Phosphomonoesterase and phosphodiesterase activities in the Eastern Mediterranean Sea during stratified versus mixed conditions

Van Wambeke, France; Conan, Pascal; Pujo-Pay, Mireille; Taillandier, Vincent; Crispi, Olivier; Pulido-Villena, Elvira

doi:https://doi.org/10.5194/egusphere-2023-2578

Preprints

https://doi.org/10.5194/egusphere-2023-2578

Preprints

30 Nov 2023

| 30 Nov 2023

Phosphomonoesterase and phosphodiesterase activities in the Eastern Mediterranean Sea during stratified versus mixed conditions

France Van Wambeke, Pascal Conan, Mireille Pujo-Pay, Vincent Taillandier, Olivier Crispi, and Elvira Pulido-Villena

Abstract. Dissolved organic phosphorus hydrolysis by marine planktonic microorganisms is a key process in the P cycle, particularly in P-depleted, oligotrophic environments. The present study assessed spatio-temporal variations of phosphomonoesterase (PME) and phosphodiesterase (PDE) activities using concentration kinetics in the eastern Mediterranean Sea during 2 contrasted situations: the end of winter (including a small bloom period), and autumn. The distribution and regulation of the maximum hydrolysis rates (Vm) and half-saturation constants (Km) of both ectoenzymes were assessed in relation to the vertical structure of the epipelagic layers. PME reached their maximum activities (Vm) after 1 µM MUF-P addition whereas, for PDE, it was necessary to add up to 50 µM bis-MUF-P to reach saturation state. On average, the Km of PDE was 33 ± 25- times higher than that of the PME. Vm of PME and Vm of PDE were linearly correlated. Conversely to the Km values, Vm were on the same order of magnitude for both ectoenzymes, their ratio (Vm PME:Vm PDE) ranging between 0.2 and 6.3). Dissolved organic phosphorus (DOP) and the phosphomonoesterease hydrolysable fraction of DOP explained mostly no variability of Vm PME nor Vm PDE. On the contrary, Vm of both phosphohydrolase enzymes was inversely correlated to DIP concentration. The particular characteristics of concentration kinetics obtained for PDE (saturation at 50 µM, high Km, high turnovertimes) are discussed with respect to possible inequal distribution of PDE and PME among organic material size continuum, and accessibility to phosphodiesters.

Received: 02 Nov 2023 – Discussion started: 30 Nov 2023

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29 May 2024

Phosphomonoesterase and phosphodiesterase activities in the eastern Mediterranean in two contrasting seasonal situations

France Van Wambeke, Pascal Conan, Mireille Pujo-Pay, Vincent Taillandier, Olivier Crispi, Alexandra Pavlidou, Sandra Nunige, Morgane Didry, Christophe Salmeron, and Elvira Pulido-Villena

Biogeosciences, 21, 2621–2640, https://doi.org/10.5194/bg-21-2621-2024,https://doi.org/10.5194/bg-21-2621-2024, 2024

Short summary

France Van Wambeke, Pascal Conan, Mireille Pujo-Pay, Vincent Taillandier, Olivier Crispi, and Elvira Pulido-Villena

