Phycosphere pH of unicellular nano- and micro- phytoplankton cells and consequences for iron speciation

Phycosphere pH of single phytoplankton cells

The pH in the phycosphere of a single cell Chlamydomonas concordia (~5 µm diameter) exposed to 140 μmol photons m⁻² s⁻¹ was 8.27 ± 0.01 (179 measurements), while the pH of bulk seawater was 8.01 ± 0.01 (160 measurements) (Fig. 1c). The observed pH variation near the cell surface was <0.03 when the probe was held in place for 45 s. Moving the nano-probe away from the cell resulted in a progressive pH decrease towards the level in the bulk medium, while the pH gradually increased when moving it towards the cell. Measurements on the same cell and different cells were repeated several times, and higher phycosphere pH values than the bulk seawater were consistently recorded (Fig. 1c, Table S3). Under dark conditions, the phycosphere pH decreased, and it increased again once exposed to light (Fig. 1d). Light is thus a major influence on phycosphere pH.

Light intensity controls the magnitude of the phycosphere pH change (Fig. 1e). A gradual increase in light intensity (i.e. 15, 36, 75, 118, 140 and 180 μmol photons m⁻² s⁻¹) progressively increased the phycosphere pH up to 8.71 ± 0.02 (13 measurements), likely due to enhanced inorganic carbon uptake by phytoplankton. In this experiment, the significant change in the phycosphere pH with light was facilitated by the low buffer capacity of the solution (0.4 mM bicarbonate, Table S1); the change was smaller in natural seawater with 2 mM bicarbonate (Table S3). About 2 min later at the highest light intensity of 180 μmol photons m⁻² s⁻¹ the phycosphere pH decreased (Fig. 1e), and this was due to the inhibition of photosynthesis of marine Chlorophyta including Chlamydomonas sp. at a high light intensity >150 µmol m⁻² s⁻¹ [33]. At light intensities <118 µmol m⁻² s⁻¹, the phycosphere pH was significantly lower than ambient seawater pH (Fig. 1e), and the decreases in pH likely resulted from weaker photosynthesis, algal respiration, and the possible presence of bacteria in the phycosphere, which would release CO₂ via respiration. Based upon these observations, we infer that the magnitude of phycosphere pH change in natural phytoplankton assemblages is depth dependent, since photosynthetically active radiation gradually decreases from a few thousands μmol m⁻² s⁻¹ at the surface to <1 μmol m⁻² s⁻¹ at a depth of several hundred metres [34].

We then investigated the mechanisms underlying the regulation of phycosphere pH, and our data indicate that both extracellular and intracellular processes associated with photosynthesis play a role (Fig. 1f). First, the extracellular transformation of bicarbonate by carbonic anhydrase at the cell surface of C. concordia and subsequent release of hydroxides contributed to an increase in pH. Specifically, upon addition of 100 μM acetazolamide (inhibitor of external carbonic anhydrase), the increase in the phycosphere pH was significantly reduced from 0.29 ± 0.06 to 0.12 ± 0.04 (n = 3–8, p = 0.000). Second, we observed no significant increase in the phycosphere pH (0.02 ± 0.01, n = 3, p = 0.074), upon a further addition of 8 μM diquat dibromide, an inhibitor of photosystem I. Similar responses to the inhibitors were observed in marine diatoms Coscinodiscus radiatus (Fig. S3) and the large diatoms Odontella sinensis [10].

Detailed phycosphere pH measurements with the diatom Coscinodiscus wailesii of ~50 µm radius were undertaken. We firstly determined pH at 8 positions along the surface of an illuminated cell to assess whether pH was uniform at the cell surface (Fig. 2a); the difference among the measured pH at the 8 positions was <0.02 (Fig. 2b). Such a small variation might link to the evenly distributed chloroplasts around this centric diatom [35]. In contrast, for the rod-shaped diatom O. sinensis, the phycosphere pH is 0.1 higher in the central region than at the tip of the cell, although the chloroplasts are evenly distributed along the length of the cell [10].

Similar to C. concordia, pH increases of 0.30 were observed when moving the pH nano-probe from the bulk medium (pH 7.97 ± 0.01, 166 measurements) to the surface of a C. wailesii cell (8.27 ± 0.02, 77 measurements) (Fig. 2c). The thickness of the pH boundary layer, defined as the distance from the cell surface to a position where the measured pH is 99–101% of the bulk seawater, was ~15 µm for C. wailesii during light exposure (Fig. 2d). We determined the phycosphere pH in stagnant seawater, but it may vary in naturally turbulent seawater. However, theoretical models suggest that turbulence can only have a significant effect on the unstirred boundary layer in microorganisms of >100 µm in diameter [36], but not in those smaller cells [37].

