# Radiative energy budgets of migrational microphytobenthic biofilms

S. Haro<sup>1,4</sup> · A. Barnett<sup>2</sup> · S. Papaspyrou<sup>1,5</sup> · A. Corzo<sup>1,5</sup> · M. Kühl<sup>3</sup> · B. Jesus<sup>2</sup>

Received: 5 July 2023 / Accepted: 11 September 2024 / Published online: 4 October 2024
© The Author(s) 2024

## Abstract

Epipelic, biofilm-forming diatoms are key drivers of the primary production of mudflats. Such primary production is strongly affected by the vertical migration of diatoms, which is modulated by diurnal photoperiods, tidal cycles, and photoprotection mechanisms. However, the role of vertical migration in the radiative energy budget (REB) of microphytobenthic biofilms remains largely unknown. Here we used microsensor measurements of temperature and O₂ in combination with reflectance spectroscopy and variable chlorophyll fluorimetry to construct the REB of intertidal, epipelic diatom-dominated biofilms for different emersion times (1, 3 and 5 h after the beginning of the *in-situ* emersion) and photon irradiance regimes, i.e., 400 and 800 µmol photon m⁻<fnref n="2" /> s⁻<fnref n="1" />. The effect of migration on REBs was studied by inhibiting diatom motility with Latrunculin A (Lat-A). Photosynthetic activity and light utilization efficiency decreased slightly, after adding Lat-A, while the amount of reflected light energy remained constant at ~23% of the incident irradiance and the majority (76–78%) of the incident light energy was dissipated as heat. Of the energy dissipated as heat, <24% was dissipated upward in Lat-A treated samples, while an increasing downward heat dissipation was observed in Lat-A treated samples, as compared to control samples under an irradiance of 800 µmol photon m⁻<fnref n="2" /> s⁻<fnref n="1" />. However, we found no statistical significant differences in the REB and physiological parameters in the different treatments. Thus, we did not find any evidence that vertical migration of diatoms affected photosynthesis and light efficiency in the microphytobenthic biofilm over an emersion cycle, and a clear effect of non-photochemical quenching in REB and heat dissipation fluxes was not observed.

**Keywords** Microphytobenthos · Photosynthesis · Energy budget · Motility · Diatom

## Introduction

Intertidal flats contribute significantly to total primary pro-
duction in coastal areas (Underwood and Kromkamp 1999),
and a large part of this production arises from photosynthetic

microbial communities encompassing diatoms, cyanobacte-
ria and green microalgae, i.e., microphytobenthos (MPB),
that colonize the sediment photic layer. These photosyn-
thetic microorganisms engage in a close spatial and func-
tional association with other microbial groups (heterotrophic
bacteria, archaea, protozoa, etc.) forming biofilms at the
sediment surface in shallow intertidal areas. The microen-
vironmental conditions at sediment-water and sediment-air
interfaces of MPB are extreme and characterised by steep
physico-chemical gradients, dynamic changes in O₂ and CO₂
diffusion coefficients, sediment temperature, optical condi-
tions, desiccation and potential contraction of the sediment
matrix (e.g. Kühl et al. 1994; Coelho et al. 2009; Faber et al.
2012; Lara et al. 2018).

Intertidal MPB growth is strongly influenced by the tidal
regime (immersion/emersion) and photoperiod (light–dark
cycles) (Round and Happey 1965) creating daily oscillations
of light and temperature at the sediment surface (Serôdio
and Catarino 1999), which are both key regulating factors
for MPB photosynthesis (Blanchard et al. 1996). Ambient

Responsible Editor: K. Yin.

<figure><img src="image_1.png" /></figure>

S. Haro
sara.haro@uma.es

<footnote n="1">¹ Department of Biology, University of Cadiz,
11510 Puerto Real, Spain</footnote>

<footnote n="2">² Institut Des Substances Et Organismes de La Mer, Nantes Université, ISOMer, UR2160, F-44000 Nantes, France</footnote>

<footnote n="3">³ Marine Biological Section, Department of Biology,
University of Copenhagen, Strandpromenaden 5,
DK 3000 Helsingør, Denmark</footnote>

<footnote n="4">⁴ Ecology and Geology Department, University of Malaga,
29010 Malaga, Spain</footnote>

<footnote n="5">⁵ Instituto Universitario de Investigación Marina (INMAR),
Campus Universitario de Puerto Real, 11510 Cadiz, Spain</footnote>
light is strongly attenuated in the first millimetres or even micrometres of the sediment, restricting the photic layer and limiting MPB photosynthetic activity to a thin top sediment layer (Kühl et al. 1994, 1997). The photic layer thickness depends on grain size, MPB biomass, incident irradiance and tidal state (immersion/emersion). Overall, the amount of light that reaches the sediment surface is higher in emersion than immersion due to light being attenuated by water column turbidity and depth in submerged sediment. Thus, higher primary production is usually observed in mudflats covered by microphytobenthic biofilm during emersion periods (Colijn et al. 1987; Spilmont et al. 2007).

