We thank Referee#1 for his/her review.

We fully agree our results cannot be used to suggest Microcystis aeruginosa synthesise nitrous oxide (N₂O) in natural environments, but we also argue we cannot infer that they do not: Significant N₂O emissions were indeed reported from outdoor cultures of C. vulgaris fed nitrate (NO₃^-, Guieysse et al., 2013; Plouviez et al., 2017), despite this alga also producing much more N₂O when fed nitrite (NO₂^-, Guieysse et al., 2013). We believe this was caused by NO₂^- intracellular accumulation under varying light, as this condition is known to have different impacts on the rate of NO₃^- reduction into NO₂^- by NR and the rate of NO₂^- reduction into NH₄⁺ by NiR (Plouviez et al., 2017). We also respectfully note that N₂O emissions under NO₃^- supply were low, but not negligible. We will clarify this potential for emissions under NO₃^- supply in the final manuscript.

Therefore, without evidence from field measurements, we cannot conclude that M. aeruginosa is or is not a major N₂O producer in lakes. As mentioned by us (Abstract, Section 2.4 and Conclusions) and the referee, further research is needed.

With regards to NirK, M. aeruginosa possess a nitrate reductase (NiR) (Chen et al., 2009; Chen et al., 2015) but no homologs of Chlamydomonas reinhardtii NirK copper-containing nitrite reductase was found. Further research is needed to determine if NiR in M. aeruginosa can also catalyse the reduction of NO₂^- into NO.

References:

Chen, W. and Liu, H.: Intracellular nitrite accumulation: The cause of growth inhibition of Microcystis aeruginosa exposure to high nitrite level, Phycol Res, 63, 197-201, 10.1111/pre.12090, 2015.

Chen, W., Zhang, Q., and Dai, S.: Effects of nitrate on intracellular nitrite and growth of Microcystis aeruginosa, J Appl Phycol, 21, 701-706, 10.1007/s10811-009-9405-1, 2009.

Guieysse, B., Plouviez, M., Coilhac, M., and Cazali, L.: Nitrous Oxide (N2O) production in axenic Chlorella vulgaris microalgae cultures: evidence, putative pathways, and potential environmental impacts, Biogeosciences, 10, 6737-6746, 10.5194/bg-10-6737-2013, 2013.

Plouviez, M., Shilton, A., Packer, M. A., and Guieysse, B.: N₂O emissions during microalgae outdoor cultivation in 50 L column photobioreactors, Algal Res, 26, 348-353, 10.1016/j.algal.2017.08.008, 2017.

We thank Referee#2 for his/her review.

We will carefully check the manuscript before final submission to improve the readability. For instance:

Li 41-42:” As can be seen in Fig. 1, N₂O was only significant in cultures supplied NO₂^- as there was no significant production in the absence of the cyanobacterium (abiotic control) or the absence of NO₂^- (negative control).” Will be modified to: ‘As can be seen in Fig. 1, N₂O production was only recorded in cultures supplied NO₂^- as there was no significant production in the absence of the cyanobacterium (abiotic control) or the absence of NO₂^- (negative control).”

Li 76-77:” Intracellular NO₂^- was not possible when NH₄⁺ was supplied as the sole exogenous N source, explaining the absence of N₂O production (p-value = 0.91, two samples t-test when compared with the negative controls)” Will be modified to: ‘Intracellular NO₂^- production and accumulation is not expected when cells assimilate NH₄⁺ (Plouviez et al., 2019), explaining the absence of N₂O production in the flasks supplied NH₄⁺ as sole exogenous N source (p-value = 0.91, two samples t-test when compared with the negative controls)’.

Regarding the major comment, we respectfully note that Figure 2 shows N₂O production normalized per g-DW^-1. Because the production rates (slope of the near-linear production curves expressed in nmole of N₂O produced per hour and per gram of cyanobacteria initially present in the flasks) are similar for the three biomass concentrations tested (Table 1), our results showed that there is a relation between biomass concentration (g-DW·L^-1) and N₂O production (nmol N₂O·hr^-1). Our statement is therefore correct. To improve clarity, we will include “normalized N₂O production” on the y axis and in the caption of Figure 2, and we will include the data presented below as supplementary information.

