Nitrogen fixation in lichens is important for improved rock weathering.

It is known that cyanobacteria in cyanolichens fix nitrogen for their nutrition.However, specific uses of the fixed nitrogen have not been examined. The present study shows experimentally that a mutualistic interaction between a heterotrophic N2 fixer and lichen fungi in the presence of a carbon source can contribute to enhanced release of organic acids, leading to improved solubilization of the mineral substrate. Three lichen fungi were isolated from Xanthoparmelia mexicana, a foliose lichen, and they were cultured separately or with a heterotrophic N2 fixer in nutrient broth media in the presence of a mineral substrate. Cells of the N2-fixing bacteria attached to the mycelial mats of all fungi, forming biofilms. All biofilms showed higher solubilizations of the substrate than cultures of their fungi alone. This finding has bearing on the significance of the origin and existence of N2-fixing activity in the evolution of lichen symbiosis. Further, our results may explain why there are N2-fixing photobionts even in the presence of non- fixing photobionts (green algae) in some remarkable lichens such as Placopsis gelida. Our study sheds doubt on the idea that the establishment of terrestrial eukaryotes was possible only through the association between a fungus and a phototroph.