Microphytobenthos can employ both behavioural (vertical migration) and physiological mechanisms for adjusting to the daily light and temperature oscillations on intertidal flats (Serôdio et al. 2005b; Prins et al. 2020). Epipelic diatom-dominated biofilms are predominant sites of benthic primary production in European estuaries (Stal 2003). During diurnal low-tide periods, the diatoms can move vertically towards the sediment surface to increase light exposure and maximize their photosynthetic rate (Coelho et al. 2011), while the diatoms move down into the sediment before night or high-tide periods to increase nutrients uptake, release extracellular polymeric substances (EPS), and carry out cellular division (mitosis) (Saburova and Polikarpov 2003; Orvain et al. 2003; Rakotomalala et al. 2019). The vertical migration of diatoms is controlled by a circadian rhythm synchronized by photoperiod, tides or both (Serôdio et al. 2005b; Haro et al. 2019a; Kromkamp et al. 2020). Vertical movements are thus responsible for diel oscillations in primary production observed in intertidal MPB biofilms (Serôdio et al. 2005b; Coelho et al. 2011; Cartaxana et al. 2016; Haro et al. 2019a), and highest primary production rates are expected during daytime emersion periods (Denis et al. 2012; Kwon et al. 2012). Diatoms vertical migration and daily changes in the photosynthesis rate have wide ecological and biogeochemical implications for the sediment-water interface. For instance, they are likely to affect nutrient concentrations in the sediment by changing MPB demand. These changes affect competition with other microbial groups (Risgaard-Petersen 2003; Longphuirt et al. 2009; Høgslund et al. 2023) as well as the oxygen availability and therefore the rates and pathways of organic matter mineralization (Glud 2008; Jørgensen et al. 2022).

At fine spatial scales, epipelic diatoms can also use vertical migration as a fine tuning mechanism for photosynthesis by constantly moving up and down within the photic layer (micromigration) to (i) fine-tune light exposure and optimize photosynthetic activity, and (ii) avoid excess light exposure by moving to deeper sediment layers, e.g. during midday-low tide periods (Kromkamp et al. 1998; Perkins et al. 2010; Cartaxana et al. 2016). Furthermore, epipelic (motile) and epipsammic (non-motile) diatoms have physiological

mechanisms to dissipate excess energy as heat, such as the xanthophyll cycle (involving the reversible de-epoxidation of the pigment diadinoxanthin, DD, into diatoxanthin, DT) that dissipates energy via non-photochemical quenching (NPQ) (Perkins et al. 2010; Cartaxana et al. 2011; Serôdio et al. 2012). We note, however, that the xanthophyll cycle is not the only physiological energy dissipating mechanism involved in NPQ (Cartaxana et al. 2013; Lavaud and Goss 2014).

Daily temperature oscillations in combination with high solar irradiance present another environmental factor affecting photosynthetic efficiency in MPB biofilms (Blanchard et al. 1996). Of the total solar light energy reaching the sediment surface, a fraction is reflected by the sediment surface and the rest is absorbed. Absorbed light energy is either used to carry out photosynthesis or is dissipated as heat. The photosynthetic efficiency is the fraction of absorbed light energy converted to chemical energy in photosynthesis (Falkowski and Raven 1997). The radiative energy budget (REB) of microbenthic phototrophs was first investigated in photosynthetic microbial mats by Al-Najjar et al. (2010) and has been further studied in cyanobacterial mats with different structure and community composition (Al-Najjar et al. 2012), different incident irradiance regimes (Lichtenberg et al. 2017) and tidal stages (Haro et al. 2019b). In addition, the REB have been studied in a coastal microbial mat dominated by motile cyanobacteria and colourless sulphur bacteria (Lichtenberg et al. 2020). Hitherto, REBs have not been calculated for migrational diatom-dominated MPB biofilms.

In the present study, we applied microsensor measurements of temperature and $O_2$ concentration and a spectroradiometer to investigate the effect of vertical migration on the radiative energy budget of MPB biofilms dominated by motile, epipelic diatoms. The REBs were calculated for different emersion times (1, 3 and 5 h after *in-situ* emersion) and under two light treatments, i.e., photon irradiances (400-700 nm) of 400 and 800 µmol photon m$^{-2}$ s$^{-1}$, as well as with ad without the diatom motility inhibitor Latrunculin A (Lat-A) (Poulsen et al. 1999). Furthermore, reflectance spectra and variable chlorophyll fluorescence measurements were used to further assess physiological responses in relation to light use efficiency.

