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<p>Pure cultures of the freshwater cyanobacterium <i>Microcystis aeruginosa</i> synthesized nitrous oxide (N<span class="inline-formula">₂</span>O) when supplied with nitrite (NO<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M4" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">2</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="9pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="8bf9e215c6faa7b6532e252fa836d722"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-20-687-2023-ie00001.svg" width="9pt" height="16pt" src="bg-20-687-2023-ie00001.png"/></svg:svg></span></span>) in darkness (198.9 nmol g-DW<span class="inline-formula">⁻¹</span> h<span class="inline-formula">⁻¹</span> after 24 h) and illumination (163.1 nmol g-DW<span class="inline-formula">⁻¹</span> h<span class="inline-formula">⁻¹</span> after 24 h), whereas N<span class="inline-formula">₂</span>O production was negligible in abiotic controls supplied with NO<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M10" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">2</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="9pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="e2f8c3d8f388ffa127fb090bff1821f2"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-20-687-2023-ie00002.svg" width="9pt" height="16pt" src="bg-20-687-2023-ie00002.png"/></svg:svg></span></span> and in cultures deprived of exogenous nitrogen. N<span class="inline-formula">₂</span>O production was also positively correlated to the initial NO<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M12" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">2</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="9pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="71c4f283853e1c63b082e22081694bf1"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-20-687-2023-ie00003.svg" width="9pt" height="16pt" src="bg-20-687-2023-ie00003.png"/></svg:svg></span></span> and <i>M. aeruginosa</i> concentrations but was low to negligible when nitrate (NO<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M13" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">3</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="9pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="a192f22c747584054322d55d69a940ca"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-20-687-2023-ie00004.svg" width="9pt" height="16pt" src="bg-20-687-2023-ie00004.png"/></svg:svg></span></span>) and ammonium (NH<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M14" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">4</mn><mo>+</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="8pt" height="15pt" class="svg-formula" dspmath="mathimg" md5hash="d0f05de3ef9fdb7d2948354df3a21cd8"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-20-687-2023-ie00005.svg" width="8pt" height="15pt" src="bg-20-687-2023-ie00005.png"/></svg:svg></span></span>) were supplied as the sole exogenous N source instead of NO<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M15" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">2</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="9pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="605864571c3dcb0b6e3cb32dc4ee1961"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-20-687-2023-ie00006.svg" width="9pt" height="16pt" src="bg-20-687-2023-ie00006.png"/></svg:svg></span></span>. A protein database search revealed that <i>M. aeruginosa</i> possesses protein homologous to eukaryotic microalgae enzymes known to catalyze the successive reduction of NO<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M16" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">2</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="9pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="59aa938310f11367d407e443cafbbbd3"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-20-687-2023-ie00007.svg" width="9pt" height="16pt" src="bg-20-687-2023-ie00007.png"/></svg:svg></span></span> into nitric oxide (NO) and N<span class="inline-formula">₂</span>O. Our laboratory study is the first demonstration that <i>M. aeruginosa</i> possesses the ability to synthesize N<span class="inline-formula">₂</span>O. As <i>M. aeruginosa</i> is a bloom-forming cyanobacterium found globally, further research (including field monitoring) is now needed to establish the significance of N<span class="inline-formula">₂</span>O synthesis by <i>M. aeruginosa</i> under relevant conditions (especially in terms of N supply). Further work is also needed to confirm the biochemical pathway and potential function of this synthesis.</p>