Interactive discussion

Status: closed

RC1:
'Comment on egusphere-2023-2578', Anonymous Referee #1, 04 Dec 2023

General Comments
This article reveals the distribution of phosphatemonoesterase (PME) and phosphatediesterase (PDE) activities in the seawater of the Mediterranean Sea in the fall and winter. Primary production in the survey area is known to be limited by low phosphors bioavailability, and the magnitude of the limitation varies seasonally, in accordance with a variation in the stratification intensity there. The activities of these two enzymes are indices of microbial phosphorus stress and they provide bioavailable inorganic phosphate to microbes via hydrolysis of organic phosphorus materials. The scientific significance of this study is obvious, and the validity of the methodology employed in this study is thoroughly examined in this article. The results and discussion provided in this article evidently contribute to understanding the biogeochemical cycling of phosphorus and relevant biogenic materials in this area.
One of the inputs that I can make to this article is a comparison with the other phosphorus limiting areas. Particularly, the western North Pacific is also known to be heavily limited by phosphorus deficiency, and PME and PDE activities have been already reported from it. Despite these similarities between the Mediterranean Sea and the western North Pacific, they are different in many respects, including trace metal supply (especially iron and copper), nitrogen fixation (high abundance of Trichodesmium and Crocosphaera in the North Pacific), and microbial community composition. The comparison should be of great significance to contrast the difference between the two areas, and possibly with other phosphorus-limiting water(s).
In addition, I recommend the authors to incorporate a discussion on the role of meso- and microscale eddies observed during the campaign, particularly in the winter season. The eddy structure can profoundly influence the vertical structure of the water column, as already described in the article, and hence the microbial community structure and their biological processes. Despite its experimental design, I do not think that the article sufficiently discusses this viewpoint. I believe that these insights can improve the quality of this article.

Specific Comments
L62 Describing typical P-diester chemical species found in seawater will be useful for the upcoming discussion.
L115 Here the authors said that they obtained “pigment distributions”, but only the distribution of total chlorophyll a was provided in this article. Describe more accurately or provide all the results of pigment analyses and discuss them.
L125 What do the figures with a ± sign mean?
L141 The species name coli should be written in lower case.
L169 Chlorophyll should be written in lower case.
L185 Does this “PRSIM” mean a statistic software? Then describe its properties more in detail.
L256 Out of these two figures, the former should be from the autumn cruise, but it is not clear from the text. Make clearer which figure is which.
L255 The sequence of the supplementary figure numbers does not match with that of appearance in the text. Correct it.
L274 More abundant than what?
L280 Including “Nanoeuk” and “crypto” into picophytoplankton groups is not natural.
L295 The authors use the word “than” here, but I am not sure what and what are compared here.
L298 Is this figure ”33 ± 25” the arithmetic average of all the ratios of PME and PDE in the same subsamples with a standard deviation?
L319 Are these rates correlated positively or negatively? It would be more informative to make it clear about that.
L325 The statistical test for a difference in slopes of correlation curves is usually done by F-test. Additionally, 0.03 is uncommon for the criterion of the level of significance.
L336 Do the authors mean V_m by “maximum rates”?
L359 As I mentioned in the General Comments, add the discussion from the more detailed comparison of the present results with those from the other phosphorus limiting areas, with special emphasis on the differences in environmental conditions, including physical, chemical, and biological conditions. In addition to such comparisons, the effects of eddy structures on microbial processes of phosphorus cycling in this area should be examined.
L377 “always largely” sounds self-contradictory. Which is correct, always (100%) or largely (>~80%)?
L378 What do the authors mean by “best”? What is “good” here (same for L416)
L424 Does it mean “From studies where both phosphatase rates were available”?
L432 Relationship between what and what?
L432 What do the authors compare here with the ratio V_m_PME:V_m_PDE?
L439 What do the authors mean by “effectively”? What is more effective to what?
L448 “In autumn cyanobacteria switched from…” “Switch” suggests a temporal change from one state to another, but in this situation, the authors only describe a vertical (spatial) variation of a plankton community. It sounds misleading.
L455 Here the authors’ speculation is unclear from the text. Rearrange the discussion line and make it clear what is suggested from the present results.
L528 Turnover times should be evaluated by “long” or “short”. The description “high turnover times” is ambivalent whether the turnover is fast or slow.
Tables. Is there any authors’ intention in providing the results from the cruise PERLE2 in the upper rows, although this cruise was conducted later and in the figures, the results from this cruise are provided in the lower panels? It may be confusing.
Fig. S4a. The near-zero abundance of Prochlorococcus in the surface water is not realistic considering the physical and chemical conditions of the water. I am afraid that very faint cellular autofluorescence of Prochlorococcus within the well-lit stratified water layer resulted in failure to detect them by flow cytometry. If any other data based on genomic analyses or microscopy are available, I recommend the authors to check the validity of these data. Otherwise, the authors can show some caveats in the materials and methods section. Anyway, it is a frequently seen situation, and it will not critically affect the discussion in the present study.

