Candidatus Scalindua, a Biological Solution to Treat Saline Recirculating Aquaculture System Wastewater

Round 1

Reviewer 1 Report

In current study author used marine anammox bacterium Candidatus Scalindua, for the removal of NH₄⁺ and NO₂^- from saline recirculating aquaculture system wastewater. Similar study had been reported by author in Processes 2021, 9, 1183. Although this study contains some new findings but will not catch significant readership.   

Compare to your previous study salinity is double in this study with higher removal of NH₄⁺ and NO₂. While the activity and growth of marine anammox bacteria are generally considered to be affected by the presence of salinity.

Were figure 1 and 2 opt from reference [27]? Reference in caption gives an impression that figures are reproduce from other study.

Concentrations for trace elements are not mentioned. Why?

What is difference between % removal and loading rate removal? How did you calculate % removal?

In this sentence “Therefore, we can conclude that the required TE must have been present in sufficient quantities in the marine RAS WW” sufficient amount means higher or lower concentration.

Explanation for figure 4 is missing inside the text.

Effect of pH. COD and PO4 on removal efficiency are not discussed. How could you maintain their concentrations throughout the experiment time?

Author Response

We would like to thank the reviewer for their time and effort with reviewing our manuscript. We have made the changes to improve our manuscript accordingly. The changes in the text are visible through ‘track changes’ and answered the reviewer’s questions/comments in bold below. We have added two figures (Figure 1 and 2) and one table (Table 1) and added some new references according to the reviewer’s suggestions.

In addition, we have had a thorough check of the English by a native English speaker (Geoffrey Ziccardi, our third author of the manuscript) and corrected the manuscript when necessary.

In current study author used marine anammox bacterium Candidatus Scalindua, for the removal of NH₄⁺ and NO₂^- from saline recirculating aquaculture system wastewater. Similar study had been reported by author in Processes 2021, 9, 1183. Although this study contains some new findings but will not catch significant readership.

In our study from 2021 (Roques, Micolucci et al., 2021), we have conducted a first pilot experiment in Japan, with RAS wastewater from Sweden, as we did not have enough biomass in our new anammox reactor in Sweden at that time. We only had a limited amount of wastewater to conduct our pilot experiment (20 L), we could only expose the anammox for very short period to wastewater in the presence/absence of trace element mix. We saw that the anammox activity, but not the bacterial community, was negatively affected by this new substrate. We concluded that Ca. Scalindua could be a good candidate, but we needed to investigate more in detail what led to this reduction/how we could improve this setup.

In the current study, we had unlimited access to RAS wastewater, as the experiment was carried out in Sweden, where we had a fully operational anammox reactor and a pilot RAS at our disposal. We then hypothesized that a slow exposure to increasing concentrations of RAS wastewater could lead to a better acclimation of the bacteria. This is was we have observed with removal rates of over 90% throughout the experimental phases 2 and 3.

In addition, we have also demonstrated that trace element mix, originally developed for freshwater anammox by van de Graaf et al., 1996 [34] and used as standard in all the anammox culture worldwide, are in fact not necessary when cultivating a marine strain of anammox, since those trace elements are already present in the saltwater. We believe this is a finding of significant interest for researchers and industrial using marine strains of anammox. This could both save money and be better for the environment, as some chemicals used to make this solution are expensive and hazardous (i.e NaSeO₄. 10H₂O; H₃BO₃).     

Compare to your previous study salinity is double in this study with higher removal of NH₄⁺ and NO₂. While the activity and growth of marine anammox bacteria are generally considered to be affected by the presence of salinity.

In our first study, we used water from the former RAS of our partner Smogenlax, who were cultivating rainbow trout in brackish water (14.5 ppt). In the anammox feed using this RAS WW, we have corrected the salinity by adding artificial sea salt in order to match the salinity of the artificial substrate the anammox reactor was fed with in order not to fiddle with too many environmental parameters and reach a salinity of 25.2 ppt. In the current study, we had access to a full-strength salinity RAS (29 ppt), hosting salt-acclimated rainbow trout and Atlantic wolffish. The salinity of the WW feed between these two studies are therefore very similar.

Awata et al. (2013) studied the impact of salinity on the N₂ production in batch experiment. The anammox activity is indeed affected by salinity, as the reviewer mention, but the authors concluded that this anammox strain is halophilic, as it had relatively high and stable activity from salinity between 1.5 and 4% (15-40 ppt) (see figure A).

Were figure 1 and 2 opt from reference [27]? Reference in caption gives an impression that figures are reproduce from other study.

No, these figures are from our results. In the caption of figure 1, we indicated the composition of the artificial wastewater (designed according to [34] (reference [27] in the first version of the manuscript)). In the caption of figure 2, we indicated that the trace element mix supplied during the phases 1 and 2 were the standard one developed by [34]. As we already mention the different feed composition and references it in the text, we have removed it from the legend for more clarity. We are sorry for the confusion.

Concentrations for trace elements are not mentioned. Why?

We have added these information in the Table 1 (L127-128). 

