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.