In the present study, surface PAB accumulation was positively influenced by blue (465 nm) and possibly by green (530 nm) wavelengths, but not by red ones (677 nm, with or without far red). Although green light generated a similar PAB accumulation than BL for the same intensity (40 µmol photons m−2 s −1 ), our experimental setup did not allow to test higher green light intensities to unambiguously conclude the responses to green light. It was previously demonstrated that green light (>510–520 nm) generally has a lower effect than BL (450 ± 20 nm) on phototactic directional cell movements (Nultsch, 1971; Cohn et al., 2004, 2015, 2016). Differently, red light of low or moderate intensity usually has no/little effect or negative effect on directional motility of diatoms and their accumulation, although this response is species-specific (Cohn and Weitzell, 1996; McLachlan et al., 2009; Cohn et al., 2015). These reports partially explain the action spectrum of MPB abundance at the surface of sediment at low irradiance (5 µmol photons m−2 s −1 ) showing a strong positive effect of blue wavelengths (440 ± 10 nm), limited effect of greens (500–550 nm), and a moderate effect of red (680 nm) (Wenderoth and Rhiel, 2004).

Here, as regards to the specific effect of blue wavelengths, we have found that (i) BL induced a ×1.8 ± 0.4 total PAB increase compared to the "white" light control at the same intensity of 120 µmol photons m−2 s −1 ; (ii) PAB accumulation was irradiance-dependent with a positive effect from 10 to 120 µmol BL photons m−2 s −1 ; (iii) PAB accumulation was species-specific with two taxa, namely, N. spartinetensis and G. fasciola (and probably other Navicula species to a lesser extent), which strongly reacted (i.e., from 0 to 10% relative presence at the surface of sediment to up to more than 80%) at 40 µmol BL photons m−2 s −1 ; (iv) their surface accumulation was independent of the season: the BL effect was observed with both winter and summer sediments for a particular species. These features fit well with previous reports. In general, blue wavelengths have a positive effect (up to a certain irradiance threshold, see Cohn et al., 2004; McLachlan et al., 2009) on surface PAB (×1.4 to ×1.8, Wenderoth and Rhiel, 2004) by modulating the speed to which epipelic diatoms move directionally (Nultsch, 1971; Cohn and Weitzell, 1996; McLachlan et al., 2009; Cohn et al., 2015). This BL photosensitivity is species-specific (McLachlan et al., 2009; Cohn et al., 2015), and it is detected at least at the tips of pennate cells (Cohn et al., 1999). The way blue wavelengths act at the cellular level and how the "BL signal" might be transduced is still unclear in diatoms, although significant recent advances have been made (Jaubert et al., 2017). It was early proposed (Nultsch, 1971) that pigments, chlorophylls and carotenoids, should act as primary harvesters and transducers to trigger the previously described action spectrum of MPB motility, and especially photokinesis (i.e., change of motility speed; Wenderoth and Rhiel, 2004). The latter would directly depend on the energy provided by photosynthesis, which itself is driven by actinic wavelengths, and obviously BL. In that respect, the optical properties of the photonic crystal-like structures of the diatom frustule are highly relevant; they have been shown to shape the availability of light and of specific wavelengths inside the cell, thereby potentiating pigment light-harvesting and photosynthetic efficiency (Goessling et al., 2018a,b). Interestingly, the frustule of one of our most BL-reactive species, G. fasciola,

was shown to be transparent when the frustule of other epipelic species significantly (up to 20%) attenuated the transmission of blue wavelengths (Goessling et al., 2018a). While BL attenuation can be advantageous to decrease potentially harmful incident irradiance, its nearly 100% transmission under low/moderate irradiance could support specific BL signaling inside the cell. These interspecies differences in frustule optical properties were claimed to additionally modulate the light availability inside the biofilm itself (Goessling et al., 2018a). Taken together with the fact that a species response to BL can be modulated by the presence of other species in its close surrounding (Cohn et al., 2016), it points out to the importance of the species consortium composition, which varies year around (this study, Haubois et al., 2005). Therefore, wavelengths, together with irradiance, would fine tune the differential rhythmic MPB migratory behavior and surface PAB accumulation versus the sediment light climate at the cell, species, and consortium (i.e., biofilm) levels.

However, in the present study, upon 24-h exposure, we did not observe any specific BL effect on (i) MPB pigment content, and especially carotenoids, independent of the irradiance, of the season, and of the sediment depth (down to 600 µm); (ii) MPB photosynthetic performances, independent of the irradiance. Therefore, the positive BL effect we observed was based on molecular cell components that were already present at the beginning of the light treatment (such as pigments), and it was not due to a surge in photosynthetic performance, that is, higher surface PAB accumulating because of more diatom cells photosynthesizing better under BL. Although we do not question the importance of the frustule optical properties and of photopigments in triggering the BL-enhanced PAB with the irradiance-dependent PAB increase being a proof of such role, we aim at seeking for alternative complementary answers. Photoreceptors have been proposed before to play a central role in the positive upward phototaxis of epipelic MPB to blue wavelengths (Wenderoth and Rhiel, 2004; McLachlan et al., 2009). In the recent years, three types of BL photoreceptors have been identified in planktonic diatom models including cryptochromes and a family unique to stramenopiles, aureochromes (Jaubert et al., 2017). Aureochromes control the onset of diatom cell division (mitosis) (Huysman et al., 2013), and BL stimulates growth rate and biofilm formation of the epipelic representative Navicula species (Cao et al., 2013). Therefore, the BL increase in surface PAB we observed could also be due to the proliferation of diatom cells. Because blue wavelengths are rapidly attenuated with sediment depth (Kuhl and Jorgensen, 1994), it is possible that BL, in conjunction with the necessary level of photochemical energy and organic carbon stored during emersion (Underwood et al., 2005; Marques Da Silva et al., 2017), could act as a trigger for mitosis when migrating downward, which, according to Saburova and Polikarpov (2003), are coupled. Furthermore, recent highlights on the role of photoreceptors in the rhythmic light response of diatoms (see Jaubert et al., 2017 for a synthesis) point out to a highly complex regulatory molecular network of lightdependent (such as the photoperiod) and independent (such as the tidal cycle) biological rhythms. It is very likely that sophisticated cross-talks between light perception (by frustule, pigments, and photoreceptors), photosynthetic metabolism, and endogenous regulators (circadian clock components) occur to finely tune the light response of diatoms beyond photoperiod (see section "What Influences the Most the Amount of PAB Present at the Surface of Sediment: Tides or Light?"). Obviously, such a network would be a strong asset for epipelic diatoms in coping with the multifaceted intertidal sediment light climate.

# CONCLUSION

The present study strengthens the view that the vertical migratory rhythm of motile MPB diatoms in intertidal sediment is of endogenous nature and is first mapped onto the tidal cycle with the migration rhythm driven by a "lunidian clock" phased on the lunar day length and cycle. Migration rhythmicity is further modulated, and even overridden in some conditions, by light, including the diurnal cycle and the irradiance level during daytime periods. Optimal light conditions can significantly "potentiate" the tidal effect on the accumulation of PAB at the surface of sediment during emersion. Additionally, we found a strong positive effect of blue wavelengths (465 nm) on surface PAB accumulation as compared to others (white, green, and red lights). This effect is intensity- and species-dependent (i.e., some species strongly react to BL, whereas others do not). We propose that, in addition to photopigments and frustule optical properties, BL photoreceptors (cryptochromes and aureochromes) might play a crucial role in regulating the presence and abundance of PAB at the surface of intertidal sediment. We found two new potential model species, N. spartinetensis and G. fasciola, which strongly react to blue wavelengths. The fact that N. spartinetensis shows a much smaller size than G. fasciola, and probably a different migratory behavior (see Wenderoth et al., 2004; Underwood et al., 2005) is an additional asset. The complementary use of the epipelic model Seminavis robusta (Blommaert et al., 2017, 2018), which genome has been sequenced, will allow to search for molecular players, such as timekeepers (Annunziataa et al., 2019), photoreceptors (Jaubert et al., 2017), and diurnal-responsive genes (see Meyer et al., 2003; Blommaert et al., 2020), of the rhythmic migratory light-tuned response of intertidal epipelic MPB.

# DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/**Supplementary Material**.

# AUTHOR CONTRIBUTIONS

AB and JL contributed to the conception and design of the study. AB performed the experiments. AB, VM, and

JL performed samples and data analyses. AB wrote the first draft of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.

# FUNDING

This work was supported by the Centre National de la Recherche Scientifique-CNRS, the University of La Rochelle-ULR, the Contrat Plant Etat Région-CPER "Littoral" 2007– 2013, the Region Poitou-Charentes-CG17, and by the Natural Sciences and Engineering Research Council-NSERC (Discovery grant to JL).

# REFERENCES

# ACKNOWLEDGMENTS

The authors thank I. Benyoucef for his help with the preparation of definitive slides, the identification and the counting of diatom cells, J.-L. Mouget for the access to the Water-PAM, X. Cousin for the access to the cryomicrotome, and D. Campbell for the critical reading of the manuscript.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmars. 2020.00212/full#supplementary-material

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Barnett, Méléder, Dupuy and Lavaud. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Effect of Light Intensity and Light Quality on Diatom Behavioral and Physiological Photoprotection

Antoine Prins1,2 \* † , Paul Deleris1 , Cédric Hubas2 and Bruno Jesus1,3†

1 Mer Molécules Santé EA 2160, Faculté des Sciences et des Techniques, Université de Nantes, Nantes, France, 2 Muséum National d'Histoire Naturelle, FRE BOREA 2030, MNHN-IRD-CNRS-SU-UCN-UA, Station Marine de Concarneau, Concarneau, France, 3 Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisbon, Lisbon, Portugal

#### Edited by:

Wim Vyverman, Ghent University, Belgium

#### Reviewed by:

Graham J. C. Underwood, University of Essex, United Kingdom Reimund Goss, Leipzig University, Germany

#### \*Correspondence:

Antoine Prins<EMAIL_ADDRESS><EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Marine Ecosystem Ecology, a section of the journal Frontiers in Marine Science

Received: 30 November 2019 Accepted: 16 March 2020 Published: 21 April 2020

#### Citation:

Prins A, Deleris P, Hubas C and Jesus B (2020) Effect of Light Intensity and Light Quality on Diatom Behavioral and Physiological Photoprotection. Front. Mar. Sci. 7:203. doi: 10.3389/fmars.2020.00203 In this study, we investigated the different photoregulation responses of diatom dominated natural biofilms to different light intensities and wavelengths, over a tidal cycle in the laboratory. We compared the overall effect of light spectral quality from its light absorption (Qphar) dependent effect. Two different conditions were compared to study photoprotective strategies: sediment (migrational) and without sediment (non-migrational). Three different colors (blue, green, and red) and two light intensities (low light, LL at 210 µmol.photons.m−2 .s−1 and high light, HL at 800 µmol.photons.m−2 .s−1 ) showed strong interactions in inducing behavioral and physiological photoprotection. Non-migrational biofilm non-photochemical quenching (NPQ) was much more reactive to blue HL than red HL while it did not differ in LL. We observed a biphasic NPQ response with a light threshold between 200 and 250 µmol.photons.m−2 .s−1 of Qphar that elicited the onset of physiological photoprotection. Similar HL differences were not observed in migrational biofilms due to active vertical migration movements that compensated light saturating effects. Our results showed that within migrational biofilms there was an interaction between light quality and light intensity on cell accumulation pattern at the sediment surface. This interaction led to inverse diatom accumulation patterns between blue and red light at the same intensity: LL (blue + 200.67%, red + 123.96%), HL (blue + 109.15%, red + 150.34%). These differences were largely related to the differential amount of light absorbed at different wavelengths and highlighted the importance of using wavelength standardized intensities. Different vertical migration patterns significantly affected the total pigment content measured at the surface, suggesting that cell could migrate downward more than 2 mm as a photoregulatory response. Colloidal carbohydrates patterns paralleled the vertical migration movements, highlighting their possible role in diatom motility. Our data strongly suggests a wavelength and Qphar dependent light stress threshold that triggers upward and downward movements to position microphytobenthic diatoms at their optimal depth.

