Tracing particulate matter and associated microorganisms in freshwaters
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Sediment resuspension represents a key process in all natural aquatic systems, owing to its role in nutrient cycling and transport of potential contaminants. Although suspended solids are generally accepted as an important quality parameter, current monitoring programs cover quantitative aspects only. Established methodologies do not provide information on origin, fate, and risks associated with uncontrolled inputs of solids in waters. Here we discuss the analytical approaches to assess the occurrence and ecological relevance of resuspended particulate matter in freshwaters, with a focus on the dynamics of associated contaminants and microorganisms. Triggered by the identification of specific physical–chemical traits and community structure of particle-associated microorganisms, recent findings suggest that a quantitative determination of microorganisms can be reasonably used to trace the origin of particulate matter by means of nucleic acid-based assays in different aquatic systems.
KeywordsTotal suspended solids Resuspended particulate Turbidity Sediment traps Particle-associated microorganisms Pathogens
Freshwater particulate matter (PM) refers to the bulk inorganic and organic non-dissolved material, which stays suspended in waters of streams, rivers, lakes and reservoirs, e.g., by turbulence (Chapman, 1996). Conventionally characterized as particles with a nominal diameter larger than 0.45 µm, PM represents a fundamental structural component in freshwaters and an important quality parameter in current monitoring plans worldwide (Bartram & Ballance, 1996). PM acts as both source and sink for nutrients and contaminants, therefore, knowledge concerning particle dynamics and inflow, suspended matter concentration, sinking flux, resuspension and settling processes has become crucial for environmental impact studies and watershed models aimed at optimizing water resource management at a catchment level (Grathwohl et al., 2013). The direct impact on human activities is obvious, since turbid waters can, e.g., reduce underwater visibility, adversely affect recreational uses, contribute to clogging tanks and pipes in industrial settings, erode impellers of pumps and can cause damage to water-treating machines. Moreover, PM sedimentation and accumulation in reservoirs gradually reduce the water storage capacity, and probably represent the most serious technical problem faced by the dam industry (Fox et al., 2016).
An increase in turbidity is also associated with potential pollution risks and human health-related issues, in particular when contaminated sediments are frequently resuspended, since pollutants of various origins can be transported in association with suspended particles and follow their remobilization/sedimentation dynamics in water (Mahler et al., 2000; Bai & Lung, 2005). Many countries and organizations have established recommended turbidity levels assessed on site by probe-based techniques, such as sensors measuring the light scattering properties of the particles in liquid. Beside the traditional assessment by Secchi disk, total suspended solids (TSS in mg/l) and turbidity (nephelometric turbidity units—NTU) are common PM-related indicators of water quality (Kemker, 2014). The most accurate but time-consuming method of quantifying suspended solids in a water sample is filtering, drying, and weighing (ISO5667-3, 2012). TSS can also be estimated directly from turbidity sensors, upon linear regression modeling, and calculation for each sampling period and location (Bertrand-Krajewski, 2004). According to European water directives, member states should strive for a parametric value not exceeding 1 NTU upon surface water treatment, and 0.2 NTU in the case of drinking water (European Union, 1998; European Communities Environmental Objectives, 2009; Official Journal of the European Union, 2009). Turbidity levels below 1 NTU are generally recommended for human consumption, while a level of 5 NTU or lower is acceptable for recreational purposes according to the American Water Works Association (Bartram & Ballance, 1996).
Unlike the impact on human-related issues, consequences of high and highly variable turbidity levels on aquatic ecosystem functioning are multiple, site-specific, and rather unpredictable from an ecological point of view (Bilotta & Brazier, 2008). Physical and chemical conditions may be completely altered by reduced light penetration, unsuitable for efficient photoautotrophic metabolic processes of planktonic and benthic organisms (Sobolev et al., 2009). It was also reported that PM-associated toxicants can be harmful for aquatic life (Gordon & Palmer, 2015; Quinteiro et al., 2015). In contrast, PM-associated nutrients and organic matter such as proteins can stimulate microbial growth and facilitate development of algal blooms (Cole et al., 2006).
The contrasting ecological effects of freshwater PM inputs are even exacerbated when referring to climate change models, by which extreme weather events (e.g., drought, storms, heavy rains) are expected to increase in intensity and frequency in near future (García-Ruiz et al., 2011; Coumou & Rahmstorf, 2012). When extended dry periods are followed by extreme rain events (i.e., a global-warming scenario in arid and semi-arid regions worldwide), both solutes and particle fluxes increase dramatically for a short period, thus impairing water quality for aquatic life and/or humans, and increasing flooding risks (Wood, 2014; Butturini et al., 2016; Ejarque et al., 2017; Vercruysse et al., 2017).