Table 1: N₂O production rates (nmol N₂O·g DW^-1·h^-1) recorded from the linear regressions performed for each M. aeruginosa biomass concentrations (0.1, 0.2 and 0.4 g-DW·L^-1)

About the minor comment, we will add Li 70: "However, O₂ production during photosynthesis could also influence N₂O synthesis. Burlacot et al. (2020) indeed reported that one of the enzymes involved in NO reduction to N₂O (Flavodiiron, as discussed in the next section) can also catalyse the reduction of O₂ into H₂O. Because of this dual activity and the reactivity of NO with O₂, N₂O production could be sensitive to O₂. Further research is therefore needed to understand if O₂ influence N₂O production by competitive NO conversion to products such as nitrogen oxides and peroxynitrite, or/and by competitive O₂ reduction into H₂O instead of its reduction to N₂O by the enzymes with nitric reductase ability.

References:

Burlacot, A., Richaud, P., Gosset, A., Li-Beisson, Y., and Peltier, G.: Algal photosynthesis converts nitric oxide into nitrous oxide, Proc Natl Acad Sci USA, 117, 2704-2709, 10.1073/pnas.1915276117, 2020.

Plouviez, M., Shilton, A., Packer, M. A., and Guieysse, B.: Nitrous oxide emissions from microalgae: potential pathways and significance, J Appl Phycol, 31, 1-8, 10.1007/s10811-018-1531-1, 2019.

We thank Referee#1 for his/her review and helpful recommendations. As can be seen in the responses to the specific comments below, we have considered all the comments and we have specifically expanded Section 2.4.

As can be seen in our response to Referee#1 we cannot conclude that M. aeruginosa (or other species) is or is not a major N₂O producer in lakes and other aquatic environments without evidence from field measurements. Indeed, high nitrite (NO₂^-) concentrations are rare in natural and engineered ecosystems environments, which would suggest insignificant microalgal N₂O production in most context. But our experience is that significant N₂O emissions can still be observed under very low exogenous NO₂^- concentration, potentially due to the intracellular accumulation of this metabolite of the nitrate assimilation pathway (Plouviez et al., 2017a,b). The aim of this work was/is indeed to raise awareness and to trigger further research in the field. The work from Weathers and Niedzielski (1986) and ours suggest that Nostoc spp., Aphanocapsa (PCC 6308), Aphanocapsa (PCC 6714) and M. aeruginosa have the ability to synthesize N₂O. Consequently, other cyanobacteria species may also have this ability. Considering the wide distribution of cyanobacteria in the environment (including in some wastewater treatment systems, Romanis et al., 2021), extensive monitoring (i.e. long-term with wide spatial coverage and high sampling frequency) of several types of microalgae-rich environments are required (see the expanded Section 2.4).

In addition, I have a few specific comments listed below:

Lines 58/59 and Figure 3 – this is about the only mention (and use) of enzyme kinetics and characteristics. I think that in Biogeosciences, this would either need some more information, or it may be moved to the supplementary material to make room for discussion of environmental consequences. You do not really discuss the kinetics anyway, and I think a supplement would not harm the overall scope of the manuscript. In Figure 3, please indicate vmax and Km.

The figure will be moved to the supplementary material, and we will include Vmax and Km on the Figure.

Typos – please change nmole to nmol (Fig 1), and check for typos, such as numerous brackets opened and not closed, e.g. lines 91, 92

We will change the y axis label (Fig 1) and we will carefully check the manuscript before final submission for typos and missing punctuation.

Line 76 – “intracellular No2- was not possible…” Odd wording. Additionally, I am not sure whether it really is “not possible”, given that cyanobacteria may always come up with O2 from somewhere. Please rephrase.

We agree that our initial sentence was unclear and as can be seen in our response to Referee#2 we will rephrase to: ‘Intracellular NO₂^- production and accumulation is not expected when cells assimilate NH₄⁺ (Plouviez et al., 2019b), explaining the absence of N₂O production in the flasks supplied NH₄⁺ as sole exogenous N source (p-value = 0.91, two samples t-test when compared with the negative controls)’

Lines  77 – 81 – I am not sure what the authors want to say here, why is the regulation with regards to light relevant? Especially given that there is so little difference in N2O production? I cannot really see what the (environmental) applications would be.

