The contents, components and geographical distribution of EPS in biofilms
EPS contents (dry weight) in periphytic biofilm varied from 1.28–2.88% of the total biofilm mass (Fig. 1a). On average, EPS accounted for 1.9% of the biomass of periphytic biofilm, which provides a direct evidence that that EPS is an important abiotic component in periphytic biofilms. In periphytic biofilms, the contents of protein are much larger than the contents of polysaccharides (Fig. 1b), and the ratio of protein to polysaccharide varied from 1.8 to 20 (Fig. 1a), with an average value of 5.4.
Here, we employed the latitudinal diversity gradient (LDG) theory to evaluate the geographical distribution pattern of EPS content in periphytic biofilm. We found that EPS content in periphytic biofilm decreased significantly with increasing latitude (r = 0.3294, p < 0.0001, red line in Fig. 2). In addition, we found that the ratio of protein to polysaccharide in periphytic biofilm increased significantly with the increasing latitudes (r = 0.2742, p = 0.0005, blue line in Fig. 2). The results showed that both the EPS content and the ratio of protein to polysaccharide in periphytic biofilm have significant geographical distribution characteristics, namely, the higher the latitude at which a periphytic biofilm grows, the lower its EPS content, and the higher its ratio of protein to polysaccharide.
Driving forces for the geographical distribution of EPS in biofilm
Partial Least Squares Path Modeling (PLS-PM) was employed to synthesize the data and analyze the forces driving the geographical distribution of EPS in periphytic biofilm focusing on climate, paddy soil composition, and floodwater characteristics. Microbes are the primary executors changing the component and content of EPS in periphytic biofilm , all the other factors such as climate, paddy soil, and floodwater are secondary: they affect the microbial components first, and (thus) only indirectly affect the geographical distribution pattern of EPS in periphytic biofilm (Fig. 3). The total effect of climate on eukaryotes and prokaryotes is 0.55 and 0.69, respectively. While the total effects of paddy soil on eukaryotes and prokaryotes are respectively 0.28 and 0.19, and the total effects of floodwater on eukaryotes and prokaryotes are 0.45 and 0.40, respectively (Fig. 3). Combined their direct and indirect effect on microbial composition together, the total effect of climate, soil and floodwater on the geographical distribution of EPS are − 0.41, -0.11, and − 0.23, respectively. By contrast, the total contribution of these three factors on the microbial components which in turn shift the geographical distribution of EPS is in the following order: Climate > floodwater > paddy soil. Thus we can conclude that climate factors are the principle forces driving the geographical distribution of EPS in periphytic biofilm. Among the analyzed climate factors, both sunshine duration (path coefficient = 0.91) and radiation intensity (path coefficient = 0.63) showed positive effect (Fig. 3). Additionally, the factor of effective accumulated temperature (EAT) showed significantly negative effect (path coefficient= -0.92, Fig. 3) on the geographical distribution of EPS in periphytic biofilm. These patterns suggest a role for physical geography in determining the geographical distribution of EPS in periphytic biofilm.
Microbes in periphytic biofilm had direct effect on the EPS composition. PLS-PM analysis showed that prokaryotes in periphytic biofilms exerted a negative effect (path coefficient=-0.63, Fig. 3) on EPS production by periphytic biofilm, suggesting that prokaryotes in periphytic biofilm may function as the main EPS consumer. While eukaryotes on the other hand exerted a positive effect (path coefficient = 0.46, Fig. 3), implying that eukaryotes may be producers of EPS in periphytic biofilms.
In our periphytic biofilm samples, a total of 130 genera of prokaryotes belonging to 16 phyla and 145 genera of eukaryotes belonging to 23 phyla were found. This indicates that a wide diversity of both prokaryotes and eukaryotes are present in periphytic biofilms, and that eukaryotes are slightly more varied than prokaryotes. For eukaryotes, Aporcelaimellus, Paratripyla, Characiopodium, Heteromita, Desmodesmus, Chlorotetraedron, Rhabdolaimus, Halteria, Pythium, and Chaetomium are the genera most frequently present in the individual top 10 of most abundant genera in a specific periphytic biofilm (Fig. 4a); while for prokaryotes the corresponding top 10 genera are Flavobacterium, Acinetobacter, Pirellula, Dinghuibacter, Massilia, UTCFX1, Bacteroides, Luteolibacter, Clostridium_sensu_stricto_13, and Proteiniclasticum (Fig. 4b).
Both prokaryotes and eukaryotes are significantly related to EPS content in periphytic biofilm (Fig. 5). Most of the prokaryotes in biofilm, except for a few bacteria (e.g. Azospira, Dechloromonas, Paludibacterium, Phreatobacter, Prevotella), had negative correlation with the EPS content. On the contrary, most of eukaryotes, except for Rhogostoma, in periphytic biofilm showed significantly positive correlation with the EPS in periphytic biofilm. In summary, the correlation analysis results (c.f. Figure 5) echo the results of eukaryotes showing a positive effect on EPS in periphytic biofilm while prokaryotes show the opposite roles (c.f. Figure 3). The correlation analysis results further support our speculation that eukaryotes are possibly EPS producers in periphytic biofilm, while prokaryotes may act as EPS consumers.
Furthermore, we found that EPS in periphytic biofilm was significantly affected by the availability of nutrients such as phosphorus in paddy soil (r=-0.155, p = 0.03, Fig. 6a) and NH4+-N in floodwater (r = 0.175, p = 0.017, Fig. 6b).
Potential environmental and agronomic effects of EPS in periphytic biofilms
As periphytic biofilm grows in paddy fields, we thus pay close attention to their possible roles in regulating nutrients (C, N, and P) cycling in paddy fields. Herein, we found that periphytic biofilm showed great potential in nutrients accumulating (Fig. 7a), and the concentration of TN (r = 0.372, p < 0.001, green line in Fig. 7b) and TP (r = 0.272, p < 0.001, red line in Fig. 7b) in periphytic biofilm were significantly related to the EPS composition, which may partly explain why periphytic biofilm could accumulate plenty of N and P. In other words, EPS contribute to assisting periphytic biofilm accumulating N and P, thus shifting the biogeochemical cycling of N and P in paddy fields. For the environmental effect, periphytic biofilm accumulating N and P increases the retention time of N and P in paddy fields and then prevents N and P loss from paddy fields in runoff .
Additionally, we found that EPS in periphytic biofilm also showed significantly positive correlation with TOC in periphytic biofilm (r = 0.302, p < 0.001, Fig. 7b). The results suggest that EPS from periphytic biofilms may be a source of TOC in paddy soils, and then improve the fertility of paddy soils, which is the agronomic effect of EPS in periphytic biofilm. In summary, we revealed two novel ecological functions of EPS in periphytic biofilms: 1) EPS in periphytic biofilms contributes to accumulating N and P and then decreasing the loss of nutrients from paddy fields; 2) EPS in periphytic biofilms helps to improve the TOC levels and hence the fertility of paddy soils.