Manifestations and environmental implications of microbially-induced calcium carbonate precipitation (MICP) by the cyanobacterium <em>Dolichospermum flosaquae</em>

Abdel-Basset, Refat; Hasssan, Elhagag Ahmed; Grossart, Hans-Peter

doi:https://doi.org/10.5194/bg-2021-146

Preprints

https://doi.org/10.5194/bg-2021-146

Preprints

08 Jul 2021

| 08 Jul 2021

Status: this discussion paper is a preprint. It has been under review for the journal Biogeosciences (BG). The manuscript was not accepted for further review after discussion.

Manifestations and environmental implications of microbially-induced calcium carbonate precipitation (MICP) by the cyanobacterium Dolichospermum flosaquae

Refat Abdel-Basset, Elhagag Ahmed Hasssan, and Hans-Peter Grossart

Abstract. The aim of this work is to explore the ability and magnitude of the temperate cyanobacterium Dolichospermum flosaquae in microbially-induced calcium carbonate precipitation (MICP). Environmentally, MICP controls the availability of calcium, carbon and phosphorus in freshwater lakes and simultaneously controls carbon exchange with the atmosphere. Cultures of flosaquae were grown in BG11 medium containing 0, 1, 1.5, 2 and 4 mg Ca²⁺ L⁻¹, as cardinal concentrations previously reported in freshwater lakes, in addition to a control culture (BG11 containing 13 mg Ca²⁺ L⁻¹). Growth (cell number, chlorophyll a, and protein content) of D. flosaquae was generally reduced by elevating calcium concentrations of the different salts used (chloride, acetate, or citrate). D. flosaquae exhibited its ability to perform MICP as carbonate alkalinity was sharply increased up to its highest level (six times that of the control) at a citrate concentration of 4 mg Ca²⁺ L⁻¹. Calcium carbonate was formed at a pre-precipitation stage as the minimum pH necessary for precipitation (8.7) has been scarcely approached under such conditions. In this work, MICP took place mostly empowered by photosynthesis and respiration. Residual calcium exhibited its lowest value at 4 mg Ca²⁺ citrate L⁻¹, coinciding with the highest alkalinity level. Precipitated calcium was increased with chlorophyll a content, but not with increasing cell numbers.

Received: 10 Jun 2021 – Discussion started: 08 Jul 2021

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Refat Abdel-Basset, Elhagag Ahmed Hasssan, and Hans-Peter Grossart

Status: closed

RC1:
'Comment on bg-2021-146', O.S. Pokrovsky, 02 Sep 2021

This paper is devoted to experimental study of cyanobacterium-induced precipitation of CaCO3. The topic is generally well studied, and fits the scope of the journal. However, the overall quality of this research, its design and interpretation are below the standards of academic journal and rather suit for some applied journal audience.

The major problems are the following:

1) There is no information on CaCO₃ saturation state during the experimental run and even at the beginning of experiments. One cannot study CaCO3 precipitation without having an idea of solution saturation state

2) There is no kinetic assessment of pH and Ca concentration evolution in the course of experiment. It is thus impossible to assess the rate of the process and the dynamics of bacterially induced precipitation. The 4-weeks duration of experiment is not justified; the growth curve Is not presented.

3) The effects of anions and Ca are not distinguished. In addition to Ca salts, Na salts should be used if the authors aim to characterize the effect of citrate, for instance.

4) 100% BG-11 used in the experiments contain unreasonably high PO4 concentration, totally irrelevant to natural settings, especially for P-limited lakes. Not only PO4 is a strong inhibitor of calcite precipitation, it also provide unrealistical conditions for cyanobacterial growth. The application of obtained results to lakes is unwarranted.

5) Alkalinity titration of unfiltered solution is not suitable. Part of H+ will be used for i) cell surface adsorption, ii) HCO3- neutralization, and iii) CaCO3 dissolution. The authors cannot distinguish between these 3 processes

Several specific comments below.

L 27-29 unclear. What is the driving force, photosynthesis or precipitation?

L32, unclear, why Chl a is not dependent on cell number in a monocultural experiment

L64 Ca does not coprecipitate. It precipitates as CaCO3

The link between 2^nd and 3^rd § of the Introduction is unclear

L179 It is unclear where these ratios are shown

L203-205 How do we know that this release is not dependent on the identity and concentration of anion? Otherwise it is inconsistent with what is stated in L 154-157

L 246-248 This is not shown in the resent work ; no phosphate analysis !

L252-253 contradicts to what is state din L203-204

L257-258 This is self-contradictory. Why 1.5 ppm if cells released 2.26 ppm?

L263265 unclear, and unsupported. What about bicarbonate level and buffering?

