**3. Results**

#### *3.1. Irradiance and In Situ Oxygen Dynamics in the Microbial Mat*

The O2 concentration and penetration from the surface green layer to the deeper orange layer in the microbial mat varied dramatically with irradiance. The whole mat was anoxic during the night, and the O2 concentration started to increase at the mat surface at around 06:00, correlating with the time of sunrise and thus the onset of cyanobacterial oxygenic photosynthesis under diffuse light (Figures 1 and 2). However, O2 did not accumulate in deeper mat layers until later in the morning at around 09:00, when the microbial mats were exposed to direct sunlight as the sun rose over the surrounding mountains. Supersaturating O2 levels were observed in the uppermost mat layers at ~12:00 (noon) during the highest solar irradiance (1531 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup>1; 400–700 nm). The maximum O2 concentration at the microbial mat surface reached >900 μmol O2 L−<sup>1</sup> and with a maximal O2 penetration of >2 mm depth under the highest irradiance between 10:00 and 14:00. Thus, there was sufficient O2 available for aerobic microbes in the upper 2 mm of the mat during this period of the day. In the afternoon, after 14:00, O2 concentration started to decrease gradually (from approx. 500 μmol O2 <sup>L</sup>−1) and no O2 was detected at a depth of 1–2 mm shortly after 15:00. The upper layer remained oxic (100–200 μmol O2 <sup>L</sup>−1) until 16:00. At this time, the microbial mats experienced a substantial decrease in solar irradiance (see Figures 3–8 and S1–S5) as the sun set behind the mountains. However, low levels of diffuse sunlight (<100 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup>1; 400–700 nm) hit the microbial mats until complete darkness was observed at 17:00. Anoxic conditions started to become established in the lower parts of the microbial mats ~2 h before sunset, potentially enabling anaerobic and microaerophilic metabolism under low-light conditions during this time interval.

**Figure 2.** A heat map of the vertical O2 concentration profiles in the cyanobacteria-dominated microbial mat of Nakabusa Hot Springs as measured over a diel cycle. The O2 concentration (μmol <sup>L</sup>−1) was measured as a function of depth in the microbial mat at 15-min intervals for 24 h from 18:00 on 3 November to 18:00 on 4 November. The mat surface is indicated by 0 mm. Positive depth values indicate the depth below the mat surface, and negative values indicate the depth above the mat surface, i.e., the distance into the overlying water column of the hot spring.

#### *3.2. Transcriptome Profiles and Differentially Transcribed Genes*

Approx. 0.26–5.23 million reads of transcripts were assigned to the *C. aggregans* genome throughout the day (Table S1). Among 3848 CDSs contained in the genome of *C. aggregans* DSM9485T, the number of genes in which more than 10 transcripts were detected in all timepoints was 2542–3506 genes. Statistical significance of transcription level changes of each gene was determined using the *p*-value (*p* < 0.05) based on the "exactTest" function in edgeR [32]. Thousands of genes were differentially transcribed during the period from 19:00 on 3 November to 15:00 on 4 November indicating a versatile and changing transcriptional activity between the different time points. During 15:00–16:00 as well as 17:00–19:00, the numbers of significantly differentially transcribed genes were considerably lower (less than 10% of all CDSs), indicating transcriptional activity of *C. aggregans* during this period was relatively stable compared with former 20 h of the day. Those times were taken together as 'afternoon' and 'evening', respectively, in which both transcriptional activity as well as environmental conditions are expected to be rather similar. Overall the statistics support the changes in relative transcriptional levels and activity of *C. aggregans* to be significant. Detailed information on the differential transcription of the genes discussed is given in Tables S2 and S3.

#### *3.3. Transcription of Photosynthesis-Related Genes*

*Chloroflexus aggregans* contains a type 2 photosynthetic reaction center complex (RC) and light-harvesting chlorosomes; the main photosynthetic pigments are bacteriochlorophylls (BChls) *c* and *a* [2]. Transcripts of *pufLMC* genes (Cagg\_1639–1640 and Cagg\_2631) encoding RC proteins in *C. aggregans* showed significant nocturnal patterns and were most abundant in the evening and at night (Figure 3). Similarly, nocturnal transcriptional patterns of chlorosome proteins encoded by *csmAMNOPY* genes (Cagg\_1222, Cagg\_1209, Cagg\_1208, Cagg\_2486, Cagg\_1206, and Cagg\_1296) were detected (Figure 3).

