Biogenic Phosphonate Utilization by Globally Distributed Diatom Thalassiosira pseudonana

Phosphonates are a class of organic phosphorus (P) compounds that contribute ~25% of dissolved organic P. Recent studies reveal the important role of phosphonates mediated by prokaryotes in the marine P redox cycle. However, its bioavailability by eukaryotic phytoplankton is under debate. 2-Aminoethylphosphonic acid (2-AEP) and 2-amino-3-phosphonopropionic acid (2-AP3) are two biogenic phosphonates in the marine environment. Here, Thalassiosira pseudonana, a common diatom species in the ocean, is able to recover growth from P starvation when provided with 2-AEP and 2-AP3. Moreover, 2-AEP cultures exhibited a more similar growth rate at 12 °C than at 25 °C when compared with inorganic P cultures. The cellular stoichiometry of 2-AEP groups was further determined, the values of which are in-between the P-depleted and DIP-replete cultures. This study provides evidence that biogenic phosphonates could be adopted as alternative P sources to support diatom growth and may provide physiological adaptation.


Introduction
Phosphorus (P) is an essential nutrient for the growth of living organisms.Dissolved inorganic P (DIP), the preferred form of P used by phytoplankton, is often a limiting nutrient for phytoplankton in marine environments [1][2][3].Thus, dissolved organic P (DOP) has emerged as a prominent alternative P source [4,5].
Phosphonates are a class of organic P with a chemically stable C-P bond.Besides synthetic compounds (e.g., herbicide glyphosate), biogenic phosphonates produced by various organisms are present in the ocean [6,7].Two biogenic phosphonate compounds, 2-aminoethylphosphonate (2-AEP) and its derivative, 2-amino-3-phosphonopropionic acid (2-AP3), are the composition of membrane phospholipids in many organisms, such as prokaryotes and mollusks [8,9].A recent study shows that Prochlorococcus likely allocates over 40% of cellular P towards phosphonate production in the ocean [10].Genome surveys suggest that de novo synthesis of 2-AEP is performed in corals [11].Therefore, exploring the bioavailability of biogenic phosphonates by phytoplankton has considerable importance.
Compared with the well-elucidated metabolism of phosphonates in prokaryotes [12][13][14], discrepancies were found in a few studies of eukaryotic phytoplankton [15,16].Picoprasinophyte Micromonas commode and coccolithophore Emiliania huxleyi are able to utilize 2-AEP, whereas diatom Phaeodactylum tricornutum failed [17].However, our recent study showed that P-starvation-treated P. tricornutum can recover growth with a 2-AEP supplement, and the utilization is mediated by endocytosis and integration into membrane phospholipids (DAG-2-AEP, diacylglyceryl-2-AEP) [16].Furthermore, an in silico analysis of the global meta-omic atlas suggests that the associated utilization functional genes are prevalent in diatom assemblages and actively expressed in the cold regions [16].
On these grounds, a hypothesis that the cosmopolitan diatom Thalassiosira pseudonana [18][19][20] is able to utilize biogenic phosphonates and the metabolic activity is temperaturesensitive is proposed.Here, the bioavailability of 2-AEP and 2-AP3, which share similar chemical structures and are components of membrane phospholipids, was investigated [8,9].Then, the physiological responses of algae grown with phosphonate supplements were examined at different temperatures.

Cell Culture and Experiment Setup
T. pseudonana was provided by the Center for Collections of Marine Bacteria and Phytoplankton of Xiamen University, China.Two batch experiments were conducted to (batch 1) examine the bioavailability of 2-AEP and 2-AP3 (Sigma-Aldrich, St. Louis, MO, USA) and (batch 2) explore the physiological response under the conditions of significant temperature differences (Table 1).As documented in other studies, 12 • C and 25 • C (comparable to those of 20 • C) represent the temperatures with the lowest growth rate and the highest growth rate, respectively [21].Before the experiments, seed cultures were subject to P starvation for 8~10 days until the ambient phosphate concentration was below ~0.3 µM and the cell growth ceased.Antibiotics were applied to inhibit the growth of bacteria (Table 1).

Determination of Cell Density and Fv/Fm
Cell density was measured daily by using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) and estimated by gating areas in the chlorophyll A versus SSC-A dot plot generated from a 1 mL cell sample.Fv/Fm was determined using a FIRe Fluorometer System (Satlantic, Halifax, NS, Canada).Prior to the measurement, 1 mL of the cell sample was subject to dark adaption for 20 min and then processed following the manufacturer's protocol [22].

Cellular C and N Content
Cells were collected using pretreated GF/F membranes.The collected cells were dried at 60 • C for 8 h.Then, 500 µL of 1% HCl was dripped onto the filters, and the filters were dried at 60 • C for 12 h again.After the cells were pretreated [16], the cellular C and N contents were determined using a Vario EL cube analyzer (Elementar Analysensysteme GmbH, Langenselbold, Germany) in accordance with the reported method [23].