L277 Not really? S.I. of CaCO3 is more important

L287-288 Irrelevant without pH/pCO2 parameters

L290 What does it mean, solid phase

L290-293 This is not correct. There are many quantitative laboratory studies of CaCO3 precipitation kinetics and mechanisms in the presence of cyanobacteria

L294-297 This is irrelevant to the discussion of results of this study

L308 This is not correct. The OH-/HCO3- exchange during photosynthesis is by far the most important process

L345-346 This is not assessed in this study

L364-366 This has been shown well fifty years before Berry

L354-382: The purpose of this § and its relevance to the present work are unclear. This is not a discussion of obtained results

L393-394 Unclear

L400-401 The cost will be quite elevated and thus commercially not interesting

Figures: legend is unclear

++++

Oleg S Pokrovsky

Citation: https://doi.org/10.5194/bg-2021-146-RC1
- AC1:
  'Reply on RC1', Refat Mostafa, 27 Sep 2021
  RC1: 'Comment on bg-2021-146', O.S. Pokrovsky, 02 Sep 2021 reply
  This paper is devoted to experimental study of cyanobacterium-induced precipitation of CaCO3. The topic is generally well studied, and fits the scope of the journal. However, the overall quality of this research, its design and interpretation are below the standards of academic journal and rather suit for some applied journal audience.
  The major problems are the following:
  1) There is no information on CaCO₃ saturation state during the experimental run and even at the beginning of experiments. One cannot study CaCO3 precipitation without having an idea of solution saturation state
  The process of CaCO3 formation, dealt with in this work, is microbially induced, which depends on (micro)biological metabolism, rather than on chemical or physical conditions including solution saturation. Microbial walls (or sheaths) act as nucleation sites, which concentrate calcium ions regardless of solution concentration and microbial metabolism (particularly urease activity) catalyze precipitation.
  2) There is no kinetic assessment of pH and Ca concentration evolution in the course of experiment. It is thus impossible to assess the rate of the process and the dynamics of bacterially induced precipitation. The 4-weeks duration of experiment is not justified; the growth curve Is not presented.
  Neither the rate nor the dynamics were targeted in this work; only the final magnitude of pH, growth and product were concerned. Rate and dynamics may need a sperate work.
  3) The effects of anions and Ca are not distinguished. In addition to Ca salts, Na salts should be used if the authors aim to characterize the effect of citrate, for instance.
  Why should Na citrate be used, for instance? Sodium salts do not precipitate. To distinguish the effect of anions, three different anions citrate, acetate, and chloride are studied. Meanwhile, only cation, calcium, is at concern.
  4) 100% BG-11 used in the experiments contain unreasonably high PO4 concentration, totally irrelevant to natural settings, especially for P-limited lakes. Not only PO4 is a strong inhibitor of calcite precipitation, it also provide unrealistical conditions for cyanobacterial growth. The application of obtained results to lakes is unwarranted.
  BG11 is a standard enrichment medium widely used for growth and enrichment of cyanobacteria and algae since a long time (since Rippka and Herdman 1993). Yes, PO4 in BG11 is high as it is used as phosphate buffer of the medium. The cyanobacterium used has been isolated from a freshwater lake (Stechlinsee, Germany) but the unialgal culture is not applicable for natural lakes but shows the ability of the organism at defined conditions of not only phosphorus but rather at constant temperature, pH, nutrients, etc.., which are not the case in nature.
  5) Alkalinity titration of unfiltered solution is not suitable. Part of H+ will be used for i) cell surface adsorption, ii) HCO3- neutralization, and iii) CaCO3 dissolution. The authors cannot distinguish between these 3 processes
  This is a standard method used in the literature for titrating carbonate; all fractions are supposed to be constant e.g. cell surface adsorption.
  Several specific comments below.
  L 27-29 unclear. What is the driving force, photosynthesis or precipitation?
  Photosynthesis was the driving force for precipitation
  L32, unclear, why Chl a is not dependent on cell number in a monocultural experiment
  The confusing part has been removed from the text.
  L64 Ca does not coprecipitate. It precipitates as CaCO3
  OK, changed in the text.
  The link between 2^nd and 3^rd § of the Introduction is unclear
  The text and information in the two paragraphs have been modified to clear confusion and be more synchronized.
  L179 It is unclear where these ratios are shown
  They were existing in a former version but removed from this version, as one of the reviewers recommended that; these are now indicated in the manuscript as “data not shown”
  L203-205 How do we know that this release is not dependent on the identity and concentration of anion? Otherwise it is inconsistent with what is stated in L 154-157
  This is an assumption that the cells of the same organism release similar amounts of calcium since the release itself has been found. No link between these two results; one for calcium release and one for growth.
  L 246-248 This is not shown in the resent work ; no phosphate analysis !
  The sentence is “Calcium precipitates carbon in the form of calcium carbonate either chemically or microbially and precipitates phosphorus in the form of calcium phosphate.”
  It does not refer to experimental analysis; it is a fundamental scientific information supported by references on several occasions in the manuscript.
  L252-253 contradicts to what is state din L203-204
  L252-253:
  The studied concentrations (0, 1, 1.5, 2 and 4 mg Ca2+ L-1) were chosen from previous records in the literature (Weyhenmeyer et al 2019), which considered these concentrations critical.
  L203-204:
  Therefore, a virtual concentration of total calcium is given to account for the externally supplemented concentration of calcium (0, 1, 1.5, 2 or 4 mg Ca2+ L-1) and the amount of calcium found at calcium-deprivation i.e., 2.26 mg Ca2+, which is assumed to be equally released by each culture.
  .
  No contradictions or even a link
  The first is cited from the literature while the second are validated values based on experimental estimates.
  
