**5. Conclusions**

This study suggests that *C. aggregans* uses its metabolic flexibility and capability for both phototrophic and chemotrophic growth to optimize its performance under the varying environmental conditions in its natural habitat, the microbial mat community at Nakabusa Hot Springs. The main ATP-generating and thus metabolically most-active times are not only the high-light hours around midday (phototrophy), but—most notably— also the early morning hours around 05:00, when the cells are hypothesized to conduct chemomixotrophic growth (Figure 9).

Genes for the biosynthesis of the photosynthetic apparatus are predominantly transcribed during the night; however, photosynthesis is active during the light hours in the morning, midday and afternoon. Under low O2 concentrations in the dim morning light (≤100 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup>1), photoauto/mixotrophic metabolism potentially using CO as an electron donor is suggested to be the major energy source for *C. aggregans* in the cyanobacteria-dominated mats. Later on, under midday high-light conditions, intense oxygenic photosynthesis by cyanobacteria renders the upper millimeters of the microbial mat highly oxic. However, O2 respiration in *C. aggregans* does not seem to take place under these conditions. Instead, photoheterotrophic growth (and the assimilation of glycolate) is most likely the dominant lifestyle, supplemented with a certain degree of anaplerotic CO2 fixation. In the afternoon, under anaerobic light conditions, photoautotrophic or photomixotrophic growth with sulfide and/or H2 as the electron donor takes place in the

deeper mat layers, and aerobic respiration and chemoheterotrophic growth are hypothesized for the cells in the upper layers. At nighttime, chemoheterotrophic fermentative growth and the production of H2 may take place. In the late-night/early morning hours, at around 05:00, *Chloroflexus* migrates to the mat surface and undergoes mixotrophic growth with H2 and O2 prior to sunrise, after which *C. aggregans* switches back to phototrophy.

**Figure 9.** Proposed diel growth modes of *C. aggregans* (indicated by brown filaments) in cyanobacterial mats based on in situ metatranscriptomic and microsensor analyses in the cyanobacterial mats of Nakabusa Hot Springs. The green area represents the green upper layer containing oxygenic phototrophs (i.e., cyanobacteria), while the orange area corresponds to the orange colored undermat. The black curvy line indicated the overflowing water surface.

> **Supplementary Materials:** The following are available online at https://www.mdpi.com/2076 -2607/9/3/652/s1, Figure S1: Relative transcription levels of paralogous respiratory complex I; Figure S2: Relative transcription levels of housekeeping genes; Figure S3: Relative transcription levels of genes related to oxidative stresses; Figure S4: Relative transcription levels of amino acid and oligopeptide transporter genes; Figure S5: Relative transcription levels of glycoside and sugar transporter genes; Table S1: Transcriptomic profiles and differentially transcribed genes; Table S2: Differential transcription analyses of genes involved in energy metabolisms of *C. aggregans*; Table S3: Summary of differentially transcribed genes; Table S4: Transcription patterns of *bch* genes; Table S5: Transcription patterns of genes involved in energy metabolisms of *C. aggregans.*

> **Author Contributions:** Conceptualization, S.K. and V.T.; Transcriptomic data analysis, S.K.; RNA extraction, J.N.M.; Microsensor measurements and analysis, M.L., E.T., and M.K.; Sample collection, J.N.M., M.T., A.N., and V.T.; Writing—original draft preparation, S.K. and V.T.; Writing—review and editing, S.K., V.T., M.T., M.K., M.L., E.T., S.H. (Shin Haruta), A.N., and J.N.M.; Supervision, V.T. and S.H. (Shin Haruta); Funding acquisition, S.H. (Satoshi Hanada), M.K., and A.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Institute for Fermentation, Osaka (IFO) to S.H. (Satoshi Hanada), grants from the Independent Research Fund Denmark (DFF-1323-00065B; DFF-8021-00308B) to M.K., and the fund from international practice course of Tokyo Metropolitan University to A.N.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The sequence collected in this study is available under NCBI BioProject accession number PRJNA715822.

**Acknowledgments:** The authors would like to thank the owner of Nakabusa Hot Spring (Takahito Momose) for the permission to collect samples from the hot spring.

**Conflicts of Interest:** The authors declare no conflict of interest.