Nowadays, PM quantification with established methodologies does not offer detailed indications on origins and risks associated with uncontrolled inputs of solids in waters. One of the on-going tasks for limnologists is to determine the dynamics and fate of suspended PM (e.g., allochthonous vs. autochthonous, allogenic vs. authigenic). The ability to distinguish settling authigenic particles from those originating from bottom sediment resuspension represents a prerequisite to assess the ecological relevance of PM in aquatic systems and to distinguish its functional relevance. However, there is a lack of literature in this regard, and consideration of PM in either fundamental ecological theories or current monitoring plans rarely exceeds the black-box parameter, termed turbidity.
This article presents an approach on how our knowledge of PM could be considerably improved by studying particle-associated microorganisms. Assuming that specific physiological traits and community structure vary depending on PM origin, their analysis could play a key role in defining potential biomarkers that will allow resuspended sediment particles to be identified within the bulk particulate load in freshwater systems.
Origin, fate, and significance of resuspended particulate matter in freshwaters
Suspended particles can originate from different processes (e.g., resuspension, erosion, runoff, discharge, flocculation, algal blooms, microbial aggregation), all of which contribute to determine their physical (e.g., particle size and morphology), chemical (e.g., organic and inorganic components), and biological properties (e.g., associated biofilm and microorganisms). The most common method to estimate the contribution of sediment resuspension to the total settling flux is to deploy sediment traps at various depths and to compare the measured particle flux in bottom and epilimnion traps (Rosa, 1985; Bloesch & Uehlinger, 1986). Gasith (1975) used the organic matter fraction of seston as an indicator for resuspension by comparing it to tripton and to the amount of inorganic settling material. A second parameter likely to reveal differences between resuspended and authigenic material is the chemical composition of collected particles. Previous studies aimed at the different particulate phosphorus (PP) fractions in seston and sediments indicating that settling matter is relatively enriched in labile and organic PP when compared to the P-distribution in the sediment (Eckert et al., 2003; Eckert & Nishri, 2014). To which extent this difference can be useful in the resuspension studies on P-pool is a matter of debate. Bloesch (1994) concluded that for reliable in situ studies a combination of sediment traps, sediment cores, near bottom current meters, and turbidity meters need to be employed to better measure the occurrence of suspended particulate matter. Other authors explained resuspension as the difference between sediment trap catch and bottom sediment accumulation (Dillon et al., 1990).
An advanced approach for measuring and modeling resuspension is based on the detection of radionuclides and stable isotopes (Bloesch, 1994; Cornett et al., 1994). Cesium (137Cs) accumulates in sediments, thus an increase of its activity in the PM fraction let infer for resuspension (Ritchie & McHenry, 1990). Beryllium (7Be) decays rapidly in sediments and it is typically indicative for fresh settled material (Fitzgerald et al., 2001). Thorium isotopes (e.g., 228Th, 230Th) give indications of autochthonous sinking particles (Yang et al., 2013). Although the list of informative PM-related isotopes is rather long, all these approaches are mainly based on empirical considerations with the lack of explicit tracers and means to specifically distinguish between authigenic and resuspended material.
In rivers and shallow lakes, bottom sediment resuspension has long been recognized as the most important factor providing PM to the water column, and its quantification as a critical precondition for the understanding of biogeochemical processes, modeling, and restoration actions (Weyhenmeyer, 1996; Hakanson, 2004; Kleeberg et al., 2013). In large deep lakes, the nearshore sediment resuspension, focusing, and settling are the main processes responsible for particle dynamics in deep layers (Bloesch, 1995). By comparing different aquatic systems all over the US, Evans (1994) estimated a long-term contribution of resuspended material to the total PM flux of about 85%. Weyhenmeyer (1996) calculated a value close to 75% in seven Swedish lakes. Koski-Vähälä et al. (2000) estimated the portion of resuspended matter in a Finnish lake from 56 to 99% of the gross sedimentation.
The extent of resuspension depends on many physical, chemical, and biological properties, and contribution to particle settling flux can vary greatly among aquatic systems with different geomorphologies and at different times of the year. In general, the effects of sediment remobilization processes are case sensitive and site-specific (Evans, 1994). For example, wind-driven surface waves can promote sediment resuspension in all exposed surfaces of shallow lakes, but only in nearshore areas of deep lakes (Hamilton & Mitchell, 1996; Reardon et al., 2016).