  L257-258 This is self-contradictory. Why 1.5 ppm if cells released 2.26 ppm?
  The “1.5 ppm” refers to the externally supplemented concentrations before cells grow and secrete.
  L263265 unclear, and unsupported. What about bicarbonate level and buffering?
  Removed
  L277 Not really? S.I. of CaCO3 is more important
  A lot of references support the notion that alkalinity is a prerequisite for microbial CaCO3 formation. e.g.:
  “Most calcite precipitation occurs under alkaline conditions of pH values from 8.7 to 9.5 (Ferris et al 2003; Dupraz et al 2009).”
  The superiority of carbonate over alkalinity may be found in physico-chemical systems as microbially induced CaCO3 precipitation may rely on collecting CO2 with water to form bicarbonate then carbonate.
  L287-288 Irrelevant without pH/pCO2 parameters
  L287-290:
  “In addition, it has been stated that calcium carbonate can be formed at very low solubility levels in pure water before precipitation; its solubility in pure water is as low as 13 mg L-1 at 25°C (Aylward et al 2008); it increases relatively with decreasing temperature and increases in rainwater saturated with carbon dioxide, due to the formation of more soluble calcium bicarbonate.”
  This is a statement from the cited references, not mine.
  L290 What does it mean, solid phase
  The sentence states: “soluble”
  L290-293 This is not correct. There are many quantitative laboratory studies of CaCO3 precipitation kinetics and mechanisms in the presence of cyanobacteria
  I could not find any publications despite several google searches. Contribution of cyanobacteria are rich but as a population in ancient structures.
  L294-297 This is irrelevant to the discussion of results of this study
  It is actually in the discussion of this study.
  L308 This is not correct. The OH-/HCO3- exchange during photosynthesis is by far the most important process
  However, this line does not deal with anything about photosynthesis.
  
  L345-346 This is not assessed in this study
  The conclusion is a “citation” supported by the reference.
  L364-366 This has been shown well fifty years before Berry
  Yes. The paragraph also contains a sentence referenced at the year 1975.
  L354-382: The purpose of this § and its relevance to the present work are unclear. This is not a discussion of obtained results
  Removed
  L393-394 Unclear
  Reformulated
  L400-401 The cost will be quite elevated and thus commercially not interesting
  OK
  
  Figures: legend is unclear
  Revised and reformulated
  ++++
  Oleg S Pokrovsky
  
  Citation: https://doi.org/10.5194/bg-2021-146-AC1
RC2:
'Comment on bg-2021-146', Anonymous Referee #2, 05 Sep 2021

This paper investigates the capability of cyanobacterium D. flosaquae to induce MICP. The results of the study rely on the measurement of the growth parameters, and a few solution chemistry parameters (Ca, pH, TA, NH3). Based on these datasets, the authors claimed that the D. flosaquae induces precipitation of CaCO3 (calcite). I find that assertion poorly supported by the presented data and paucity of other (necessary) datasets (saturation state, microscopy/spectroscopy observations, abiotic controls) needed to support the findings claimed in the paper. In light of these issues, I find that study is not up to the standards of the journal and will be more appropriate for another journal. Below are my more elaborate comments:

1) The introduction lacked a clear motivation for the study. Several statements in the introduction were not properly referenced, which is surprising given that MICP is a very well-studied field.

2) Lack of any abiotic control in the experiment design makes it difficult to infer whether the formation of CaCO3 was due to D. flosaquae or merely due to changes in the solution chemistry.

3) No information on the saturation state of the solution with respect to carbonate and other minerals is included in the results and discussion.

4) The time evolution data on the growth parameters, and dissolved Ca concentrations are not included, which again makes it challenging to fully understand the evolution of the system under different treatments

5) In the current design of the incubations, the effect of Ca concentrations and anions cannot be decoupled. This results in confusing interpretations of the Figures.

6) Lastly, the authors refer to the possible CaCO3 phase as calcite, but yet provide no data that confirms whether the carbonate is calcite.

Specific comments:

Line 41: references missing.

Line 52-55: The phrase is difficult to understand at first. Recommended to revise it

Line 56: why is “ate” in carbonate in parentheses?

Line 56: The use of co-precipitation to define CaCO3 is incorrect. Co-precipitation implies that 2 or more phases precipitating simultaneously. In CaCO3, it’s just one phase.

Line 58: “Calcium and phosphate also coprecipitate…” phrasing unclear. Do Ca-phosphate co-precipitates with CaCO3 forming a solid solution? Please revise

Line 62: it would be helpful to add some references or some examples of key industrial applications.

Line 64: misuse of word co-precipitation (see comment on Line 56)

Line 76-77: Why is the cyanobacterium D. flosaquae chosen for this study? What is its environmental importance? A brief description of the cyanobacterium itself and reference to any previous studies would be helpful to better understand the context of this manuscript.

Line 87: where was the strain procured from? Is it axenic?

Line 90-93: There is no sterile control for the experiment (i.e. BG11 with no cells). This is a crucial problem as the authors are proposing the D. flosaquae is able to induce CaCO3 precipitation.

Line 151-153: The way the incubations are set up, it’s not possible to discriminate between the effects of Ca concentrations and the effect of citrate, acetate, or chloride anions, on the growth of D. flosaquae. Using perhaps, sodium salts to assess the effect of anions would be a more logical choice.

Line 160: how was pH set?

Line 260-261: I don’t agree with the authors that solely based on the alkalinity levels, pH, and Ca concentrations; it could be asserted that D. flosaquae induces MICP. These datasets don’t eliminate the possibility of the formation of other Ca phases (such as phosphates). A simple saturation index calculation (for e.g using Visual Minteq or PHREEQC) would be easiest to confirm what mineral phases would be over-saturated under the experimental condition. Alternatively, microscopic observation could also confirm with confidence what mineral is actually precipitated.

