicated that polar bacterial populations are able to respond to the detrimental effects of simulated hydrocarbon pollution, by developing into a more specialized active oil degrading community.

Microbial community composition of water samples stored inside the International Space Station.

During the VIABLE ISS project (eValuatIon And monitoring of microBiofiLms insidE International Space Station), water samples subjected to two different silver treatments were sent and kept on board the International Space Station (ISS) from 2011 to 2016. In this note we report data on the viable and total bacterial load and on the composition of the microbial communities of the VIABLE ISS samples.

Disentangling natural and anthropogenic impacts on groundwater by hydrogeochemical, isotopic and microbiological data: Hints from a municipal solid waste landfill.

Within human-impacted areas, high levels of inorganic compounds in groundwater are broadly and preventively attributed to local anthropogenic pollution, thoroughly disregarding geogenic natural background levels. Particularly in landfills, a proper evaluation of the significant adverse environmental effects should be completed through a detailed groundwater characterization, and appropriate reference values established prior to landfill onset. However, the monitoring network may lack a full hydrogeological representativeness of the site and of the background conditions of groundwater. This study aimed at disentangling natural and anthropogenic impacts through a synoptic analysis of hydrogeochemical, isotopic and microbiological characteristics of groundwaters from a municipal solid waste landfill area in Central Italy. Samples were collected during four seasonal monitoring surveys from the mostly anoxic aquifer underlying the target area. Field parameters, inorganic and organic compounds, environmental isotopes, faecal contamination, and microbial community characteristics were determined, along with a detailed hydrogeological conceptual model. Key inorganic contaminants (As, Fe and Mn) exceeded the local threshold values in most of the sampling points, while organic contamination was generally very low. Stable isotopes suggested that groundwater originated mainly from local rainfall, except at one monitoring points where tritium levels might indicate moderate impact. Microbiological data and the microbial community characterization, assessed by flow cytometry and BIOLOG assays, provided further supportive information, also highlighting fundamental effects of groundwater quality alterations. Overall, an integrated multi-parametric approach proved suitable to distinguish geogenic and anthropogenic impacts, thus improving strategies and schemes for protection and management of groundwaters in landfills and waste related industrial areas.

Biological As(III) oxidation in biofilters by using native groundwater microorganisms.

Arsenic (As) contamination in drinking water represents a worldwide threat to human health. During last decades, the exploitation of microbial As-transformations has been proposed for bioremediation applications. Among biological methods for As-contaminated water treatment, microbial As(III)-oxidation is one of the most promising approaches since it can be coupled to commonly used adsorption removal technologies, without requiring the addition of chemicals and producing toxic by-products. Despite the As(III) oxidation capability has been described in several bacterial pure or enrichment cultures, very little is known about the real potentialities of this process when mixed microbial communities, naturally occurring in As contaminated waters, are used. This study highlighted the contribution of native groundwater bacteria to As(III)-oxidation in biofilters, under conditions suitable for a household-scale treatment system. This work elucidated the influence of a variety of experimental conditions (i.e., various filling materials, flow rates, As(III) inflow concentration, As(III):As(V) ratio, filter volumes) on the microbially-mediated As(III)-oxidation process in terms of oxidation efficiency and rate. The highest oxidation efficiencies (up to 90% in 3 h) were found on coarse sand biofilters treating total initial As concentration of 100 µg L-1. The detailed microbial characterization of the As(III) oxidizing biofilms revealed the occurrence of several OTUs affiliated with families known to oxidize As(III) (e.g., Burkholderiaceae, Comamonadaceae, Rhodobacteraceae, Xanthomonadaceae). Furthermore, As-related functional genes increased in biofilter systems in line with the observed oxidative performances.

Water Quality and Total Microbial Load: A Double-Threshold Identification Procedure Intended for Space Applications.