We found that the phycosphere pH (i.e. H⁺ concentration) in C. wailesii was sensitive to the pH of the bulk seawater (Fig. 2e). The increase in phycosphere pH (i.e. the decrease in H⁺ concentration) of diatoms exposed to seawater of a lower pH (i.e. pH 7.78) was significantly higher than those exposed to seawater of higher pH (i.e. pH 8.10 or 8.40) (p = 0.000). Similarly, the giant marine diatom O. sinensis experiences much greater pH increases within the phycosphere at bulk seawater pH 7.60 than pH 8.20 [10]. The experimental observations generally agree with the previous modelling work which predicts that the difference in pH between phycosphere and bulk seawater will increase in future as the buffering capacity of seawater decreases [15]. This is in line with the buffering capacity of our seawater solutions decreasing with decrease of the seawater pH (Table S1).

In addition to the seawater buffering capacity, our data indicate that the biological processes responsible for the phycosphere pH are sensitive to ambient seawater pH. Specifically, if there were no changes in such biological processes, we would have seen a bigger decrease in the phycosphere H⁺ concentration at pH 8.10 than at pH 8.40, in agreement with the reduced buffering capacity at the lower pH. But we observed no significant difference in average phycosphere H⁺ shift between the diatoms exposed to seawater of pH 8.40 and 8.10 (Fig. 2e).

The nutritional status of algae cells also plays an important role in the magnitude of the phycosphere pH change. Following a 24 h starvation of nutrients (i.e. N, P, Si and micronutrients), the measured changes in the phycosphere pH of C. wailesii cells exposed to different seawater pH were consistently smaller than the nutrient-replete cells (Fig. 2e versus Fig. S8). Similarly, a previous study reports a higher pH in the phycosphere of Fe-replete diatoms Thalassiosira weissflogii than Fe-limited cells [11]. Overall, the limitation or starvation by nutrients would have decreased photosynthesis and/or extracellular carbonic anhydrase of these diatoms, and hence reduced the overall phycosphere pH change.

The thickness of the pH boundary layer is sensitive to ambient bulk seawater pH. For C. wailesii in the light, the thickness of the layer at bulk seawater pH 7.78 was 122 ± 17 µm, which was sevenfold thicker than that at a bulk seawater pH of 8.00 (18 ± 4 µm, p = 0.014, Fig. 2f). The large increase in thickness of the layer at a lower bulk seawater pH arose from the smaller buffering capacity of the exposure solution (Table S1); the H⁺ would travel a longer distance in seawater of a lower pH buffering capacity, leading to a thicker diffusive boundary layer around a cell. When bulk seawater was fixed at pH 8.00, but buffering capacity reduced by altering the bicarbonate concentration, we found the thickness of phycosphere pH layer in diatoms increased (Fig. S4), consistent with the longer transport distance calculated for CO₂ in seawater with a lower pH and buffering capacity [38]. Hence, we show that both seawater pH and buffering capacity play an important role in setting the phycosphere thickness.

Increases of the phycosphere pH were observed in the coccolithophore Emiliania huxleyi upon exposure to 140 μmol photons m⁻² s⁻¹ (Fig. 3). However, the phycosphere pH of E. huxleyi did not always increase, and decreases were also observed in the light. We suspect the decrease of local pH in the light likely resulted from biogenic calcification of this species, as CO₂ or protons are released in the course of biomineralisation [2]. Indeed, a gradual reduction in pH was observed during foraminiferal calcification in the microenvironment surrounding a calcifying specimen of Ammonia sp [39]. Hence, we suspect that the overall pH change in the phycosphere of coccolithophores should be a combined effect of photosynthesis, calcification and respiration; a significant increase in pH in the phycosphere would only be observed when their photosynthesis is stronger than calcification and respiration. Further experimental and modelling work [38] on the interactions between biogenic calcification and phycosphere pH is required.

Our observations on the nano- and micro- phytoplankton species are consistent with those on giant phytoplankton species and algal colonies [10, 12, 40]. Overall, in the seawater of 2 mM bicarbonate at pH 8.0 under 140 μmol m⁻² s⁻¹ light exposure, we observed that the phycosphere pH significantly increased by 0.15 ± 0.20 for C. wailesii (n = 32, p = 0.000), 0.11 ± 0.07 for C. concordia (n = 7, p = 0.005), 0.41 ± 0.04 for C. radiatus (n = 3, p = 0.002) and 0.20 ± 0.09 for E. huxleyi (n = 5, p = 0.008) (Table S3). Moreover, we found that there was a clear variation of the phycosphere pH even within a population of cells. For instance, amongst the 32 individual cells of C. wailesii, the phycosphere pH of one cell was 1.12 higher than the bulk seawater while the pH of two cells was not higher than the bulk seawater (data sheet of Table S3, https://doi.org/10.6084/m9.figshare.19576477.v1). Such inter-individual differences would be due to their differences in photosynthesis, respiration and/or carbonic anhydrase activity.

