A multi-disciplinarily designed mesocosm to address the complex flow-sediment-ecology tripartite relationship on the microscale

Biofilm growth within the new sophisticated mesocosm

The main purpose of the present study was to test this new design of straight flumes in terms of nature-like biofilm settlement and cultivation. Despite the absolutely identical setup of the six flumes, biofilm development could still differ due to smallest, possibly undetected variations in, e.g. the flow field. Moreover, biofilm growth is per se characterized by spatial heterogeneity. Thus, for the new flume design, it remained to be shown whether deviations in biofilm growth increase strongly along the test sections of one flume or between the individual flumes. This is very important since the comparability of biofilm growth and composition in the different straight flumes as well as within different flume regions is an essential prerequisite for further studies on the impact of environmental parameters on biofilm ecology and functionality.

The high spatial heterogeneity in biofilms and their complex mutual interactions with the environment have been described manifold (e.g. [19-21]). Biofilms have even been described as microbial landscapes that not only are shaped in their spatial configuration by multiple physico-chemical factors but also alter their environment in considerable dimensions due to their growth [22]. Thus, not surprisingly, biofilms settling in the flumes exhibited high small-scale heterogeneity (on a single cartridge), but these pattern were clearly similar in all flume regions and flumes. This reflects the exact same settings of temperature, illumination and, most importantly, hydrodynamics in all flumes to provide the same settling and growth conditions for biofilms. Most common, an integral approach (Reynolds) is applied to account for the flow conditions within a flume as a precisely controlled hydrodynamic regime is either not necessary or not practicable. However, an exact determination of near-bed turbulences and bed shear stress directly at the sites of interest is essential for this study because various boundary effects such as eddy developments along the walls of a flume may influence hydrodynamics and near-bed turbulence significantly. Consequently, this might impact the erosive forces acting on biofilms as well as their nutrient replenishment to affect biofilm morphology and activity. Hence, in this study, the turbulence distribution was determined via high-resolution laser Doppler anemometry (LDA) before this experiment and later on checked with acoustic Doppler velocimetry (ADV) [17]. In addition, the long inlet flow section as well as the design of the flumes (with a sufficient distance of the biofilm cartridges to the flume walls) ensures a homogenous turbulence distribution over the biofilm growth section. Another essential fact to keep in mind is that bed shear stress is an exponential function of the flow velocity emphasizing the importance of constant discharges within the flumes. This is why high-resolution discharge measurements were performed in all flumes throughout the experiment. These data gave evidence on the constant and similar discharges in all six flumes and thus on the identical bed shear stress levels over all biofilm growth sections in the presented experiment. This is one important prerequisite for the high reproducibility of biofilm cultivation that could be shown for the new experimental setup in this study.

Biomass and EPS

Several studies investigating intertidal biofilms suggested a complex non-linear relationship between microbial biomass and EPS production [23-25]. The occasionally fast shifting nutritional and physiological states of the biofilm microbes might be of major importance for EPS production and secretion. This was underpinned by Underwood et al. [26] who described that diatom EPS production was, among others, dependent on the nutritional condition of the cells. The natural riverine water used in the flumes contained moderate nutrient concentrations comparable to oligotrophic habitats that remained stable over the experiment. Besemer et al. [27] and Artigas et al. [12] reported microbial biomass similar to the values determined in the present experiment under comparable conditions of nutrient availability (e.g. bacterial cell densities up to 1.13 × 10⁸cells cm⁻² or chlorophyll a concentrations of 0.03 to 6.28 μg cm⁻²). In contrast, biofilms in a eutrophic river (Neckar) were characterized by high microbial biomass and EPS production [28], e.g. an up to 16-fold higher chlorophyll a content than in this study emphasizing the effects of different water qualities upon biofilm development, corresponding metabolic rates and possibly, functionality. Low supply of nutrients may have restricted EPS production during biofilm development in the present experiment; still, the biostabilization effect (as discussed later) was impressive.