What is difference between % removal and loading rate removal? How did you calculate % removal?

We are sorry, there was a typo in the legend, in the second graph, it should be written ‘Loading and removal rates’, and not ‘Loading removal rates’, as we present both rates on the figure. It has been corrected. These formula for calculating the removal rates and the HRT were added to the manuscript (L205-213).

In this sentence “Therefore, we can conclude that the required TE must have been present in sufficient quantities in the marine RAS WW” sufficient amount means higher or lower concentration.

Here, we meant that there were enough trace elements for the normal functioning of the anammox bacteria. We have changed ‘sufficient’ by ‘appropriate’. By this we want to stretch that the trace elements all present in good quantity, it could be just what was needed, it could be higher than what is necessary.

Explanation for figure 4 is missing inside the text.

We are sorry about that; we would like to thank the reviewer for pointing it out. We have added a reference to the figure 6 (figure 4 in the previous version of the manuscript) in the text L296-298. ‘This stable population of Ca. Scalindua throughout all the different experimental phases was also supported by FISH observations (Figure 6).’

Effect of pH. COD and PO4 on removal efficiency are not discussed. How could you maintain their concentrations throughout the experiment time?

Regarding the pH:

The pH was maintained to ca. 7.0 throughout the experiment with the addition of few drops of H₂SO₄, as it was done by [34], we have added this information in the text (L189-192). Furthermore, Awata et al. (2013) also have demonstrated that the optimum pH for the functioning if this anammox strain was between 6.5 and 8.0. The pH of our RAS wastewater (and the ones from marine RAS in general is usually comprised between 7.0 and 8.0, which is matching the optimal range for this bacterium (see figure B).

We would like to also add that we changed ‘PO₄^3-’ in table 1 and in the text to ‘Total P’ (Total phosphorous), since we realized that there was a mistake in the original manuscript, our kit measured Total P, not PO₄^3-, we are sorry for the confusion.

Regarding the phosphorus

In this study, we have added 6.2 mg-P L^-1 of phosphorus as the form of KH₂PO₄ in the synthetic wastewater (Phase 1) as previously reported by van de Graaf et al. [34]. During Phase 3, the phosphorus concentration in the influent (100% RAS wastewater) decreased to 2.6 mg-P L^-1 (0.08 mmol L^-1). The low concentration of phosphorus did not affect the anammox activity. We have previously checked this by batch experiments under different phosphorus concentrations (see figure C, unpublished data).

Figure C. The impact of phosphorous on Total nitrogen removal efficiency of Ca. Scalindua

The removal of phosphorus does not occur during the experiments. Once we have checked phosphorus concentrations in the influent and effluent; we have measured very similar concentrations throughout the experiment. This suggests that the uptake of phosphorus into cells is a small part and does not affect the concentration.

Regarding the COD:

It is possible that COD can be removed by the coexisting heterotrophic bacteria (Figure 6). In this study, we did not focus on the COD removal during the experiments because the COD concentrations is not as high compared with the other industrial wastewaters. We have previously worked with (Here, we have checked the COD (and TSS) concentrations in the RAS wastewater used in this study from the wastewater before the experiments.

References:

Awata, Takanori, et al. "Physiological characterization of an anaerobic ammonium-oxidizing bacterium belonging to the “Candidatus Scalindua” group." Applied and environmental microbiology 79.13 (2013): 4145-4148.

https://doi.org/10.1128/AEM.00056-13

Roques, Jonathan AC, et al. "Effects of recirculating aquaculture system wastewater on anammox performance and community structure." Processes 9.7 (2021): 1183.

https://doi.org/10.3390/pr9071183

Van de Graaf, Astrid A., et al. "Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor." Microbiology 142.8 (1996): 2187-2196.

https://doi.org/10.1099/13500872-142-8-2187

Author Response File: Author Response.docx

Reviewer 2 Report

The current manuscript the authors investigated the potential of Ca. Scal- indua to treat marine RAS WW in a 3 phases experiment. In the first phase (control, 83 26 days), Ca. Scalindua was fed a synthetic feed, enriched in NH4+, NO2- and trace element (TE) mix. Removal rates of 98.9% and 99.6% for NH4+ and NO2- respectively were 28 achieved. In the second phase (116 days), they slowly exposed Ca. Scalindua to nitrogen- enriched RAS WW by gradual increases over a period of about 80 days. In the last phase (79 days) they investigated the needs of TE supplementation for the Ca. Scalindua after they were fully acclimated to 100% RAS WW. Their results show that the gradual exposure of Ca. Scalindua resulted in a successful acclimation to 100% RAS WW, with maintained high removal rates of both NH4+ and NO2- throughout the experiment. The manuscript is well written and the topic is interesting. It can be reconsidered after addressing the following concerns:

1.       Line 28 abstract: “, respectively” is correct.

2.       How major the topic can be respect to the human health (in the first paragraph of the introduction) and a general statement on the importance of wastewater treatment and removal technologies making profit from: (https://link.springer.com/article/10.1134/S0036024417050028, https://link.springer.com/article/10.1007/s11356-012-1333-y , https://link.springer.com/article/10.1007/s11356-012-1333-y )

3.       How about adding a schematic diagram of the reactor?

4.       Mechanism of action through the removal process should be comprehensively discussed (would be great if make use of a Scheme).