BChl synthesis-related *bch* genes in *C. aggregans* in the mats showed inconsistent transcription patterns, with the highest relative transcription levels either under oxic conditions during the daytime (11:00) and/or under microoxic conditions in the afternoon (15:00; Table S4). In total, four different groups of transcription patterns could be distinguished for *bch* genes. Group I (*bchH*-III, *bchI*-I and II, *bchY*, *bchZ*, *acsF*, *bchL*, *bchB*, *bchN*, *bchM*) showed highest relative transcription during the daytime (midday, 11:00). Group II (*bchH*-I and II, *bchJ*, *bchF*, *bchX*, *bchC*) showed maximal values in the afternoon around 15:00, with more or less pronounced lows at 11:00. The two other groups showed highest relative transcription levels under anaerobic conditions. Four genes (*bchG*, *bchK*, *bchU*, and *bchP*) showed peaks at 18:00. *bchD* and *bchI*-III had maximal relative transcription in the early morning at 05:00.

Paralogs with different patterns were present for the genes encoding Mg-chelatase, i.e., *bchH* (three paralogs; Cagg\_0239, 0575, 1286) and *bchI* (three paralogs; Cagg\_1192, 2319, 3123). In all three cases, the paralogs showed differences in absolute read abundance as well as different temporal peaks in relative transcription over the day (Table S4).

Two different genes encoding for Mg-protoporphyrin monomethylester cyclase are present in *C. aggregans*. AcsF and BchE both catalyze the synthesis of divinylprotochlorophyllide from Mg-protoporphyrin IX 13-monomethyl ester (one of the intermediates in the BChl synthesis pathway) under aerobic and anaerobic conditions, respectively [34–36]. *acsF* (Cagg\_1285) was transcribed throughout the diel cycle with relative transcription levels about four times higher during day, when the mat was (super)oxic than under anoxic conditions at night (Figure 3). In contrast to *acsF*, the *bchE* (Cagg\_0316) showed significantly lower relative transcription levels (only 1/32 of the diel average) under high-light/high-O2 conditions in the mat and higher transcription levels during the night as well as during low-light transition times in the morning and afternoon (Figure 3).

**Figure 3.** Relative transcription levels of genes encoding photosynthetic reaction center and chlorosome proteins, as well as genes involved in bacteriochlorophyll biosynthesis. Mean values of genes encoding the type 2 reaction center (RC) (*pufLMC*: Cagg\_1639–1640 and Cagg\_2631) and chlorosome proteins (*csmAMNOPY*: Cagg\_1222, Cagg\_1209, Cagg\_1208, Cagg\_2486, Cagg\_1206, and Cagg\_1296) are represented by a blue line and an orange line, respectively, with standard deviations. The values of Mg-protoporphyrin IX monomethyl ester aerobic cyclase (*acsF*, Cagg\_1285, yellow line) and anaerobic cyclase (*bchE,* Cagg\_0316, green line) are shown. The downwelling photon irradiance (photosynthetically active radiation [PAR]; 400–700 nm) is indicated in white. The asterisk indicates the transcription of a particular gene corresponding to the color in a timepoint differed significantly (*p* < 0.05) from that in the previous timepoint.

#### *3.4. Phototrophic and Respiratory Electron Transport*

Electron transport chains are involved in both phototrophic and chemotrophic (respiratory) metabolism. *Chloroflexus* spp. contain paralogs of some of the major enzyme complexes involved in the electron transport chain, namely, NADH:menaquinone oxidoreductase (Complex I) [11,37,38] alternative complex III (ACIII) [39–41] and the soluble electron carrier auracyanin [42–45] in their genomes. Single-copy genes are present encoding succinate dehydrogenase (Complex II) [46,47] and F-type ATP synthase (Complex V) [48].

The respiratory complex I carries out the transfer of electrons between soluble cytoplasmic electron carriers and membrane-bound electron carriers coupled to proton translocation generating a transmembrane proton motive force. *C*. *aggregans* DSM9485<sup>T</sup> contains two sets of genes encoding Complex I (NADH:menaquinone oxidoreductase, *nuo*): one (Cagg\_1620–1631) represents a cluster comprising 12 genes with an additional *nuoM* (2-M complex) inserted between the original *nuoM1* and *nuoN* genes (*nuoABCDHJKLMMN*, 3); the second is a complete cluster comprising 14 genes (Cagg\_1036–1049). The additional proton-pumping subunit NuoM has been speculated to lead to a higher stoichiometry of protons translocated per 2e− reaction cycle [49]. Both sets of *nuo* genes were transcribed and showed the highest relative transcription during the daytime (Figure S1). The 14-gene set showed a transcriptional peak at 11:00 and the 12-gene peak came slightly later (in the afternoon at 15:00–16:00). Both *nuo* gene sets showed a small transcription peak in the early morning at 05:00.

