|After reviewing the authors’ responses to the reviewers’ comments on the original submission and the revised manuscript, the authors addressed some of the reviewers’ comments and suggestions adequately but there are several remaining issues which must be addressed. Most of these issues will not be listed in this review because there are a few items on which the manuscript’s hypotheses and data interpretations are based that cannot be supported. These major or core issues are the input data and constraints for the geochemical modeling using PHREEQC and the lack of data showing definitive lysis of the cyanobacterial cells by cyanophage. Based upon the statements in the Abstract and Discussion the reliability of geochemical data and active lysing of cyanobacterial cells by cyanophage are the two most critical aspects of this study and on which the authors base their conclusions. Comments on the issues associated with these concepts are listed below:|
1. All experiments were conducted in a complex medium (i.e., f/2 medium). Therefore, all data from the geochemical modeling using PHREEQC and seawater carbonate chemistry using CO2calc are suspect when extending the data to natural seawater or freshwater. A reviewer’s comment on the original submission requested the actual PHREEQC code and variable entries and respective concentrations (or activities) (i.e., the basis) be included in the revised manuscript. The code and basis entries were not included in the revised manuscript. This is critically important because the resulting data and interpretations are dependent upon what was entered into PHREEQC and the options the authors used to constrain the program’s outputs. For example, the relevance and reliability of PHREEQC outputs are totally dependent upon how the components of f/2, as listed in Table 1, were entered into PHREEQC. The saturation indices (SI) for the calcium carbonate polymorphs listed in Table 2 and Table S1 are not relevant to natural freshwater or seawater, only to f/2 medium. With regard to using the CO2calc, the use of this program is not appropriate for this study because it is optimized for calculations of equilibrium carbonate chemistry variables in seawater and, to a lesser degree, freshwater.
2. Cyanobacteria vs cyanophage data (Figure 2A): One of the recurring concepts in the original and revised manuscript is the role cyanophage play in changing the carbonate chemistry in a culture of cyanobacteria. By lysing the host cyanobacterial host cell, the infecting cyanophage not only release the intracellular contents of the host cell (e.g., HCO3-) but also relatively high numbers of progeny cyanophage. As this cycle of (1) host cell infection, (2) cyanophage controlling host cell synthesis processes, (3) synthesis of infectious progeny cyanophage and (4) host cell lysis and release of more cyanophage progresses through a contained culture as described in this study, there should be a point in the cyanobacterial growth curve where the cell numbers significantly decrease with a concomitant increase in the number of cyanophage. The graphed data in Figure 2A does not show these trends in the respective counts. Therefore, the contribution of cyanophage lysis of host cyanobacterial cells to the changes in carbonate chemistry variables cannot be supported as Figure 2A shows no significant reduction in cyanobacterial counts (i.e., no significant viral lysis) over a 14 day period of incubation.
3. Table S1: For natural freshwater and seawater systems the values listed for pH, pCO2, CO3 and HCO3 are unrealistic when compared to data from the same variables from natural seawater. For example, the typical range for pCO2 should be between 140-770 micro-atm. The large deviations of these data sets from what is typically measured may be the result of improper geochemical modelling with PHREEQC. (See Comment #1)
The authors have not described a laboratory system from which the data can be reliably extended to natural seawater or freshwater systems as they propose. The experimental design is more applicable to an industrial setting where cyanobacteria are used to scrub CO2 from product streams and/or precipitate carbonate-based minerals on the surfaces of the cells and/or viruses in those product streams or perhaps a solid phase reactor.