Keywords: diatoms, fluorescence, PAM, migration, photoprotection

Mycrophytobenthos dominated mudflats are highly productive coastal ecosystems (MacIntyre et al., 1996; Underwood and Kromkamp, 1999) and diatom biofilms forming at the sediment surface during each diurnal tidal cycle are often at the source of this high productivity (e.g., Paterson et al., 2003). These biofilms are also responsible for a range of important biogeochemical processes including sediment stabilization (Paterson, 1989; Yallop et al., 1994), modulation of oxygen availability (Walpersdorf et al., 2017), as well as influencing carbon turnover and availability through the production of polymeric exudates (Perkins et al., 2001; Taylor et al., 2013) which in turns affects microbial diversity (Haynes et al., 2007). Epipelic diatoms, i.e., motile diatoms that move around sediment particles (Round, 1965), exhibit marked vertical movements inside the sediment matrix (Round and Happey, 1965; Consalvey et al., 2004b) that can be divided in two types: an endogenous circadian cycle synchronized with daily emersion periods whereby cells migrate to the sediment surface forming biofilms; and smaller fine tuning vertical adjustments positioning cells at optimal light levels, functioning as a photo-regulation mechanism (Perkins et al., 2001; Jesus et al., 2006; Cartaxana et al., 2011; Serôdio et al., 2012). The circadian rhythms are endogenously controlled and are kept for several days in laboratory conditions without artificial tides and exposed to continuous light (Round and Happey, 1965; Paterson, 1986; Serôdio et al., 1997; Mitbavkar and Anil, 2004; Coelho et al., 2011). The smaller photo-regulation movements are one of the main photo-regulation mechanisms of epipelic diatoms (Jesus et al., 2009; Perkins et al., 2010; Cartaxana et al., 2011; Laviale et al., 2016), while epipsammic diatoms (strongly associated to sediment grains) depend mainly on the thermal dissipation of excessive light energy through the xanthophyll cycle (XC) to photo-regulate (Cartaxana et al., 2011; Barnett et al., 2015; Blommaert et al., 2018). The XC relies on the presence of a transthylakoidal proton gradient to deepoxidise diadinoxanthin (Diad) into its energy-dissipation form diatoxanthin (Diat). It presumably induces a conformational shift in the light harvesting antennas that leads to excess energy being dissipated by heat through a non-photochemical quenching (NPQ) process (e.g., Olaizola and Yamamoto, 1994; Lavaud et al., 2004; Lavaud and Goss, 2014). Several studies have tried to quantify the relative importance of the two photo-regulation mechanisms (e.g., Perkins et al., 2010; Serôdio et al., 2012), notably by using migration inhibitors (Cartaxana and Serôdio, 2008) to separate the photo-regulatory effect of the XC from the vertical movements. Overall, published data shows that microphytobenthos depend on both photo-regulation mechanisms but that the two strategies are strongly dependent on their life forms (i.e., epipelic vs. epipsamic) (Cartaxana et al., 2011; Barnett et al., 2015). A few studies have investigated the trade-off between vertical migration (VM) and NPQ (Perkins et al., 2010; Serôdio et al., 2012; Barnett et al., 2015; Laviale et al., 2016; Blommaert et al., 2018). Diatoms movements have been differentiated in phototaxis and photokinesis (Nultsch, 1971; Häder, 1986). The former describing an oriented movement toward or away from a light source and the latter describing the speed of the movement as a response to light fluence rate (McLachlan et al., 2009). Both positive and negative phototaxis, toward and away from light, have been reported for different diatoms and different light intensities (Nultsch, 1971; Cohn et al., 1999; Du et al., 2010; Ezequiel et al., 2015). Benthic diatoms, in the absence of light cues, may also exhibit negative gravitaxis inducing upward movements to the sediment surface (Frankenbach et al., 2014). Most studies on diatom movement, growth and photosynthetic properties have investigated the effect of light intensity regardless of light spectra (Perkins et al., 2001; Paterson et al., 2003; Underwood et al., 2005; Jesus et al., 2006; Chevalier et al., 2010; Jauffrais et al., 2015). Light intensity is a wavelength weighted energy measurement that refers to the total number of photons received per unit area in a given time. Its use has been widely spread in photosynthetic studies because photosynthetic photochemical reactions are driven by total amount of photon received rather than the amount of energy received by each photon (Kume, 2018). In the aforementioned studies (Perkins et al., 2001; Paterson et al., 2003; Underwood et al., 2005; Jesus et al., 2006; Chevalier et al., 2010; Jauffrais et al., 2015) light has been integrated over the photosynthetic active radiation (PAR) spectrum, not accounting for the spectral variations within the light spectrum. However, these changes in light composition can affect diatom photo-protective capacity and photo-acclimation to high light intensities (Phaeodactylum tricornutum) (Schellenberger Costa et al., 2013a; Brunet et al., 2014). Light composition can also affect locomotion speed thereby affecting diatom vertical migration (Wenderoth and Rhiel, 2004; McLachlan et al., 2009). Spectral differences affect the relative proportion of photons available at each wavelength per unit area in a given time. In turn this modify on one hand the total amount of energy and on the other hand specific interactions, i.e., absorptions, with different structures such as diatoms' pigments and chromophores. It is unknown whether light quality changes only modulate the amount of light being absorbed by diatoms or if there are other effect, possibly due to differential energy levels and other confounding factors. To differentiate between chromatic differential absorption and other effects we compared intensity with Qphar (Gilbert et al., 2000) which is the photosynthetically absorbed radiation weighing in specific absorption coefficients of the major pigments. Currently, no studies exist on the effect of light quality regarding the two main diatom photo-regulation mechanisms. The objective of the current study was to investigate the effect of light spectral composition and light intensity on diatom physiological and behavioral photo-regulation mechanisms throughout a tidal cycle, using MPB assemblages in natural sediment and assemblages immobilized on petri dishes. Light quality effects will firstly be studied from an intensity perspective before considering their effect from an absorbance point of view.

# MATERIALS AND METHODS

# Site and Sampling

The sampling site was located in La Couplasse mudflats (Bourgneuf Bay, 47.015753, −2.024148) and sampling was carried out on February 2018 during low tide. It is a nonprotected area that did not require a permit for this kind of biological material sampling. Superficial sediment (upper 5 mm) was scrapped and transported back to the laboratory where it was spread out evenly in trays and submerged with water from the site in a room with natural lighting, allowing for the reconstitution of the biofilm the next day. The sediment at this site is dominated by benthic epipelic diatoms (e.g., Hernández Fariñas et al., 2017).

# Experimental Setup

fmars-07-00203 April 19, 2020 Time: 13:44 # 3

Water was drained from the trays on the day after the sampling and 3 h prior to the expected low tide we added two layers of lens tissue on top of the sediment. The sediment was then exposed to natural light from a window to facilitate biofilm vertical migration. Lens tissues were recovered at the middle of the virtual emersion time (low-tide peak), thereby harvesting the microalgae that had migrated to the sediment surface during the first 3 h (Eaton and Moss, 1966). Cells were extracted from the lens tissues by gently scrapping them into site-filtered sea water and kept in darkness before further use. On the second day, 5 h before expected low tide and 2 h before the beginning of the experiment 48 black microplates wells (22 mm diameter and 18 mm depth) were filled with either the same mud used for extraction or clean lens tissue inoculated with algal suspension. Mud samples contained both migratory and non-migratory diatoms from the field. Half were filled with one lens tissue layer and inoculated with 2 mL of algal suspension; the other half was filled with mud (15 mm) and 0.5 mL of algal suspension. To facilitate handling the 12 wells microplates were cut in rows of 4 wells containing each 3 replicates and a dark control (**Figure 1**). Mud and lens tissue samples will henceforth respectively be referred as migrational and non-migrational biofilms.

# Light Treatments

LEDs light sources (SL 3500 – PSI) were calibrated with a light sensor (MSC15 – Gigahertz-Optik) to deliver 800 µmol.photons.m−2 .s−1 from a 30 cm distance onto the microplates. Three monochromatic lights were used to set up three light conditions: Red (635 nm, FWHM 20 nm), Green (528 nm, FWHM 34 nm) and Blue (444 nm, FWHM 20 nm). To produce low intensities condition neutral filters were applied on each color to reduce light intensity to 210 µmol.photons.m−2 .s−1 . Dark condition was obtained by covering microplate wells with dark tape. All treatments were replicated three times.

# PAM Chlorophyll Fluorescence

Fluorescence was measured with an imaging-PAM (WALZ – Germany) using blue Luxeon LEDs (450 nm) for both actinic illumination and saturating pulses. Absorptivity was measured with the same instrument using a different set of incorporated LEDs, red (660 nm) and infrared (780 nm) performing a pixel by pixel comparison of images recorded under the two lights using the equation: Absorptivity = 1 – (Red/Infrared). The microplates were placed at 20 cm from the camera (CCD IMAG-K4) to optimize lens focus and illumination. Fluorescence measurements consisted of rapid light curves (RLC) from which all photosynthetic parameters were calculated. RLCs were carried in sequences, starting with migrational 800 µmol.photons.m−2 .s−1 RGB followed by nonmigrational 800 µmol.photons.m−2 .s−1 RGB then migrational 210 µmol.photons.m−2 .s−1 RGB and finally non-migrational 210 µmol.photons.m−2 .s−1 RGB. The time elapsed between the first and last measurement was 25 min. The first RLC measurement (T0) was done 3 h before expected low tide and in darkness, it was followed by three time measurements: T1, done 1.5 h before low tide and after 1.5 h of illumination; T2, synchronized with low tide and after 3 h of illumination; and T3, 1.5 h after low tide with 4.5 h of illumination. RLCs consisted of 12 lights levels (0, 4, 18, 41, 74, 142, 279, 518, 745, 986, 1302, 2342 µmol.photons.m−2 .s−1 ) with 30 s steps. RLC parameters α-slope and rETRm were estimated by fitting Silsbe and Kromkamp (2012) modified equation of Eilers and Peeters (1988).

$$
\phi I I\_i = \frac{Fm^{'} - F^{'}}{Fm^{'}} \tag{1}
$$

$$\text{s}\Phi II(E) = rETRm + \alpha - \text{slope} + \frac{\alpha - \text{slope}}{E \stackrel{\*}{\text{E}} \stackrel{\*}{\text{E}} \text{exppt \*}(\text{Eopt} \stackrel{\*}{\text{E}} - 2)} \tag{2}$$

Where F 0 and Fm0 are respectively the fluorescence yield under actinic light and the maximum fluorescence yield after a light saturating pulse at each RLC step; E is the light intensity in µmol.photons.m−2 .s−1 ; φIIi is the photosystem II effective quantum efficiency at each i steps of the RLC. φII1 correspond to the optimal photosystem II quantum efficiency measured during the first step of the RLC. φII(E) refers to lightdependent changes of φII. From fitting φII(E) the following photosynthetic parameters were estimated: the initial slope of the RLC (α-slope) under limiting irradiance, the maximum relative electron transport rate of the RLCs (rETRm) and the optimal light parameter (Eopt) of φII versus E curves. Eopt corresponds to the irradiance (µmol.photons.m−2 .s−1 ) at which ETR saturates and where further increment of light induces photoprotection. Continuous light exposure prevented measurements of the fully oxidized photosystem II at the different time of the tide. In these conditions direct NPQ measurements proved to be impractical due to the cofounding effect of NPQ relaxation and migration. Instead α-slope was used as a proxy for NPQ induction and relaxation. It has been shown to be inversely proportional to NPQ allowing to trace short-term changes in NPQ levels without dark incubation (Cruz and Serôdio, 2008).

# Pigments

At the end of the experiment (T3) the migrational samples were sampled by freezing with liquid nitrogen and contact cores (1 cm diameter and 2 mm deep). They were then stored in a −80◦C freezer and freeze dried before further processing. Pigment extraction was performed in a dark chamber using acetone (90%), sonicated for 90 s, followed by overnight extraction at 4◦C. HPLC analysis was performed according to Van Heukelem method (Heukelem and Thomas, 2001) using a 1 ml/min flow for 36 min with methanol (solvent B) and

methanol/TBAA-28 mM (70/30 – solvent A) going from 5 to 95% of solvent B and vice versa for solvent A. Injection volume was 100 µL with 28.5 µL sample and 71.5 µL TBAA 28 mM. An Agilent Eclipse XDB-C8 column was used at 60◦C. Detection was done at 450 and 665 nm using a diode array detector. Approximately 0.17 g of sediment and 1.5 ml of solvent were used for each sample. Identification and pigment concentrations were calibrated using the response factors (RF) of pigments standards (DHI-Danemark). Data analysis was conducted on relative percentage of pigments. The de-epoxidative state (DES) was calculated as Diat/(Diat + Diad). Pigment determination was not performed on non-migrating biofilms due to lack of biological material for the different analysis.

# Absorption Spectra

To compare whether differences in light color responses were due to total light absorbed or other chromatic effect a pigment weighted light absorption (Qphar) was calculated. Qphar(λ) correspond to the amount and proportion of light absorbed by the cells at different wavelengths. It is calculated according to a modified Eq. (4) of Gilbert et al. (2000). The specific in vitro absorption coefficients of microphytobenthos samples ampb were reconstructed according to Eq. (3).

$$a\_{mpb}(\lambda) = \sum\_{i}^{n} a\_{i}(\lambda)^{\*} C\_{i} \tag{3}$$

$$Qphar(\lambda) = Q(\lambda) - (Q(\lambda)^\* e^{-a\_{\mathbb{H}}(\lambda)}) \tag{4}$$

a(λ) is the concentration specific absorption coefficient in m2 g −1 , its values were obtained from the literature (Clementson and Wojtasiewicz, 2019). C is the pigment specific average concentration within all contact cores in mg.g−1 . Q(λ) is the photosynthetically available radiation at the top of the sediment or in lens tissue in µmol.photons.m−2 .s−1 . Pigments used for reconstruction of the light absorption spectrum were chlorophyll a, chlorophyll c2, fucoxanthin, β-carotene, diadinoxanthin and diatoxanthin.

# Carbohydrates and Proteins

At T3, migrational and non-migrational samples were frozen and stored at −80◦C for carbohydrate and protein quantification. Colloidal (soluble) fractions were extracted from thawed samples. They were first mixed in 2 mL of artificial sea water for 1.5 h (Orbital shaker, YELLOWline), then the supernatant was removed and the remaining sediment was mixed again with 2 mL of artificial sea water and 300 mg of Dowex Marathon Cex change resin to extract the bound (attached to sediment particles) fraction. Carbohydrate and protein quantification were done by spectroradiometry (Genesys 10S Uv/Vis) using the colorimetric reactions methods of DuBois et al. (1956) for carbohydrates and a modified Lowry method for proteins (Frølund et al., 1996).

