Orals

Through their special way of life, biofilms have several advantages over organisms in planktonic growth. By being surface-attached and producing a mass of extracellular polymeric substances (EPS), microorganisms possess inherent self-immobilization, which decreases the expenditure of downstream processing in industrial applications. Furthermore, they are more resilient against environmental stress and toxic substances, such as antibiotics. An important factor here is diffusion, of substrate into the biofilm and metabolites through and out of the biofilm; however, these mechanisms are still poorly understood. By utilizing a specially developed diffusion model and CLSM FRAP microscopy, diffusion constants in the living, fully hydrated biofilm of L. lactis during development can be assessed. With it, heatmaps of diffusional constants and finally a diffusion profile encompassing a true 3D space of the living biofilm in growth can be generated. With those, possible hotspots and changes of diffusion inside the biofilm structure with regard to changing cultivation conditions and the substratum can be identified, thus furthering our understanding of diffusion in biofilms and how they react to their environment.

The project is funded by the DFG (UL 170/14-1) and the collaborative research center (SFB) 926 on “microscale morphology of component surfaces” (MICOS).

How to cite: Chodorski, J., Hauth, J., Wirsen, A., and Ulber, R.: Morphological and diffusional changes in L. lactis biofilms, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-31, https://doi.org/10.5194/biofilms9-31, 2020.

Biofilms are now considered to be the most abundant form of microbial life on Earth, playing critical roles in biogeochemical cycles, agriculture, and health care. Phenotypic and genotypic variations in biofilms generally occur in three-dimensional space and time, and biofilms are therefore often investigated using microscopy. However, the quantitative analysis of microscopy images presents a key obstacle in phenotyping biofilm communities and single-cell heterogeneity inside biofilms. Here, we present BiofilmQ, a comprehensive image cytometry software tool for the automated high-throughput quantification and visualization of 3D and 2D community properties in space and time. Using BiofilmQ does not require prior knowledge of programming or image processing and provides a user-friendly graphical user interface, resulting in editable publication-quality figures. BiofilmQ is designed for handling fluorescence images of any spatially structured microbial community and growth geometry, including microscopic, mesoscopic, macroscopic colonies and aggregates, as well as bacterial biofilms in the context of eukaryotic hosts.

How to cite: Jeckel, H., Hartmann, R., Jelli, E., and Drescher, K. and the BiofilmQ team: BiofilmQ, a software tool for quantiative image analysis of microbial biofilm communities, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-26, https://doi.org/10.5194/biofilms9-26, 2020.

            More than 30 years have passed now since the pioneering work of Costerton and co-workers^e.g.,1. We have learned that the biological functions of the cells embedded in the complex, self-produced polymeric extracellular matrix, differ radically from the ones of the planktonic cells. Emergent properties such as enhanced antimicrobial resistance appear.  Biofilms are widely spread in different habitats, both in the environment and the living organisms. Mostly, the characterization of this bacterial specific phenotype has been carried out using mono-species lab models. Yet, these systems are in marked contrast to the biofilms found in the environment. Those are usually complex and contain multiple bacterial species and, in many cases, also fungi, algae, and protozoa². To take this into account, researches have recently turned to multispecies communities, aiming at describing the interspecies interactions in order to decipher the mechanisms underlying the properties of these complex consortia.

            We present here a simplified model community consisting of 4 species — Bacillus thuringiensis, Kocuria salsicia, Pseudomonas fluorescens, Rhodocyclus sp. — elaborated from a natural environment to investigate the mechanisms supporting the formation of a multispecies consortium. We have been able to grow the 4-species biofilm under flow in a millimetric channel made of PDMS, which enabled to monitor the biofilm settlement and development using video-microscopy³. We found a deterministic development which follows defined kinetics and spatial distribution, suggesting that the formation of this adherent community is dominated by the self-induced modulation of the environmental parameters. To clarify this hypothesis, we focused our attention on the spatio-temporal distribution of oxygen and we devised an original experiment to map in situ and in real-time the evolution of oxygen level within the 4-species biofilm.

            We used an O₂ fluorescent probe made of a Ruthenium complex encapsulated in lipidic micelles to overcome the metal toxicity. We derived local oxygen concentration in the biofilm from fluorescent-lifetime imaging microscopy (FLIM) measurements of the probe in situ. The setup was equipped with a light sheet to ensure the optical sectioning for a 3D mapping. We will show here the spatial and temporal characteristics of the method and the first O₂ map obtained on a growing biofilm.