Consequences of phycosphere pH change for Fe speciation and bioavailability

Iron availability to phytoplankton is influenced by seawater chemistry and cell physiology [6, 41, 42]. However, most experiments have not assessed the influence of the phycosphere on Fe speciation and bioavailability. No analytical technique is currently available for direct measurements of Fe speciation in the phycosphere, and even modelling Fe speciation as a function of pH in seawater was previously challenging due to a lack of intrinsic chemical binding parameters for marine DOM [23].

Here, we took advantage of newly derived proton and Fe-binding parameters for marine DOM [17, 18], and calculated the effect of a 0.26 pH change in the phycosphere on Fe speciation for phytoplankton living in coastal and open ocean environments (Fig. 4). For both scenarios, when the phycosphere pH increases by 0.26, the fraction of inorganic Fe species, which is considered to be directly available for biological uptake [43], increases by ~2 fold. These increases arise because, with the organic matter binding parameters predicted with our model, the hydroxide ion (OH⁻) competes more effectively for Fe as pH increases. On the other hand, the fraction of inorganic Fe species decreases by 50% when the phycosphere pH decreases by 0.26. Such changes in inorganic Fe species are not trivial and might have significant impacts on Fe bioavailability and growth of marine phytoplankton, because even dissociation of 2% of organic Fe complexes can markedly improve the growth of many oceanic algae species [44].

In addition to pH, the concentrations of DOM, siderophores and other Fe-binding ligands in the phycosphere might differ from bulk seawater as a result of algae and associated bacteria metabolism [14, 32]; under such scenarios our calculations show that the phycosphere Fe speciation is largely different from that in bulk seawater (Figs. S5 and S6). For instance, when the concentrations of DOM and siderophores increase by tenfold in this microenvironment as a result of intensive algal/bacterial exudation (Fig. S6), the fraction of inorganic Fe species in the phycosphere becomes negligible (<0.01% of total dissolved Fe) and is 100-fold lower than the bulk seawater. Further increases in pH in the phycosphere results in a higher proportion of Fe bound to siderophore but less binding to DOM. On the other hand, when the local concentrations of DOM and siderophores decrease by tenfold via e.g. bacterial consumption, some dissolved Fe precipitates as Fe(OH)₃ and a higher pH in the phycosphere then leads to the formation of more Fe precipitates (Fig. S5).

Based on our results, we propose that the pH change in the phycosphere alters Fe availability to phytoplankton. An increase in the phycosphere pH enhances Fe bioavailability via three pathways (Fig. 5): (a) a higher pH in the phycosphere increases the abundance of inorganic Fe species and hence Fe bioavailability; (b) a higher local pH increases the availability of carbonate in the phycosphere and hence will facilitate Fe(III) uptake by carbonate sensitive transferrins in certain diatoms [45]; and (c) elevated phycosphere pH increases the amount of algal surface-bound Fe [46] and hence facilitates Fe bio-uptake. We suggest that the light-induced increase in pH in the phycosphere is likely an important component of Fe acquisition in phytoplankton.

In contrast to the effect of the pH increase in the phycosphere, dark and low light intensities likely reduce Fe availability to phytoplankton cells. This arises because a decrease in the phycosphere pH decreases the concentration of inorganic Fe species, carbonate and surface bound Fe. In addition, altered abundance/chemistry of Fe-binding ligands in the microenvironment as a consequence of algal/bacterial metabolisms could further change the Fe speciation and hence Fe bioavailability. At present, very few studies have investigated the influences of organic ligands and bacteria in the phycosphere on Fe bioavailability; one study [47] shows that an algal-associated bacterium Marinobacter sp. increases the Fe uptake by 70% and dinoflagellate partner Scrippsiella trochoidea by >20-fold with a light radiation of 450 μmol m⁻² s⁻¹.

This study shows that even in the cells of ~5 µm diameter, the pH in the phycosphere is consistently different from bulk seawater. For the first time, our data show that the thickness of the pH boundary layer is largely amplified by ocean acidification. Moreover, our modelling results suggest that the local pH alters Fe speciation in this microenvironment, and in a future more acidic ocean, a much thicker boundary layer will result in a larger deviation of the Fe speciation in the phycosphere from bulk seawater. In addition, we suspect that the local pH microenvironment would influence biogenic calcification; for instance, higher phycosphere pH likely favours extracellular precipitation of CaCO₃ in certain holococcolith-forming species such as Coccolithus pelagicus and Calyptrosphaera sphaeroidea [48].

Precise quantification of chemical conditions in the phycosphere is crucial for better understanding how phytoplankton will respond to environmental changes. Evidence is emerging that interactions between phytoplankton and abiotic/biotic environments are governed by micro- and nano- scale interfacial processes [13, 47, 49, 50], which cannot be determined using bulk water analyses. Small changes in the phycosphere likely translate into large impacts on the oceanic carbon and nitrogen cycle (Fig. 5). Even a minor increase in Fe availability could result in a large amount of biological CO₂ and N₂ fixation, on the order of 400,000 atoms of C and/or 60,000 atoms of N per Fe atom [44, 51].

Source: Ecology - nature.com