In situ modelling of biofilm formation in a hydrothermal spring cave

Abstract

Attachment of microorganisms to natural or artificial surfaces and the development of biofilms are complex processes which can be influenced by several factors. Nevertheless, our knowledge on biofilm formation in karstic environment is quite incomplete. The present study aimed to examine biofilm development for a year under controlled conditions in quasi-stagnant water of a hydrothermal spring cave located in the Buda Thermal Karst System (Hungary). Using a model system, we investigated how the structure of the biofilm is formed from the water and also how the growth rate of biofilm development takes place in this environment. Besides scanning electron microscopy, next-generation DNA sequencing was used to reveal the characteristic taxa and major shifts in the composition of the bacterial communities. Dynamic temporal changes were observed in the structure of bacterial communities. Bacterial richness and diversity increased during the biofilm formation, and 9-12 weeks were needed for the maturation. Increasing EPS production was also observed from the 9-12 weeks. The biofilm was different from the water that filled the cave pool, in terms of the taxonomic composition and metabolic potential of microorganisms. In these karstic environments, the formation of mature biofilm appears to take place relatively quickly, in a few months.

Figure 1

Geographic location of the sampling site (A). The arrangement of the in situ experimental model system in the RT spring cave (B,C).

Figure 2

SEM micrographs of biofilm samples developing on the glass slides during a one year period (3 weeks (A,B), 9 weeks (C,D), 12 weeks (E), 21 weeks (F), 30 weeks (G), 1-year-old biofilm (H)) in the RT thermal spring. Scale bar (A,B,D,F) 2 µm, (C,E,G) 10 µm, (H) 20 µm.

Figure 3

Percentile distribution of amplicon sequences on genus level revealed from the RTW sample and changes in the bacterial composition of biofilm communities in the one year period based on the 16S rRNA gene amplicon sequence data. Genera having < 2% relative abundance are combined in the “Other” category.

Figure 4

NMDS ordination based on Bray–Curtis distance of the bacterial OTUs from biofilm developed for years on the rock (RTB), glass slides (3–30 week and 1 year) and water samples (RTW) of the RT spring (stress: 0.03). Based on SIMPER analysis, OTUs responsible for 60% dissimilarity among samples are shown in gray. Figure is based on the combination the results of this study and Enyedi et al. (2019).