Determination of DIP and P Content
Cells were filtered onto GF/F membranes and resuspended in 25 mL of distilled water.The suspension was digested by adding 4 mL of 50 g/L potassium persulfate and autoclaving at 121 • C for 30 min [24].Then, the cellular P content of the digested suspension and the DIP concentration of the filtrate were determined using the molybdenum method [24].

Statistical Analysis
Differences among different P and temperature treatment groups were measured with t-tests.

Differential Growth-Promoting Effects between 2-AEP and 2-AP3
Different growth-promoting effects were observed in the batch 1 culture.After P starvation, T. pseudonana was able to recover growth significantly in the medium supplied with 2-AEP (36 µM) and 2-AP3 (72 µM), respectively (p < 0.05), while failing to grow in the 2-AP3 supplement (36 µM, Figure 1a).The cells in the 2-AEP (36 µM) group continued growth and peaked at the maximum cell concentration of 8.24 × 10 5 cells mL −1 at Day 3, then declined gradually, and Fv/Fm exhibited a similar pattern accordingly (Figure 1a,b).In comparison, we observe mild growth promotion in the 2-AP3 group.After the supplement of 72 µM 2-AP3, instant cell growth was recovered in 24 h, showing a growth rate of 0.24 µ d −1 , which is about half of that in the 2-AEP (36 µM) group (Figure 1a).After then, cells ceased growth and declined towards the end of this experiment, accompanied by an abrupt decline in Fv/Fm (Figure 1b).Maximum cell density observed on D2 was 6.26 × 10 5 cells mL −1 , about half of that in the 2-AEP (36 µM) group.
Cells were filtered onto GF/F membranes and resuspended in 25 mL of distilled water.The suspension was digested by adding 4 mL of 50 g/L potassium persulfate and autoclaving at 121 °C for 30 min [24].Then, the cellular P content of the digested suspension and the DIP concentration of the filtrate were determined using the molybdenum method [24].

Statistical Analysis
Differences among different P and temperature treatment groups were measured with T-tests.

Differential Growth-Promoting Effects between 2-AEP and 2-AP3
Different growth-promoting effects were observed in the batch 1 culture.After P starvation, T. pseudonana was able to recover growth significantly in the medium supplied with 2-AEP (36 μM) and 2-AP3 (72 μM), respectively (p < 0.05), while failing to grow in the 2-AP3 supplement (36 μM, Figure 1a).The cells in the 2-AEP (36 μM) group continued growth and peaked at the maximum cell concentration of 8.24 × 10 5 cells mL −1 at Day 3, then declined gradually, and Fv/Fm exhibited a similar pattern accordingly (Figure 1a,b).In comparison, we observe mild growth promotion in the 2-AP3 group.After the supplement of 72 μM 2-AP3, instant cell growth was recovered in 24 h, showing a growth rate of 0.24 μ d −1 , which is about half of that in the 2-AEP (36 μM) group (Figure 1a).After then, cells ceased growth and declined towards the end of this experiment, accompanied by an abrupt decline in Fv/Fm (Figure 1b).Maximum cell density observed on D2 was 6.26 × 10 5 cells mL −1 , about half of that in the 2-AEP (36 μM) group.DIP was barely detected (lower than the detection limit; ~0.3 mM) in the 2-AEP and 2-AP3 groups, demonstrating that 2-AEP and 2-AP3 can be utilized as an alternative P source by T. pseudonana.Though provided in the same or higher concentration, lower cell density acquired in both phosphonate groups suggests limited utilization efficiency compared with DIP, which is common according to previous reports [15,17].Furthermore, lower Fv/Fm indicates repressed photosynthesis, suggesting that T. pseudonana cells were under P stress in both phosphonate groups.
2-AP3 is the derivative of 2-AEP, known as a component of phospholipids in cell membranes.Studies have demonstrated that 2-AEP and its derivatives can be incorporated into phospholipids in cell membranes [25][26][27].2-AP3 can be decomposed via a transamination reaction and decarboxylation to 2-AEP in Tetrahymena [28].Given the limited utilization of 2-AP3 observed in the present study, the other possible metabolic pathways cannot be completely excluded.The underlying mechanism of 2-AP3 utilization by T. pseudonana and its potential bioavailability need to be further investigated to address the knowledge gap in eukaryotic phytoplankton.