# Species Identification

Algal suspensions were preserved with glutaraldehyde (4%) and sediment cores were frozen with liquid nitrogen, respectively stored at 4 and −80◦C. Three vials were filled with 1.5 mL of algal suspension while sediment cores were first placed in ludox ( R HS-40 coloidal silica) to separate cells from the

sediment and were then resuspended in a known volume of water and cleaned up following the protocol by Consalvey (2002). Succinctly, it consisted of a 24 h oxidation in saturated potassium permanganate solution, followed by the addition of hydrochloric acid for 2 h at 70◦C. Lastly, cells were rinsed seven times with ultrapure water in a final volume of 1.5 mL. A 50 µl subsample was permanently mounted on microscope slides using NaphraxTM. Cell counting, observation and identification of diatom valves were made with an optical microscope (Olympus). At 1000 magnification, 10 zones were chosen using randomly generated numbers and counted in five different slides until more than 400 individuals were counted. Identification was performed using the literature (Paulmier, 1997; Ribeiro, 2010; Mertens et al., 2014). Biovolumes were retrieved from the literature (Olenina et al., 2006; Ribeiro, 2010) and calculated according to morphologically based equations (Hillebrand et al., 1999; Olenina et al., 2006).

# Statistical Analysis

All statistical analyses were performed using R (R Core Team, 2017). Two-ways analysis of covariance (ANCOVA) were performed to compare datasets possessing a time variable with a linear regression. This comprised analysis of absorptivity, φII1 and α-slope over time for the different treatments of either same color or same light intensity. Pigment compositions were tested using a permutational multivariate analysis of variance using distance matrices based on Bray–Curtis dissimilarity index. Multivariate homogeneity of group dispersions was tested with a permutation-based test. Pigment and EPS differences between treatments were tested using two-way analysis of variance (ANOVA), after normality and homogeneity of variance had been verified using Shapiro–Wilk and Barlett tests, respectively. Individual differences were tested using Post hoc Tukey HSD tests. A principal components analysis (PCA) was used to visualize the pigment variance between treatments.

# RESULTS

# Species Composition

The dominant species in the non-migrational biofilms were: Navicula meulemansii (71.8%), Navicula spartinetensis (17.3%), Gyrosigma limosum (5.3%), Gyrosigma wansbecki (3.8%), and Pleurosigma angulatum (1.3%) (**Figure 2**). The migrational biofilm was dominated by Navicula meulemansii (30.42%), Thalassiosira cf. proschkinae (11.47%), Navicula spartinetensis (10.7%), Gyrosigma limosum (7.0%), Gyrosigma wansbeckii (5.7%), Thalassiosira cf. pseudonana (4.5%), Odontella aurita (3.74%), Raphoneis amphiceros (2.49%), Tryblionella sp. (2.0%), Coconeis speciosa (2.0%), Nitzschia cf. lorenziana (2.0%), Thalassiosira cf. angulata (1.7%), Pleurosigma angulatum (1.7%) (**Figure 2**).

# Photosynthetic Activity as a Function of Color-Dependent Light Intensity (Qphar)

Photosynthetic active radiation intensity is a widely used measure of standardized light dosage that can be compared across a variety of experiments. However, when light spectral composition is taken into account this measurement becomes flawed due to the fact that photosynthetic organisms absorb light differently, depending on their pigment composition. Thus direct comparisons of similar light intensities at different wavelengths are hindered by potentially different photoprotective reactions. Recalculating the total intensity absorbed at each wavelength (Qphar) produced six different intensities. These Qphar intensities were respectively 63.8 (R210), 76.2 (G210), 198.3 (B210), 255.02 (B800), 305.01 (G800) and 793.29 (B800) µmol.photons.m−2 .s−1 (**Figure 3**). Migrational and nonmigrationnal biofilms (**Figure 3**) displayed a Qphar threshold where alpha values switched from a NPQ relaxation state to a light induced NPQ production. Alpha is inversely proportional to NPQ intensity and a characteristic shift from values over 100% to values below 100% indicated a threshold where light started inducing physiological photoprotection throught NPQ increase. This threshold was observed between 200 and 250 µmol.photons.m−2 .s−1 of Qphar (**Figure 3**) effectively separating our treatments in low light (LL) when it is below 200 µmol.photons.m−2 .s−1 and high light (HL) when above 250 µmol.photons.m−2 .s−1 of Qphar.

# Vertical Migration Absorptivity Changes at the Surface

Absorptivity increased over time in all treatments subjected to light and decreased in the dark control (**Figure 4**). Light intensity had a significant effect on the absorptivity changes in red, blue and green wavelengths (ANCOVA, p < 0.05). Time also had a significant effect on absorptivity changes in red, green, blue, and dark treatments (ANCOVA, p < 0.05), with no significant interaction between time and light intensity. Within each set of light intensities there was also a significant effect of light color (ANCOVA, p < 0.001), and light color over time (ANCOVA, p < 0.05) which differed in the two conditions (HL and LL). Red and blue treatments showed different effects at the two light intensities, with blue absorptivity increasing 101% in LL and only 9% in HL; while red absorptivity increased 50% in HL and only 24% in LL (**Figure 4**). The dark control showed a decrease in absorptivity over time.

# Optimal PSII Quantum Efficiency (φII1)

On average, optimal PSII quantum efficiency (φII1) in a dark adapted state at T0 were significantly higher (p < 0.001 t-test) for migrational biofilms (mean = 0.607) than non-migrational biofilms (mean = 0.401). Non-migrational treatments were characterized by a φII1 increase in LL and φII1 decrease in HL, with significant effects of time (ANCOVA, p < 0.01), light intensity (ANCOVA, p < 0.001) and intensity over time (ANCOVA, p < 0.001). Non-migrational biofilms φII1 was affected differently by the light color in LL and HL treatments (**Figure 5**). With the exception of dark and red, φII1 in HL showed significant differences between colors (p < 0.001), time (p < 0.001) and color over time (p < 0.001). Contrastingly, φII1 in LL samples increased significantly over time (p < 0.001) and remained constant in dark controls. There were no significant

differences in LL φII1 between colors and colors over time (**Figure 5**). φII1 of all migrational biofilms increased in LL and decreased in HL, with significant effects of time (p < 0.001), intensity (p < 0.001), and intensity over time (p < 0.001) (**Figure 5**). All migrational HL biofilms (apart from control) showed a significant φII1 decrease over time (p < 0.001) and no significant differences between colors and colors over time. With the exception of green and blue samples, the LL migrational φII1 differed significantly between color treatments with significant effect of color (p < 0.001), time (p < 0.05), and color over time (p < 0.01).

# RLC Parameters

Changes in α-slope were strongly correlated with φII1 (Pearson correlation of 0.99, p < 0.001) with α-slope increasing in all LL treatments and decreasing in all HL treatments over time (**Tables 1**, **2**). There was a significant effect of light intensity and light color in all α-slope values that were always significantly different from the dark controls over time (ANCOVA, p < 0.001) (**Figure 6**). α-slope significantly decreased with time in HL migrational biofilms and significantly increased with time in migrational LL biofilms (ANCOVA, p < 0.001), with no significant differences between color and light color over time (**Figure 6**). In migrational LL biofilms only red samples showed significantly higher α-slope values than the other two colors (ANCOVA, p < 0.05). The biggest significant difference in α-slope values was observed in non-migrational HL samples were α-slope significantly decreased between colors (p < 0.001), over time (p < 0.001) and between colors over time (p < 0.001) (**Figure 6**).

Changes in rETRm over the course of the experiment were strongly influenced by light color and light intensity (**Tables 1**, **2**). In all samples and all conditions rETRm systematically increased from T0 to T1 and then depending on the conditions either continuously decreased or increased. LL non-migrational biofilm was the only condition where rETRm kept increasing throughout

FIGURE 3 | α-slope changes for differing Qphar intensities. α-slope are displayed as percentages of the initial value prior to illumination. Qphar recalculated intensities are 63.8 (R210), 76.2 (G210), 198.3 (B210), 255.02 (R800), 305.01 (G800), and 793.29 (B800) µmol.photons.m−2 .s−1 . The area below 200 µmol.photons.m−2 .s−1 (yellow) correspond to non-stressful light conditions where NPQ is relaxing while the area above 250 µmol.photons.m−2 .s−1 (purple) correspond to stressful light inducing NPQ. The boundary and threshold between LL Qphar and HL Qphar is represented in pink. n = 3 for each point.

the whole experiment (with the exception of blue T3). rETRm color treatments were significantly higher than dark controls (p < 0.01) while not significantly differing between each color. On the contrary in HL all treatments significantly differed (p < 0.01) with the exception of red and dark. At the end of the light exposure in non-migrational biofilms there was a significant effect of intensity (p < 0.001) on rETRm values exemplified by a 44% decrease in blue HL and a 224% increase in LL compared to initial values. In all migrational biofilms rETRm increased to their maximum value at T1 and then slowly decreased over

TABLE 1 | Photosynthetic parameters and φII1 values obtained from rapid light curve fitting of φII(E) within the non-migrational biofilm.

Equation derived from Silsbe and Kromkamp (2012) modified equation of Eilers and Peeters (1988). The entire well was used as a region of interest. n = 3. ± : standard deviations.

time. Only red light displayed a significant rETRm difference between intensities (p < 0.05). Overall blue light treatments had a significant higher rETRm than samples of the same intensity with the exception of red LL.

# Pigments and EPS

Mean total pigment content is a proxy of the biomass within the 2 upper mm (**Figure 7**). At T3 the mean total pigment content (**Figure 7**) resembled the patterns observed in absorptivity measured at T3 (**Figure 4**) that were marked by an interaction of light quality and light intensity (p < 0.05), producing opposite effects at LL and HL. In the LL treatment, the highest mean total pigment content was found under blue light while the lowest content was found in red. The opposite trend was observed in HL with the highest mean total pigment content observed under red light and lowest in blue light.

The average pigment composition of migrational biofilms at the end of the experiment was: chlorophyll a (Chl a, 40.8%), fucoxanthin (Fuco, 37.8%), diadinoxanthin (Diad, 8.8%), chlorophyll c1 (Chl c1, 3.8%), diatoxanthin (Diat, 2.54%),

TABLE 2 | Photosynthetic parameters and φII1 values obtained from rapid light curve fitting of φII(E) within the migrationnal biofilm.

Equation derived from Silsbe and Kromkamp (2012) modified equation of Eilers and Peeters (1988). The entire well was used as a region of interest. n = 3. ± : standard deviations.

chlorophyll c2 (Chl c2, 2%), alpha and beta-carotene (αβ car, 1.9%), chlorophyll b (chl b, 1%) and antheraxanthin (anth, 0.8%). Color, light intensity and their interaction had a significant effect (p < 0.001) on the relative percentage of pigments (**Figure 8**). The PCA results (**Figure 8**) show that the two main axes explained 57.5% of the variance between pigments, with color treatments aligned along the two axes. Light intensity accounted for 23.1% of the total variance (Between Class Analysis) with significant percentage differences in Diat (p < 0.001), Diad (p < 0.01), Fuco (p < 0.01), and Chl c1 (p < 0.01). A post hoc test showed a higher percentage of Diat (p < 0.001) in all HL treatments (**Figure 8**).This was further exemplified by the significant higher (p < 0.001) DES (**Table 3**) in HL compared to LL. Color treatments accounted for 27.4% of the total variance with significant pigment percentage differences of Chl a (p < 0.001), Fuco (p < 0.001), Anth (p < 0.001), Diad (p < 0.01), and Diat (p < 0.01). Post hoc tests showed significant higher Chl a pigment percentage in blue treatments compared to red ones (p < 0.001) and higher Fuco percentage in red light (p < 0.05) compared to the other two colors. The interaction effect of color and intensity accounted for 67.3% of the total variance with significant pigment percentage differences of Fuco (p < 0.01), Diad (p < 0.01), Anth (p < 0.05), Diat (p < 0.01), Chl a (p < 0.05). Amongst these interaction effects there was a significantly lower Diat (p < 0.01) in blue HL compared to the other two colors.

Non-migrational biofilms showed significant differences in colloidal carbohydrates (CC) with higher CC content observed in HL (p < 0.05) (**Figure 9**). In HL, there was no significant difference between colors for any carbohydrate fraction nevertheless the variance was very high in blue and red CC. Overall, the carbohydrate contents measured in nonmigrational biofilms were very small, which led to values below the detection threshold in bound carbohydrates (BC) and to high variability in CC. There were no significant differences between treatments and controls for all protein measurements (data not shown). The average protein concentration was 0.0114 (± 0.0025) µg.mm−2 for non-migrational biofilms and 0.0608 (± 0.010) µg.mm−2 for migrational biofilms.

Migrational biofilm CC contents were significantly different between color (p < 0.001), light intensity (p < 0.05) and their interaction (p < 0.05) (**Figure 9**). All color treatments with the exception of red LL had significantly higher CC contents than the dark controls (p < 0.05). The interaction effect led to contrasting results at the different light intensities with no significant CC differences between colors in HL while at LL lower CC concentrations were observed in red in comparison to blue (p < 0.01). The BC concentrations of the migrational biofilms were also significantly affected by color (p < 0.001), with blue treatment having higher BC contents than dark control (p < 0.05) in both LL and HL while red treatment only had a higher BC contents than dark control under HL intensities (p < 0.05).

# DISCUSSION

Both light intensity and light quality had a significant effect on diatom photo-regulation mechanisms (migration and NPQ), with clear differences being observed between migrational and non-migrational biofilms, confirming that vertical migration movements within the sediment matrix plays a major role in diatom photo-regulation.