Line 311: “The high ability of D. flosaquae to shift the pH to alkalinity …” high compared to what? There’s no comparison of D. flosaquae versus other cyanobacteria inducing extracellular carbonates. It would be useful to clarify how authors calculate or assign the “high ability” to D. flosaquae.

Citation: https://doi.org/10.5194/bg-2021-146-RC2
- AC2: 'Reply on RC2', Refat Mostafa, 27 Sep 2021
  
  RC2: 'Comment on bg-2021-146', Anonymous Referee #2, 05 Sep 2021 reply
  This paper investigates the capability of cyanobacterium D. flosaquae to induce MICP. The results of the study rely on the measurement of the growth parameters, and a few solution chemistry parameters (Ca, pH, TA, NH3). Based on these datasets, the authors claimed that the D. flosaquae induces precipitation of CaCO3 (calcite). I find that assertion poorly supported by the presented data and paucity of other (necessary) datasets (saturation state, microscopy/spectroscopy observations, abiotic controls) needed to support the findings claimed in the paper. In light of these issues, I find that study is not up to the standards of the journal and will be more appropriate for another journal. Below are my more elaborate comments:
  
  1) The introduction lacked a clear motivation for the study. Several statements in the introduction were not properly referenced, which is surprising given that MICP is a very well-studied field.
  
  Done
  2) Lack of any abiotic control in the experiment design makes it difficult to infer whether the formation of CaCO3 was due to D. flosaquae or merely due to changes in the solution chemistry.
  
  BG11 medium is daily used in our lab and doesn’t contain carbonate to precipitate calcium.
  3) No information on the saturation state of the solution with respect to carbonate and other minerals is included in the results and discussion.
  
  The process of MICP, which under study at this work is (micro)biological; all other physical and chemical components are equal at all experimental samples i.e. their impact is equal, if any.
  4) The time evolution data on the growth parameters, and dissolved Ca concentrations are not included, which again makes it challenging to fully understand the evolution of the system under different treatments
  Kinetics of the process was not targeted in this work; only the net product or net result was the aim. Once again, a microbiological system is different from the physico-chemical system.
  5) In the current design of the incubations, the effect of Ca concentrations and anions cannot be decoupled. This results in confusing interpretations of the Figures.
  
  It has been actually decoupled. Three different anions are included to show the effect of each anion at a certain calcium concentration and different calcium concentrations to show the effect of calcium.
  6) Lastly, the authors refer to the possible CaCO3 phase as calcite, but yet provide no data that confirms whether the carbonate is calcite.
  We have never referred to our possible CaCO3 phase as calcite since we did not plan to identify it; the word calcite has been mentioned only in the cited literature and reference list.
  
  Specific comments:
  
  Line 41: references missing.
  Added.
  
  Line 52-55: The phrase is difficult to understand at first. Recommended to revise it
  Reformulated
  
  Line 56: why is “ate” in carbonate in parentheses?
  Carbon(ate) refers to carbon or carbonate.
  Now, it is changed to carbon/carbonate in the submitted version.
  
  Line 56: The use of co-precipitation to define CaCO3 is incorrect. Co-precipitation implies that 2 or more phases precipitating simultaneously. In CaCO3, it’s just one phase.
  OK, changed to “precipitation”.
  This is although the two phases, in the course, are calcium and carbonate, which coprecipitate forming calcium carbonate.
  
  Line 58: “Calcium and phosphate also coprecipitate…” phrasing unclear. Do Ca-phosphate co-precipitates with CaCO3 forming a solid solution? Please revise
  
  Yes, but the terminology used in this case is “binding” as follows:
  Furthermore, any calcium carbonate precipitate is a good binder of phosphate (Yanamadala 2005), mentioned in Line 247-248.
  Line 62: it would be helpful to add some references or some examples of key industrial applications.
  Reference and some applications have been added.
  
  Line 64: misuse of word co-precipitation (see comment on Line 56)
  
  OK, changed
  Line 76-77: Why is the cyanobacterium D. flosaquae chosen for this study? What is its environmental importance? A brief description of the cyanobacterium itself and reference to any previous studies would be helpful to better understand the context of this manuscript.
  
  Done
  The hypothesis of this work is to explore whether Dolichospermum flosaquae is able to perform MICP in freshwater lakes.
  Line 87: where was the strain procured from? Is it axenic?
  
  D. flosaquae is a major temperate cyanobacterium; the strain used in this work has been isolated from Stechlinsee lake (IGB Berlin). It was not axenic.
  Line 90-93: There is no sterile control for the experiment (i.e. BG11 with no cells). This is a crucial problem as the authors are proposing the D. flosaquae is able to induce CaCO3 precipitation.
  
  Done
  BG11 medium is daily used in our lab and doesn’t contain carbonate to precipitate calcium.
  Line 151-153: The way the incubations are set up, it’s not possible to discriminate between the effects of Ca concentrations and the effect of citrate, acetate, or chloride anions, on the growth of D. flosaquae. Using perhaps, sodium salts to assess the effect of anions would be a more logical choice.
  Sodium salts do not precipitate.
  
  Line 160: how was pH set?
  The pH of BG11 medium was adjusted to pH 7.0 using acid(HCl)/alkali(NaOH).
  