## Material and methods

### Sampling and experimental set-up

Epipelic, diatom-dominated MPB biofilm samples and seawater were collected from La Couplasse mudflat in Bourgneuf Bay, France (47° 0'57.39"N; 2° 1'14.11"W). The bay is characterized by semi-diurnal tides with maximum tidal amplitudes of 6 m during spring tides. The intertidal zone occupies ~100 km$^2$ of the total bay surface area (340

<figure><img src="image_2.png" /></figure>
km²) (Méléder et al. 2007; Kazemipour et al. 2012). The sediment is composed of > 60% particles with a grain size of < 63 µm (Méléder et al. 2007) and had a porosity of 0.778 ± 0.016 (mean ± standard deviation, n=5), as determined from the water content loss of a known volume of sediment sampled from the uppermost mm. Microscopic analysis revealed that the MPB community was mainly composed of epipelic diatoms, as previously described by Méléder et al. (2007). Further details on the benthic communities in the area can be found in Reddin et al. (2022). The main consumers of MPB in the area include the grazer *Peringia ulvae*, and the facultative suspension feeders *Macoma balthica* and *Scrobicularia plana*.

Twelve sediment cores (i.d. 5 cm; 7 cm length) were collected during low tide (spring tide). In the laboratory, the sediment cores were maintained in darkness (approximately between 18 and 90 h depending on treatment) and kept fully hydrated inside an aquarium (salinity 32) but without submersion in order not to disturb the sediment surface until the experimental day. PVC tubes with small millimetric cuts in the upper part were used. Measurements were done on 4 consecutive days after the sampling day (Table S1). The entire experiment was repeated on three different occasions with cores from each sampling day being regarded as biological replicates. During the experiment, the samples were illuminated with an incident photon irradiance (400–700 nm) of either 400 or 800 µmol photons m⁻² s⁻¹ during 6 h, in synchronization with the *in-situ* emersion period (Fig. 1), using LED lights (SL 3500; Photon System Instruments). The incident photon irradiance was measured with a calibrated

spectroradiometer (MSC15; Gigahertz-Optik GmbH). For each light level, half of the samples were treated with Lat-A and half were kept as controls. Each sample was measured at 1, 3 and 5 h during the virtual emersion period, i.e., 2 h before low tide (BLT), at low tide hour (LT), and 2 h after low tide (ALT), respectively. In summary, four experimental conditions were carried out randomly over four consecutive days during three independent experiments (n=3): control at 400 µmol photon m⁻² s⁻¹ (C_E400), control at 800 µmol photon m⁻² s⁻¹ (C_E800), Lat-A at 400 µmol photon m⁻² s⁻¹ (Lat_E400) and Lat-A at 800 µmol photon m⁻² s⁻¹ (Lat_E800).

For the Lat-A treatments, 200 mL of 15 µM Lat-A was added to a sediment sample 1 h before *in-situ* LT, to ensure that the biofilm had time to migrate to the surface before adding the movement inhibitor (Perkins et al. 2010; Cartaxana et al. 2011; Serôdio et al. 2012). The 15 µM Lat-A solution was prepared by diluting an aliquot from a 1mM Lat-A stock solution with filtered seawater from the sampling site. The 1 mM Lat-A solution was prepared by dissolving purified Lat-A (Sigma-Aldrich) in dimethylsulfoxide and storing at – 20 °C following Cartaxana and Serôdio (2008).

## Oxygen and temperature profiles

Oxygen and temperature profiles were measured using retractable needle-sensors with optical isolation (OXR50-OI and TPR430-SUB; PyroScience) connected to a FireSting O₂ II (PyroScience). Signals were recorded via an USB-interfaced A/D-converter (DCR16, PyroScience GmbH, Germany) through dedicated PC-controlled data acquisition software

<figure><img src="image_3.png" /><figcaption>**Fig. 1** Oxygen profiles and reflectance spectra were measured over the emersion period of 6 h, coinciding with the *in-situ* emersion period at the sampling site. Temperature profiles and the radiative energy budget (red sticks) were measured at different emersion times: 1 h after the start of the *in-situ* emersion, i.e. 2 h before lowest tide</figcaption></figure>

