Characterization and comparison of microbial communities in biofilms on different substrates in tropic lakes and hot springs of Rwanda

Abstract

Biofilms are complex and dynamic structures, mainly composed of viable cells (bacteria, archaea, algae, fungi, protozoa, and other metazoans) embedded in a self-produced hydrated extracellular polymer substance (EPS) matrix that binds them together and adhere to the solid surface or liquid-solid interface of various substrates. Based on the attachment surface, biofilms in aquatic ecosystems can be categorized as, for example, epipelon/ epipsammon (sediment/sand), mats (rock), or epiphytic (plant surface) biofilms. The submerged and floating macrophyte leaves provide unique niches and nutrients for microbial growth, forming plant-biofilm symbiotic systems that represent the basic components of aquatic ecosystems and play important roles in transforming pollutants and maintaining ecological balance. The ecological functions of biofilms are closely linked to nutrient chelating, (re) cycling, and the detoxification of environmental pollutants by biofilm-dwelling communities. Previous studies explored the planktonic microbial diversity and composition in the water column and rarely in epiphytic biofilms. However, the influence of environmental variables (e.g., seasons and water chemistry) and microhabitat types (e.g., submerged and floating macrophytes, surface sediments, and mats) on the microbial biodiversity, assemblages, interactions, and ecological functions are poorly understood, especially in the tropical Lakes and hot springs. In this study, epiphytic bacterial and eukaryotic biofilm communities in submerged and floating macrophytes and surface sediments were investigated in two tropical Lakes (Rumira and Cyohoha North), Rwanda in August and November 2019, and mat samples from two hot spring regions (Bugarama hot pool and Gisenyi hot springs); the water quality and environmental parameters were also determined, and the differences in microbial communities were compared under these environments. The main findings are as follows: 1) The exploration of microbial composition in Rumira Lake revealed that eight phyla, including Firmicutes, Proteobacteria, Cyanobacteria, Actinobacteria, Chloroflexi, Bacteriodetes, Verrumicrobia, and Myxomycota, dominated bacterial communities, while the microeukaryotic communities were dominated by Unclassified (uncl) SAR (Stramenopiles, Alveolata, Rhizaria), Rotifers, Ascomycota, Gastrotricha, Platyhelminthes, Chloroplastida, and Arthropoda. Interestingly, the eukaryotic OTUs (operational taxonomic units) number and Shannon indices were significantly higher in sediments and epiphytic biofilms on Eichhornia crassipes than Ceratophyllum demersum (P < 0.05), while no differences were observed in bacterial OTUs number and Shannon values among substrates. Redundancy analysis (RDA) shows that water temperature, pH, dissolved oxygen (DO), total nitrogen (TN), and electrical conductivity (EC) were the most important abiotic factors closely related to the microbial community on C. demersum and E. crassipes. Furthermore, co-occurrence networks analysis (|r|>0.7, P < 0.05) and functional prediction revealed more complex interactions among microbes on C. demersum than on E. crassipes and sediments, and those interactions include cross-feeding, parasitism, symbiosis, and predatism among organisms in biofilms. 2) The influence of Ceratophyllum demersum, Eichhornia crassipes, and surface sediments on the microbial structures, co-occurrence patterns, and community assembly mechanisms of epiphytic bacterial and eukaryotic biofilm communities in Lake Cyohoha North was investigated. Illumina sequencing method reveals that phylum Cyanobacteria, Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Chloroflexi dominated bacterial communities microeukaryotic communities were dominated by Rotifera, SAR (Stramenopiles, Alveolata, and Rhizaria), Platyhelminthes, Chloroplastida, Phragmoplastophyta, and Ascomycota. There were no significant differences in microbial alpha diversity indices (except OTU richness in microeukaryotes) among Abstract 5 substrates across seasons. Interestingly, the bacterial community dissimilarity was significantly different among substrates (P < 0.05). The null model analysis shows that stochastic processes dominated the microbial community assembly in epiphytic (except for bacteria on E. crassipes) and surface sediment biofilms. Notwithstanding the dominance of stochastic processes in microbial community assembly, the deterministic processes still represented a significant part of the assembly processes. Thus, the stochastic and deterministic processes drove microbial community assembly on aquatic plants and sediments. Moreover, co-occurrence network analysis revealed stable and complex food chains in surface sediments compared to other substrates. 3) The study on microbial assemblage and interaction has been conducted in hot springs Bugarama (40.2-47.3℃) and Gisenyi (58-71.4 ℃). The results indicate that bacterial community β-diversity in moderate temperature was strongly driven by stochastic processes, whereas in hightemperature springs, the variable selection in microeukaryotic and bacterial communities was potentially shaped by temperature. For example, some Amoebozoa (e.g., Echinamoeba, BOLA868, and Telaepolella) and SAR taxa have adapted to high-temperature in hot spring microbial mats. RDA revealed that microeukaryotic communities were strongly driven by temperature in hightemperature microbial mats. Furthermore, co-occurrence networks and functional prediction showed more stable interactions among microbes in high-temperature mats, including crossfeeding, symbiosis, parasitism, and predation. Even though microeukaryotic communities are often overlooked in hot spring mats and other extreme environments, our findings shed new insights into that microeukaryotic communities harbor complex interactions and adaptations to high-temperature in tropical hot spring microbial mats, which may provide a basis to understand better microbial communities inhabiting extreme environments. 4）Lastly, we compared of influence of environmental parameters on microbial interactions and predicted bacterial functions on microbial communities in epiphytic, epipelic, and mat biofilms in shallow lakes and hot springs. Bacterial and microeukaryotic communities on C. demersum and E. crassipes were positively associated with DO, pH, EC, TN, TP, and temperature in tropical lakes, whereas bacterial communities in C. demersum and mats were commonly positively related to temperature, TDS, pH, and EC. Co-occurrence networks of possible interactions among microbial genera in aquatic macrophytes, surface sediment and mat biofilms were different. However, there were similarities of top microbial phyla in networks among lakes and hot springs. Specifically, all microbial networks shared phylum Ascomycota and SAR super group, whereas GHS mat network displayed the higher microeukaryotic taxa and Cyanobacteria. Furthermore, this study suggests that all microbial networks share organic matter decomposition, predation and parasitism relationships, and primary production. The predicted metabolic functions (FAPROTAX) of the bacterial communities to all substrates were related to carbon and nitrogen cycling and xenobiotic degradation. Phototrophic functions were significantly dominant in epiphytic and mat bacterial communities, whereas methanogenesis dominated the sediment bacteria. The higher bacterial functional abundance of sediment and C. demersum bacteria was detected in the wet season, while high-temperature mats exhibited the high bacterial functional abundance The findings provide new insights or cues to understand better the influence of microhabitat/substratum type and environmental variables on bacterial and microeukaryotic biodiversity, interactions, assemblages, and ecological functions in epiphytic, surface sediment, and mat biofilms from tropical lakes and hot springs. Furthermore, this study not only attempts to fill the knowledge gaps but also acts as a basis for future studies – with a particular emphasis on Doctoral Dissertation of Hohai University 6 including epiphytic, sediment, and mat biofilm to understand, maintain, and improve aquatic ecosystem health and integrity.