Differences in growth of A. flos-aquae in various P supply
The biomass of A. flos-aquae was represented by the cell density. At the beginning, the three A. flos-aquae strains incubated in the four treatments gained similar biomass increasing from around 5.0×108 cells L-1 to 1.5×109 cells L-1 after 8 days (Fig. 1). The cells in the -P and +DIP treatments were then rapidly growing until the day 20 in terms of cell densities and active values of specific growth rate (Fig. 1 A, D; Fig. 2 A, D). What’s more, the average specific growth rates in the -P treatments had the highest values among all the treatments in the first 20 days (Fig. 2 A), after that they grew more slowly reaching the peak at day 31 (with cell densities at 4.6, 6.6 and 6.5×109 cells L-1 in A. flos-aquae 1249, A. flos-aquae 1171 and A. flos-aquae 1260, respectively), followed by remarkable drop in biomass (Fig. 1 A) together with negative specific growth rates (Fig. 2 A), whereas the growth didn’t cease and were at constant positive growth rate in the +DIP treatments (Fig. 2 D), which made the biomass amassed more and more to be the highest (1.75 , 2.55 and 2.86 ×1010 cells L-1 in A. flos-aquae 1249, A. flos-aquae 1171 and A. flos-aquae 1260, respectively) compared with other treatments (Fig. 1).
The lowest biomass was that in the treatments of +DOPα and +DOPβ with the maximum value at 2.3 ×109 cells L-1 for the three strains. Specifically, the maximum biomass in the +DOPα and +DOPβ treatments were about 50% for A. flos-aquae 1249, 30% for A. flos-aquae 1171 and 1260 of that in -P treatments. While compared with +DIP treatments, they were 12.42 % and 13.38 % for A. flos-aquae 1249, 7.64 % and 8.04 % for A. flos-aquae 1171, and 6.1% and 6.66% for A. flos-aquae 1260 of that, respectively. Noticeably, the biomass in the +DOPα and +DOPβ kept increased before day 11, then the cell densities stabilized at a ranging of 1.5×109 cells L-1 to 2.0 ×109 cells mL-1 (Fig. 1 B, C). Moreover, the specific growth rates were also the lowest before day 20 compared with other two treatments, after which they showed down tendencies from the highest at the beginning towards nearly none, with sometimes positive or opposite and very low absolute values near zero (Fig. 2 B, C). All three strains performed generally consistent in these growth parameters.
Photochemical efficiency of photosystem II in different treatments
The photochemical efficiency of photosystem II (Fv/Fm) values remained relatively consistent at 0.400 to 0.538 during the whole incubation time in the +DIP treatments. While in the -P treatments, it showed descending after day 31 from consistent values with that in the +DIP treatments and dropped strongly to very low values at the end of the incubation time (0.014, 0.077 and 0.037 for A. flos-aquae 1249, A. flos-aquae 1171 and A. flos-aquae 1260, respectively). On the other hand, the Fv/Fm values in the +DOPα and +DOPβ treatments were about 0.1 less compared with that in the +DIP treatments, they had no significant differences among the whole incubation time despite a little decreased after day 35 (Fig. 3).
SRP concentrations in different treatments
For all selected strains, the -P treatments always showed low SRP concentrations in the media (Fig. 4 A). Thus, growth conditions in the -P treatments regarding P supply didn’t markedly change during the experiment. The SRP concentrations in the +DOPα and +DOPβ treatments appeared rapid increasing to the peak at the value of around 3.5 mg L-1 for the first 15 days with the DOP concentrations decreased to near none, then the SRP concentrations stabilized at around 2.7 to 3.0 mg L-1 (Fig. 4 B, C). In the +DIP treatments, the SRP concentrations declined gradually before day 20 and more rapidly thereafter. Moreover, they were decreased into the levels at the limit of detection in the strains of A. flos-aquae 1171 and A. flos-aquae 1260 (Fig. 4 D).
Expression of APase and its relationship with ambient P
The ELF signals in cells of A. flos-aquae were observed from different subtropical and temperate water bodies in Czechia, Poland and China, as well as in the studied strains (Fig. 5). Noticeably, the ambient SRP concentration was high at 0.681 mg L-1 in Stefanski Reservoir in Poland (Table. S1). In addition, the SRP concentration was negatively relative with the ELFp of A. flos-aquae in Lake Chaohu, similar trend was also observed in the cultures among four treatments in the lab-culture (Fig. 6).
In the laboratory experiment, the ELFp exhibited significantly positive relationship with APA>3.0μm (r=0.7768, P<0.01) (Fig. S2). In the culture media of the -P treatments, APA>3.0μm accounted for the largest part of the APAT (67.6-70.5%) (Fig. S2), which together with the percentages of cells with ELF labeling (ELFp) were both significantly higher compared to those in the other treatments (P<0.01) (Fig. S3, S4). In addition, though the ELFp in +DOPα and +DOPβ treatments were strongly lower than that in the -P treatments (10.5 to 15.2 - fold greater in -P treatments versus DOP treatments, P < 0.01), they showed remarkably higher than that in +DIP treatments (17.8 to 25.6 - fold, P < 0.01) (Fig. S4).
Cellular P content and PPB containing cells in different treatments
There were great differences in specific cellular TP content among the four P treatments, which showed remarkable lower values in the -P treatments compared to that in the other ones (P < 0.01) (Fig. 7). The polyphosphate granules in the cells showed the P storage in A. flos-aquae (Fig. 8). In addition, the percentages of cells with PPB staining (PPBp) in the -P treatments also exhibited the lowest (P < 0.01) (Fig. 9). What’s more, the initial percentages of cells with PPB staining (PPBp) was around 0.05% in -P treatments, and it could be observed PPB containing cells before day 15 (Fig. 9 A), with an up-ward trend began at day 1 or 2 to the highest at day 5, and then went down to the none within strains A. flos-aquae 1171 and 1260, meanwhile, the strain A. flos-aquae 1249 showed the same pattern with the other two but harbored lesser PPBp, the peak is invisible in the figure (Fig. 9 A). In the +DIP treatments, PPBp increased from day 1 and reached maximum at day 2 then remained constant (85-93%), less variation was observed in PPBp (Fig. 9 D). Additionally, numbers of polyphosphate granules within one cell were also relatively constant in the +DIP treatments (Fig. 8 E and F). Furthermore, the PPBp lessened in the strain A. flos-aquae 1171 and A. flos-aquae 1260 at day 46 to 74% and 78%, respectively (Fig. 9 D). In the +DOPα and +DOPβ treatments, the PPBp exhibited fluctuations. In details, the PPB containing cells achieved to the most with percentages comparable to that in the +DIP treatments, but with time incubated, the percentages decreased gradually and sometimes even to extremely low levels (Fig. 9 B, C). Furthermore, number of granules with one cell also obviously fluctuated in +DOPα and +DOPβ treatments (Fig. 8 A-D).