# Species Composition

The diatoms that were present in both migrational and nonmigrational biofilms were typical epipelic diatoms from the Bourgneuf bay site, similar to the assemblages observed by Méléder et al. (2007). Navicula spartinetensis is found exclusively in intertidal muddy sediments and is one of the most commonly found epipelic species in European Atlantic coasts (Ribeiro, 2010). The only species that had not yet been described for this site was Navicula meeulmansii. However, N. meeulmansii has only recently been described as a cosmopolitan species tolerating wide range of salinities (Mertens et al., 2014), which

may have been previously described as Navicula phyllepta or Navicula microdigitoradiata in the other studies from this ecosystem (Méléder et al., 2007; Hernández Fariñas et al., 2017). These epipelic diatoms are known to photo-regulate using both physiological and behavioral photoprotection (Cartaxana et al., 2011; Laviale et al., 2015). The species differences observed between the two treatments were mainly related to the non-migrational nature of Thalassiosira cf. proschkinae, Thalassiosira cf. pseudonana, Thalassiosira cf. angulata, Odontella aurita, Raphoneis amphiceros, Coconeis speciosa, and Nitzschia lorenziana which will not be captured by our sampling method (Eaton and Moss, 1966). Nevertheless, the two biofilms were strongly dominated by migratory species: Navicula meulemansii, Navicula spartinetensis, Gyrosigma limosum, Gyrosigma wansbecki and Pleurosigma angulatum and their biovolumes were strongly dominated by Gyrosigma limosum, Gyrosigma wansbecki and Pleurosigma angulatum that, albeit present only in relatively small numbers, correspond to 18.5, 48.1, 22.6 and 17.3, 51.7, 22.2% of the total biovolume (non-migrational and migrational, respectively).

# Physiological Photoprotection

In the non-migrational biofilms cells were artificially constrained to immobility and thus had to rely solely on physiological photoprotection to cope with changing light environment. This immobility was characterized by an increase in φII1 and α-slope in the LL condition (**Figures 5**, **6**). A φII1 increase indicates that a higher proportion of absorbed light was being used for photosystem II (PSII) photochemistry implying a concomitant

decrease in NPQ from the start of the experiment. This is coherent with the α-slope increase observed in the LL nonmigrational biofilms (**Figure 6**) as RLC α-slope increases have been shown to be proportional to NPQ reversal (Cruz and Serôdio, 2008). An α-slope increase over time indicates that a significant amount of NPQ had accumulated overnight in all conditions and was being dissipated over the course of the experiment in the LL conditions. Diatom dark NPQ induction has been previously observed and attributed to a transthylakoidal pH-gradient dependent activation of the XC cycle, due to chlororespiration or to the reverse operation of ATP synthase (Ting and Owens, 1993; Jakob et al., 1999; Dijkman and Kroon,

2002; Mouget et al., 2004). The decrease of NPQ and the consequent increase of both α-slope and φII1 under LL could be explained by the gradual dissipation of the transthylakoidal proton gradient under low fluence rates (Consalvey et al., 2004a; Serôdio et al., 2005). This NPQ dissipation attests that the LL intensity condition was indeed perceived, for each color, as a non-stressful factor and did not require the development of photoprotective mechanisms hence a gradually increasing rETRm (**Table 1**).Within the LL treatments there was no significant differences between NPQ dissipation despite varying Qphar values (**Figure 3**, non-migrational) indicating that below 200 µmol.photons.m−2 .s−1 of Qphar, NPQ build up was not modulated by the amount of light absorbed. This was further exemplified by the absence of noteworthy differences in EPS that were characterized by the same amount of carbohydrates (**Figure 9**) and proteins. Under HL exposure in non-migrational biofilms photosynthetic parameters φII1 and α-slope showed less NPQ induction under red light (**Figure 6**) with no significant differences between red and dark treatments. Comparatively, green and especially blue light induced much more NPQ buildup in non-migrational HL samples (**Figure 6**). CC concentrations were also significantly higher in HL than LL. These changes in CC production/secretion could reflect a need to readjust the carbohydrate:protein ratio within the cell as an "overflow metabolism" that helps coping with the stressful environmental conditions imposed by exposure to high light intensities (Staats et al., 2000; Orvain et al., 2003; Underwood et al., 2004; Takahashi et al., 2009). At HL there was a strong inverse correlation of r = −0.92 (p < 0.001) between α-slope and Qphar (**Figure 3**). The same correlation was not observed between CC and Qphar as CC content were not significantly different in HL (**Figure 9**). The physiological role of CC in photoprotection therefore seemed limited to whether or not NPQ was induced but not proportional to the amount of NPQ induction. There was a threshold between 200 and 250 µmol.photons.m−2 .s−1 of Qphar where light started inducing physiological photoprotection. Despite not having pigment data for the non-migrational biofilms, α-slope and φII1 values observed in HL non-migrational biofilms suggested that blue light, notably due to its higher Qphar value would have had a higher effect in inducing the xantophyll cycle as observed by the strong decrease in α-slope (**Figure 6**) and rETRm (**Table 1**), in comparison to the other two light colors. Previous observations, using similar Qphar intensities (Schellenberger Costa et al., 2013a) or using wavelength dependent absorption cross-section of PSII (PSII effective quanta.s−1 ) (Schreiber et al., 2012), have shown that despite these standardization growing diatoms in blue light induced significant higher NPQ and ETRm values compared to red light (Schreiber et al., 2012; Schellenberger Costa et al., 2013a,b; Jungandreas et al., 2014). Diatoms possess a number of photoreceptors, including blue light sensing aureochrome and red/far-red light sensing phytochrome, hypothesized to mediate photoprotective

Pigments were sampled on the top 2 mm of the sediment using contact cores. n = 3. ± : standard deviations.

responses (Depauw et al., 2012 and reference therein). The blue photoreceptor Aureochrome 1a was found to repress HL acclimation of Phaeodactylum tricornutum in both blue and red light implying a mediated pathway between these lights and aureochrome (Schellenberger Costa et al., 2013b). Similarly, but without correcting for wavelength specific absorption, Brunet et al. (2014) found that Pseudo-nitzschia multistriata could not induce HL acclimation with monochromatic wavelengths and required both blue and red light sensing to regulate DES and NPQ production. All aforementioned studies used monospecific culture grown under very different light regime favoring long term acclimation. To our knowledge the effect of light quality on physiological photoprotection has not been investigated on scales of minutes to hours making comparison between studies extremely challenging. Besides, specific absorption and Qphar measurements are highly variable, species specific and susceptible to changes due to DES variation (Fujiki and Taguchi, 2001), cell volume (Fujiki and Taguchi, 2001), "pigment package" effect (Bidigare et al., 1990) and in vivo bathochromic shifts (Bidigare et al., 1990). Ideally Qphar should be monitored continuously but is not the only chromatic effect of light and may sometime prove to be unadapted to quantitatively measure light. For instance, frustule nanostructure may change upon exposure to different monochromatic wavelengths (Su et al., 2015) consequently modifying photonic properties either in increasing blue light absorption by the frustule (Yamanaka et al., 2008) or in increasing blue light scalar irradiance thus enhancing the effective intensity of blue light (Goessling et al., 2018). Such effect would not be integrated in Qphar calculation. Consequently, it is uncertain whether different physiological responses at different color wavelengths were only a reflection of different Qphar values or if other chromatics differences would exist.

# Biofilm Movement

Light quality effects on migration were strongly dependent on light intensity, with certain wavelengths inducing variable migration responses. Namely, blue and red colors induced intense upward migration movements in LL and HL, respectively. A stronger migration response and diatom accumulation in blue light has been supported by Nultsch (1971) early work on Nitzschia communis as well as McLachlan et al. (2009) on Navicula perminuta where wavelengths up to 540 µm induced

positive phototropism while red wavelengths did not. Similarly, Wenderoth and Rhiel (2004) observed cell accumulations 1.8 times higher in blue compared to white light (low fluence rate 5 µmol.photons.m−2 .s−1 ). The cited studies were done at low light levels and are consistent with the absorptivity increases we observed in blue LL in comparison to green, red and dark (**Figure 4**), supporting the hypothesis that at low fluence rates blue wavelengths are more efficient at stimulating diatom movements. In diatoms, several light sensors have been evidenced and hypothesized to be involved in the light quality responsive changes in motility (McLachlan et al., 2009; Cohn et al., 2015). Changes in light intensities have been shown to be most sensitive in modulating the whole cell movement near the distal ends of the raphes (Cohn et al., 1999, 2015) and a putative photo-detection system could involve an aureochrome. However, biochemical and functional characterization of these photoreceptors and their chromophores is still very limited (Depauw et al., 2012; Wilhelm et al., 2014). The existence of different species specific motile responses to light quality (Cohn et al., 2015) as well as the lack of current identified receptors at the tips of the cells hinder further conclusions on the role of these photoreceptors. The high correlation, observed in LL, between absorptivity and Qphar (r = 0.74, p < 0.001) suggests that the different migrational responses were a response to the amount of Qphar rather than selective effect of different wavelength and their specific interaction with photoreceptors. Changes observed in absorptivity (**Figure 4**) were paralleled by mean total pigment content (**Figure 7**) for different intensity and treatments. Although this correlation (r = 0.75) was not statistically significant (p = 0.09) it could indicate that the absorptivity parameter, which was measured at the sediment surface, was linked to the biomass (pigments) measured in the 2 mm deep contact core. This strong correlation could imply that cells would have migrated deeper than 2 mm in the lower absorptivity samples. Diatom speeds have not been often measured in natural sediments but the few available datasets supports the possibility that diatoms could have migrated more than 2 mm during the course of the experiment (4.5 h). Namely, using values presented by Hay et al. (1993) the biofilm would have been able to move at least 2.7 mm in the 4.5 h. The downward migration in blue HL could be related to a light-stress response due to higher Qphar values resulting in less accumulation of cells at the sediment surface. This light stress would be accompanied by an increase in NPQ build-up, which would induce a photoprotective downward response or a reduced upward migration in blue light from the start of the experiment. This would be consistent with previous observations that have described light thresholds between 500 and 1000 µmol.photons.m−2 .s−1 for inducing an avoidance response for both blue (450 nm) and green (550 nm) lights (Cohn, 2001). Furthermore, there is a general consensus that diatom photoaccumulation and photodispersal are intensity dependent whereby low to moderate white light stimulate upward migration and high light promotes downward migration (Serôdio et al., 2008; Du et al., 2010, 2018; Perkins et al., 2010; Coelho et al., 2011; Laviale et al., 2016). The absorptivity changes followed a typical biphasic dose response to Qphar shifting from a positive phototaxis at a theoric maximum between 200 and 250 µmol.photons.m−2 .s−1 to a negative or downregulated positive phototaxis. Our hypothesis is that above 250 µmol.photons.m−2 .s−1 of Qphar the onset and amount of NPQ – proportional to Qphar – (**Figure 3**) downregulated behavioral positive phototaxis. However, the light intensity thresholds and the spectral limits described in previous works are very variable and it is possible that the recorded differences are partially the result of the complexity of measuring light environments inside the sediment. Many factors will affect the light received by a diatom incorporated in microphytobenthic biofilms, e.g., organic matter, type of sediment particles, biofilm density, species composition, water content, etc. All these factors will produce different absorption and scattering effects that will affect the intensity and light spectral quality (e.g., Kühl et al., 1994). Nonetheless, regardless of light color, absorptivity increased significantly with time in both LL and HL treatments in comparison to dark controls, which suggests that light intensities were never high enough to completely inhibit upward migration in our conditions. Benthic diatoms are capable of positioning themselves in a light gradient at sediment depths where light exposure is optimal. This optimal depth may vary with photoacclimation status and light intensity tolerance tresholds that induce photoinhibition (Ezequiel et al., 2015) and our data supports the hypothesis that diatom optimal depth will vary as an interaction between light intensity and light quality to adjust to the optimal Qphar light.