Citation: https://doi.org/10.5194/egusphere-2023-2578-RC1
- AC1: 'Reply on RC1', F. Van Wambeke, 19 Feb 2024
  
  The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2023/egusphere-2023-2578/egusphere-2023-2578-AC1-supplement.pdf
  
  Citation: https://doi.org/10.5194/egusphere-2023-2578-AC1
RC2:
'Comment on egusphere-2023-2578', Anonymous Referee #2, 30 Jan 2024

General comments
The authors investigate phosphomonoesterase (PME) versus phosphodiesterase (PDE) activities in a well-known P-depleted oligotrophic environment, the Eastern Mediterranean Sea, at two contrasted season. They characterize maximum hydrolysis rates (Vm) and half-saturation constants (Km) of both PME and PDE activities in relation to dissolved stocks of Phosphorus : DIP, DOP and the enzymatically hydrolysable fraction of DOP. Although phosphomonoesterase, also known as alkaline phosphatase, activities have been extensively studied in P deplete oligotrophic and coastal environments during past decades, the measurements of both PME and PDE have been achieved only recently. The authors have paid particular attention on the methodology for the measurement of these activities. The results of this paper confirm the results found elsewhere that PDE activities (Vm) could be in the same order of magnitude than PME activities. PDE seem to be regulated as PME, by the availability of DIP. However the regulation of PDE by NOx:DIP ratio is also discussed as well as the occurrence of different microbial communities having different PDE expression pattern. This paper has a significant contribution to the understanding of the Phosphorus fluxes through the microbial food web, participating to the biogeochemical cycle of Phosphorus. Obviously, it is within the scope of EGUsphere.
Specific comments
The scientific approach and applied methods to the studies of PME and PDE activities in relation to the dissolved phosphorus pools are particularly well adapted. The measurements of nanomolar concentrations of DIP and labile DOP in such oligotrophic environments as the Mediterranean Sea are achieved with the LWCC method having a very low detection limit (1 nM). The measurements of kinetic parameters of enzymatic activities need a particular attention since methodological biases can lead to misestimated Vm and Km. The most notable divergences in existing methodologies on enzymatic activity assays in natural environment is the substrate concentrations used for assays. The range of substrate concentration significantly affect kinetic parameters estimation and it is generally recommended to use a large substrate concentration range, up to 10 Km at least. A specific literature exists on this particular bias which could be cited by authors in the Discussion part. However, the authors discussed their results with published literature, considering these methodological aspects, which is scarcely made while necessary for meaningful comparison.
Details
Line 27: Define the significance of DIP the first time it appears rather than Line 42
Line 49, 692: Labry et al. 2016 rather than 2021
Line 58: precise under optimal conditions of concentrations of what ? enzyme ?
Line 120: nitrite rather than « nitrites » and use « DOP » rather than its significance
Line 141,637: Djaoudi et al. 2018 rather than 2017
Line 225 : concerning winter depth of Pcline, refer to Fig. 3b,c
Line 395 : a little more exhausted literature on P diesters composition would be informative
Line 408-412 : The difficult comparison with previous studies also comes from the different substrates used, MUF- derivates (Thomson et al. 2020, Sato et al. 2013) vs paranitrophenyl- derivatives (Huang et al. 2022), corresponding to different enzyme affinity. Conditions of incubation, particularly temperature may also differ between studies, optimal versus in situ temperature.
Line 425 : Thomson et al., 2020 rather than 2019
Line 478 – 483 : discussion on Km PME >> LDOP : do the authors mean that enzymes experience locally higher substrate concentrations due to intermittent and patchy distribution of organic Phosphorus ? Could the authors explain it more precisely.
Figure 7 : The frame around the legend on the Km versus DIP graphe could be removed.