            To conclude, we will discuss how the monitoring of oxygen spatio-temporal distribution in a model community can help to elucidate basic interspecies interactions and reveal general mechanisms likely to govern number of more complex natural systems.

How to cite: Ben Youssef, W., Monmeyran, A., Sureau, F., Panier, T., and Henry, N.: Oxygen spatio-temporal distribution in a 4-species adherent community of bacteria , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-64, https://doi.org/10.5194/biofilms9-64, 2020.

Marine fouling biofilms typically have diverse community assemblages in which microalgae are strongly represented.  The visible light absorption properties of microalgal photosynthetic pigments typically drive the overall visible light reflectance spectra of these biofilms.  In some cases diagnostic spectral features can be used to infer algal taxonomy, while in mixed communities the overlapping pigment signatures of the constituent species often blur together.  In this study, we apply methods common in remote sensing approaches to spectral data to extract information from subtle variations in the reflectance spectra of mixed composition marine biofilms.  We demonstrate that marine biofilm community composition, as evidenced by their reflectance spectra, is both spatially heterogenous and spatially structured.

Visible-NIR hyperspectral images (3.3nm x 200 bands) of biofilms grown on 7.5cm x 7.5cm panels (n=9), immersed in a coastal marina at ~1m depth for 13 months, were captured with a benchtop line-scan imager.   The hyperspectral data were smoothed and transformed to consolidate the major aspects of spectral variability.  A novel active learning spectral classification method incorporating iterative spectral library building by k-means clustering and spectral angle mapping, followed by hierarchical clustering by spectral similarity, discovered more than 70 distinct spectral classes present in the biofilms.  Accordingly, the hyperspectral images of the fouling biofilms were converted to spatially explicit spectral class maps, where each class was assumed representative of a distinct community compositional mix.  Hyperspectral indexing calibrated to chl a surface area density was used to map biomass for the same images. 

Cross-tabulating the spectral class and biomass data, it was apparent that for these biofilms, different biomass density levels were consistently associated with specific community compositions (spectral classes.)  Only a small number of the possible classes were represented in the densest areas of biofilm, suggesting that these species composition mixes have a competitive advantage.  In contrast, the full diversity of class types was present in the low biomass areas. 

Our hyperspectral approach does not convey exact species composition, as would pooled metagenomic sampling or in-depth microscopy.  However it does allow for the examination of spatially explicit changes in biofilm composition at relatively large scales (the landscape), and so may be a useful tool in hypothesis generation, long term monitoring, and other environmental biofilm applications.

How to cite: Longyear, J. and Stoodley, P.: Landscape-level patterns in photosynthetic marine fouling biofilm compositional heterogeneity as revealed by hyperspectral classification, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-66, https://doi.org/10.5194/biofilms9-66, 2020.

Bacteria live in sprawling communities forming complex dynamic biofilm structures. The spatial heterogeneity found in biofilms may be a driver for the selection for optimized biofilm variants. Bacterial species that increase their matrix production can position favourably in the exterior biofilm regions in order to compete for valuable substrates. Here, we study both the spatial organisation and growth of bacterial cells in different bacterial communities over time to determine links between the structure of interspecies biofilms and selection for phenotypes adapted to growth and persistence in biofilms. This leads to the identification of driving mechanisms behind the dynamic spatial organisation combined with the performance of individual species over time in the spatial biofilm structure.

Four species, previously co-isolated from soil, were used in different combinations. We examined the formation and spatial organisation of biofilms in distinct experimental model systems, as we hypothesized that their interaction would change dependent on the specific environment. The biofilm models used included the static Calgary biofilm device assay and two flow systems: the microfluidic BioFlux model (liquid bulk flow), and the drip flow reactor (liquid-air interphase). Both chromosomal fluorescent markers and FISH were used to visualize the organisation within biofilms by confocal laser scanning microscopy.

We reveal how the changes in biofilm structure affect the overall performance of the biofilm community as well as the individual species in the biofilm. Our data indicates that a favourable localization of the individual species in a multispecies biofilm reduces selection for competitive phenotypes. Furthermore, we also observed that changes in matrix production could serve to stabilise the interspecific interaction between bacteria. This highlights the specific structural composition of a biofilm community as important for explaining biofilm dynamics.

How to cite: Røder, H. L., Liu, W., Russel, J., Herschend, J., Andersen, M. F., Madsen, J. S., Sørensen, S. J., and Burmølle, M.: Impact of spatial heterogeneity for selection regimes in multispecies biofilms, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-104, https://doi.org/10.5194/biofilms9-104, 2020.