Alternative complex III (ACIII) transfers electrons from menaquinol to water-soluble proteins such as auracyanin, the blue copper electron carrier protein found in *Chloroflexus* spp. [44]. Two types of ACIII have been reported in *Chloroflexus* spp.: Cp, which is thought to be involved in cyclic phototrophic electron transport, and Cr, which is predicted to be related to the reduction of oxygen in respiratory electron transport [11,23,39]. In the present study, clear diel patterns as well as differences between the two sets of genes were observed. The genes encoding Cp showed high relative transcription levels all day with the highest level at 11:00 and a significant decrease in the afternoon as the sunlight vanished. The Cr genes were not highly transcribed under the daytime high O2 conditions in the mat, but they significantly increased and exhibited maximal relative transcription levels in the microoxic low-light afternoon hours at 15:00 and 16:00 (Figure 4). Genes for both Cp and Cr were highly transcribed in the early morning at 5:00.

Two homologs encoding the soluble electron carrier protein auracyanin, *aurA* and *aurB*, are present in the genome of *C. aggregans*. The transcriptional profile of *aurA* in *C. aggregans* in the cyanobacterial mats in the present investigation showed patterns similar to those of Cr and other genes involved in the respiratory electron transport chain, with one significant peak in the early morning at 05:00 and another under microoxic and anaerobic conditions in the afternoon and evening (15:00 until 18:00; Figure 4). In contrast, *aurB* was significantly higher transcribed during a high-light period (11:00) as well as at 05:00.

**Figure 4.** Relative transcriptional levels for alternative complex III (ACIII) and auracyanin. The mean values of ACIII (Cp, Cagg\_3382–3383, and 3385–3387) for phototrophic electron transfer and ACIII (Cr, Cagg\_1523–1527) for chemotrophic electron transfer are represented by a blue line and an orange line, respectively, with standard deviations. The values of *aurA* and *aurB* (Cagg\_0327 and 1833) encoding auracyanin are respectively displayed as yellow (*aurA*) and green (*aurB*) lines. The downwelling photon irradiance (PAR; 400–700 nm) is indicated in white. The asterisk indicates the transcription of a particular gene corresponding to the color in a timepoint differed significantly from that in the previous timepoint.

Respiratory Complex IV—i.e., the cytochrome *c* oxidase complex—plays a key role in the reduction of O2 to H2O in the respiratory electron transport chain. The oxygen profiles over the diel cycle indicated high O2 concentrations in the mats during the daytime (Figure 2). In the laboratory, *Chloroflexus* spp. can grow chemoheterotrophically using respiration under aerobic dark conditions. In *Chloroflexus* spp., the cytochrome c oxidase (COX, or Complex IV; EC 1.9.3.1) genes are clustered with the Cr operon (Cagg\_1519– 1522). Similar to Cr, the average relative transcription levels of these COX genes reached their highest values in the early morning at 05:00 and in the afternoon at 15:00 and 16:00 (Figure 5).

*Chloroflexus aggregans* possesses a type-B succinate dehydrogenase (Complex II) which comprises one polypeptide and two hemes for a transmembrane cytochrome *b* (*sdhC*) in addition to a flavoprotein subunit (*sdhA*) and iron-sulfur subunit (*sdhB*) [50,51]. Complex II encoded by *sdhCAB* (Cagg\_1576–1578) is involved in electron transport as well as in the TCA cycle and the 3-OHP bi-cycle. They showed significant high relative transcription levels in the morning at 05:00 and during the daytime, and were significantly low throughout the night (Figure 5).

F-type ATP synthase is involved in the production of ATP based on the proton motive force obtained by phototrophic as well as respiratory electron chain activity. The ATP-synthase consists of two parts: F1, which is a catalytic part, and F0, which is a transmembrane proton channel part [48]; *C*. *aggregans* DSM9485<sup>T</sup> contains a complete gene set for both parts in the genome [11]. Similar to Complex I-1, II, and ACIII Cp, the relative transcription of ATP-synthase showed a diurnal pattern with two peaks, one at 05:00 and the other at 11:00 (Figure 5).