Citation: https://doi.org/10.5194/egusphere-2023-2578-RC2
- AC2: 'Reply on RC2', F. Van Wambeke, 19 Feb 2024
  
  The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2023/egusphere-2023-2578/egusphere-2023-2578-AC2-supplement.pdf
  
  Citation: https://doi.org/10.5194/egusphere-2023-2578-AC2

Interactive discussion

Status: closed

RC1:
'Comment on egusphere-2023-2578', Anonymous Referee #1, 04 Dec 2023

General Comments
This article reveals the distribution of phosphatemonoesterase (PME) and phosphatediesterase (PDE) activities in the seawater of the Mediterranean Sea in the fall and winter. Primary production in the survey area is known to be limited by low phosphors bioavailability, and the magnitude of the limitation varies seasonally, in accordance with a variation in the stratification intensity there. The activities of these two enzymes are indices of microbial phosphorus stress and they provide bioavailable inorganic phosphate to microbes via hydrolysis of organic phosphorus materials. The scientific significance of this study is obvious, and the validity of the methodology employed in this study is thoroughly examined in this article. The results and discussion provided in this article evidently contribute to understanding the biogeochemical cycling of phosphorus and relevant biogenic materials in this area.
One of the inputs that I can make to this article is a comparison with the other phosphorus limiting areas. Particularly, the western North Pacific is also known to be heavily limited by phosphorus deficiency, and PME and PDE activities have been already reported from it. Despite these similarities between the Mediterranean Sea and the western North Pacific, they are different in many respects, including trace metal supply (especially iron and copper), nitrogen fixation (high abundance of Trichodesmium and Crocosphaera in the North Pacific), and microbial community composition. The comparison should be of great significance to contrast the difference between the two areas, and possibly with other phosphorus-limiting water(s).
In addition, I recommend the authors to incorporate a discussion on the role of meso- and microscale eddies observed during the campaign, particularly in the winter season. The eddy structure can profoundly influence the vertical structure of the water column, as already described in the article, and hence the microbial community structure and their biological processes. Despite its experimental design, I do not think that the article sufficiently discusses this viewpoint. I believe that these insights can improve the quality of this article.

Specific Comments
L62 Describing typical P-diester chemical species found in seawater will be useful for the upcoming discussion.
L115 Here the authors said that they obtained “pigment distributions”, but only the distribution of total chlorophyll a was provided in this article. Describe more accurately or provide all the results of pigment analyses and discuss them.
L125 What do the figures with a ± sign mean?
L141 The species name coli should be written in lower case.
L169 Chlorophyll should be written in lower case.
L185 Does this “PRSIM” mean a statistic software? Then describe its properties more in detail.
L256 Out of these two figures, the former should be from the autumn cruise, but it is not clear from the text. Make clearer which figure is which.
L255 The sequence of the supplementary figure numbers does not match with that of appearance in the text. Correct it.
L274 More abundant than what?
L280 Including “Nanoeuk” and “crypto” into picophytoplankton groups is not natural.
L295 The authors use the word “than” here, but I am not sure what and what are compared here.
L298 Is this figure ”33 ± 25” the arithmetic average of all the ratios of PME and PDE in the same subsamples with a standard deviation?
L319 Are these rates correlated positively or negatively? It would be more informative to make it clear about that.
L325 The statistical test for a difference in slopes of correlation curves is usually done by F-test. Additionally, 0.03 is uncommon for the criterion of the level of significance.
L336 Do the authors mean V_m by “maximum rates”?
L359 As I mentioned in the General Comments, add the discussion from the more detailed comparison of the present results with those from the other phosphorus limiting areas, with special emphasis on the differences in environmental conditions, including physical, chemical, and biological conditions. In addition to such comparisons, the effects of eddy structures on microbial processes of phosphorus cycling in this area should be examined.
L377 “always largely” sounds self-contradictory. Which is correct, always (100%) or largely (>~80%)?
L378 What do the authors mean by “best”? What is “good” here (same for L416)
L424 Does it mean “From studies where both phosphatase rates were available”?
L432 Relationship between what and what?
L432 What do the authors compare here with the ratio V_m_PME:V_m_PDE?
L439 What do the authors mean by “effectively”? What is more effective to what?
L448 “In autumn cyanobacteria switched from…” “Switch” suggests a temporal change from one state to another, but in this situation, the authors only describe a vertical (spatial) variation of a plankton community. It sounds misleading.
L455 Here the authors’ speculation is unclear from the text. Rearrange the discussion line and make it clear what is suggested from the present results.
L528 Turnover times should be evaluated by “long” or “short”. The description “high turnover times” is ambivalent whether the turnover is fast or slow.
Tables. Is there any authors’ intention in providing the results from the cruise PERLE2 in the upper rows, although this cruise was conducted later and in the figures, the results from this cruise are provided in the lower panels? It may be confusing.
Fig. S4a. The near-zero abundance of Prochlorococcus in the surface water is not realistic considering the physical and chemical conditions of the water. I am afraid that very faint cellular autofluorescence of Prochlorococcus within the well-lit stratified water layer resulted in failure to detect them by flow cytometry. If any other data based on genomic analyses or microscopy are available, I recommend the authors to check the validity of these data. Otherwise, the authors can show some caveats in the materials and methods section. Anyway, it is a frequently seen situation, and it will not critically affect the discussion in the present study.

