Scarcity of fixed carbon transfer in a model microbial phototroph-heterotroph interaction

Although the green alga Chlamydomonas reinhardtii has long served as a reference organism, few studies have interrogated its role as a primary producer in microbial interactions. Here, we quantitatively investigated C. reinhardtii's capacity to support a heterotrophic microbe using the established coculture system with Mesorhizobium japonicum , a vitamin B ₁₂ -producing α-proteobacterium. Using stable isotope probing and nanoscale secondary ion mass spectrometry (nanoSIMS), we tracked the flow of photosynthetic fixed carbon and consequent bacterial biomass synthesis under continuous and diurnal light with single-cell resolution. We found that more ¹³ C fixed by the alga was taken up by bacterial cells under continuous light, invalidating the hypothesis that the alga's fermentative degradation of starch reserves during the night would boost M. japonicum heterotrophy. ¹⁵ NH ₄ assimilation rates and changes in cell size revealed that M. japonicum cells reduced new biomass synthesis in coculture with the alga but continued to divide - a hallmark of nutrient limitation often referred to as reductive division. Despite this sign of starvation, the bacterium still synthesized vitamin B ₁₂ and supported the growth of a B ₁₂ -dependent C. reinhardtii mutant. Finally, we showed that bacterial proliferation could be supported solely by the algal lysis that occurred in coculture, highlighting the role of necromass in carbon cycling. Collectively, these results reveal the scarcity of fixed carbon in this microbial trophic relationship (particularly under environmentally relevant light regimes), demonstrate B ₁₂ exchange even during bacterial starvation, and underscore the importance of quantitative approaches for assessing metabolic coupling in algal-bacterial interactions.