(BLT); 3 h, i.e. at the lowest tide hour (LT); and 5 h, i.e. 2 h after the lowest tide (ALT). In addition, rapid light-response curves of relative PSII electron transport versus photon irradiance were measured at BLT, LT and ALT (red sticks). Lat-A was added 1 h before LT
(PyroScience). All microsensors were mounted on a motorized micromanipulator (MU1, PyroScience) and were calibrated according to the manufacturer's specifications. A temperature probe (TSUB21; PyroScience) was also connected to the FireSting O₂ II and introduced in the water surface to enable automatic temperature compensation of the oxygen sensor signal. Vertical O₂ concentration profiles were measured every 10 min with a vertical depth resolution of 100 µm, as controlled with the Profix software (Pyro Science). Oxygen profiles were measured during the 6 h of the experiments, in all experimental conditions. All the profiles were measured in the same point to ensure the comparison between different emersion times. Additionally, volumetric rates of gross primary production (P₉) were measured using the light–dark shift method (Kühl et al. 1996) in steps of 100 µm throughout the photic zone at different emersion times (1, 3 and 5 h) (Fig. 1). Manual temperature profiles were also measured every 1, 3 and 5 h with a vertical depth resolution of 200 µm. P₉ and temperature profiles were recorded with the software Pyro Oxygen Logger (PyroScience). The positions of the sensor tip were observed on the sediment surface using a digital endoscope camera (Flylinktech USB portable endoscope).

## Reflectance measurements

Changes in the MPB biomass at the sediment surface were estimated from reflectance spectra, as measured with a 400 µm diameter, flat cut optical fiber (P400-2-UV–VIS, Ocean Optics) connected to a fiber-optic spectrometer (USB 2000-VIS, Ocean Optics). The optical fiber was positioned obliquely (45°) at a fixed distance of 2 cm from the sediment surface to measure reflectance from the same area where O₂ and temperature measurements were performed. Reflected light spectra were measured each minute during the 6 h experiment. The reflected light spectra from MPB biofilm (Rₘₐt) were normalized with the reflected light spectrum

from a 99% white reflectance standard (Rₐf; Spectralon; Labsphere, North Sutton, NH, USA) to calculate reflectance for photosynthetically active radiation (PAR; 420–700 nm) in % of incident photon irradiance (R; Eq. 1). For these measurements, the samples were exposed during 30 s at 1, 3, and 5 h under a halogen lamp for obtaining reflectance measurements (Fig. 2a). The continuous utilization of the halogen lamp during the entire experiment would have resulted in an excessive increase in temperature on the sediment surface.

$$R(PAR) = \int_{420}^{700} \frac{R_{mat}}{R_{ref}} d\lambda \quad (1)$$

MPB biomass at the sediment surface was monitored using the green–red reflectance ratio index (R570/R635) (see definitions of abbreviations in Appendix). The percentages change in the R570/R635 ratio from the initial measurement value at time 0 min (t₀) were plotted. The rationale for using this ratio instead of the more common normalized difference vegetation index (NDVI) is that the LED light source did not extend into the near infrared region (Fig. 2b). Thus, we used the reflectance at 635 nm that corresponds to the absorption maximum of chlorophyll c and looked for a region in the spectra that changed very little (i.e., 570 nm) to normalize the measurements between samples. This chlorophyll c proxy is not as sensitive as NDVI, but it has the advantage of enabling use of light sources without infrared radiation that can enhance the drying and temperature profiles in air-exposed sediments.

## Variable chlorophyll fluorescence measurements

We used an underwater variable chlorophyll fluorometer (Diving-PAM, Walz GmbH) with a pulse-amplitude-modulated red measuring light (emission peak 665 nm; Fig. S1)

<figure><img src="image_4.png" /><figcaption>**Fig. 2** a Spectrum of reflectance of the halogen lamp used to measure the reflectance, here shown for a control sample under an incident photon irradiance of 800 µmol photon m⁻² s⁻¹; b Incident spectral</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>irradiance (in energy units) of the LED lamp used in the experiments, here shown for a photon irradiance of 800 µmol photon m⁻² s⁻¹</figcaption></figure>
to assess the PSII-related photosynthetic electron transport activity of the MPB using the saturation pulse method (Vieira et al. 2013) at BLT, LT and ALT, corresponding to 1, 3, and 5 h into the experiment (Fig. 1). Fluorescence was measured via a 5.5 mm diameter optical fiber, which was positioned 0.5 cm from the sediment surface to avoid shadowing. The fluorometer was programmed to supply 30 s of actinic light (in defined photon irradiance steps from 0 to 1686 µmol photon m⁻² s⁻¹) before applying a saturation pulse to record the minimum fluorescence yield after dark incubation (F₀) or under illumination (Fₛ), and the maximum fluorescence yield after dark incubation (Fₘ) or under illumination (Fₘ') for each applied photon irradiance level (E).

Relative electron transport rates (rETR) for PSII activity were calculated from the product of E and the PSII effective quantum yield, F₀/Fₘ' (Genty et al. 1989), with F₀ = Fₘ'Fₛ. The parameter α (initial RLC slope under light limitation) was estimated using the slope of a linear regression to the first five points (E=0, 97, 130, 188 and 255 µmol photon m⁻² s⁻¹), rETRₘₐₓ (maximum relative electron transport rate) was extracted from fitting the model of Eilers and Peeters (1988) and Eₖ, i. e., the photon irradiance at onset of light saturation coefficient, was calculated as rETRₘₐₓ/α.

