4. Discussion
The interactions between clay minerals and microorganisms have significant environmental implications in the biogeochemical cycle. For example, microbial activities could alter mineral compositions and enrich potassium (K), leading to a significant K sink that helps to equilibrate the unbalanced K budget in the ocean [42]. In addition, the algae-mineral interactions could also be used to remediate heavy metal pollution owing to large surface area to volume ratio of algae-mineral composites [43]. However, it has not been documented as to the effects of different clay minerals on microalgal cells.
In this study, Pal greatly inhibited algal photosynthesis, whereas Mt showed mild stimulation of algal growth. In particular, due to its large surface area (136.35 ± 0.31 m2/g) and interwoven fibrous structure (Fig. 6D), the particles of Pal were bound tightly to the cell surface and some of them even penetrated into algal cells, inducing tight and dense aggregation on the cell surface (Fig. 6E&6F, Fig. 7C&7D). Therefore, this mineral could prevent light from entering the cells, resulting in decreased photosynthesis parameters such as Fv/Fm and rETRmax (Fig. 1). Fv/Fm describes the reduction of the primary electron acceptor and may indicate the loss of function of PSII to capture enough light and interruption of photosynthetic electron transfer from PSI to PSII [44]. The value of rETRmax can be used to evaluate the change of the capacity of the electron transport chain [45]. The decline of these two parameters indicated that photosynthesis of C. reinhardtii was inhibited by the particles of Pal. In order to compensate for the decline of photosynthesis, the content of chlorophyll increased (Fig. 4C) and several related genes, e.g., CAB4, CAB7 and CHLG, were upregulated (Fig. 8), which was consistent with previous literature [46-49]. In contrast to Pal, the addition of Mt did not significantly change the photosynthesis of algal cells (Fig. 1). Although Mt adsorbed on the cell surface and also prevented some light from entering the cell, energy could still move across the physical barrier from Mt on the cell surface [50]. Therefore, Mt did not negatively affect the cell photosynthesis.
The presence of Pal inhibited the P uptake by cells at P concentration of 3.15 mg/L (Fig. 2A). Lower supply of P affected the synthesis of metabolites (e.g., component of sugar phosphates) and reactions that involved the coenzyme ATP [51]. Therefore, photophosphorylation, rubisco activity, and reactions of the Calvin cycle were suppressed, which further affected the photosynthesis of algae and downregulated IF2CP, CPLS1, psbH, and OHP1 genes (Fig. 8). In addition, the metabolic reactions were not effective in supporting the cell growth under P limitation [52], finally leading to a decrease in cell numbers (Fig.2B). However, cell respiration increased even though the P was deficient (Fig. 2A). The cells were then forced to upregulated odhA and SAMC1 genes involved in cellular respiration and consume more energy to survive. Furthermore, when the P level in the culture was 31.5 mg/L, the P uptake increased to resist the negative effects [53]. In contrast, Mt did not cause much damage to cells, which did not absorb too much P to promote their growth and metabolism and to resist the adverse environment [54].
Our results showed that proteins in EPS were the key organic compounds that reacted with Pal (Fig. 4A), This finding is consistent with previous research [55]. More specifically, Mt did not lead to significant changes of EPS components while Pal reduced the contents of proteins and polysaccharides in EPS (Fig. 4A&B). In order to resist the dense aggregation of Pal on the surface of algae and prevent further interaction between cells and Pal, functional groups (such as C=O and -COOH) were declined (Fig. 5). These ligands in EPS are critical in binding clay minerals [15]. The 3D-EEM results further illustrated that tryptophan-like substance was the major substance in response to mineral treatments (Fig 3&4). This may be due to complexation between Pal and benzene ring contained in tryptophan-like substance. These responses can be explained by the self-protection mechanism of the algae [56].
Pal particles can penetrate the cell walls based on TEM results (Fig. 7C&D). With the increase of Pal concentration, the cell membrane could be damaged and the intracellular proteins flowed out and the soluble proteins contents increased (Fig. 4A). The DNA might also be leaked into the bacterial cytoplasm due to Pal stress [57]. To resist this stress, the cells may switch intracellular carbon flux from lipids to polysaccharides [23]. Therefore, intracellular polysaccharides contents were significantly increased (Fig. 4B). When Pal concentration was 200 ml/L, the cells could survive but at the concentration of 500 mg/L, almost all the cells died (Fig. 2B). Therefore, the damage of the whole cells, together with the impacted functions discussed above, finally led to the reduction of activities in C. reinhardtii by fibrous clay minerals. Compared with Pal, the content of protein and polysaccharide did not change significantly after Mt addition (Fig. 4A&B), suggesting that Mt did not need to activate its self-protection mechanism.