  Line 260-261: I don’t agree with the authors that solely based on the alkalinity levels, pH, and Ca concentrations; it could be asserted that D. flosaquae induces MICP. These datasets don’t eliminate the possibility of the formation of other Ca phases (such as phosphates). A simple saturation index calculation (for e.g using Visual Minteq or PHREEQC) would be easiest to confirm what mineral phases would be over-saturated under the experimental condition. Alternatively, microscopic observation could also confirm with confidence what mineral is actually precipitated.
  The process of MICP, studied in this work, is (micro)biological; all other physical and chemical components are equal at all experimental samples i.i. their impact is equal.
  
  Line 311: “The high ability of D. flosaquae to shift the pH to alkalinity …” high compared to what? There’s no comparison of D. flosaquae versus other cyanobacteria inducing extracellular carbonates. It would be useful to clarify how authors calculate or assign the “high ability” to D. flosaquae.
  It is high, not higher; no comparison was there. It is high because the pH elevated from 7.0 at the beginning of the experiment to pH 12 at the end of the experiment.
  
  Citation: https://doi.org/10.5194/bg-2021-146-AC2

Status: closed

RC1:
'Comment on bg-2021-146', O.S. Pokrovsky, 02 Sep 2021

This paper is devoted to experimental study of cyanobacterium-induced precipitation of CaCO3. The topic is generally well studied, and fits the scope of the journal. However, the overall quality of this research, its design and interpretation are below the standards of academic journal and rather suit for some applied journal audience.

The major problems are the following:

1) There is no information on CaCO₃ saturation state during the experimental run and even at the beginning of experiments. One cannot study CaCO3 precipitation without having an idea of solution saturation state

2) There is no kinetic assessment of pH and Ca concentration evolution in the course of experiment. It is thus impossible to assess the rate of the process and the dynamics of bacterially induced precipitation. The 4-weeks duration of experiment is not justified; the growth curve Is not presented.

3) The effects of anions and Ca are not distinguished. In addition to Ca salts, Na salts should be used if the authors aim to characterize the effect of citrate, for instance.

4) 100% BG-11 used in the experiments contain unreasonably high PO4 concentration, totally irrelevant to natural settings, especially for P-limited lakes. Not only PO4 is a strong inhibitor of calcite precipitation, it also provide unrealistical conditions for cyanobacterial growth. The application of obtained results to lakes is unwarranted.

5) Alkalinity titration of unfiltered solution is not suitable. Part of H+ will be used for i) cell surface adsorption, ii) HCO3- neutralization, and iii) CaCO3 dissolution. The authors cannot distinguish between these 3 processes

Several specific comments below.

L 27-29 unclear. What is the driving force, photosynthesis or precipitation?

L32, unclear, why Chl a is not dependent on cell number in a monocultural experiment

L64 Ca does not coprecipitate. It precipitates as CaCO3

The link between 2^nd and 3^rd § of the Introduction is unclear

L179 It is unclear where these ratios are shown

L203-205 How do we know that this release is not dependent on the identity and concentration of anion? Otherwise it is inconsistent with what is stated in L 154-157

L 246-248 This is not shown in the resent work ; no phosphate analysis !

L252-253 contradicts to what is state din L203-204

L257-258 This is self-contradictory. Why 1.5 ppm if cells released 2.26 ppm?

L263265 unclear, and unsupported. What about bicarbonate level and buffering?

L277 Not really? S.I. of CaCO3 is more important

L287-288 Irrelevant without pH/pCO2 parameters

L290 What does it mean, solid phase

L290-293 This is not correct. There are many quantitative laboratory studies of CaCO3 precipitation kinetics and mechanisms in the presence of cyanobacteria

L294-297 This is irrelevant to the discussion of results of this study

L308 This is not correct. The OH-/HCO3- exchange during photosynthesis is by far the most important process

L345-346 This is not assessed in this study

L364-366 This has been shown well fifty years before Berry

L354-382: The purpose of this § and its relevance to the present work are unclear. This is not a discussion of obtained results