We used variations in α (Δα) as a proxy for in-situ non-photochemical quenching (NPQ) (Serôdio et al. 2006):

$$ \Delta\alpha = \alpha_{\text{Start}} - \alpha_{t} \qquad (2) $$

which avoids the problem of Fₘ' changes caused by vertical migration movements rather than NPQ processes.

## Flux calculations and radiative energy budgets

The radiative energy budget (REB) was calculated according to the approach of Al-Najjar et al. (2010). Of the total incoming light energy that reach the sediment surface, a fraction is reflected by the sediment surface (Eq. 1) and the rest is absorbed by the biofilm Jₐₓₐₓ (Eq. 3), which in turn can be used for photosynthesis (Jₚₚ, Eq. 4) or dissipated as heat (Jₘ, Eq. 7) (Al-Najjar et al. 2010).

The total incoming light energy (Jₐₘ), i.e., the absolute downwelling irradiance, Eₐₘₐₓ(λ) (in W m⁻² nm⁻¹), was quantified with a calibrated spectroradiometer (MSC15; Gigahertz-Optik GmbH).

The absorbed light energy in the MPB biofilm (Jₐₘₐₓ in J m⁻² s⁻¹) was estimated as the net downwelling irradiance:

$$ J_{|ABS|} = \int_{400}^{700} E_{|ABS|(\lambda)}(1 - (\lambda))d\lambda \qquad (3) $$

The absorbed light energy used in photosynthesis (J m⁻² s⁻¹) was calculated as:

$$ J_{PS} = P_G E_G \qquad (4) $$

where, Pₚ is the areal rate of gross photosynthesis (nmol O₂ cm⁻² s⁻¹) calculated by integrating the depth-specific volumetric rates (Pₚ(z) in nmol O₂ cm⁻³ s⁻¹) over the euphotic zone, and Eₐₗ [482.9 kJ (mol O₂)⁻¹] is the Gibbs energy produced when O₂ is formed by splitting water (Al-Najjar et al. 2010).

The amount of absorbed light energy that was not photochemically conserved was dissipated as heat, resulting in a temperature increase in the MPB biofilm and the air layer just above the biofilm surface forming a temperature boundary layer (TBL) (Jimenez et al. 2008). The heat dissipation fluxes (J m⁻² s⁻¹) from the euphotic zone of the biofilm to the air (upward heat dissipation flux, Jₘₐₗ) was calculated from Fourier's law of conduction:

$$ J_{H_{up}} = k \frac{dT}{dz} \qquad (5) $$

where, dT/dz is the temperature gradient in the TBL (Lichtenberg et al. 2017; Haro et al. 2019b) and k is the thermal conductivity of air (0.025 J m⁻¹ s⁻¹ K⁻¹; Balandin et al. 2008).

The downward heat flux from the photic zone into the aphotic sediment (Jₘₐₗ) was estimated in accordance to Brodersen et al., (2014) assuming that autofluorescence from the MPB biofilm is negligible:

$$ J_{H_{down}} = J_{|ABS|} - J_{PS} - J_{H_{up}} \qquad (6) $$

The sum of upward and downward heat dissipation fluxes (Eq. 5; Eq. 6) provides the total amount of light energy dissipated as heat in the biofilm (Eq. 7). Thus, the total amount of light energy absorbed by the biofilm (Eq. 8) is given by the sum of energy conserved by photosynthesis Jₚₚ (Eq. 4) plus the energy dissipated as heat Jₘ (Eq. 7) (Lichtenberg et al. 2017).

$$ J_{H} = J_{H_{up}} + J_{H_{down}} \qquad (7) $$

$$ J_{|ABS|} = J_{PS} + J_{H} \qquad (8) $$

Finally, the radiative energy budget (REB) of the intertidal MPB biofilms was calculated as a percentage of incident light energy (Eq. 9; Eq. 10):