Microbial community

As Marzorati et al. [29] stated, RWR, dynamics and functional organization calculated from the DGGE fingerprints should be seen as qualitative indicators, not as absolute measures due to the known drawbacks of molecular fingerprinting techniques. Nevertheless, they are an important tool to describe, compare and interpret different DGGE band patterns in order to obtain a higher level of information about ecological processes in biofilms. Generally, observing the microbial community of biofilms in this study revealed two distinct stages during development. The first phase could be described as an initial colonization of the abiotic substrate by planktonic bacteria. The findings of Beier et al. [30] or Crump and Hobbie [31], e.g. 40 bacterial TGGE bands in comparable riverine water, support the initially high bacterial RWR in this experiment. During the subsequent establishment of a bacterial biofilm within the first 11 days, competition and specialization of the bacterial community became visible by decreasing RWR and increasing functional organization. Manz et al. [32] and Araya et al. [33] described similar shifts within the bacterial community composition during the formation of lotic biofilms. In the period between days 11 and 14, severe changes within the bacterial community could be observed. Along with significantly decreasing cell counts and RWR, increasing dynamics and functional organization of the bacteria might be due to bacterial biofilm detachment, the initial algal colonization or settlement of grazing ciliates as described by Wey et al. [34] - or a combination of these processes.

Besemer et al. [27] highlighted the role of diatoms as key players in river biofilms as they shape the bacterial community in a combination of physical and biological processes. The importance of diatoms in this experiment became obvious since parallel to the development of the diatom community (from day 14 on), a steady increase of vertical and horizontal heterogeneity of the biofilms could be visually observed. As described by Besemer et al. [16], this spatial small-scale heterogeneity was a major driver affecting the development of the microbial community. Thus, diatoms apparently influenced the bacterial community in different ways. On one hand, their photosynthetic activity and EPS production may have supported the growth of associated bacteria as indicated by high increase rates of the total cell counts after day 14 of the experiment, a phenomenon already described by Espeland et al. [35]. On the other hand, the pronounced three-dimensional structures of their colonies may have allowed the formation of various physical and biochemical transition/gradient zones, e.g. O₂ gradients as described by Glud et al. [36] or Fenchel and Glud [37]. Due to this formation of many microenvironments, accumulation of different metabolic products of the diatoms as well as their associated bacteria in the biofilm matrix and several metabolic cascades, various niches can develop for a broad range of bacteria with different physiologies [38]. In this context, specialized bacteria may have found optimal conditions in the matured biofilms as reported by Manz et al. [32]. Summarizing, the diatom development apparently led to dominance of a few bacterial species but may also have led to the establishment of various niches where diverse bacterial species might occur in low abundances, indicated by steadily decreasing dynamics within the bacterial community and parallel increasing bacterial RWR.

Biostabilization

Due to its high economic and ecological importance, sediment dynamics in lotic systems plays a major role in hydraulic engineering. Different modelling approaches approximated the highly complex sediment-water system. Briefly, the Shields model [39] is commonly applied to determine the stability of sediments versus erosional forces by defining characteristic diameters and the density of the sediment grains. However, this approach is not applicable for sediments with strong biological influence. Righetti et al. [40] described the first model based on the Shields equation introducing adhesiveness/adhesion as a new parameter in order to incorporate the influence of biofilms on sediment stability. Finally due to the development of the MagPI device that has been applied successfully in the marine habitat [6,7,41], the determination of biological-induced adhesiveness could be achieved at high temporal and spatial resolution. While most studies focused on brackish/intertidal areas (e.g. [23,42,43]), up to now, biostabilization of lotic fine sediments is virtually unaddressed. Spears et al. [44] suggested the major importance of biostabilization in marine/brackish habitats supporting the current doctrine that high quantities of strong ionic bounds significantly strengthen the EPS matrix. Nevertheless, despite observing significantly lower EPS values as well as microbial biomass (as compared to brackish/marine biofilms), a significant biostabilization effect was detected. This emphasized the importance of biofilm-induced stabilization of fine sediments in lotic waters (and contradicted the current doctrine).

Furthermore, this study gave insights in the development of various geochemical and (micro-) biological parameters during biofilm growth affecting this important ecosystem function. In this context, the content of colloidal EPS compounds could be seen as an approximate marker for biostabilization capacity. Further investigation of the quality of these polymers could lead to the identification of single carbohydrate or protein moieties with high gluing and stabilizing capability. While biofilm stability appeared to be related to total cell counts in nascent biofilm stages, the development of the bacterial community composition turned out to be even more crucial. Thereby, short and long-term shifts within the bacterial and algal community occurred simultaneously to significantly change biofilm stability. This emphasizes the importance of the molecular biology tools used in this study to address biofilm composition and diversity. Identification of bacterial key players in biostabilization and their interactions with algae could be the next step to further elucidate the contribution of microbiology to sediment stability. Future studies should also consider the role of the protozoa community in shaping microbial biofilm community and functionality, here biostabilization.