Structure and Composition of Aggregates in Two Large European Rivers,Based on Confocal Laser Scanning Microscopy and Image and Statistical Analyses

Floating riverine aggregates are composed of a complex mixture of inorganic and organic components from their respective aquatic habitats. Their architecture and integrity are supplemented by the presence of extracellular polymeric substances of microbial origin. They are also a habitat for virus-like particles, bacteria, archaea, fungi, algae, and protozoa. In this study we present different confocal laser scanning microscopy strategies to examine aggregates collected from the Danube and Elbe Rivers. In order to collect multiple types of information, various approaches were necessary. Small aggregates were examined directly. To analyze large and dense aggregates, limitations of the technique were overcome by cryo-sectioning and poststaining of the samples. The staining procedure included positive staining (specific glycoconjugates and cellular nucleic acid signals) as well as negative staining (aggregate volume) and multichannel recording. Data sets of cellular nucleic acid signals (CNAS) and the structure of aggregates were visualized and quantified using digital image analysis. The Danube and Elbe Rivers differed in their aggregate composition and in the relative contribution of specific glycoconjugate and CNAS volume to the aggregate volume; these contributions also changed over time. We report different spatial patterns of CNAS inside riverine aggregates, depending on aggregate size and season. The spatial structure of CNAS inside riverine aggregates was more complex in the Elbe River than in the Danube River. Based on our samples, we discuss the strengths and challenges involved in scanning and quantifying riverine aggregates.In rivers, primary particles are frequently and perhaps characteristically transported as larger flocculated aggregates. They are structurally very stable because they are exposed to a constant shear force, resulting in relatively small aggregates compared to aggregates in lakes and marine systems (41, 49). Abiotic mechanisms such as physical coagulation, collision frequency, and stickiness are involved in particle aggregation (12). These aggregates, which may be regarded as mobile biofilms (e.g., 8, 9), can be very heterogeneous in their composition. Up to 97% of the biofilm matrix is actually water (46). Apart from water, biofilms may consist of dissolved, colloidal, and particulate materials varying in size and composition (12). They are composed of a complex mixture of components including inorganic (minerals), living organic (bacteria, archaea, fungi, algae, protozoa, and viruses), and nonliving organic (extracellular polymeric substances [EPS], allochthonous and autochthonous detritus, lignins, tannins, etc.) material from the respective aquatic habitat and its terrestrial environment (41). Cellular material within a biofilm can vary greatly. Measurements of organic carbon content suggest that cellular material represents 2 to 15% of the biofilm (46). Up to 95% of the biofilm is composed of EPS (13, 23, 42). The actual structure of the biofilm matrix varies greatly depending on the microbial cells present, their physiological status, the nutrients available, and the prevailing physical conditions (46).Knowledge about the structure and the function of aggregates, both in environmental and engineered systems, is very important (12). In engineered systems such as wastewater treatment plants, understanding flocculation can help in the management of that process. In environmental systems, such structure-function relationships can provide ecologically relevant information about material transfers between particulate and dissolved matter or about spatial distribution of microorganisms, with the related impacts on the aquatic food web. Numerous methods are available to help characterize aggregate properties. Microscopic as well as photographic techniques have been used to analyze aggregate structure. In recent years, confocal laser scanning microscopy (CLSM) data sets have allowed the visualization and quantification of three-dimensional (3-D) structures (18, 31).In this study, we analyzed aggregates from the Danube and Elbe Rivers by collecting reflection, nucleic acid, glycoconjugate, and negative stain signals using CLSM. In order to receive multifarious information about the aggregates, various approaches were necessary: small aggregates were examined directly, and large and dense aggregates were physically sectioned and poststained. Although most of the detected nucleic acid signals derive from bacteria, we refer to them as cellular nucleic acid signals (CNAS) including potential archaea and virus signals. Nucleic acid signals can potentially also be obtained from fungi, algae, and protozoa. But the detection of extracellular DNA can be excluded due to its type of appearance (7). Data sets of specific glycoconjugates, CNAS, and aggregate structure were visualized and quantified by using digital image analysis. The distribution of CNAS within riverine aggregates was determined by autocorrelation. Based on our samples, we describe the strengths and challenges in scanning and quantifying riverine aggregates using CLSM. Additionally, we discuss structure, function, and potentially important differences in aquatic aggregates from these two large European rivers.