Different Growth Strategies under Variable P Nutrients and Temperatures
A recent study of the global ocean gene atlas shows an enriched distribution of representative genes of the proposed 2-AEP utilization mechanism by diatoms in lowtemperature waters [16].Therefore, the batch 2 experiment was conducted to further explore the cellular physiological response grown with different P nutrients and temperatures, in which 72 µM of 2-AEP was provided to obtain more significant differences for the comparative analysis.
Different growth patterns that are temperature-dependent were observed (Figure 1c,d).T. pseudonana cells exhibited sustained growth at 12 • C with higher cell density, and a shorttime rapid growth at 25 • C with lower cell density regardless of P condition.At 12 • C, the cell density almost showed no change during the first 24 h and then increased steadily in the 2-AEP and DIP groups until D6, sharing no difference in the first 4 days (Figure 1c).When cultured at 25 • C, the cell density exhibited a rapid growth in the first 48 h and then entered the stationary phase after D4 in the DIP group.The cells ceased growth after D3 in the 2-AEP group and then decreased significantly.Consistently, the growth rate of T. pseudonana is higher under 8 • C~17 • C than that under 17 • C~25 • C with sufficient DIP supply [29].
A significant difference was found in the promotion effect under different P conditions (Figure 1c).In the DIP-replete groups, the final cell concentration at 12 • C was slightly higher than at 25 • C.Meanwhile, in the 2-AEP groups, the final cell concentration in the 12 • C culture was about three times higher than that in the 25 • C culture.When cultured at 12 • C, the highest growth rate on D6 in the 2-AEP group was 0.152 ± 0.049 µ d −1 , which is 66.4% of that in the DIP-replete group (0.228 ± 0.034 µ d −1 , Figure 1c).
Variations of Fv/Fm showed a comparable pattern in accordance with the growth curve (Figure 1d).The dramatic increase in Fv/Fm values in the first 24 h indicated that instant recovery of photosynthetic capacity accounts for the rapid growth in both cultures under 25 • C.After day 3, the Fv/Fm values declined continuously along with the cells entering the stationary phase in 25 • C cultures.In contrast, the Fv/Fm values increased steadily to meet sustained cell growth under 12 • C, and they were higher in the DIP-replete group than in the 2-AEP group.
These results showed that T. pseudonana cells cultured with 2-AEP exhibited better physiological adaptability under lower temperatures, resulting in significantly increased cell density.Meanwhile, the Fv/Fm value represents a good consensus of higher photosynthetic capacity and a higher growth rate.

Changes in Cellular Elemental Stoichiometry of T. pseudonana
Many studies have revealed that marine phytoplankton elemental stoichiometric ratios deviate from the empirical Redfield ratio of 106C:16N:1P [30], thus playing a major role in shaping the environmental stoichiometry ratio [31].

Stoichiometry Variation under Different P Conditions
In this study, the N:P ratio of the DIP-replete group (~6:1) (Figure 2b) was far below that of the Redfield ratio and the group-specific optimal value of 14:1 [32].This finding can be explained by the luxury uptake of DIP and storage in the form of polyP after P starvation, which is typical in diatoms [33,34].Regarding the initial P starvation state, the C:P and N:P ratios were 117.72 ± 23.14 and 24.25 ± 7.94, respectively, consistent with reported cellular stoichiometry in diatoms under insufficient nutrient conditions [35].Afterwards, the C:P and N:P ratios decreased significantly to 36.93 ± 3.96 and 5.63 ± 0.56, respectively, at D5 in the DIP-replete group, whereas they barely changed or increased under other different P conditions (Figure S2a).
synthetic capacity and a higher growth rate.

Changes in Cellular Elemental Stoichiometry of T. pseudonana
Many studies have revealed that marine phytoplankton elemental stoichiometric ratios deviate from the empirical Redfield ratio of 106C:16N:1P [30], thus playing a major role in shaping the environmental stoichiometry ratio [31].

Stoichiometry Variation under Different P Conditions
In this study, the N:P ratio of the DIP-replete group (~6:1) (Figure 2b) was far below that of the Redfield ratio and the group-specific optimal value of 14:1 [32].This finding can be explained by the luxury uptake of DIP and storage in the form of polyP after P starvation, which is typical in diatoms [33,34].Regarding the initial P starvation state, the C:P and N:P ratios were 117.72 ± 23.14 and 24.25 ± 7.94, respectively, consistent with reported cellular stoichiometry in diatoms under insufficient nutrient conditions [35].Afterwards, the C:P and N:P ratios decreased significantly to 36.93 ± 3.96 and 5.63 ± 0.56, respectively, at D5 in the DIP-replete group, whereas they barely changed or increased under other different P conditions (Figure S2a).The stoichiometry of 2-AP3 groups (36 μM and 72 μM) was similar to that of the Pdepleted group (Figure 2), in line with the growth pattern.The P-depleted cells and those treated with 2-AP3 were grouped together, showing higher C:P and N:P ratios, mainly The stoichiometry of 2-AP3 groups (36 µM and 72 µM) was similar to that of the P-depleted group (Figure 2), in line with the growth pattern.The P-depleted cells and those treated with 2-AP3 were grouped together, showing higher C:P and N:P ratios, mainly because of higher cellular C and N contents and lower cell P content than that in the DIP-replete groups (Figure S3a).