# Behavioral and Physiological Interplay

The LL migrational biofilms showed a similar trend of increasing φII1 as the LL non-migrational biofilms albeit with less intensity (**Figure 5**). The smaller φII1 increase in migrational biofilms is likely due to an already higher absolute φII1 value at the onset of the experiment. φII1and α-slope in LL migrational biofilms increased significantly more under red light, perhaps as a positive tradeoff for having produced little CC (**Figure 9**) and migrated less than other colors (**Figure 4**). In LL intensities there was significant carbohydrate production in blue and green light which was not seen in the non-migrational biofilm. Colloidal carbohydrate concentration (**Figure 9**), mean total pigment (**Figure 7**) and absorptivity (**Figure 4**) followed the same pattern, being highest in blue and lowest in red. These similar patterns could be the result of polysaccharide secretion produced by diatoms during their vertical movements (Pniewski et al., 2015). At these non-stressful LL intensities photo-regulation was mainly dominated by behavioral positive phototaxi to increase light exposure as a response to increasing Qphar values. Exposing migrational biofilms to HL induced a decrease in α-slope and φII, which implied physiological photoprotection due to stress exerted by the light intensities. Analysis of the relative pigment percentage showed significant differences (**Figure 8**). HL samples were characterized by a significant higher Diat relative concentration and a lower relative concentration of Diad which is consistent with the xanthophyll cycle operation (Olaizola and Yamamoto, 1994; Lavaud et al., 2004). Colorwise there were no significant DES differences between color treatments (**Table 3**) despite very different Qphar amount. Similarly, at

HL in migrational biofilms, despite a differential migration between colors there were no significant differences in either α-slope (**Figure 3**) or EPS secretion for different colors. This implies that, even if blue light has the potential to induce an higher NPQ and DES (see physiological photoprotection discussion), diatoms can migrate to different sediment depths. Diatoms seemed to position themselves at an optimal depth as a response to the interaction between light intensity and spectral composition. At these depths they might maximize their CC production for both behavioral and physiological photoprotection, thus showing no obvious differences in CC concentration between the different colors (**Figure 9**). These result contrasts with previous observations (Perkins et al., 2001) that did not find significant colloidal carbohydrates accumulation differences at the end of the migration between shaded and unshaded biofilms. It is noteworthy to stress that samples from natural sediments will contain EPS from a range of sources (bacteria, detritus, and dissolved organic matter) (Smith and Underwood, 1998) and while our extraction technique has been proposed as a standardized method for microphytobenthos (Takahashi et al., 2009) other extraction techniques may have had different EPS fractions making comparison with previous studies difficult (Underwood et al., 1995, 2004; Smith and Underwood, 2000; De Brouwer and Stal, 2002). It is possible that blue and red photoreceptors are involved in tailoring the specific behavioral photoprotective responses. In natural conditions blue/red ratio is a very informative cue for diatoms, changing during tides, twilight and with depth (Ragni and D'Alcalà, 2004; Spitschan et al., 2016). However the exact pathway and signal cascading remain to be elucidated. We hypothesize that the trigger for the vertical migration movements and behavioral photoprotection is linked to an NPQ mechanism or some other light-stress induced mechanism, e.g., ROS production. This would be in agreement with other observations of diatoms using motility to select their optimal light exposure based on their photophysiological status (Ezequiel et al., 2015; Cartaxana et al., 2016) emphasizing the behavioral role of migration in regulating photosynthesis. Nevertheless, while migration compensated the different NPQ and DES levels observed in nonmigrational biofilm between the different color treatments we still observed higher rETRm values in blue light migrational biofilms (**Table 2**). These differences show that despite an active behavioral photoprotection mechanism, light specific wavelengths affected photosynthesis differently. Further investigation on the role of photoreceptors, wavelength specific chloroplast aggregation (Furukawa et al., 1998; Noyes et al., 2008) or frustule waveguiding properties (Shihira-Ishikawa et al., 2007) could shed some light on the underlying mechanisms between these two types of photoprotection.

# REFERENCES

# CONCLUSION

Overall there was a strong interaction between light intensity and spectral quality in inducing diatoms migration and behavioral photoprotection. The difficult task of disentangling the respective role of light inherent properties showcased how relevant considering both light quality and intensity helped understanding the underlying mechanisms of biofilm photoprotection. While not accounting for all chromatic effects of light, the use of Qphar proved to finely integrate and correctly correlate to the light quality and intensity interactions. The higher impact of blue light in stimulating ETR and NPQ development in comparison to red remains to be elucidated but was largely dependent on how much light was being absorbed. These differences are largely reduced in the sediment biofilms due to finely tuned vertical migration movements, supporting the hypothesis of epipelic diatom photoprotection being governed by behavioral mechanisms. Furthermore, it strongly suggests a wavelength and Qphar dependent light stress threshold that triggers NPQ development and consequently override or downregulate upward movements. Our data supports the hypothesis that diatoms accumulation and migration can extend deeper than 2 mm. The absence of light cues at these depths and the fast migration in a matter of few hours could have implication for future experimental design of microphytobenthos migrational studies.

# DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/supplementary material.

# AUTHOR CONTRIBUTIONS

AP, BJ, PD, and CH: conceptualization. AP, CH, and BJ: formal analysis, visualization, and writing – review and editing. AP and BJ: investigation and writing – original draft. BJ and CH: methodology and validation.

# FUNDING

This research was supported under the BIO-Tide project, funded through the 2015–2016 BiodivERsA COFUND call for research proposals, with the national funders BelSPO, FWO, ANR, and SNSF.

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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Prins, Deleris, Hubas and Jesus. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Vertical Migration Optimizes Photosynthetic Efficiency of Motile Cyanobacteria in a Coastal Microbial Mat

#### Mads Lichtenberg† , Paulo Cartaxana† and Michael Kühl\*

Marine Biological Section, Department of Biology, University of Copenhagen, Copenhagen, Denmark

#### Edited by:

Vona Meleder, Université de Nantes, France

#### Reviewed by:

Bo Barker Jørgensen, Aarhus University, Denmark Dirk de Beer, Max-Planck-Gesellschaft (MPG), Germany

### \*Correspondence: Michael Kühl

<EMAIL_ADDRESS>
#### †Present address:

Mads Lichtenberg, Costerton Biofilm Center, University of Copenhagen, Copenhagen, Denmark Paulo Cartaxana, Departamento de Biologia & CESAM & ECOMARE, Universidade de Aveiro, Aveiro, Portugal

#### Specialty section:

This article was submitted to Marine Ecosystem Ecology, a section of the journal Frontiers in Marine Science

Received: 24 September 2019 Accepted: 27 April 2020 Published: 25 May 2020

#### Citation:

Lichtenberg M, Cartaxana P and Kühl M (2020) Vertical Migration Optimizes Photosynthetic Efficiency of Motile Cyanobacteria in a Coastal Microbial Mat. Front. Mar. Sci. 7:359. doi: 10.3389/fmars.2020.00359 Microbial mats are diverse and stratified microbial biofilm communities characterized by steep gradients in light, temperature and chemical parameters. Their high optical density creates a competition for light among phototrophic microalgae and bacteria residing in the uppermost mat layers. Strategies to counter such resource limitation include metabolic investment in protective and light-harvesting pigments enabling exploitation of separate niches in terms of irradiance and spectral composition, or investment in motility to enable migration to an optimal light microenvironment. We used microsensor measurements of light, temperature and gross photosynthesis in coastal microbial mats dominated by motile cyanobacteria and colorless sulfur bacteria to study how migration affected their radiative energy-budgets and relative photosynthetic efficiency. The optical density of the microbial mat was extremely high, and >99% of incident irradiance of visible light (400–700 nm) was attenuated <0.4 mm below the surface. While energy conservation efficiency did not change dramatically with previous light acclimation, vertical profiles of photosynthetic efficiency showed a shift in the position of maximum efficiency of ∼0.2 mm, depending on light treatment. Besides avoidance of unfavorable chemical conditions such as high sulfide levels, vertical migration over short distances thus enable cyanobacteria to track zones with optimal light exposure thereby efficiently counteracting detrimental effects of excessive light at the surface and insufficient light deeper in the mat.

### Keywords: biofilm, light, microenvironment, microsensors, photosynthesis, cyanobacteria

# INTRODUCTION

Light-exposed coastal sediments in shallow waters and intertidal areas are often colonized by benthic microalgae and cyanobacteria, which under the absence of animal grazing (typically under environmental extremes such as desiccation, high salinity or sulfide levels) can form complex stratified microbial biofilm communities, i.e., microbial mats (Stal, 1995), that stabilize the sediment by excretion of exopolymers. Microbial mats are densely populated and highly compacted, vertically stratified microbial communities characterized by steep gradients of physical (light and temperature) and chemical parameters (Kühl et al., 1996; Dillon et al., 2009; Al-Najjar et al., 2012; De Beer and Stoodley, 2013). The uppermost layers of coastal microbial mat layers are typically dominated by diatoms on top of a dense green cyanobacterial layer that is often dominated

by Microcoleus chtonoplastes and various other motile, filamentous cyanobacteria (Wieland et al., 2003; Fourcans et al., 2004; Dillon et al., 2009). Often, purple sulfur bacteria and green filamentous anoxygenic phototrophs are found below the cyanobacteria followed by a reduced black layer of precipitated iron sulfide (Jørgensen, 1982). Besides light-driven sulfide oxidation by anoxygenic phototrophs, sulfide can also be oxidized efficiently by colorless sulfur bacteria such as filamentous Beggiatoa spp. (Nelson and Castenholz, 1981) that are motile and produce white patches in the microbial mat at the oxygen-sulfide interface (Jørgensen and Revsbech, 1983).

Light is the primary energy source for photosynthetic microbial mats. Due to the high density of photopigments, organic matter, and sediment particles, light is subject to intense scattering and absorption within microbial mats (Kühl and Jørgensen, 1994; Kühl et al., 1994). This can lead to an extremely narrow photic zone (Kühl et al., 1997) and a rapid change in spectral composition with depth (Lassen et al., 1992; Cartaxana et al., 2016b). Ploug et al. (1993) related changes in light quality in a coastal microbial mat to the vertical zonation of a population of diatoms over a dense filamentous cyanobacteria layer that largely sustained their oxygenic photosynthesis via phycobiliproteins with absorption characteristics complementary to chlorophylls. Similarly, complementary use of visible and near-infrared radiation by chlorophylls/phycobilins vs. bacteriochlorophylls enables coexistence of dense populations of oxygenic phototrophs on top of anoxygenic phototrophs (Kühl and Fenchel, 2000). Apart from light, other parameters such as nutrient availability or the presence of sulfide may vertically limit photosynthesis in microbial mats (Stal, 1995; Kühl et al., 1996; Wieland et al., 2003).

The ecological success of benthic microbes in optically dense and vertically stratified communities has recurrently been linked to cell motility allowing individual microbes to search for optimal environmental conditions regarding crucial parameters such as light, temperature, O2 or nutrient availability (Whale and Walsby, 1984; Bebout and Garcia-Pichel, 1995; Bhaya, 2004; Serôdio et al., 2006). Complex migratory rhythms determined by day/night cycles, tidal regimes, UV exposure and changes in irradiance levels have been described for both diatomand cyanobacteria-dominated phototrophic mat communities (Bebout and Garcia-Pichel, 1995; Serôdio et al., 2006; Coelho et al., 2011). Similar strategies to optimize photon capture are known in terrestrial plants, where the position of chloroplast in palisade and mesophyll layers in leaves can change depending on light levels and light field directionality, i.e., diffuse versus collimated light (Vogelmann, 1993; Gorton et al., 1999; Wada et al., 2003). Raphidic diatoms, filamentous cyanobacteria and Beggiatoa spp. are able to glide within an extracellular polymeric matrix at speeds of 0–10 µm s−1 (Glagoleva et al., 1980; Richardson and Castenholz, 1987; Hoiczyk, 2000; Gupta and Agrawal, 2007; Kamp et al., 2008; Tamulonis et al., 2011). Because of the steep light gradient, migration and the resultant vertical redistribution of the productive biomass have important consequences for both the photobiology of the phototrophs and the net primary productivity of the microbial mat ecosystem (Bebout and Garcia-Pichel, 1995; Cartaxana et al., 2016b).

Recent studies have focused on the efficiency with which light is utilized and converted to chemical energy via photosynthesis in cyanobacterial mats and mixed cyanobacteria-diatom biofilms (Al-Najjar et al., 2010, 2012). In these studies, relatively low photosynthetic efficiencies were estimated for microbial mats compared with ecosystems with a more open canopy-like organization such as coarse sediments (Lichtenberg et al., 2017), macroalgal stands (Sand-Jensen et al., 2007), corals (Brodersen et al., 2014), or terrestrial forest communities (Terashima et al., 2016), where light propagation is not hindered to the same extent by self-shading and photosynthetic inactive components. How the photosynthetic efficiency of biofilms and microbial mats is modulated by the migration of motile phototrophic populations remains to be studied in detail. In this study, we used fiber-optic scalar irradiance and field radiance microprobes in combination with O2 and temperature microsensors to investigate the radiative energy budget for the euphotic zone in a coastal microbial mat dominated by motile cyanobacteria and colorless sulfur bacteria. We investigated how photosynthetic efficiency of oxygenic phototrophs in the microbial mat was affected by changes in vertical biomass distribution imposed by acclimating the microbial mat to different light conditions.

# MATERIALS AND METHODS

# Sample Collection and Preparation

Microbial mats were collected from a periodically desiccated sand flat in Aggersund, Limfjorden, Denmark. The water level at the sample site is mainly determined by wind and local current patterns, and the mats can experience desiccation for extended periods. The sampled mats were dark green/black in appearance and were dominated by filamentous cyanobacteria (Microcoleus spp. and other motile morphotypes). Beneath the cyanobacterial band, a population of the sulfur bacteria Beggiatoa spp. was found. See Nielsen et al. (2015) for more details of the microbial mat and sampling site.

Mat samples were collected using small plastic trays (7 × 2 × 5 cm) and were brought back to a field laboratory (Rønbjerg Marine Research Station, Aarhus University, Denmark), where they were incubated submerged in seawater (20◦C; Salinity = 27) under a low photon irradiance (∼75 µmol photons m−2 s −1 ) of photosynthetically active radiation (PAR, 400–700 nm) provided by a halogen lamp. Within few hours of incubation, the mat turned dark green as motile cyanobacteria aggregated and extensive bubble formation from oxygenic photosynthesis appeared on the surface of the mat.