Our results showed that N₂O synthesis was lower in light than in darkness as previously reported for other species in the laboratory (Guieysse et al., 2013, Plouviez et al., 2017b). However, N₂O production was positively correlated with light supply in C. vulgaris grown outdoors and supplied NO₃^- (Plouviez et al., 2017a). Plouviez et al., 2017a suggested that NR activation by light generated intracellular NO₂^- from NO₃^- reduction (as part of the normal nitrate assimilation pathway) and that a small amount of this intracellular NO₂^- was converted to N₂O (see reply to Referee#1), the main fraction being ‘normally’ further reduced and assimilated into proteins and other biomolecules. The gene encoding NR in M. aeruginosa has the same function than in C. vulgaris i.e. convert NO₃^- to NO₂^-. Because NR activity is influenced by light and the availabilities of NO₃^- and NO₂^- in M. aeruginosa (Chen et al., 2009; Ohashi et al., 2011; Chen and Liu, 2015), it is possible that light influences NR activity (i.e. the rate of intracellular NO₂^- production) and, thereby, the rate of N₂O synthesis under outdoor conditions. In addition, as suggested by Referee#2, light might also indirectly influence N₂O synthesis by influencing the activity of FLVs via O₂ synthesis during photosynthesis (as can be seen in the response to Referee#2, this will be discussed in the new version of the manuscript).

Lines 94/95 – this is your result, correct? The mix of results and discussion section makes this sometimes hard to distinguish, please clarify.

This is indeed our results. We will re-phrase as follow:’ Interestingly, NO₂^- reduction into NO by nitrate reductase (narB) has been demonstrated in M. aeruginosa (Tang et al., 2011; Song et al., 2017) and here we found that M. aeruginosa possesses homologs of the CYP55, FLVs, and HCPs found in C. reinhardtii (Table. 2).’

Line 107 – as a biogeochemist, the allelopathic response is unclear to me. Please add a short explanation/definition.

To improve clarity, we will modify the sentence to: “Interestingly, NO stimulates the production of secondary metabolites (e.g. linoleic acid) by M. aeruginosa that inhibit the growth of competitors (Song et al., 2017). NO also promotes the growth of this cyanobacteria (Tang et al., 2011).”

Lines 108 – 110 – is this hypothesis yours, or can you back it up with references? The reference to further research should be deleted here, this is rather suitable for conclusions.

This hypothesis is ours and we will remove “Further research is needed.”

Line 113 – which groups of microalgae have been found to synthesize N2O? Please specify.

In section 2.4 we already indicated that: “the N₂O synthesis rates reported during our study are in the same order of magnitude as the rate previously reported for members of the green microalgae, cyanobacteria, and diatoms (Bauer et al., 2016; Plouviez et al., 2019b).”

However, we will modify section 2.4 (see below) and will include the following sentence:

‘Microalgae species from at least 3 divisions (Chlorophyta, Bacillariophyta, Cyanobacteria) have the ability to synthesise NO (Kim et al., 2008; Kumar et al., 2015; Plouviez et al., 2017b; Tang et al., 2011) and/or N₂O (Weathers, 1984; Weathers and Niedzielski, 1986; Guieysse et al., 2013; Kamp et al., 2013; Plouviez et al., 2017a, b, this study).’

Generally, I think this section 2.4 might be expanded, please see my general comments above.

This section will be expanded:

Microalgae species from at least 3 divisions (Bacillariophyta, Chlorophyta, Cyanobacteria) have the ability to synthesise NO (Kim et al., 2008; Kumar et al., 2015; Plouviez et al., 2017b; Tang et al., 2011) and/or N₂O (Weathers, 1984; Weathers and Niedzielski, 1986; Guieysse et al., 2013; Kamp et al., 2013; Plouviez et al., 2017a, b, this study). All these observations suggest that the ability to synthesise N₂O is widely distributed among microalgae. Critically, N₂O emissions from aquatic environments where microalgae abound, such as oceans, lakes and engineered cultivation systems, have been repeatedly reported (Bauer et al., 2016; Plouviez et al., 2019b; Zhang et al., 2022) even under very low exogenous NO₂^- concentrations (Plouviez et al., 2019b). These emissions can be explained by intracellular NO₂^- production during reductive nitrate assimilation (Plouviez et al., 2017a, b, 2019b) under conditions when excess NO₂^- production (Bristow et al., 2015; French et al., 1983; Mortonson and Brooks, 1980; Schaefer and Hollibaugh, 2018) could support N₂O synthesis. Based on the data available, DelSontro et al. (2018) and Plouviez and Guieysse, (2020) estimated that global N₂O emissions from eutrophic lakes alone could represent 110 to 450 kt N-N₂O·yr^-1, which represent 14-56% of the natural and anthropogenic N₂O emissions reported from inland and coastal waters (Tian et al., 2020). Importantly, Delsontro et al. (2018) predicted that N₂O emissions from lakes and impoundments would increase with lake size and chlorophyll a concentration. The N₂O synthesis rates reported during our study are in the same order of magnitude as the rate previously reported for members of the Bacillariophyta, Chlorophyta and Cyanobacteria (Bauer et al., 2016; Plouviez et al., 2019b). Because microalgae are ubiquitous in the environment and often associated with anthropogenic pollution, the potential significance of microalgal N₂O biosynthesis should be recognised. Our findings support past predictions of the global relevance of photosynthetic N₂O emissions from eutrophic aquatic bodies as Microcystis is globally found and often the dominant genus in these ecosystems (Qian et al., 2010; Kataoka et al., 2020; Zhou et al., 2020). Further research is now needed to quantify N₂O emissions from eutrophic aquatic ecosystems where M. aeruginosa abounds. This is especially timely considering that the frequency and geographic distribution of harmful algae blooms have increased due to anthropogenic activities (Paerl et al., 2018; Kataoka et al., 2020). In addition, algae blooms can lead to the decrease of O₂ in oceans, coastal waters and lakes (Jenny et al., 2015; Rabalais and Turner, 2019), a condition that can increase the accumulation of NO₂^- in aquatic ecosystems (Schaefer and Hollibaugh, 2018; Bristow et al., 2015). Because microalgal N₂O synthesis is rapid and influenced by factors such as the cell biology (Plouviez et al., 2019b) and, as observed during our study, the type and concentration of the nitrogen source microalgae receive, extensive monitoring (i.e. long-term with wide spatial coverage and high sampling frequency) of several types of microalgae-rich environments are required (e.g. hypoxic waters).

Conclusions section – as it is, this section is a bit weak and merely a repetition of the abstract. This would be another good location to discuss environmental consequences.

We will modify the conclusions as:

We herein present the first demonstration that M. aeruginosa synthesizes N₂O. M. aeruginosa synthesized N₂O when supplied with NO₂^- in darkness (198.9 nmol·g-DW^-1·h^-1 after 24 hours) and illumination (163.1 nmol∙g-DW^-1∙h^-1 after 24 hours), and this production was positively correlated to the initial NO₂^- and M. aeruginosa concentrations. A protein database search also revealed M. aeruginosa possesses proteins homologues to eukaryotic microalgae known to catalyse the successive reduction of NO₂^- into NO and N₂O. Further studies are needed to confirm the genes/proteins involved as a better understanding of the biochemical pathway involved during microalgal N₂O synthesis is critical to efficiently monitor (i.e. identify the source) and mitigate N₂O emissions.

Our study is another evidence of the ability of photosynthetic microorganisms, especially cyanobacteria, to synthesize N₂O. Preliminary estimation showed that N₂O emissions from eutrophic lakes alone could represent 110 to 450 kt N-N₂O·yr^-1, which represent 14-56% of the natural and anthropogenic N₂O emissions reported from inland and coastal waters. However, how much microalgae contribute to these emissions is currently unknown. As M. aeruginosa is globally distributed, further research (including field monitoring with wide spatial coverage, high sampling frequency and water type) is now needed to evaluate the significance of N₂O synthesis by these cyanobacteria under relevant conditions (especially in terms of N supply).

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Chen, W., Zhang, Q., and Dai, S.: Effects of nitrate on intracellular nitrite and growth of Microcystis aeruginosa, J Appl Phycol, 21, 701-706, 10.1007/s10811-009-9405-1, 2009.

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Plouviez, M., Shilton, A., Packer, M. A., and Guieysse, B.: Nitrous oxide emissions from microalgae: potential pathways and significance, J Appl Phycol, 31, 1-8, 10.1007/s10811-018-1531-1, 2019b.

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