L393-394 Unclear

L400-401 The cost will be quite elevated and thus commercially not interesting

Figures: legend is unclear

++++

Oleg S Pokrovsky

Citation: https://doi.org/10.5194/bg-2021-146-RC1
- AC1:
  'Reply on RC1', Refat Mostafa, 27 Sep 2021
  RC1: 'Comment on bg-2021-146', O.S. Pokrovsky, 02 Sep 2021 reply
  This paper is devoted to experimental study of cyanobacterium-induced precipitation of CaCO3. The topic is generally well studied, and fits the scope of the journal. However, the overall quality of this research, its design and interpretation are below the standards of academic journal and rather suit for some applied journal audience.
  The major problems are the following:
  1) There is no information on CaCO₃ saturation state during the experimental run and even at the beginning of experiments. One cannot study CaCO3 precipitation without having an idea of solution saturation state
  The process of CaCO3 formation, dealt with in this work, is microbially induced, which depends on (micro)biological metabolism, rather than on chemical or physical conditions including solution saturation. Microbial walls (or sheaths) act as nucleation sites, which concentrate calcium ions regardless of solution concentration and microbial metabolism (particularly urease activity) catalyze precipitation.
  2) There is no kinetic assessment of pH and Ca concentration evolution in the course of experiment. It is thus impossible to assess the rate of the process and the dynamics of bacterially induced precipitation. The 4-weeks duration of experiment is not justified; the growth curve Is not presented.
  Neither the rate nor the dynamics were targeted in this work; only the final magnitude of pH, growth and product were concerned. Rate and dynamics may need a sperate work.
  3) The effects of anions and Ca are not distinguished. In addition to Ca salts, Na salts should be used if the authors aim to characterize the effect of citrate, for instance.
  Why should Na citrate be used, for instance? Sodium salts do not precipitate. To distinguish the effect of anions, three different anions citrate, acetate, and chloride are studied. Meanwhile, only cation, calcium, is at concern.
  4) 100% BG-11 used in the experiments contain unreasonably high PO4 concentration, totally irrelevant to natural settings, especially for P-limited lakes. Not only PO4 is a strong inhibitor of calcite precipitation, it also provide unrealistical conditions for cyanobacterial growth. The application of obtained results to lakes is unwarranted.
  BG11 is a standard enrichment medium widely used for growth and enrichment of cyanobacteria and algae since a long time (since Rippka and Herdman 1993). Yes, PO4 in BG11 is high as it is used as phosphate buffer of the medium. The cyanobacterium used has been isolated from a freshwater lake (Stechlinsee, Germany) but the unialgal culture is not applicable for natural lakes but shows the ability of the organism at defined conditions of not only phosphorus but rather at constant temperature, pH, nutrients, etc.., which are not the case in nature.
  5) Alkalinity titration of unfiltered solution is not suitable. Part of H+ will be used for i) cell surface adsorption, ii) HCO3- neutralization, and iii) CaCO3 dissolution. The authors cannot distinguish between these 3 processes
  This is a standard method used in the literature for titrating carbonate; all fractions are supposed to be constant e.g. cell surface adsorption.
  Several specific comments below.
  L 27-29 unclear. What is the driving force, photosynthesis or precipitation?
  Photosynthesis was the driving force for precipitation
  L32, unclear, why Chl a is not dependent on cell number in a monocultural experiment
  The confusing part has been removed from the text.
  L64 Ca does not coprecipitate. It precipitates as CaCO3
  OK, changed in the text.
  The link between 2^nd and 3^rd § of the Introduction is unclear
  The text and information in the two paragraphs have been modified to clear confusion and be more synchronized.
  L179 It is unclear where these ratios are shown
  They were existing in a former version but removed from this version, as one of the reviewers recommended that; these are now indicated in the manuscript as “data not shown”
  L203-205 How do we know that this release is not dependent on the identity and concentration of anion? Otherwise it is inconsistent with what is stated in L 154-157
  This is an assumption that the cells of the same organism release similar amounts of calcium since the release itself has been found. No link between these two results; one for calcium release and one for growth.
  L 246-248 This is not shown in the resent work ; no phosphate analysis !
  The sentence is “Calcium precipitates carbon in the form of calcium carbonate either chemically or microbially and precipitates phosphorus in the form of calcium phosphate.”
  It does not refer to experimental analysis; it is a fundamental scientific information supported by references on several occasions in the manuscript.
  L252-253 contradicts to what is state din L203-204
  L252-253:
  The studied concentrations (0, 1, 1.5, 2 and 4 mg Ca2+ L-1) were chosen from previous records in the literature (Weyhenmeyer et al 2019), which considered these concentrations critical.
  L203-204:
  Therefore, a virtual concentration of total calcium is given to account for the externally supplemented concentration of calcium (0, 1, 1.5, 2 or 4 mg Ca2+ L-1) and the amount of calcium found at calcium-deprivation i.e., 2.26 mg Ca2+, which is assumed to be equally released by each culture.
  .
  No contradictions or even a link
  The first is cited from the literature while the second are validated values based on experimental estimates.
  
  L257-258 This is self-contradictory. Why 1.5 ppm if cells released 2.26 ppm?
  The “1.5 ppm” refers to the externally supplemented concentrations before cells grow and secrete.
  L263265 unclear, and unsupported. What about bicarbonate level and buffering?
  Removed
  L277 Not really? S.I. of CaCO3 is more important
  A lot of references support the notion that alkalinity is a prerequisite for microbial CaCO3 formation. e.g.:
  “Most calcite precipitation occurs under alkaline conditions of pH values from 8.7 to 9.5 (Ferris et al 2003; Dupraz et al 2009).”
  The superiority of carbonate over alkalinity may be found in physico-chemical systems as microbially induced CaCO3 precipitation may rely on collecting CO2 with water to form bicarbonate then carbonate.
  L287-288 Irrelevant without pH/pCO2 parameters
  L287-290:
  “In addition, it has been stated that calcium carbonate can be formed at very low solubility levels in pure water before precipitation; its solubility in pure water is as low as 13 mg L-1 at 25°C (Aylward et al 2008); it increases relatively with decreasing temperature and increases in rainwater saturated with carbon dioxide, due to the formation of more soluble calcium bicarbonate.”
  This is a statement from the cited references, not mine.
  L290 What does it mean, solid phase
  The sentence states: “soluble”
  L290-293 This is not correct. There are many quantitative laboratory studies of CaCO3 precipitation kinetics and mechanisms in the presence of cyanobacteria
  I could not find any publications despite several google searches. Contribution of cyanobacteria are rich but as a population in ancient structures.
  L294-297 This is irrelevant to the discussion of results of this study
  It is actually in the discussion of this study.
  L308 This is not correct. The OH-/HCO3- exchange during photosynthesis is by far the most important process
  However, this line does not deal with anything about photosynthesis.
  