Resuspended particles as vehicle for microbial propagation
Kleeberg et al. (2013) reported that numbers of particle-associated bacteria directly followed PM dynamics in a resuspension experiment of river sediments. Consequently, hydrodynamic forces to entrain particle-associated bacteria equaled those necessary to resuspended cohesive sediments. Moreover, when rapidly settling particles overtake and intercept more slowly sinking particles, both will collide if the distances of their flow streamlines are smaller than the sum of the particle radii. As a result, scavenging of small particles will occur (Kepkay, 1994) and free-living bacteria will be lost via settling. On the other hand, sediment-borne microorganisms can even survive in open waters when associated to particles thanks to favorable ecological conditions (e.g., limited competition for nutrients, optimal light exposure, cooperation favored by cells proximity), thus finding transitorily opportunities for dispersal (Drummond et al., 2015). Sediment resuspended particles can constitute “hot-spots” for microbial growth and intimate associations among microorganisms with different functions, as it was reported for planktonic microbial aggregates (Simon et al., 2002; Wotton, 2007; Lyons et al., 2010). Compared to the pelagic zone (free-water), particle-associated bacteria might have a selective advantage due to higher nutrient and organic matter availability similar to biofilm-like structures (Hall-Stoodley & Stoodley, 2005; Grossart, 2010). It was found that stream biofilms and aggregates form distinct communities under varying hydraulic conditions, with higher number of resident taxa associated with aggregate communities (Niederdorfer et al., 2016). Owing to improved light exposure, photoautotrophic cells can grow on particle surfaces and contribute to foraging heterotrophic microbial biomass development (Battin et al., 2003; Romani et al., 2013).
Moreover, the vicinity in which bacteria can grow on particles allows horizontal transferring of genetic material between microorganisms and might enable a persistence of resistance genes, including those of human health-related concern (Costerton, 1999; Allen et al., 2010). The spread and persistence of antibiotic-resistant bacteria and resistance genes in suspended aggregates was reported recently (Corno et al., 2014). Such multi-resistant bacteria might be remobilized with sediments, since sediments harbor heavy metal contaminations, which can select for heavy metal resistance genes that can co-occur with antibiotic resistance genes (Di Cesare et al., 2016a, b). It is worth noting that suspended aggregates and particles may also offer protection to microbial species susceptible to stressing factors, such as chemical disinfectants, high PAR radiation, UV radiation, and predation pressure (Mamane, 2008; Callieri et al., 2011; Tang et al., 2011; Callieri et al., 2016a).
Consequently, typical PM-associated microorganisms may also comprise human pathogens such as Vibrio cholerae, Salmonella spp., Shigella spp., and diarrheagenic strains of Escherichia coli, and toxic cyanobacteria that can grow and persist when attached to particles (Du Preez et al., 2010; Singh et al., 2010; Walters et al., 2014). Turbid waters, whether due to organic or inorganic material, cannot be easily sanitized by conventional treatments (e.g., chlorination, UV irradiation, heating), as the suspended particles can “hide” microbial pathogens and invasive species (Amalfitano et al., 2015), thus causing great concerns from PM contaminated waters (Tang et al., 2011; Edge et al., 2013; Jacob et al., 2015). Higher concentrations of Enterococcus sp. and E. coli coincided with the increase in particles following resuspension of sediments during storm events (Fries et al., 2006). Stormwater-suspended particles were found to prolong survival of fecal indicator organisms for several days prior to reduction to background levels (Jeng et al., 2005). It was postulated that the risk of an E. coli infection increases approximately tenfold if there is a disturbance of sediments, because attached bacteria are more persistent within the aquatic environment than free-floating bacteria (Abia et al., 2016). A further critical factor in determining human health risk is the partitioning of bacterial pathogenic organisms between particle-attached and free-living cells in the water column (Colwell et al., 1985; Characklis et al., 2005; Fries et al., 2006). Regrettably, such effects are largely disregarded because current regulations for microbiological quality evaluations are based on standardized cultivation techniques and set on the number of microorganisms (e.g., heterotrophic plate count and coliforms), regardless of how many cells are found attached on particles.