Effect of Temperature on Cellular Stoichiometry
In the DIP-replete groups, no significant difference was identified between 12 • C and 25 • C (Figures 2 and S2b).In the 2-AEP group, the C:P and N:P ratios were lower than those in P-depleted T. pseudonana and higher than those in the DIP-replete group.In the 72 µM 2-AEP cultures (12 • C and 25 • C), the C:P and N:P values were 111.2 ± 11.1~115.8± 12.1 and 13.9 ± 1.8~12.1 ± 0.6, respectively, closer to the Redfield ratio with higher 2-AEP concentration and lower temperature.
The interactions between environmental conditions and cell growth are the key factors driving stoichiometric variation [36].Temperature is the major factor due to its direct effect on cell growth [34,37].Global research on phytoplankton stoichiometry has found that C:P and N:P ratios decrease with temperature [37][38][39], but laboratory evidence regarding species differences is insufficient.The findings of the present study show the synergetic effect of temperature and phosphonates on cellular stoichiometry in T. pseudonana, higher N:P ratios when cultured with 2-AEP under lower temperature.In the DIP-replete group, the lower temperature significantly decreased the C:P and N:P ratios of T. pseudonana, consistent with previous reports [37,40,41].
Cells increase ribosome concentration and cellular P content to compensate for low translation efficiency of ribosomes at low temperature [42].Such a hypothesis is consistent with the observation of the strong temperature dependency of C:P and N:P in high-latitude ecosystems [43].In this study, the cellular P content of the DIP-replete groups was significantly higher at 12 • C than at 25 • C (Figure S3b), indicating that low temperature may increase the P demand and promote the P absorption of T. pseudonana.In addition, temperature had a significant effect on the C:P and N:P ratios in the DIP group.In the 2-AEP group, temperature effects were barely observed on the stoichiometry of T. pseudonana.This finding may be attributed to the stable chemical properties of 2-AEP, which is hard to be hydrolyzed into phosphate during cellular P determination.

High Variability of Stoichiometry in Diatom
Nutrient availability is considered to be the major driver shaping phytoplankton stoichiometry; for example, P limitation accounts for the increase in C:P and N:P ratios of phytoplankton [44,45].In this research, the C:P and N:P ratios of T. pseudonana declined rapidly after 36 µM DIP supplementation.The C:P and N:P in this study were much lower under the same or similar P conditions than those in other studies (Figure 3) [37,[45][46][47].In agreement with the previous results, the C:P and N:P ratios of DIP-depleted T. pseudonana were higher than the Redfield ratio [45,48,49].According to the observation data from ALOHA and BATS stations, the C:P ratio of suspended particles varies mostly from 100 to 200 and below the P-stressed threshold [50].Our results show that T. pseudonana grown with 2-AEP is close to the classical Redfield ratio.

Conclusions
Overall, this study has three major findings.(1) Biogenic phosphonates 2-AEP and 2-AP3 can be utilized by diatom T. pseudonana to support cell growth, and 2-AEP is more preferable than 2-AP3, as evidenced by higher cell density.(2) Disparate growth strategies are identified under different temperatures, and the significantly promoted cell growth of

Figure 1 .
Figure 1.Physiological responses of T. pseudonana to different phosphorus levels ((a), growth curve; (b), Fv/Fm) and temperatures ((c), growth curve; (d), Fv/Fm).The culture temperature was 20 • C (a,b) and 12/25 • C (c,d).Each culture group was set up in biological triplicate.The error bar represents the standard deviation of the mean values.The inner panel of (c) represents the growth rate on D6 at 12 • C, and * represents a significant difference (p < 0.05) between P-depleted and other groups.

Microorganisms 2024 , 9 Figure 3 .
Figure 3.Comparison between the elemental stoichiometry of T. pseudonana in this study and that in previous research.Normal represents the nutrients in adequate culture conditions, P-replete represents DIP resupplied to the phosphorus-starvation cells, P-depleted represents the phosphorusstarvation condition, and low P represents a very low phosphorus concentration in the medium [37,45-49,51-53].

Figure 3 .
Figure 3.Comparison between the elemental stoichiometry of T. pseudonana in this study and that in previous research.Normal represents the nutrients in adequate culture conditions, Preplete represents DIP resupplied to the phosphorus-starvation cells, P-depleted represents the phosphorus-starvation condition, and low P represents a very low phosphorus concentration in the medium [37,45-49,51-53].

Table 1 .
Culture conditions (a, b: control groups in batch 1, b: control group in batch 2).