During measurements, the mat samples were transferred to a flow chamber (25 × 8 × 8 cm) that provided a stable laminar flow (∼2 cm s−1 ) of aerated seawater (room temperature = 21–23◦C; Salinity = 27) (see also Brodersen et al., 2014; Lichtenberg et al., 2017) that minimized bubble formation due to enhanced mass transfer between the microbial mat and water. The flow chamber was connected to a 25 L aquarium where water was recirculated through. Light was provided vertically from above by a white LED lamp (KL-2500 LED, Schott, Germany; color temperature of 5600K) equipped with a collimating lens. The incident irradiance

(400–700 nm) from the lamp was regulated electronically without spectral distortion. The downwelling photon irradiance was measured with a calibrated photon irradiance meter (ULM-500, Walz GmbH, Germany) equipped with a factory-calibrated photon irradiance detector (LI-192S, LiCor, United States). Incident spectral irradiance was also measured in radiometric units (W m−2 nm−1 ) with a calibrated spectroradiometer (Jaz ULM, Ocean Optics, United States). All sensors were mounted in a 45◦ angle (relative to the vertically incident light) on a motorized micromanipulator (MU-1, PyroScience, Germany), which could be controlled by the manufacturer's software (Profix, PyroScience, Germany). All measurements were performed under an incident photon irradiance (400–700 nm) of 1000 µmol photons m−2 s −1 as provided by the white LED lamp. This light level was chosen as a saturating irradiance based on a previously measured photosynthesis – irradiance curve (PI). For each profile a new horizontal position in a mat was randomly chosen. Prior to measurements, the vertical distribution of biomass was modulated by incubating the biofilm samples for at least 3 h in either darkness, low light (∼75 µmol photons m−2 s −1 ) or high light (1000 µmol photons m−2 s −1 ), respectively. Upon increasing the irradiance to 1000 µmol photons m−2 s −1 , scalar irradiance and gross photosynthesis were measured immediately, reflecting the systems immediate response to the new irradiance. Profiles of O2 and temperature were illuminated for 10 min at 1000 µmol photons m−2 s −1 before measurements, and we note that the mats were therefore not measured under steady state (see also discussion).

# Light Measurements

Spectral scalar irradiance was measured with a fiber-optic scalar irradiance microprobe (spherical tip diameter ∼70 µm) (Rickelt et al., 2016) connected to a fiber-optic spectrometer (USB2000+, Ocean Optics, United States). Spectral downwelling irradiance, Ed(λ), was measured with the microprobe tip positioned in a black, non-reflective, light-well at the same distance from the light source as the mat surface (see **Table 1** for definition of abbreviations). In the mat samples, spectral scalar irradiance, E0(λ) was measured in vertical increments of 0.1 mm. These measurements were then corrected for exposure time of the spectrophotometer and normalized to similarly corrected downwelling irradiance spectra yielding scalar irradiance transmittance spectra in different mat layers in % of Ed(λ), i.e., 100 · E0(λ)/Ed(λ ).

Spectral attenuation coefficients of scalar irradiance, K0(λ) were calculated for specific depth intervals in the microbial mats as (Kühl, 2005):

$$\mathcal{K}\_0(\lambda) = -\ln \frac{(E\_0(\lambda)\_1/E\_0(\lambda)\_2)}{z\_2 - z\_1}$$

where E0(λ)1 and E0(λ)2 are the spectral scalar irradiances measured at depth z1 and z2, respectively.

Similarly, depth profiles and attenuation coefficients of PAR, K0, were calculated from integrated values over the spectral region of interest. Light measurements <420 nm exhibited noisy signals and increasing amounts of stray light from within the detector and integrations were therefore carried out from 420–700 nm (henceforth mentioned as PAR). In deeper layers of the mat, integration of the measurements encompassed regions in the blue part of the spectrum exhibiting very noisy signals and an increasing contribution from stray light in the spectrometer, and these noisy signals were therefore excluded.

Reflectance of the microbial mat surface was measured with a fiber-optic field radiance miniprobe (flat cut tip diameter = 1 mm) connected to the same fiber-optic spectrometer used for scalar irradiance measurements. The PAR reflectance (RPAR) was calculated from the upwelling irradiance [Eu(λ)], here measured as the backscattered spectral radiance assuming Lambertian (diffuse) backscatter from the mat surface (Kühl and Jørgensen, 1994), and the downwelling irradiance [Ed(λ)] measured as the backscattered spectral radiance measured over a white Lambertian reflectance standard (99%; Spectralon, Labsphere, United States) as (Kühl, 2005):

$$R\_{PAR} = \int\_{420}^{700} \frac{E\_u(\lambda)}{E\_d(\lambda)} d\lambda$$

The acceptance angle of light collection through the fiber depends on the numerical aperture (NA) of the fiber and the refractive index of the medium. Since Ed(λ) was estimated in air, and Eu(λ) was measured in water we corrected for the acceptance angle differences by the relation 2a = sin−1 NA RIw , where 2a is the acceptance angle, NA is the numerical aperture of the fiber (0.22) and RIw is the refractive index of water (1.33).

# Microsensor Measurements of O2 Concentration and Gross Photosynthesis

Vertical depth profiles of O2 concentration were measured using Clark-type O2 microelectrodes (tip diameter = 25 µm, OX-25, Unisense A/S, Aarhus, Denmark) with fast response time (<0.5 s) and low stirring sensitivity (<1–2%) (Revsbech, 1989), connected to a pA-meter (Unisense A/S, Aarhus, Denmark) and interfaced through an A/D converter (DCR-16, PyroScience, Germany) to data acquisition software (Profix, PyroScience, Germany). The O2 signals were also recorded on a strip-chart recorder (BD 12E; Kipp & Zonen BV, Netherlands) connected to the pA-meter. Sensor signals were linearly calibrated at experimental temperature and salinity from measurements in the aerated free flowing water in the flow-chamber and in anoxic zones in the sediment. Depth profiles of O2 concentration were measured in 0.1 mm increments relative to the mat surface position determined by placing the sensor tip at the mat surface (z = 0 mm), as observed through a USB microscope (AM7013MZT Dino-Lite, AnMo Electronics Corporation, Taiwan). The sensor tip was then moved to 1.5 mm above the mat surface (z = −1.5 mm) and from here profiles were measured in steps of 100 µm until reaching anoxic mat layers (z∼1.5 mm).

The volume-specific rate of gross photosynthesis [PS(z) in nmol O2 cm−3 s −1 ] was measured using the light/dark shift method (Revsbech and Jørgensen, 1983) in vertical steps of 0.1 mm starting from just above the microbial mat surface until the depth where no immediate O2 signal changes were observed upon darkening. Areal rates of gross photosynthesis [PS(a) in

nmol O2 cm−2 s −1 ] were calculated by integrating the volumetric rates over depth.

# Temperature Microsensor Measurements

Temperature profiles were measured using thermocouple microsensors (tip diameter = 50 µm, TP-50, Unisense A/S, Aarhus, Denmark) connected to a thermocouple meter (Unisense A/S, Aarhus, Denmark) and interfaced through an A/D converter (DCR-16, PyroScience, Germany) to data acquisition software (Profix, PyroScience, Germany). Signals were linearly calibrated against a high precision digital thermometer (± 0.2◦C; Testo 110, Testo AG, Germany) in seawater of different temperatures. The areal heat dissipation from the microbial mat was calculated using Fourier's law of conduction using the linear temperature gradient in the thermal boundary layer (TBL) and the thermal conductivity of seawater (k = 0.6 W m−1 K −1 ):

$$J\_H = k \frac{\partial T}{\partial z}$$

The downward heat dissipation could not be directly calculated from the measured temperature profiles, and were estimated from the principle of energy conservation as the difference between the absorbed light energy and the sum of photosynthesis and upward heat flux (both in energy units).

# Energy Calculations

To obtain absolute scalar irradiance spectra, we multiplied the measured transmittance spectra for each depth with the measured incident radiometric spectra (in W m−2 ). By using Planck's equation:

$$E\_{\lambda} = h \stackrel{c}{\underset{\lambda'}{\times}},$$

where Eλ is the energy of a photon with a wavelength λ, h is Planck's constant (6.626·10−34 W s2 ) and c is the speed of light in vacuum (in m s−1 ), we then converted absolute scalar irradiance spectra to absolute spectra of photon scalar irradiance [E0(z) in µmol photons m−2 s −1 nm−1 ].

The total absorbed light energy in the mat (JABS in J m−2 s −1 ), i.e., the vector irradiance, was calculated from the downwelling spectral irradiance Ed(λ) and the irradiance reflectance as:

$$J\_{ABS} = \int\_{420}^{700} E\_d(\lambda)(1 - R\left(\lambda\right))d\lambda.$$

The amount of energy dissipated via photosynthesis was calculated by multiplying the areal photosynthesis rates [PS(a)] with the Gibbs free energy from the light reactions, where O2 is produced by the photolysis of water. Including the formation of ATP this yields 482.9 kJ (mol O2) −1 (Thauer et al., 1977).

Energy budgets for the entire photic zone were calculated under the assumption that the total energy stored in photosynthesis (JPS), and dissipated via heat (JH) and reflection (R) balanced the incoming radiative energy (JIN):

$$J\_{\rm IN} = J\_H + J\_{\rm PS} + R \text{ and } J\_{\rm IN} - R = J\_{\rm ABS} = J\_H + J\_{\rm PS}$$

To investigate how photosynthetic quantum efficiency varied with depth in the microbial mat, we calculated relative photosynthetic efficiencies (in mol O2 (mol photons) −1 mm−1 ) by dividing the photosynthetic rates in a specific depth layer with the photon scalar irradiance just above that layer (Lassen et al., 1992). We note that the calculated efficiency parameter does not reflect the true quantum efficiency, where depth specific photosynthesis is related to the number of absorbed photons in that particular depth (see section Discussion).

# HPLC Pigment Analysis

Sediment samples of the 0-0.5 mm surface layer were collected using the "crème brûlée" sampler described by Laviale et al. (2015) and stored at −80◦C. Approximately 100 mg of sampled mat material were ground with a micro pestle and extracted in a mixture of acetone and methanol (7:2). Samples were sonicated (S-4000, Branson Ultrasonic Corporation, United States) for 20 s to improve pigment extraction and were then centrifuged for 60 s at 13,400 rpm. Supernatants were filtered through 0.45 µm PTFE-membranes and immediately injected in a HPLC (1260 Infinity, Agilent Technologies, United States). Fifteen microliter of 1 M ammonium acetate was added to each HPLC vial prior to injection as a resolution-improving agent. The solvent gradient was set up according to Frigaard et al. (1997) with a 69 min elution program, a flow rate of 1.0 mL min−1 and an injection volume of 100 µL. Chromatographic separation was carried out using a C18 Ascentis column for reverse phase chromatography (5 µm particle size; L × I.D: 25 cm × 4.6 mm). Pigments were identified from their characteristic retention times and absorbance spectra.

# RESULTS

# Light, Temperature, and Photosynthesis

Microprofiles of photon scalar irradiance in the dense microbial mat were measured after incubation in dark, low light (75 µmol photons m−2 s −1 ), and high light (1000 µmol photons m−2 s −1 ), respectively, which yielded a different spatial organization of motile microbes in the mat (**Figure 1**). In the low lightacclimated state, a cyanobacterial layer formed near the surface, while in the high light-acclimated state the cyanobacteria were found in deeper layers. In the dark-acclimated mat, a dense whitish layer of colorless Beggiatoa spp. formed at the surface with the cyanobacteria located just below the colorless sulfur bacteria (**Figure 1**).

The vertical attenuation of photosynthetically active radiation (PAR, 400–700 nm) with depth in the microbial mat did not follow a mono-exponential decay, but exhibited variable attenuation in different layers depending on light acclimation and the distribution of biomass in the mat (**Figures 2A–C**). In the low light acclimated state, the strongest attenuation of PAR was found in the top 0.2 mm (K0 = 13.9 mm−1 ), whereas the underlying part of the microbial mat (0.3–0.5 mm) showed a slightly lower attenuation of PAR scalar irradiance (K0 = 10.3 mm−1 ). In the high light-acclimated state this trend was reversed, where the attenuation of PAR in the top layer (K0 = 10.6 mm−1 ) was lower

photic zone measured under an incident photon irradiance of 1000 µmol photons m−2 s . The surface layers of the microbial mat were dominated by dense populations of motile cyanobacteria (Microcoleus sp. and other filamentous species) and filamentous sulfide oxidizing bacteria (Beggiatoa spp.). Beneath the narrow photic zone, a dark FeS-containing sediment layer was present. In low light, the upper layers became oxidized driving the O2-H2S interface and thus Beggiatoa deeper into the mat, while the motile cyanobacteria moved toward the mat surface under non-inhibitory light levels. In high light, a larger part of the biofilm is oxidized, Beggiatoa moved further down, while motile cyanobacteria in the top layers started to move downwards to avoid high photoinhibitory light levels at the microbial mat surface. In darkness, the entire mat became anoxic and the Beggiatoa moved to the mat surface forming a white layer on top of the cyanobacteria, to avoid the high levels of H2S and remain at the O2-H2S interface (Preisler et al., 2007), whereas the cyanobacteria remained in a dense layer underneath.

than in deeper layers of the microbial mat (K0 = 16.5 mm−1 ). In the dark-acclimated state, a lower attenuation of PAR was found in the top 0.2 mm (K0 = 5.8 mm−1 ) of the microbial mat followed by a very steep attenuation from 0.2 to 0.4 mm depth (K0 = 22.2 mm−1 ).

Spectral attenuation of scalar irradiance in the PAR region was enhanced around absorption maxima of most abundant photopigments commonly found in cyanobacterial mats, e.g., Chl a (440 nm; 675 nm), phycocyanin (620 nm) and phycoerythrin (565 nm) (**Figures 2D–F**). HPLC analysis revealed the presence of other cyanobacterial pigments such as myxoxanthophyll, zeaxanthin, oscillaxanthin and β,ε-carotene in the upper 0.5 mm of the mat, along with BChl a indicative of anoxygenic phototrophs (**Figure 3**). Due to the strong light attenuation in the mat, <1% of PAR surface scalar irradiance remained just 0.4 mm below the biofilm surface (**Figure 2**). The very high lightattenuation of PAR (400–700 nm) and dense pigmentation of the microbial mat resulted in low surface reflection (**Figure 1**). In the low-light acclimated state, only 0.75% of the incident light was reflected, whereas the reflection from the mat surface in the high light-acclimated state was twice as high (1.6%). In the dark-acclimated state, where the surface of the microbial mat was covered by a whitish layer of motile, filamentous colorless sulfur bacteria (Beggiatoa spp.), the reflection increased ∼10-fold to 9.2% of the incident irradiance.