**Figure 5.** Relative transcription levels of respiratory complex II, IV and ATP synthase. The mean values of the relative transcripts of respiratory complex II (Cagg\_1576–1578, blue line), complex IV (Cagg\_1519–1522, orange line) and ATP synthase (Cagg\_0984—991, yellow line) are shown with standard deviations. The downwelling photon irradiance (PAR; 400–700 nm) is indicated in white.

#### *3.5. 3-Hydroxypropionate Bi-Cycle and Anaplerotic Carbon Fixation*

*Chloroflexus* spp. contain all genes for the 3-hydroxypropionate (3-OHP) bi-cycle, a carbon fixation pathway found only in members of filamentous phototrophic *Chloroflexota* [11,52–57]. The number of transcripts per million (TPM) for genes encoding key enzymes of the 3-OHP bi-cycle, i.e., malonyl-CoA reductase (Cagg\_1256) and 3- hydroxypropionyl-CoA synthase (Cagg\_3394) [23], were considerably higher than the average of all genes and appeared relatively stable over the diel cycle (Table S5). Although transcription was detected at all times, the relative transcripts of the two key enzyme genes on average peaked at 15:00. The second highest peak of 3-OHP bi-cycle key enzyme genes was detected at 05:00 before sunrise, and again at 07:00. Under the oxic, high-light conditions, at 11:00, the relative transcriptional levels of the genes encoding key enzymes of 3-OHP bi-cycle were the lowest (Figure 6).

Filamentous anoxygenic phototrophs also contain anaplerotic pathways for incorporating inorganic carbon, such as phosphoenolpyruvate carboxylase (*ppc*, Cagg\_0399), which catalyzes the unidirectional production of oxaloacetate from phosphoenolpyru-

vate [11,58,59]. Transcripts of *ppc* were abundant during the daytime under aerobic, highlight conditions (Figure 6).

**Figure 6.** Relative transcription levels of genes encoding key enzymes of the 3-OHP bi-cycle and related enzymes of the anaplerotic pathway in cyanobacterial mats. The mean values of the relative transcription levels of key 3-OHP enzymes, i.e., malonyl-CoA reductase (Cagg\_1256) and propionyl-CoA synthase (Cagg\_3394), plus that of phosphoenolpyruvate carboxylase (*ppc*, Cagg\_0058 and 0399) are represented by a blue line and an orange line, respectively, with standard deviations. The downwelling photon irradiance (PAR; 400–700 nm) is indicated in white.

#### *3.6. Electron Donors: Hydrogenase, Sulfide: Quinone Reductase and CO-Dehydrogenase*

Recent studies demonstrated that *C. aggregans* has the capability to use sulfide as well as H2 as an electron donor for photoautotrophic growth [9,10]. Genome analyses have suggested that carbon monoxide can also serve as a potential electron donor [11]. The correlation between the relative transcriptions of genes encoding hydrogenases, sulfide:quinone reductase and carbon monoxide dehydrogenase and the genes encoding key enzymes in the 3-OHP bi-cycle was analyzed in order to predict autotrophic metabolism.

*C. aggregans* contains two Ni-Fe hydrogenases: a bidirectional hydrogenase (Cagg\_2476–2480) and an uptake hydrogenase (*hyd*, Cagg\_0470–0471)–that can provide electrons for autotrophic growth [60,61]. Relative transcription levels of *hyd* genes encoding the uptake hydrogenase significantly increased in the afternoon (at 15:00) shortly after the direct solar illumination of the mats ended around 14:00 and anaerobic conditions were established in the deeper layers of the mat, as well as in the early morning at 05:00 (Figures 2 and 7). The relative transcription levels for genes encoding the bidirectional hydrogenase (Cagg\_2476–2480) peaked a little later in the evening after sunset (17:00, PAR=0), stayed high throughout the night, and then decreased during the day. Nickel transporter genes showed the same high relative transcription pattern as the *hyd* genes, with peaks in the early morning and the afternoon (Figure 7).

*Chloroflexus* spp. contain type-II sulfide:quinone oxidoreductase (SQR), which oxidizes sulfide to elemental sulfur, but lack the *dsr* genes, which encode genes involved in the oxidation of elemental sulfur to sulfate as observed in green and purple sulfur bacteria [11,62], and also lack the *sox* system, which oxidizes elemental sulfur and thiosulfate to sulfate and is widespread in chemoautotrophic sulfur oxidizers [63]. In the present study, a significant increase of relative transcription levels with the highest peak of *sqr* was detected in the afternoon, at 15:00 (Figure 7) under microaerobic to anaerobic low-light conditions in the mat.