$$ J_{IN} = R + J_{|ABS|} \qquad (9) $$

$$ J_{IN} - R = J_{PS} + J_{H} \qquad (10) $$
## Statistical analysis

Significant differences between treatments were tested using a permutational analysis of variance (PERMANOVA) (Anderson 2001; McArdle and Anderson 2001) based on a Euclidean distance resemblance matrix, followed by post hoc pairwise test where appropriate. Lat-A (presence/ absence) and incident photon irradiance (400/800) were used as fixed factors. The replicate experiments (labeled as experiment 1, 2 and 3) were used as a random block factor. Data estimated as % or ratios were arcsin transformed. To account for differences in each individual core, all data from each core were normalized to the initial values (BLT). Normalized data were analysed separately for low tide data (LT) and after low tide (ALT). A total of 1000 unrestricted permutations of raw data were used in all tests. When the number of permutations was small (<100), p-values were obtained through Monte Carlo random draws from the asymptotic permutation distribution (Anderson and Robinson 2003) between treatments for any of the factors for any variable studied. Moreover, Student test were used to study significance differences in photophysiological parameters, i.e. F_q/F_m', and Δα, at a photon irradiance of 400 and 800 µmol photon m⁻² s⁻¹; and Pearson coefficient were also used to study the significant linear regression between physiology parameters (F_q/F_m', and Δα) and radiative energy budget, mainly its relation to light use efficiency (J PS(J ABS)) and heat dissipation fluxes (J Hup and J Hdown). Results are presented as suggested by Dushoff et al., (2019) in terms of statistical clarity.

## Results and discussion

### Vertical migration and primary production

Oxygen concentration in the sediment (penetration depth and maximum concentration) increased from the beginning of emersion time, reaching a maximum close to the LT period, and then decreased progressively until the end of emersion time (Fig. S2a; Fig. S3). Maximum peaks of O₂ concentration (reaching up to 600 µmol O₂ L⁻¹) were also observed at low tide by Cartaxana et al. (2016) and Denis et al. (2012) in intertidal sediments cores, where oxygen concentration was measured over a diel emersion period. This is in line with diel vertical migrations of diatoms associated to photoperiod and tides as a major factor regulating benthic primary production, with the highest photosynthetic activity being recorded at low tides and midday (Serôdio et al. 2005b; Coelho et al. 2011; Haro et al. 2019a). During the *in-situ* lowest tide, a decrease in oxygen penetration

<table><caption>**Table 1** Average temperature increase (°C) of the microphytobenthic biofilm surface at a depth of 600 µm for different treatments, i.e. control at 400 µmol photon m⁻² s⁻¹ (E400), Latrunculin A (Lat) at 400 µmol photon m⁻² s⁻¹, control at 800 µmol photon m⁻² s⁻¹ (E800), and Latrunculin A at 800 µmol photon m⁻² s⁻¹. Temperature increase is shown over emersion period at three time points: 2 h before lowest tide, BLT; *in-situ* lowest tide hour, LT; 2 h after lowest tide, ALT)</caption><thead><tr><th></th><th>From BLT to LT</th><th>From LT to ALT</th><th>From BLT to ALT</th></tr></thead><tbody><tr><td>Control E400</td><td>0.8 °C</td><td>0.3 °C</td><td>1.1 °C</td></tr><tr><td>Lat E400</td><td>0.6 °C</td><td>0.2 °C</td><td>0.8 °C</td></tr><tr><td>Control E800</td><td>1.5 °C</td><td>0.3 °C</td><td>1.8 °C</td></tr><tr><td>Lat E800</td><td>0.9 °C</td><td>0.5 °C</td><td>1.4 °C</td></tr></tbody></table>

depth was observed in the average profiles of oxygen concentration for the Lat-A treated samples at 400 µmol photon m⁻² s⁻¹ (as illustrated in Fig. S2b); however, this decrease was not observed at an irradiance of 800 µmol photon m⁻² s⁻¹ (Fig. S2c). Similarly, no clear changes were observed by Cartaxana et al., (2016) in the oxygen concentration profile for Lat-treated biofilm at 150 and 2000 µmol photon m⁻² s⁻¹. Note that the oxygen solubility was not affected by temperature changes (Table 1). This was made possible because a temperature probe was introduced in the aquarium, enabling automatic temperature compensation of the oxygen sensor signal. Moreover, the oxygen concentration would have been underestimated by only 3.8% when the temperature rose from BLT (21 °C, 32 ‰, oxygen solubility = 230.7 µmol L⁻¹) to ALT (23 °C, 32 ‰, oxygen solubility = 222.0 µmol L⁻¹) for the control samples at 800 µmol photon m⁻² s⁻¹ (Ramsing and Gundersen 1994).

