Respiration rates of marine prokaryotes and implications for the in vivo INT method

<p>The balance between the uptake of CO<span class="inline-formula">₂</span> by phytoplankton photosynthesis and the production of CO<span class="inline-formula">₂</span> from prokaryoplankton, zooplankton and phytoplankton respiration controls how much carbon can be stored in the ocean and hence how much remains in the atmosphere to affect climate. Yet, despite its crucial role, knowledge on the respiration of plankton groups is severely limited because traditional methods cannot differentiate the respiration of constituent groups within the plankton community. The reduction of the iodonitrotetrazolium salt (INT) to formazan, which when converted to oxygen consumption (O<span class="inline-formula">₂</span>C) using an appropriate conversion equation, provides a proxy for both total and size fractionated plankton respiration. However, the method has not been thoroughly tested with prokaryoplankton. Here we present respiration rates, as O<span class="inline-formula">₂</span>C and formazan formation (INT<span class="inline-formula">_R</span>), for a wide range of relevant marine prokaryoplankton including the gammaproteobacteria <i>Halomonas venusta</i>, the alphaproteobacteria <i>Ruegeria pomeroyi</i> and <i>Candidatus Pelagibacter ubique</i> (SAR11), the actinobacteria <i>Agrococcus lahaulensis</i>, and the cyanobacteria <i>Synechococcus marinus</i> and <i>Prochlorococcus marinus</i>. All species imported and reduced INT, but the relationship between the rate of O<span class="inline-formula">₂</span>C and INT<span class="inline-formula">_R</span> was not constant between oligotrophs and copiotrophs. The range of measured O<span class="inline-formula">₂</span>C <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M9" display="inline" overflow="scroll" dspmath="mathml"><mo>/</mo></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="8pt" height="14pt" class="svg-formula" dspmath="mathimg" md5hash="880d1b22cfae9b4167ff115d05c6894c"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-22-6225-2025-ie00001.svg" width="8pt" height="14pt" src="bg-22-6225-2025-ie00001.png"/></svg:svg></span></span> INT<span class="inline-formula">_R</span> conversion equations equates to an up to 40-fold difference in derived O<span class="inline-formula">₂</span>C. These results suggest that when using the INT method in natural waters, a constant O<span class="inline-formula">₂</span>C <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M13" display="inline" overflow="scroll" dspmath="mathml"><mo>/</mo></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="8pt" height="14pt" class="svg-formula" dspmath="mathimg" md5hash="8550e2e9970f84100ffbfa4da4f4f543"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-22-6225-2025-ie00002.svg" width="8pt" height="14pt" src="bg-22-6225-2025-ie00002.png"/></svg:svg></span></span> INT<span class="inline-formula">_R</span> relationship cannot be assumed, but must be determined for each plankton community studied.</p>