As a third possibility, based on the presence of genes encoding carbon monoxide dehydrogenase (*coxGSML*, Cagg\_0971–0974) in the genome, the capability of *Chloroflexus* spp. to utilize CO as an electron donor and/or carbon source during aerobic or microaerobic growth has been discussed [11]. In the present investigation, the *coxGSML* genes were significantly higher transcribed during high-light conditions around noon (Figure 7) as well as during the afternoon as the mats turned anoxic. A significant increase in the relative transcription of *cox* genes was seen under anaerobic, low-light conditions in the morning at 07:00 together with a spike in the relative transcription for genes encoding key enzymes of the 3-OHP bi-cycle. The relative transcription of hydrogenase and *sqr* genes were clearly and significantly decreased at that time.

**Figure 7.** Relative transcription levels of genes encoding hydrogenases, sulfide:quinone oxidoreductase and nickel transporter in cyanobacterial mats. The mean values of the relative transcription levels for type-I uptake Ni-Fe hydrogenase genes *hydAB* (Cagg\_0470–0471, blue line), bi-directional Ni-Fe hydrogenase genes homologous to *frhA*, *frhG*, *hoxU*, *nuoF* and *hoxE* (Cagg\_2476–2480, orange line), carbon monoxide (CO) dehydrogenase genes *coxGSML* (Cagg\_0971–0974, gray line), and nickel transporter genes (Cagg\_1273–1276, light blue line) are shown with standard deviations. The relative transcription levels of the type-II sulfide:quinone oxidoreductase gene (*sqr*, Cagg\_0045) are represented by the black line. The downwelling photon irradiance (PAR; 400–700 nm) is indicated in *white*. The asterisk indicates the transcription of a particular gene corresponding to the color in a timepoint differed significantly from that in the previous timepoint.

#### *3.7. Carbohydrate Metabolism and the TCA Cycle*

*Chloroflexus aggregans* contains the gene set for the pentose phosphate pathway (PP), including the key enzymes for the oxidative phase involved in anabolic pathways, i.e., glucose-6-phosphate dehydrogenase (Cagg\_3190) and 6-phosphogluconate dehydrogenase (Cagg\_3189) [23]. Herein, the transcription levels of the two key enzyme genes showed a significant diurnal pattern with the highest relative transcription under high-light conditions (11:00) and an additional peak at 05:00 (Figure 8). Similar to the genes of the oxidative pentose phosphate pathway, high relative transcription levels were also detected at those times for other genes that are indicative of active metabolism and growth, such as DNA gyrase, DNA polymerase, and RNA polymerase (Figure S2).

Because many of the enzymes involved in glycolysis are bi-directional and similarly used in gluconeogenesis, the transcriptional patterns of genes related to glycolysis were analyzed by focusing on the unidirectional enzyme, 6-phosphofructokinase (Cagg\_3643), which irreversibly catalyzes the reaction from fructose-1,6-phosphate to

fructose-6-bisphosphate to predict chemoheterotrophic growth and catabolism. In *C. aggregans*, two genes are annotated as genes encoding 6-phosphofructokinase (Cagg\_3643 and Cagg\_2702). These two genes differ considerably in length, with Cagg\_2702 encoding a protein identified as a member of the 6PF1K\_euk superfamily, the eukaryotic type of the 6- phosphofructokinase, which is almost twice as long as the 'bacterial' 6-phosphofructokinase version, represented by Cagg\_3643 (747 aa vs. 356 aa) [64]. Homologs of the Cagg\_2702 gene are present in many *Chloroflexota* genomes as identified by a BLAST search, but are not generally present in many other bacteria. The two genes differed in the relative transcription levels and patterns. Cagg\_2702 showed the same diel transcription pattern as other genes involved in glycolysis, with its highest relative transcription during high-light conditions at 11:00 (see Table S5). In contrast, Cagg\_3643 showed the highest relative transcription under anaerobic conditions in the evening and the lowest transcription levels during superoxic, high-light conditions (Figure 8), but it showed considerably higher absolute transcription levels (TPM average of 1312.12 vs. 9.28 for Cagg\_2702). Because Cagg\_3643 represents the 'bacterial' type of the enzyme (with higher similarity to the 6-phosphofructokinase in *E. coli*) and since it showed higher absolute transcription levels, it is hypothesized that it represents the unidirectional gene involved in the oxidative activity of glycolysis in *C. aggregans*.