The depth of the maximum oxygen concentration and the thickness of the production peak in the oxygen profiles measured in MPB biofilm are linked with vertical migration movements in the photic layer (Cartaxana et al. 2016; Meresse et al. 2023). In our work, the diel maximum oxygen concentration pattern coincided with the vertical migration pattern observed here using the percentage of change in the R570/R635 ratio in comparison to the value observed at the beginning of the measurements, i.e. in comparison to the ratio value at t₀ (Fig. 3). For both control and Lat-A samples, MPB moved upwards from BLT to LT, reaching a maximum around 30% at 400 µmol photon m⁻² s⁻¹ and slightly lower, 20%, at 800 µmol photon m⁻² s⁻¹, respectively. The 10% lower upward migration at the higher irradiance of E800 compared to E400 for both treatments (Fig. 3b, d) confirms previous findings, where a high light exposure induces downward vertical movements in diatom-dominated biofilms (at lowest tide) in order to avoid photoinhibition (Cartaxana and Serôdio 2008; Perkins et al. 2010; Serôdio
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 3** Temporal evolution of the percentage changes in the R570/R635 index from the initial measurement value at time 0 min, i.e. a proxy for Chl *c* used to study the changes of microphytobenthic biomass at the sediment surface over 6 h of treatments: at lowest tide hour (LT), 2 h before lowest tide (BLT) and 2 h after lowest tide (ALT). **a, b** Control samples at irradiances of 400 and 800 µmol photon m⁻² s⁻¹, and **c, d** Latrunculin A treated samples at photon irradiances of 400 and 800 µmol photon m⁻² s⁻¹. Latrunculin A was added at t=120 min of the experiment, as indicated by the red lines. Three experiments were plotted: Ex I (light dot), Ex II (black dot) and Ex III (grey dot)</figcaption></figure>

et al. 2012). In addition to exposure to accumulated light dose, carbon and nitrogen requirements (not measured in our experiment), could be provoking the downward migration of epipelic diatoms from LT to ALT for both irradiances (Hochard et al. 2010; Marques da Silva et al. 2017; Rakotomalala et al. 2019). From LT to ALT, MPB migrated downwards in control cores, although this movement was more evident in some cores than in others. This difference in diatom migration between sediment cores could be due to a circadian rhythm, which is associated with *in-situ* photoperiod, tidal cycles (immersion/emersion, and spring/neap tides) and carbon and nitrogen requirements (Coelho et al. 2011; Rakotomalala et al. 2019; Haro et al. 2019a; Kromkamp et al. 2020). This endogenous migration behaviour was maintained for at least three days after the environmental stimulus, i.e., the photoperiod, ceases. Even though the cores were collected during low tide (spring tides) and the experiments were synchronized with the *in-situ* emersion

period, the vertical movements could have been influenced by changes in the timing of the experimental light switching on/off, particularly after the third day. As a result, we observed a greater dispersion of data than expected, even though the treatments were randomly distributed. Moreover, the observed dispersion in control samples could be a consequence of significant microheterogeneity and patchiness within the MPB biofilm (Jesus et al. 2005; Daggers et al. 2020). In contrast, in Lat-A treated samples the response was similar across all cores showing only a slight decrease of the R570/R635 ratio at E400 or none at E800 (Fig. 3c, d). These observations are consistent with previous ones, where motile cells migrated upwards to the sediment surface at the beginning of the emersion period, and stayed there until the end of the emersion period when they migrate downwards again (Kromkamp et al. 2020). The absence of the latter in Lat-A treated samples (Fig. 3c, d), confirmed that Lat-A addition successfully inhibited the majority of diatom movements
and that our experimental set up could discern the effect of
vertical migrations on primary production and heat exchange
balances. However, clear differences were not found in the
R570/R635 ratio during LT or ALT regardless of treatment
(p-values > 0.05; PERMANOVA). The lack of statistically
clear differences might be attributed to the fact that these
changes occur at a microscale (micrometre scale), making
them challenging to detect statistically. Increasing the num-
ber of replicates might potentially help in identifying these
minor differences at the microscale between treatments.

The depth of the euphotic zone (determined as the maxi-
mum depth with gross production volumetric rates, P<sub>G</sub>(z))
was restricted to the uppermost 0.4 to 0.8 mm. Notably, in
the Lat-A treated biofilms during the LT period, this depth
was approximately 100 µm deeper (p-values = 0.037; PER-
MANOVA) (Fig. 4). Cartaxana et al. (2016) measured an
increase in both the euphotic zone depth and P<sub>G</sub>(z) approxi-
mately 2 h after the start of the emersion period, an effect
that was not clearly observed in our experiment. Here, the
P<sub>G</sub>(z) was, as expected, higher at E800 (up to ~10 nmol O<sub>2</sub>
cm<sup>-3</sup> s<sup>-1</sup>) than E400 (up to ~8 nmol O<sub>2</sub> cm<sup>-3</sup> s<sup>-1</sup>) in the
control samples (Fig. 4). However, this increment in P<sub>G</sub>(z)
was not observed in the Lat-A treated samples (values up
to ~8 nmol O<sub>2</sub> cm<sup>-3</sup> s<sup>-1</sup>). Probably, due to the inhibition of
vertical migration, individual MPB cells could not move to