  L345-346 This is not assessed in this study
  The conclusion is a “citation” supported by the reference.
  L364-366 This has been shown well fifty years before Berry
  Yes. The paragraph also contains a sentence referenced at the year 1975.
  L354-382: The purpose of this § and its relevance to the present work are unclear. This is not a discussion of obtained results
  Removed
  L393-394 Unclear
  Reformulated
  L400-401 The cost will be quite elevated and thus commercially not interesting
  OK
  
  Figures: legend is unclear
  Revised and reformulated
  ++++
  Oleg S Pokrovsky
  
  Citation: https://doi.org/10.5194/bg-2021-146-AC1
RC2:
'Comment on bg-2021-146', Anonymous Referee #2, 05 Sep 2021

This paper investigates the capability of cyanobacterium D. flosaquae to induce MICP. The results of the study rely on the measurement of the growth parameters, and a few solution chemistry parameters (Ca, pH, TA, NH3). Based on these datasets, the authors claimed that the D. flosaquae induces precipitation of CaCO3 (calcite). I find that assertion poorly supported by the presented data and paucity of other (necessary) datasets (saturation state, microscopy/spectroscopy observations, abiotic controls) needed to support the findings claimed in the paper. In light of these issues, I find that study is not up to the standards of the journal and will be more appropriate for another journal. Below are my more elaborate comments:

1) The introduction lacked a clear motivation for the study. Several statements in the introduction were not properly referenced, which is surprising given that MICP is a very well-studied field.

2) Lack of any abiotic control in the experiment design makes it difficult to infer whether the formation of CaCO3 was due to D. flosaquae or merely due to changes in the solution chemistry.

3) No information on the saturation state of the solution with respect to carbonate and other minerals is included in the results and discussion.

4) The time evolution data on the growth parameters, and dissolved Ca concentrations are not included, which again makes it challenging to fully understand the evolution of the system under different treatments

5) In the current design of the incubations, the effect of Ca concentrations and anions cannot be decoupled. This results in confusing interpretations of the Figures.

6) Lastly, the authors refer to the possible CaCO3 phase as calcite, but yet provide no data that confirms whether the carbonate is calcite.

Specific comments:

Line 41: references missing.

Line 52-55: The phrase is difficult to understand at first. Recommended to revise it

Line 56: why is “ate” in carbonate in parentheses?

Line 56: The use of co-precipitation to define CaCO3 is incorrect. Co-precipitation implies that 2 or more phases precipitating simultaneously. In CaCO3, it’s just one phase.

Line 58: “Calcium and phosphate also coprecipitate…” phrasing unclear. Do Ca-phosphate co-precipitates with CaCO3 forming a solid solution? Please revise

Line 62: it would be helpful to add some references or some examples of key industrial applications.

Line 64: misuse of word co-precipitation (see comment on Line 56)

Line 76-77: Why is the cyanobacterium D. flosaquae chosen for this study? What is its environmental importance? A brief description of the cyanobacterium itself and reference to any previous studies would be helpful to better understand the context of this manuscript.

Line 87: where was the strain procured from? Is it axenic?

Line 90-93: There is no sterile control for the experiment (i.e. BG11 with no cells). This is a crucial problem as the authors are proposing the D. flosaquae is able to induce CaCO3 precipitation.

Line 151-153: The way the incubations are set up, it’s not possible to discriminate between the effects of Ca concentrations and the effect of citrate, acetate, or chloride anions, on the growth of D. flosaquae. Using perhaps, sodium salts to assess the effect of anions would be a more logical choice.

Line 160: how was pH set?

Line 260-261: I don’t agree with the authors that solely based on the alkalinity levels, pH, and Ca concentrations; it could be asserted that D. flosaquae induces MICP. These datasets don’t eliminate the possibility of the formation of other Ca phases (such as phosphates). A simple saturation index calculation (for e.g using Visual Minteq or PHREEQC) would be easiest to confirm what mineral phases would be over-saturated under the experimental condition. Alternatively, microscopic observation could also confirm with confidence what mineral is actually precipitated.

Line 311: “The high ability of D. flosaquae to shift the pH to alkalinity …” high compared to what? There’s no comparison of D. flosaquae versus other cyanobacteria inducing extracellular carbonates. It would be useful to clarify how authors calculate or assign the “high ability” to D. flosaquae.

Citation: https://doi.org/10.5194/bg-2021-146-RC2
- AC2: 'Reply on RC2', Refat Mostafa, 27 Sep 2021
  
  RC2: 'Comment on bg-2021-146', Anonymous Referee #2, 05 Sep 2021 reply
  This paper investigates the capability of cyanobacterium D. flosaquae to induce MICP. The results of the study rely on the measurement of the growth parameters, and a few solution chemistry parameters (Ca, pH, TA, NH3). Based on these datasets, the authors claimed that the D. flosaquae induces precipitation of CaCO3 (calcite). I find that assertion poorly supported by the presented data and paucity of other (necessary) datasets (saturation state, microscopy/spectroscopy observations, abiotic controls) needed to support the findings claimed in the paper. In light of these issues, I find that study is not up to the standards of the journal and will be more appropriate for another journal. Below are my more elaborate comments:
  
  1) The introduction lacked a clear motivation for the study. Several statements in the introduction were not properly referenced, which is surprising given that MICP is a very well-studied field.
  
  Done
  2) Lack of any abiotic control in the experiment design makes it difficult to infer whether the formation of CaCO3 was due to D. flosaquae or merely due to changes in the solution chemistry.
  