Citation: https://doi.org/10.5194/egusphere-2023-2578-RC1
- AC1: 'Reply on RC1', F. Van Wambeke, 19 Feb 2024
  
  The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2023/egusphere-2023-2578/egusphere-2023-2578-AC1-supplement.pdf
  
  Citation: https://doi.org/10.5194/egusphere-2023-2578-AC1
RC2:
'Comment on egusphere-2023-2578', Anonymous Referee #2, 30 Jan 2024

General comments
The authors investigate phosphomonoesterase (PME) versus phosphodiesterase (PDE) activities in a well-known P-depleted oligotrophic environment, the Eastern Mediterranean Sea, at two contrasted season. They characterize maximum hydrolysis rates (Vm) and half-saturation constants (Km) of both PME and PDE activities in relation to dissolved stocks of Phosphorus : DIP, DOP and the enzymatically hydrolysable fraction of DOP. Although phosphomonoesterase, also known as alkaline phosphatase, activities have been extensively studied in P deplete oligotrophic and coastal environments during past decades, the measurements of both PME and PDE have been achieved only recently. The authors have paid particular attention on the methodology for the measurement of these activities. The results of this paper confirm the results found elsewhere that PDE activities (Vm) could be in the same order of magnitude than PME activities. PDE seem to be regulated as PME, by the availability of DIP. However the regulation of PDE by NOx:DIP ratio is also discussed as well as the occurrence of different microbial communities having different PDE expression pattern. This paper has a significant contribution to the understanding of the Phosphorus fluxes through the microbial food web, participating to the biogeochemical cycle of Phosphorus. Obviously, it is within the scope of EGUsphere.
Specific comments
The scientific approach and applied methods to the studies of PME and PDE activities in relation to the dissolved phosphorus pools are particularly well adapted. The measurements of nanomolar concentrations of DIP and labile DOP in such oligotrophic environments as the Mediterranean Sea are achieved with the LWCC method having a very low detection limit (1 nM). The measurements of kinetic parameters of enzymatic activities need a particular attention since methodological biases can lead to misestimated Vm and Km. The most notable divergences in existing methodologies on enzymatic activity assays in natural environment is the substrate concentrations used for assays. The range of substrate concentration significantly affect kinetic parameters estimation and it is generally recommended to use a large substrate concentration range, up to 10 Km at least. A specific literature exists on this particular bias which could be cited by authors in the Discussion part. However, the authors discussed their results with published literature, considering these methodological aspects, which is scarcely made while necessary for meaningful comparison.
Details
Line 27: Define the significance of DIP the first time it appears rather than Line 42
Line 49, 692: Labry et al. 2016 rather than 2021
Line 58: precise under optimal conditions of concentrations of what ? enzyme ?
Line 120: nitrite rather than « nitrites » and use « DOP » rather than its significance
Line 141,637: Djaoudi et al. 2018 rather than 2017
Line 225 : concerning winter depth of Pcline, refer to Fig. 3b,c
Line 395 : a little more exhausted literature on P diesters composition would be informative
Line 408-412 : The difficult comparison with previous studies also comes from the different substrates used, MUF- derivates (Thomson et al. 2020, Sato et al. 2013) vs paranitrophenyl- derivatives (Huang et al. 2022), corresponding to different enzyme affinity. Conditions of incubation, particularly temperature may also differ between studies, optimal versus in situ temperature.
Line 425 : Thomson et al., 2020 rather than 2019
Line 478 – 483 : discussion on Km PME >> LDOP : do the authors mean that enzymes experience locally higher substrate concentrations due to intermittent and patchy distribution of organic Phosphorus ? Could the authors explain it more precisely.
Figure 7 : The frame around the legend on the Km versus DIP graphe could be removed.