Most of the incident irradiance in the PAR region was thus readily absorbed in the mat, and the majority of the absorbed light was dissipated as heat as quantified from the heat flux over a ∼1.2 mm thick thermal boundary layer (TBL) (**Figure 4**). Under similar flow and thus TBL thickness, the mat surface heating differed slightly depending on light acclimation, where the surface temperature of the high light-acclimated mat increased by 0.3◦C as compared to the low light-acclimated mat showing a mat surface temperature increase of 0.24◦C. Lowest surface heating was measured when illuminating the dark-acclimated mat reaching a surface temperature increase of 0.2◦C.

Due to the high light-attenuation, the euphotic zone of the microbial mat was restricted to the uppermost 0.6 to 0.8 mm (**Figure 5**). The depth distribution of gross photosynthesis rates varied with light acclimation, and very high rates were observed

the spectrometer at shorter wavelengths and spectra were therefore truncated.

in all treatments (up to 30 nmol O2 cm−3 s −1 ). In the low lightacclimated state, peaks of gross photosynthesis were found at the mat surface and 0.3 mm beneath the surface. In the high light-acclimated state, the photosynthetic rates near the surface were diminished, while a stronger sub-surface peak in gross photosynthesis was found around 0.2 mm below the mat surface. The dark-acclimated mat showed a small photosynthesis peak near the mat surface and a sub-surface peak at 0.3–0.4 mm depth (**Figure 5**). However, the O2 concentration profiles between light acclimations were quite similar and did not show any clear differences in e.g., O2 penetration depth (**Figure 5**).

# Energy Budgets

The vector irradiance, i.e., the net downwelling radiative energy flux (400–700 nm), was very similar between light acclimations and amounted to 218.0 ± 5.6 W m−2 . However, the heat dissipation from the mat to the water differed between light incubations, where the dark-acclimated mat exhibited the lowest upward heat dissipation of 140 W m−2 , the heat dissipation in the low light-acclimated mat was 144 W m−2 , and the highest heat dissipation was measured in the high light-acclimated mat reaching 179 W m−2 .

Depth integrated gross (oxygenic) photosynthesis only accounted for a small part of the absorbed light energy. The highest amount of radiative energy stored via photosynthesis was measured in the low light-acclimated microbial mat and amounted to 5.4 ± 0.7 W m−2 , while the high light- and the darkacclimated mat conserved 4.06 ± 1.6 W m−2 and 4.8 ± 1.1 W m−2 , respectively.

analysis.

Relative to the incident energy, the photosynthetic energy conservation efficiency for the entire photic zone thus only accounted for ∼2% (2.1, 2.4, and 1.8% for the dark-, low light-, and high light-acclimated biofilm, respectively), while the majority of incident light energy was dissipated as heat (**Figure 1**).

# Relative Photosynthetic Efficiencies

Depth profiles of relative photosynthetic efficiencies in the microbial mat were calculated by normalizing the measured volumetric gross photosynthesis rates at each depth to the scalar irradiance incident to that depth. Such profiles showed subsurface peaks in relative photosynthetic efficiency in all light acclimation states of the microbial mat (**Figure 6**). In the low light-acclimated mat, a peak in relative efficiency was located closest to the surface at 0.3 mm depth. The dark-acclimated mat exhibited highest relative photosynthetic efficiencies 0.4 mm beneath the surface, while the highest relative photosynthetic efficiencies in the high light acclimated mat were found near the lower boundary of the photic zone at 0.5 mm below the mat surface.

# DISCUSSION

Radiative energy budgets in microbenthic systems have previously been studied in cyanobacterial mats and biofilms (Al-Najjar et al., 2010, 2012), sediments (Lichtenberg et al., 2017), and corals (Brodersen et al., 2014), albeit under the assumption of a homogenous depth distribution of biomass. In the studied microbial mat, dense populations of phototrophic microalgae and cyanobacteria reside in steep gradients of resource stratification shaped by communities of photo-, chemo-, and heterotrophic microorganisms creating a steep redox gradient (van Gemerden, 1993). Consequently, the physical and chemical landscape in such microbial mats can change, within less than one mm, from intense sun exposure (>1000 µmol photons m−2 s −1 ) and O2 supersaturation (up to 2 mM) to complete darkness and a reduced anoxic sediment high in H2S (Jørgensen, 1982).

The studied microbial mat exhibited an extremely high optical density and attenuation of PAR, where >99% of the surface irradiance was effectively absorbed <0.4 mm below the microbial mat surface. Light was thus the primary limiting resource for microbial phototrophs in the mat. One strategy to remain competitive under light limitation is to complement light absorption by Chl a, the main light harvesting pigment in oxygenic photosynthesis, by metabolic investment in producing a range of accessory pigments absorbing a broader part of the available light spectrum and channeling it to the photosynthetic reaction centers (Stomp et al., 2007; Trampe and Kühl, 2016; Kühl et al., 2020). HPLC pigment analysis of the upper 0.5 mm of the microbial mat revealed that very efficient light absorption was achieved by a mixture of light harvesting pigments from cyanobacteria (Chl a, mixoxanthophyll, zeaxanthin, oscillaxanthin and β,ε-carotene) and anoxygenic phototrophs (BChl a), able to absorb light from the UV-B well into the NIR region (**Figure 3**). We did not find any signs of typical diatom light harvesting pigments (fucoxanthin, diadinoxanthin or Chl c) or the far-red absorbing chlorophylls d or f in either measurements of scalar irradiance or by HPLC analysis.

The attenuation of light was stratified with depth and varied between the different acclimation states. In all acclimations, we found strong spectral attenuation from typical cyanobacterial pigments confirmed by the HPLC pigment analysis. In low light acclimated mats the strongest spectral attenuation was in the top 0.2 mm while the strongest spectral attenuation was found deeper (0.3–0.5 mm) in the high light acclimated mats.

Consequently, low surface reflection was observed in the low and high light-acclimated microbial mats. In the low light-acclimated mat, the cyanobacterial population was located near the surface and only 0.75% of the incident irradiance was backscattered, while in the high light-acclimated state, the motile cyanobacteria migrated downward changing the surface biomass composition leading to higher reflectance. During darkacclimation, the surface of the biofilm became anoxic and a layer of the motile, filamentous colorless sulfur bacteria Beggiatoa formed on the mat surface. Beggiatoa spp. are sulfide oxidizing bacteria known to store granules of elemental sulfur (Nelson and Castenholz, 1981), which make the filaments appear white due to strong light scattering, which in our measurements increased the surface reflectance to 9.2% of the incident irradiance. Multiple scattering in sediments and coral tissues have previously been shown to increase the scalar irradiance in top layers (Kühl and Jørgensen, 1994; Wangpraseurt et al., 2012) which could explain the very low attenuation of light in the top layers of the dark-acclimated mat (**Figure 2**). Light-dependent migration

irradiance (means ± 1 SD; n = 3). Data was fitted with an exponential saturation model (Webb et al., 1974).

patterns of cyanobacteria and Beggiatoa thus clearly modulated the radiative energy input to the microbial mat.

The strong light absorption in the uppermost mat layers, resulted in very high local photosynthetic rates reaching >30 nmol O2 cm−3 s −1 . This is about 2–5 fold higher than rates measured in most other cyanobacteria-dominated microbial mats (Al-Najjar et al., 2012), but e.g., comparable to rates measured in a highly stratified intertidal mudflat community dominated by motile diatoms (Cartaxana et al., 2016b) and in a cultivated diatom biofilm (Jensen and Revsbech, 1989). We investigated photosynthesis under high light exposure of microbial mats exhibiting different distributions of motile microbes in response to different light history. Investigations of photosynthesis under increasing irradiances in motile microbial systems are complex as migrating cells under changing light can change the spatial characteristics of the biofilm/mat and thereby potentially affect light exposure and distribution in different mat layers (Kühl et al., 1997). In the present study, the main aim was thus to investigate how the migration of motile cyanobacteria affected photosynthetic efficiency under different spatial organization of phototrophs in the microbial mat.

This is experimentally challenging, and we note that these measurements were not performed under steady-state conditions. The change to higher light than the acclimation irradiance will start to move the mat toward a new steady-state and rates and profiles will thus be measured under transient "quasi steady-state" conditions. The measurements of scalar irradiance and gross photosynthesis reflect the community's response to high light in the current distribution of biomass. The distribution of O2 and temperature is, however, more complex to interpret, as the distribution of O2 is determined by the interplay between diffusion, production and consumption, while the temperature profiles are determined by the local heat dissipation and heat transfer properties. These are slower processes and will move toward a steady state on the same time scale as motility driven biomass redistribution in such compact systems as the studied mats.

Areal rates of oxygenic photosynthesis, integrated across the entire euphotic zone, did not differ markedly between different light acclimations (0.99, 1.11, and 0.84 nmol O2 cm−2 s −1 for dark-, low light- and high light-acclimated samples, respectively) under the high saturating photon irradiance used for the energy budget measurements. It is however noted that the lowest areal photosynthetic rates were observed in the high light-acclimated state. One could speculate that the longer acclimation period to high light could have given the phototrophs, in this treatment,

time to adjust their balance between photosynthesis and nonphotochemical quenching (NPQ) processes. One of the most important short-term regulatory mechanisms to avoid photo damage is NPQ, where excess energy decays from singlet excited chlorophyll (1Chl∗ ) into heat dissipation (Müller et al., 2001). This mechanism significantly lowers the effective quantum yield of photosynthesis but avoids the formation of reactive singlet oxygen (1O2 ∗ ) by the triplet state of chlorophyll (3Chl∗ ) (Müller et al., 2001), which can have long term detrimental effects on the photosystems by degradation of the D1 protein; an important component in photosystem II (Nymark et al., 2009). The higher rates displayed by the top population for the dark- and low-light acclimated mats could therefore be a product of the more open reaction centers before regulating the different NPQ components that operate on time scales from a few minutes to a few hours (Kress and Jahns, 2017).

Photophobic responses or phototaxis enabling movement along a light gradient (Jekely, 2009) are alternative strategies of photoprotection, enabling motile phototrophs to align their position at optimum irradiance in light gradients (Tamulonis et al., 2011) depending on the status of the electron transport chain and time of the day (Burns and Rosa, 1980). Microcoleus chtonoplastes displays light dependent migration preferring moderate to low light levels (Ramsing and Prufert-Bebout, 1994), and other studies have found that as little as a 4% difference in light intensity between "head" and "tail" of a cyanobacterial filament can trigger their movement (Häder, 1987). From our measured depth profiles of gross oxygenic photosynthesis, it appears that the distance of vertical migration was limited to only 0.2 to 0.3 mm. However, due to the steep light gradients in the investigated microbial mat, the changes in the light field over such short distances are dramatic, and cyanobacteria migrating 200 µm deeper into the mat experienced a >84-fold decrease in scalar irradiance (**Figure 2C**; 0.4–0.2 mm) from >350 µmol photons m−2 s −1 to < 10 µmol photons m−2 s −1 . Such strong changes in light exposure upon migration distances of a few 100 µm have previously been demonstrated in hypersaline cyanobacterial mats (Kühl et al., 1997). Consequently, the spatial distribution of gross photosynthesis varied with light acclimation, probably as a result of migrations of the cyanobacteria and Beggiatoa.

Other triggers for migration could also play a role in the observed redistribution of biomass. Sulfide is well known to cause migration due to its cytotoxicity (Whale and Walsby, 1984). Sulfide was most likely present in high concentrations in the studied mats due to i) the black FeS layer below the photic zone, ii) the presence of BChl a, indicative of anoxygenic phototrophs that oxidize hydrogen sulfide to sulfur, and the presence of a dense population of Beggiatoa known to thrive at the interface of strong O2 and sulfide concentration gradients. Earlier microsensor measurements in microbial mats from the same sampling site also demonstrated significant sulfide production and light-driven dynamics of O2, pH and sulfide in the uppermost mat layers (Nielsen et al., 2015). As stated above, Microcoleus prefer moderate to low light levels and their migratory behavior is thus probably due to a combination of photophobic movement and sulfide avoidance, although they can grow in H2S concentrations up to 974 µM (de Wit and van Gemerden, 1987).

Compared to earlier studies of heat dissipation in corals and microbial mats (Jimenez et al., 2008; Al-Najjar et al., 2010; Brodersen et al., 2014), we found a relatively small heat dissipation from the biofilm surface into the water column and the surface temperature of the microbial mat increased by only ∼0.3◦C. However, the heat dissipation in deeper mat layers was larger than expected. The zone of maximum temperature increase was expected to be found in the same zone as the largest energy deposition, i.e., in the upper few hundred micrometers of the microbial mat, where >90% of the incident light was attenuated. However, the heat dissipation below 0.5 mm depth was in the same order of magnitude as the upward heat dissipation and reached higher temperatures (**Figure 4**). We note that below the dense photic zone, a black layer of precipitated iron sulfide was found, and absorption of light energy in this sediment layer apparently contributed significantly to the heating of the microbial mat, but this remains speculative. While we did not measure temperature deep enough in the sediment to be able to calculate the downward transport of heat, we estimated from the principle of energy conservation that the downward heat flux contributed about 20–50% of the total heat dissipation.