  BG11 medium is daily used in our lab and doesn’t contain carbonate to precipitate calcium.
  3) No information on the saturation state of the solution with respect to carbonate and other minerals is included in the results and discussion.
  
  The process of MICP, which under study at this work is (micro)biological; all other physical and chemical components are equal at all experimental samples i.e. their impact is equal, if any.
  4) The time evolution data on the growth parameters, and dissolved Ca concentrations are not included, which again makes it challenging to fully understand the evolution of the system under different treatments
  Kinetics of the process was not targeted in this work; only the net product or net result was the aim. Once again, a microbiological system is different from the physico-chemical system.
  5) In the current design of the incubations, the effect of Ca concentrations and anions cannot be decoupled. This results in confusing interpretations of the Figures.
  
  It has been actually decoupled. Three different anions are included to show the effect of each anion at a certain calcium concentration and different calcium concentrations to show the effect of calcium.
  6) Lastly, the authors refer to the possible CaCO3 phase as calcite, but yet provide no data that confirms whether the carbonate is calcite.
  We have never referred to our possible CaCO3 phase as calcite since we did not plan to identify it; the word calcite has been mentioned only in the cited literature and reference list.
  
  Specific comments:
  
  Line 41: references missing.
  Added.
  
  Line 52-55: The phrase is difficult to understand at first. Recommended to revise it
  Reformulated
  
  Line 56: why is “ate” in carbonate in parentheses?
  Carbon(ate) refers to carbon or carbonate.
  Now, it is changed to carbon/carbonate in the submitted version.
  
  Line 56: The use of co-precipitation to define CaCO3 is incorrect. Co-precipitation implies that 2 or more phases precipitating simultaneously. In CaCO3, it’s just one phase.
  OK, changed to “precipitation”.
  This is although the two phases, in the course, are calcium and carbonate, which coprecipitate forming calcium carbonate.
  
  Line 58: “Calcium and phosphate also coprecipitate…” phrasing unclear. Do Ca-phosphate co-precipitates with CaCO3 forming a solid solution? Please revise
  
  Yes, but the terminology used in this case is “binding” as follows:
  Furthermore, any calcium carbonate precipitate is a good binder of phosphate (Yanamadala 2005), mentioned in Line 247-248.
  Line 62: it would be helpful to add some references or some examples of key industrial applications.
  Reference and some applications have been added.
  
  Line 64: misuse of word co-precipitation (see comment on Line 56)
  
  OK, changed
  Line 76-77: Why is the cyanobacterium D. flosaquae chosen for this study? What is its environmental importance? A brief description of the cyanobacterium itself and reference to any previous studies would be helpful to better understand the context of this manuscript.
  
  Done
  The hypothesis of this work is to explore whether Dolichospermum flosaquae is able to perform MICP in freshwater lakes.
  Line 87: where was the strain procured from? Is it axenic?
  
  D. flosaquae is a major temperate cyanobacterium; the strain used in this work has been isolated from Stechlinsee lake (IGB Berlin). It was not axenic.
  Line 90-93: There is no sterile control for the experiment (i.e. BG11 with no cells). This is a crucial problem as the authors are proposing the D. flosaquae is able to induce CaCO3 precipitation.
  
  Done
  BG11 medium is daily used in our lab and doesn’t contain carbonate to precipitate calcium.
  Line 151-153: The way the incubations are set up, it’s not possible to discriminate between the effects of Ca concentrations and the effect of citrate, acetate, or chloride anions, on the growth of D. flosaquae. Using perhaps, sodium salts to assess the effect of anions would be a more logical choice.
  Sodium salts do not precipitate.
  
  Line 160: how was pH set?
  The pH of BG11 medium was adjusted to pH 7.0 using acid(HCl)/alkali(NaOH).
  
  Line 260-261: I don’t agree with the authors that solely based on the alkalinity levels, pH, and Ca concentrations; it could be asserted that D. flosaquae induces MICP. These datasets don’t eliminate the possibility of the formation of other Ca phases (such as phosphates). A simple saturation index calculation (for e.g using Visual Minteq or PHREEQC) would be easiest to confirm what mineral phases would be over-saturated under the experimental condition. Alternatively, microscopic observation could also confirm with confidence what mineral is actually precipitated.
  The process of MICP, studied in this work, is (micro)biological; all other physical and chemical components are equal at all experimental samples i.i. their impact is equal.
  
  Line 311: “The high ability of D. flosaquae to shift the pH to alkalinity …” high compared to what? There’s no comparison of D. flosaquae versus other cyanobacteria inducing extracellular carbonates. It would be useful to clarify how authors calculate or assign the “high ability” to D. flosaquae.
  It is high, not higher; no comparison was there. It is high because the pH elevated from 7.0 at the beginning of the experiment to pH 12 at the end of the experiment.
  
  Citation: https://doi.org/10.5194/bg-2021-146-AC2

Refat Abdel-Basset, Elhagag Ahmed Hasssan, and Hans-Peter Grossart

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Short summary

The aim of this work is to explore the ability of the cyanobacterium Dolichospermum flosaquae in microbially-induced calcium carbonate precipitation (MICP). Environmentally, MICP controls the availability of calcium, carbon and phosphorus in freshwater lakes and carbon exchange with the atmosphere. Citrate, at 4 mg/L, induced the highest carbonate alkalinity, the highest calcium consumption, the highest urease activity along with the lowest photosynthetic and respiratory oxygen exchange.


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