Citation: https://doi.org/10.5194/egusphere-2023-2578-RC2
- AC2: 'Reply on RC2', F. Van Wambeke, 19 Feb 2024
  
  The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2023/egusphere-2023-2578/egusphere-2023-2578-AC2-supplement.pdf
  
  Citation: https://doi.org/10.5194/egusphere-2023-2578-AC2

Peer review completion

AR: Author's response | RR: Referee report | ED: Editor decision | EF: Editorial file upload

ED: Publish subject to minor revisions (review by editor) (28 Feb 2024) by Perran Cook

AR by F. Van Wambeke on behalf of the Authors (01 Mar 2024) Author's response Author's tracked changes Manuscript

ED: Publish as is (24 Mar 2024) by Perran Cook

AR by F. Van Wambeke on behalf of the Authors (29 Mar 2024) Manuscript

Post-review adjustments

AA: Author's adjustment | EA: Editor approval

AA by F. Van Wambeke on behalf of the Authors (17 May 2024) Author's adjustment Manuscript

EA: Adjustments approved (21 May 2024) by Perran Cook

Journal article(s) based on this preprint

29 May 2024

Phosphomonoesterase and phosphodiesterase activities in the eastern Mediterranean in two contrasting seasonal situations

France Van Wambeke, Pascal Conan, Mireille Pujo-Pay, Vincent Taillandier, Olivier Crispi, Alexandra Pavlidou, Sandra Nunige, Morgane Didry, Christophe Salmeron, and Elvira Pulido-Villena

Biogeosciences, 21, 2621–2640, https://doi.org/10.5194/bg-21-2621-2024,https://doi.org/10.5194/bg-21-2621-2024, 2024

Short summary

France Van Wambeke, Pascal Conan, Mireille Pujo-Pay, Vincent Taillandier, Olivier Crispi, and Elvira Pulido-Villena

Supplement

https://doi.org/10.5194/egusphere-2023-2578-supplement

France Van Wambeke, Pascal Conan, Mireille Pujo-Pay, Vincent Taillandier, Olivier Crispi, and Elvira Pulido-Villena

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Distribution of phosphomonoesterase (PME) and phosphodiesterase (PDE) activities over the epipelagic zone are described in the eastern Mediterranean Sea, in winter and spring. The type of concentration kinetics obtained for PDE (saturation at 50 µM, high Km, high turnovertimes) compared to those of PME (saturation at 1 µM, low Km, low turnovertimes) are discussed in regard to possible inequal distribution of PDE and PME among organic material size continuum, and accessibility to phosphodiesters.


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