The amount of photochemically conserved energy did not change markedly between different light acclimation states of the microbial mat when investigated at similar high irradiance, despite the higher reflection and lower heat dissipation observed in the dark-acclimated biofilms. Consequently, the photosynthetic energy conservation was 2.1, 2.4, and 1.8% of the incident energy, for dark-, low light-, and high light-acclimated

biofilms, respectively. This indicates that by migration, the phototrophic community can apparently sustain similar energy conservation efficiencies, while avoiding the detrimental effects of excessive light when situated near the surface.

The vertical stratification of relative photosynthetic efficiencies (**Figure 6**) can either be ascribed to (i) a higher density of biomass contributing to photosynthesis, or (ii) an overall higher efficiency caused by higher cell specific pigment content and/or higher absorption cross section, in that area (Iglesias-Prieto and Trench, 1994, 1997; Falkowski and Raven, 2007; Al-Najjar et al., 2010). However, in our measurements we found differences in the vertical positions of the peaks of relative photosynthetic efficiency depending on light acclimation. At the cellular level, regulation of the pigment density and the absorption cross section of antennae pigments occurs on a longer time scale than the acclimation time used in these experiments, although the formation of zeaxanthin from β-carotene is a short term photoprotective mechanism (Falkowski and Raven, 2007). Given the time scale, the difference in vertical position of the peaks in relative photosynthetic efficiencies, can thus primarily be ascribed to migration.

In a fluctuating light environment, phototrophs can modulate light exposure/harvesting either by regulating pigment densities or by moving to a different light environment. For this mechanism to be energetically successful, the cost of motility must present an advantage compared to employment of regulation of light harvesting or photoprotective pigments. The cost of motility was estimated for Oscillatoria to be 0.2–5% of the energy generated by oxidative phosphorylation (Halfen and Castenholz, 1971). This is a relatively low cost considering that in a dense microbial system, where oxygenic phototrophs not only have to cope with alternating light environments but also steep and dynamic chemical gradients of e.g., pH and sulfide. Motility may thus be an important trait for phototrophs in microbial mats enabling rapid optimization of their light exposure and chemical microenvironment. Migration of cyanobacterial populations can also mitigate effects of continuous sedimentation and overgrowth by other microbes (Whale and Walsby, 1984).

Previously, photosynthetic quantum efficiencies in microbial mats have been calculated by applying a model that estimates the local quantity of absorbed light, which is then correlated to the local rates of gross photosynthesis (Al-Najjar et al., 2010). However, this model assumes a homogenous distribution of light absorption (i.e., biomass), which makes the application of such a model problematic in a motile community exhibiting photomovement. Calculations using the model of Al-Najjar et al. (2010) on our data showed apparent photosynthetic quantum efficiencies, about 2-fold higher than the theoretical maximum of 0.125 (i.e., 8 photons needed to produce 1 O2 molecule). We speculate that the presence of a black ferrous sulfide layer in close proximity to the phototrophs also interfered with such estimations of the local density of absorbed light. However, the distribution of sand particles, organic matter and photosynthetic biomass was not further quantified, and a correction for this possible artifact was not attempted. Therefore, we estimated relative quantum efficiencies of photosynthesis by relating the depth specific gross photosynthesis rates to the scalar irradiance incident to that point following the approach of Lassen et al. (1992), who also found that the position of maximum quantum efficiencies changed with light exposure. The relative quantum efficiencies estimated in our mat samples were, however, up to three times higher than values reported by Lassen et al. (1992) for a less compacted microbial mat.

The photosynthetic efficiency depends on photopigmentation and other biotic/abiotic substances contributing to light absorption (Al-Najjar et al., 2012) but also on complex interplay between cyanobacteria and sulfur oxidizers (Klatt et al., 2016). Thus, the microscale 3D structure of the microbial mat will have an influence on the light availability and the energy balance of the system, where more open systems will display larger photic zones and different responses to changes in light environment as compared to more compacted systems

(Lichtenberg et al., 2017). How such microscale canopy-like effects in microbial mats are affected by the presence of much higher densities of abiotic matter in comparison to e.g., plant canopies remain to be studied in more detail.

# CONCLUSION

We conclude that cyanobacteria can sustain high relative photosynthetic efficiencies by vertical migration in response to shifting light conditions, thereby optimizing the overall capacity of light absorption and photosynthetic activity against a backdrop of dynamic chemical gradients of O2, pH and sulfide. This was evident from the overall photosynthetic energy conservation efficiency that did not change with light acclimation but showed vertical differences in the distribution of maximum relative photosynthetic efficiency. Further studies could investigate how distribution and efficiency of gross photosynthesis respond to changing light environment in microbial mats with vertically fixed populations of phototrophs in different depth horizons, e.g., by applying a motility inhibitor (Cartaxana et al., 2016a). In very compact systems such as the mats studied here it is, however, difficult to resolve local differences due to the spatial resolution of the microsensors and methodology to noninvasively monitor biomass distributions. In less compacted systems (e.g., mats like studied by Wieland and Kühl, 2000; Saenger et al., 2006; Lichtenberg et al., 2017) with more open structures and thereby less steep gradients of the physicochemical parameters, local differences in energy deposition would be easier to describe in more detail.

Our study adds novel insight to the regulation of photosynthesis in compacted microbial mats and biofilms. In terms of energy conservation and photosynthetic efficiency, such systems are often regarded less efficient due to their more or less flat topography (e.g., Al-Najjar et al., 2012). However, there is increasing evidence that microstructural changes can modulate light harvesting and photosynthesis in dense photosynthetic biofilms and tissues, and that such systems can exhibit canopylike properties at a microscale, where a more open microstructure

# REFERENCES

and/or modulation of the scattering and lateral distribution of incident light seems to govern higher photosynthetic efficiency (e.g., Wangpraseurt et al., 2014; Lichtenberg et al., 2016, 2017; Lyndby et al., 2016). In the present study, we show that reorganization of the phototrophs via migration is yet another functional trait that helps optimize overall light harvesting and photosynthesis in biofilms and microbial mats.

# DATA AVAILABILITY STATEMENT

The datasets generated for this study are available on request to the corresponding author.

# AUTHOR CONTRIBUTIONS

ML, PC, and MK designed the experiments and analyzed the data. ML and PC performed the experiments. ML wrote the manuscript with editorial input from PC and MK.

# FUNDING

This study was supported by grants from the Independent Research Fund Denmark (DFF-1323-00065B and DFF- 8021- 00308B) (MK). PC acknowledges support from FCT/MCTES through CESAM (UIDB/50017/2020+UIDP/50017/2020).

# ACKNOWLEDGMENTS

We thank Lars Rickelt for construction of scalar irradiance microprobes, and Sofie Jakobsen for excellent technical assistance. The members of the Microenvironmental Research Group provided useful feedback, help and suggestions during the experimental and analysis part of this study. Parts of this manuscript were included in the Ph.D. thesis of ML (Lichtenberg, 2017).

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Lichtenberg, Cartaxana and Kühl. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Microscale Variability in Biomass and Photosynthetic Activity of Microphytobenthos During a Spring-Neap Tidal Cycle

Jacco C. Kromkamp1 \*, Ed Morris2 and Rodney M. Forster3 \*

1 Royal Netherlands Institute for Sea Research, Department of Estuarine and Delta Systems, Utrecht University, Utrecht, Netherlands, 2 Vizzuality, Madrid, Spain, 3 Hull Marine Laboratory, Department of Biological and Marine Sciences, University of Hull, Hull, United Kingdom

Carbon fixation by microphytobenthic algae of intertidal flats often dominates the total primary production of turbid, temperate estuaries. Whilst remote sensing can accurately measure the spatial distribution of photosynthetic biofilms at the mesoscale (1–300 m), variability at smaller scales requires in-situ investigation. Here, changes in biomass and photosynthetic activity of microphytobenthos (MPB) at the micro-scale (<1 m) were investigated. Biomass of MPB was estimated from repeated high resolution spectral measurements carried out at intervals along a short transect. Whilst the mean concentration of MPB remained constant over time, considerable variability in the spatial and temporal dimensions was measured. The biofilm grew and diminished along the transect over time, and net growth and loss rates could be established under natural conditions. Daily vertical migration was observed for the first time in an undisturbed sediment, and was modeled as a function of solar elevation and tidal angle. Top down factors such as grazing, or physical disturbance, played an important role in the changes in biomass. Photosynthetic activity was measured with portable fluorometers: one device used an artificial light source, the other measured the effective photosystem-II quantum efficiency (1F/Fm 0 ) in-situ with solar irradiance. No evidence was found for photoinhibition, with Fv/Fm values consistently high, indicating an ability of the diatom biofilm to remain productive under periods of high irradiance. The maximum rate of photosynthesis reached with artificial light was rarely achieved in-situ, even with full solar exposure. There was, however, a very good agreement between the predicted rate at ambient irradiance, and that actually measured. Composite photosynthesis-irradiance curves over the whole experimental period were similar, and showed the same initial slopes, but the maximum rate of relative photosynthetic electron transport (rETRm) based on the in-situ measurement was lower. When total depth-averaged primary production during low tide was calculated, the differences between methods varied between 5 and 13%, and were smallest when surface irradiance values were highest. These results show that the protocol used to measure the photosynthetic activity of the MPB had only minor importance on the overall productivity estimate compared to accurate knowledge of photosynthetically active biomass and irradiance.

Keywords: microphytobenthos, microscale variability, surface biofilm, photosynthetic activity, chlorophyll fluorescence, vertical migration, photosynthetic electron transport, estuary

### Edited by:

Vona Meleder, Université de Nantes, France

# Reviewed by:

Elena Maggi, University of Pisa, Italy James L. Pinckney, University of South Carolina, United States

#### \*Correspondence:

Jacco C. Kromkamp<EMAIL_ADDRESS>Rodney M. Forster<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Marine Ecosystem Ecology, a section of the journal Frontiers in Marine Science

Received: 06 December 2019 Accepted: 18 June 2020 Published: 22 July 2020

#### Citation:

Kromkamp JC, Morris E and Forster RM (2020) Microscale Variability in Biomass and Photosynthetic Activity of Microphytobenthos During a Spring-Neap Tidal Cycle. Front. Mar. Sci. 7:562. doi: 10.3389/fmars.2020.00562

# INTRODUCTION

fmars-07-00562 July 20, 2020 Time: 12:14 # 2

Benthic microalgal communities are important ecological players on intertidal estuarine sediments. They can be responsible for a significant fraction of the total estuarine primary production (MacIntyre et al., 1996; Underwood and Kromkamp, 1999; Christianen et al., 2017), providing a range of ecosystem services (Hope et al., 2019), such as provision of a highly nutritious food source that can be used directly or indirectly by higher trophic levels including important wading bird populations (Kuwae et al., 2008). Microphytobenthos (MPB) can also be considered important ecosystem engineers as the presence of surface biofilms gives enhanced resistance against erosion (Paterson, 1989; Paterson and Black, 1999; Decho, 2000; Underwood and Paterson, 2003). This effect is most prominent on muddy, cohesive diatom-dominated sediments, where the excretion of extracellular polymeric substances improves the stabilization of mudflats (Smith and Underwood, 2000; Tolhurst et al., 2003). Of particular note here are microbial communities dominated by epipelic, pennate diatoms – biofilms of this type can accumulate multiple layers of cells with areal chlorophyll concentrations of over 200 mg m−2 (Forster et al., 2006; Sahan et al., 2007; Jesus et al., 2009).

Whilst the seasonal appearance of intertidal MPB blooms can display a remarkable synchronicity at the sea-basin scale (van der Wal et al., 2010), geostatistical analysis of in-situ data and remote sensing images has shown that MPB show spatial patchiness at a range of smaller distances (Ibrahim et al., 2014). Semivariograms constructed on MPB samples typically show a range (distance at which samples are not spatially autocorrelated) of 50–500 m (Morris, 2005; Orvain et al., 2012; Ibrahim et al., 2014). There are few estimates of microscale variability in MPB biomass i.e., at distances less than the typical spread of replicate samples at a point station (1–3 m).

In addition to spatial variability in the horizontal dimension, MPB of mud-dominated tidal flats exhibit a pronounced vertical variability due to migrations of diatom cells toward and away from the surface. The vertical migration may be driven by an endogenous clock as cells only come to the surface during low tide, and only when the low tide is during the day: at night cells remain below the sediment surface (Pinckney and Zingmark, 1991; Serôdio et al., 1997; Jesus et al., 2006). Due to the changes in photosynthetic biomass exposed to the light, vertical migration is a strong driver of daily primary production (Kwon et al., 2012; Haro et al., 2019). In addition to the tidally driven migration, epipelic diatoms may also change depth in relation to the light intensity and avoid too high light conditions (Cartaxana et al., 2008). The fact that, even after prolonged exposure to high light, effective photosynthetic quantum efficiencies can still be high prompted Kromkamp et al. (1998) to propose the concept of vertical "micro-migration" where cells at the surface migrate to slightly deeper layers in the sediment in order to prevent overexposure to damaging irradiances or CO2-limitation, whereas others cells migrate to the surface. Thus, the biofilm at the sediment surface is a dynamic, productive system where cells at the very surface are continuous being replaced by others (Marques da Silva et al., 2017). In contrast to vegetated coastal environments such as seagrass beds and salt marshes, very few estimates of the contribution of MPB to coastal primary production, or long-term carbon storage ("blue carbon") are available (Cahoon, 1999 for global MPB; Howard et al., 2017 for coastal vegetation).