**Figure 8.** Relative transcription levels of genes for carbohydrate metabolism. The mean values of the relative transcriptional levels for the TCA cycle (Cagg\_3738, 3721, 2500, and 2290) and common enzymes of the TCA cycle and the 3-OHP bi-cycle that is labeled as 'TCA+3-OHP' (Cagg\_2086, 2819, and 1576–1578) are represented by a blue line and an orange line with standard deviations. The mean values of the relative transcripts of genes encoding key enzymes of the pentose phosphate pathway (Oxidative PP)—i.e., glucose-6-phosphate dehydrogenase (Cagg\_3190) and 6-phosphogluconate dehydrogenase (Cagg\_3189)— are represented by the yellow line with standard deviation. The values of the relative transcription levels are displayed for genes encoding the 'bacterial' type of the 6-phosphofructokinase that is labeled as "Bacterial" PFK (Cagg\_3643, green line). The downwelling photon irradiance (PAR; 400–700 nm) is indicated in white. The asterisk indicates the transcription of a particular gene corresponding to the color in a timepoint differed significantly from that in the previous timepoint.

The oxidative TCA cycle is important for oxygen-respiring heterotrophic organisms, and all *Chloroflexus* species are known to have the ability to grow chemoheterotrophically [1–3] with oxygen as the terminal electron acceptor. Some of the reactions involved in the TCA cycle—e.g., the conversions from succinyl-CoA to malate—are also part of the 3-OHP bicycle [65]. Therefore, the transcriptional patterns of succinyl-CoA synthase (Cagg\_2086 and

Cagg\_2819), succinate dehydrogenase (Cagg\_1576–1578), and fumarate lyase (Cagg\_2500) are labeled as 'TCA+3-OHP' and the genes that are exclusively present in the TCA cycle are labeled as 'TCA-only' (Figure 8). Genes involved in the TCA cycle were transcribed relatively evenly over the diel cycle, with two peaks: one in the early morning at 05:00 and the other increasing during the day from 07:00 to 15:00. At 05:00, genes encoding acetate/CoA ligase (Cagg\_3789), which catalyzes the production of acetyl-CoA from acetate, also showed significantly higher relative transcription levels (Table S5). The two TCA-affiliated gene groups showed only small differences in their relative transcription patterns. After a small peak at 06:00 for all TCA-related genes, the relative transcription of the 'TCA+3-OHP' genes had already increased at 07:00, whereas the relative transcription levels of the 'TCA-only' genes were low at that time and showed a small increase later in the morning (Figure 8).

#### *3.8. Transcription of Oxygen Protection Genes*

Genes for two oxidative stress-protection enzymes present in the *C. aggregans* genome were analyzed in this study: superoxide dismutase (Cagg\_2494) and two copies of a glutathione peroxidase (1: Cagg\_0324 and 2: Cagg\_0446). Transcripts for the enzymes showed their highest relative transcription levels during the daytime, when both O2 and light were present (Figure S3). The gene encoding superoxide dismutase, i.e., an enzyme-detoxifying reactive oxygen species, exhibited a second peak of relative transcripts in the early morning (at 05:00), when cytochrome c oxidase genes were also highly transcribed (see Section 3.4 above, "Phototrophic and Respiratory Electron Transport"). Glutathione peroxidase reduces lipid hydroperoxides to their corresponding alcohols and reduces free hydrogen peroxide to water. Significant high relative transcription levels of glutathione peroxidaseencoding genes in *C. aggregans* were observed at 11:00 (Figure S3), thus indicating the possible presence of not only O2 but also hydrogen peroxide.

Hydrogen peroxide is produced as a by-product in the oxidation of glycolate to glyoxylate by glycolate oxidase [66]. The encoding genes (*glcDEF*, Cagg\_1528, Cagg\_1530–1531, and Cagg\_1892–1893) showed patterns similar to that of glutathione peroxidase, with the highest relative transcription levels during midday, indicating both the presence of glycolate in the mat environment and its oxidation by *C. aggregans* (Figure S3). The oxidation of glycolate may be linked to a photoheterotrophic metabolism, which is further supported by the high relative transcript levels for genes encoding glycoside, sugar, and amino acid transporter genes during this time under high-light conditions (Figures S4 and S5).