During longer-lasting future space missions, water renewal by ground-loaded supplies will become increasingly expensive and unmanageable for months. Space exploration by self-sufficient spacecrafts is thus demanding the development of culture-independent microbiological methods for in-flight water monitoring to counteract possible contamination risks. In this study, we aimed at evaluating total microbial load data assessed by selected early-warning techniques with current or promising perspectives for space applications (i.e., HPC, ATP-metry, qPCR, flow cytometry), through the analysis of water sources with constitutively different contamination levels (i.e., chlorinated and unchlorinated tap waters, groundwaters, river waters, wastewaters). Using a data-driven double-threshold identification procedure, we presented new reference values of water quality based on the assessment of the total microbial load. Our approach is suitable to provide an immediate alert of microbial load peaks, thus enhancing the crew responsiveness in case of unexpected events due to water contamination and treatment failure. Finally, the backbone dataset could help in managing water quality and monitoring issues for both space and Earth-based applications.

Deconvolution model to resolve cytometric microbial community patterns in flowing waters.

Flow cytometry is suitable to discriminate and quantify aquatic microbial cells within a spectrum of fluorescence and light scatter signals. Using fixed gating and operational settings, we developed a finite distribution mixture model, followed by the Voronoi tessellation, to resolve bivariate cytometric profiles into cohesive subgroups of events. This procedure was applied to outline recurrent patterns and quantitative changes of the aquatic microbial community along a river hydrologic continuum. We found five major subgroups within each of the commonly retrieved populations of cells with Low and High content of Nucleic Acids (namely, LNA, and HNA cells). Moreover, the advanced analysis allowed assessing changes of community patterns perturbed by a wastewater feed. Our approach for cytometric data deconvolution confirmed that flow cytometry could represent a prime candidate technology for assessing microbial community patterns in flowing waters. © 2017 International Society for Advancement of Cytometry.

Bacterial community structure along the subtidal sandy sediment belt of a high Arctic fjord (Kongsfjorden, Svalbard Islands).

Open fjords are subject to contrasting environmental conditions, owing to meltwater glacial inputs, terrestrial runoff, and marine water mass exchanges, which are exacerbated by anthropogenic and climate perturbations. Following a slope-dependent water circulation, the subtidal sandy sediment belt regulates the convergent transport of nutrients downward the fjord depths, and the effective entrapment of suspended particles and microorganisms. In this study, we aimed at testing how glacial and seawater inputs may influence the bacterial community structure of subtidal sand deposits in the Kongsfjorden. Through total and viable cell counting and an amplicon sequencing approach, we found relevant differences in bacterial community structure along the glacio-marine sampling transect. Viable and high nucleic acid content (HNA) cells represented an important fraction of the total community, generally decreasing toward the glacier front. Besides the predominance of Alpha- and Gammaproteobacteria, Bacteroidetes, Firmicutes and Parcubacteria, the bacterial community structure was likely affected by the glacial activity in the inner fjord, with the occurrence of distinctive phylotypes belonging to Gemmatimonadates, Nitrospirae, Acidobacteria, and Chloroflexi. Overall, our outcomes highlighted that exploring the bacterial community distribution and structure can provide new insights into the active role of sand deposits in coastal cold environments.

The microbiome associated with two Synechococcus ribotypes at different levels of ecological interaction.

Planktonic cyanobacteria belonging to the genus Synechococcus are ubiquitously distributed in marine and fresh waters, substantially contributing to total carbon fixation on a global scale. While their ecological relevance is acknowledged, increasing resolution in molecular techniques allows disentangling cyanobacteria's role at the micro-scale, where complex microbial interactions may drive the overall community assembly. The interplay between phylogenetically different Synechococcus clades and their associated bacterial communities can affect their ecological fate and susceptibility to protistan predation. In this study, we experimentally promoted different levels of ecological interaction by mixing two Synechococcus ribotypes (MW101C3 and LL) and their associated bacteria, with and without a nanoflagellate grazer (Poterioochromonas sp.) in laboratory cultures. The beta-diversity of the Synechococcus-associated microbiome in laboratory cultures indicated that the presence of the LL ribotype was the main factor determining community composition changes (41% of total variance), and prevailed over the effect of protistan predation (18% of total variance). Our outcomes also showed that species coexistence and predation may promote microbial diversity, thus highlighting the underrated ecological relevance of such micro-scale factors.