5.       Please make sure your conclusions' section underscores the scientific value-added of your paper, and/or the applicability of your results.

6.       What would be the consequences, in the real world, in changing the observed situation or improve it.

Author Response

Response to the reviewers

We would like to thank the reviewer for their time and effort with reviewing our manuscript. We have made the changes to improve our manuscript accordingly. The changes in the text are visible through ‘track changes’ and answered the reviewer’s questions/comments in bold below. We have added two figures (Figure 1 and 2) and one table (Table 1) and added some new references according to the reviewer’s suggestions.

In addition, we have had a thorough check of the English by a native English speaker (Geoffrey Ziccardi, our third author of the manuscript) and corrected the manuscript when necessary.

The current manuscript the authors investigated the potential of Ca. Scal- indua to treat marine RAS WW in a 3 phases experiment. In the first phase (control, 83 days), Ca. Scalindua was fed a synthetic feed, enriched in NH4+, NO2- and trace element (TE) mix. Removal rates of 98.9% and 99.6% for NH4+ and NO2- respectively were achieved. In the second phase (116 days), they slowly exposed Ca. Scalindua to nitrogen- enriched RAS WW by gradual increases over a period of about 80 days. In the last phase (79 days) they investigated the needs of TE supplementation for the Ca. Scalindua after they were fully acclimated to 100% RAS WW. Their results show that the gradual exposure of Ca. Scalindua resulted in a successful acclimation to 100% RAS WW, with maintained high removal rates of both NH4+ and NO2- throughout the experiment. The manuscript is well written and the topic is interesting. It can be reconsidered after addressing the following concerns:

1. Line 28 abstract: “, respectively” is correct.

Thank you for pointing that out, we have added the coma before ‘respectively’

2. How major the topic can be respect to the human health (in the first paragraph of the introduction) and a general statement on the importance of wastewater treatment and removal technologies making profit from: (https://link.springer.com/article/10.1134/S0036024417050028, https://link.springer.com/article/10.1007/s11356-012-1333-y , https://link.springer.com/article/10.1007/s11356-012-1333-y )

We have added a changed the first paragraph of our manuscript, focusing on the need for more healthy and nutritious food for human, and explaining how aquaculture is important to provide healthy and nutritious food to a growing world population. We have also mentioned the importance to develop of wastewater treatments in aquaculture systems and quoted Padervand and Gholami (2013).

3. How about adding a schematic diagram of the reactor?

Thank you for the suggestion. We have added it in the manuscript, as Figure 2 (L169-171).

3. Mechanism of action through the removal process should be comprehensively discussed (would be great if make use of a Scheme).

We have added a scheme process describing the anammox process in the introduction, described in detail L96-97 and added the stoichiometry of this process L87-89.

4. Please make sure your conclusions' section underscores the scientific value-added of your paper, and/or the applicability of your results.

We have rewritten our conclusion part to lift up the added value of our scientific results (Ca. Scalindua can be slowly acclimated to treat RAS WW under these controlled conditions, and this marine strain doesn’t need the addition of TE supplementation for good functioning) and we also have rewritten our perspective. L404-432.

5. What would be the consequences, in the real world, in changing the observed situation or improve it.

In this project, aiming at implementing alternative WW treatment technologies, we are progressing step by step. The first step (Roques, Micolucci et al., 2021) showed that this anammox strain could be a good candidate but its activity was somehow affected by the quick change to RAS WW. In this second step, we wanted to see 1) if a slow adaptation to RAS WW, without changing the other optimal conditions for this organism (high ammonium and nitrite, anaerobic conditions) and 2) if TE supplementation was necessary for the normal functioning of this strain.

With this experiment, we have shown that this anammox strain could be slowly adapted to enriched RAS WW in laboratory conditions, i.e. with ammonium and nitrite supplementation and in a strictly anaerobic environment.

The next (and final) step before we can validate the use of this method as alternative RAS WW treatment technology will be to investigate it in ‘real’ commercial scale condition, not in the laboratory (i.e. with lower ammonium concentration, with the presence of high nitrate and the presence of some oxygen. We are currently working on implementing an anammox reactor in a pilot scale RAS. We have changed the perspective part of our conclusion mentioning these.

References:

Padervand, Mohsen, and Mohammad Reza Gholami. "Removal of toxic heavy metal ions from waste water by functionalized magnetic core–zeolitic shell nanocomposites as adsorbents." Environmental Science and Pollution Research 20 (2013): 3900-3909.
https://doi.org/10.1007/s11356-012-1333-y

 Roques, Jonathan AC, et al. "Effects of recirculating aquaculture system wastewater on anammox performance and community structure." Processes 9.7 (2021): 1183.

https://doi.org/10.3390/pr9071183

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Authors have revised the mauscript well.

Accepted.