Detection of particle-associated microorganisms and indicator taxa of sediment resuspension
Although several studies have identified association with PM as a critical factor in predicting the transport and fate of aquatic microorganisms, no generally accepted method exists for identifying the particle-associated microbial fraction emanating from sediment resuspension. A large body of studies has traditionally attempted to classify planktonic microorganisms into particle-attached and free-living cells by using a one-size filtration to separate the two fractions (Crump et al., 1999; Riemann & Winding, 2001; Grossart, 2010; Ortega-Retuerta et al., 2013; Rieck et al., 2015). By claiming the lack of consensus on the filter pore-size, studies tested different sized filters to fractionate bacterioplankton samples and showed that the phylogenetic composition of the particle-associated bacterial community differed among the filtered size fractions (Zhang et al., 2016; Mestre et al., 2017). The centrifugation approach was also reported as a reasonable means of separating settling particles and associated microbes from free-living ones, and allowed assessing that the fraction of PM-associated microorganisms varied by the type of microbes (Characklis et al., 2005). Overall, numerous findings suggest that existing methodologies and data published to-date might not be definitive with respect to all of the states in which PM-interacting microbial cells may exist in aquatic systems. Thus, an urgent task will be to test novel advanced approaches to physically separate floating particles with different properties and origins prior to any further microbiological analysis.
Among the promising technologies readily applicable to partly address this methodological gap, flow cytometry has been proven as a suitable tool to monitor the dynamics of free-living microbes and microbially colonized particles in natural and engineered aquatic systems (Boi et al., 2016; Casentini et al., 2016; Liu et al., 2016). Because hundreds of thousands of events can be analyzed in a few minutes with a large statistical significance, flow cytometry can reduce the analytical time and increase the accuracy needed for cell and particle quantification. The capability of resolving particle-associated fluorescence induced by attached microbial cells, simultaneously offering the possibility to physically identify targeted events (Gasol & Moran, 2015), makes the technique promising for the characterization of suspended sediment particles. Similarly, but yet with very limited applicability to environmental studies, rapid microfluidic-based technologies, in combination with emulsion oil droplet and other entrapping procedures, were used to characterize particles in a liquid suspension according to their size and chemical properties (Sajeesh & Sen, 2014). Discrimination and separation of different PM fractions can also be critical to identify specific interactions between microbes and particles by the large suite of microscopy applications, spanning from epifluorescence microscopy to further high-resolution techniques including electron microscopy (transmission, TEM; and scanning, SEM) and scanning-proximity probe microscopy (atomic force microscopy, AFM) (Ransom et al., 1999; Malfatti & Azam, 2009).
The presence of sulfate-reducing bacteria (SRB belonging to Delta-Proteobacteria) was verified either in aggregates or in the uppermost oxic sediment layers of different environments (Sass et al., 1997; Grossart & Ploug, 2000; Freese et al., 2008; Freixa et al., 2016). Studies on the microbial community structure in the anoxic sulfate-reducing and methanogenic sediment layers of deep lakes demonstrated PCR-based techniques as useful methods to rapidly quantify bacterial and archaeal cell numbers in surface layers of bottom sediments (Schwarz et al., 2007a, b; Frindte et al., 2016). The results pointed towards Delta-Proteobacteria (i.e., sulfate reducers and syntrophs), and to Methanomicrobiales and Methanosaeta (i.e., hydrogenotrophic and acetoclastic methanogens) as the dominant groups within the sediment bacterial and archaeal communities (MacGregor et al., 2001; Briée et al., 2007; Frindte et al., 2015). These findings suggest that a quantitative determination of a bottom sediment specific phylogenic group of microorganisms, such as SRB or Methanogens, can be used to trace the origin of PM by means of nucleic acid-based assays. By using metagenomic analysis of PM-associated microbial communities together with chemical characterization of the particles themselves, a specific or universal indicator may emerge that will allow for the distinction between authigenic and resuspended particles.
Despite the growing awareness of the importance of suspended particles in aquatic systems, the knowledge on factors and conditions favoring sediment resuspension and, hence, the presence of PM-associated bacteria is still very limited. Consequently, it is necessary to further investigate the dynamics of suspended particles and their associated microorganisms in aquatic systems, and those factors potentially able to promote growth and distribution of pathogenic bacteria. When exploring aquatic systems increasingly subjected to contrasting local and climate conditions, PM-targeting surveys can contribute to address fundamental ecological assumptions in linking water and sediment processes, to provide a novel glimpse of potential physical, chemical, and microbiological threats associated with sediment resuspension events, and to help the implementation of control measures for improved resource management actions.
This work was partially supported by the Short-Term Mobility programme of the CNR (Italy). HPG was supported by two Grants from the German Science Foundation (DFG GR1540/23-1 and GR1540/28-1).
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