their optimum depth along the vertical axis, decreasing the
photosynthetic efficiency. In addition, despite the indications
for vertical migration in the control samples, surprisingly
the highest P<sub>G</sub>(z) values on average were always observed
at 300 µm depth regardless of treatment. Unfortunately,
vertical biomass data are not available to normalise P<sub>G</sub>(z).
Regardless, the areal rate of gross production (P<sub>G</sub>) values for
all treatments ranged between 0.08 and 0.67 nmol O<sub>2</sub> cm<sup>-2</sup>
s<sup>-1</sup>, which falls within the range of similar studies (Kühl
et al. 1996; Haro et al. 2019b). During LT and ALT period
(i.e. 3 and 5 h into the experiment), no statistically clear
differences in P<sub>G</sub> between treatments (studied irradiances,
and with/without Lat-A) were observed (p-values > 0.05;
PERMANOVA), as illustrated in Fig. S4. However, mar-
ginally clear differences were detected between the Lat-A
and control samples across the three experiments during
the LT period, as determined through a Monte Carlo fit
(p-value=0.039; analysed using PERMANOVA). The vari-
ability in results between experiments could be attributed
to the patchiness within the mudflat covered by MPB (Bar-
ranguet and Kromkamp 2000; Jesus et al. 2005).

<figure><img src="image_10.png" /><figcaption>**Fig. 4** Depth profiles of gross production volumetric rates, expressed as nmol O₂ cm⁻³ s⁻¹, in microphytobenthic biofilms at 400 and 800 µmol photon m⁻² s⁻¹ at different emersion times (2 h before lowest</figcaption></figure>

tide, BLT; *in-situ* lowest tide hour, LT; and 2 h after lowest tide, ALT) in control samples and samples treated with Latrunculin A (Lat-A). Mean ± SE (n=3). Lat-A was added between BLT and LT
<figure><img src="image_11.png" /><figcaption>**Fig. 5** Radiative energy budgets in an intertidal microphytobenthic biofilm in percentage of incident light energy under 400 and 800 µmol photon m⁻² s⁻¹ in control samples and Latrunculin A treated samples a at 2 h before lowest tide, b at lowest tide hour and c 2 h after lowest tide. Mean values ± standard error (n=3)</figcaption></figure>

## Radiative energy budget

Reflected light energy (R) accounted for 17.5 – 32.2% on average of the total incident energy (J<sub>IN</sub>) in the control samples, depending on the emersion phase. The percentage of R did not increase proportionally with J<sub>IN</sub> though. The irradiance doubled from E400 to E800, while R was 1.84 times higher at E800 compared to E400 (Fig. 5). However, in Lat-A treated samples, mean R was ~23%, independent of photon irradiance levels and emersion time. The percentage of R in cyanobacteria-dominated microbial mats under emersion was similar (18.5%) to the one measured here at E400 and lower (19.5%) at E800, respectively (Haro et al. 2019b). Under immersion, R was even lower; ~7% at 400

and 800 µmol photons m⁻² s⁻¹ (Haro et al. 2019b), and as low as 1.6% at 1000 µmol photons m⁻² s⁻¹ (Lichtenberg et al. 2020). All these studies determine the impact of biofilm composition (diatoms versus cyanobacteria), incident irradiance intensity, and emersion time in the amount of R from the biofilm surface. However, other physical parameters, such as sediment grain size and water content, also have a direct effect on the R from the sediment surface (Thuy et al. 2022). Additionally, grain size is commonly utilized to model the MPB biomass, and indices calculated from R, such as NDVI or the ratio proposed in this work (R570/R635), are frequently employed as proxies for estimating MPB biomass (Haro et al. 2019a; Zhang et al. 2023).

The amount of incident light energy used in photosynthesis (J<sub>PS</sub> (J<sub>IN</sub>), %) decreased with increasing irradiance – independently of emersion time – and varied between 0.5% (ALT in Lat-A samples at E800; Fig. 5c) and 1.2% (BLT in control samples at E400; Fig. 5b). These values were slightly higher than similar measurements in cyanobacteria-dominated microbial mats under emersion (0.7 and 0.5% at 400 and 800 µmol photons m⁻² s⁻¹, respectively (Haro et al. 2019b)). On the other hand, average J<sub>PS</sub> values were slightly higher in the control biofilms (range 1.1–1.7 J m⁻² s⁻¹) than in the Lat-A treated biofilms (1.0–1.1 J m⁻² s⁻¹), independently of emersion time and photon irradiance (Table S2). This points to onset of photoinhibition of immobilized photosynthetic cells on the sediment surface after Lat-A adding (Fig. 5). However, no clear statistical differences were found between Lat-A treated biofilm and controls (p-values > 0.05; PERMANOVA) and these results need to be interpreted with caution. A decrease of J<sub>PS</sub> with increasing incident irradiance was also observed by Al-Najjar et al., (2010, 2012), Lichtenberg et al., (2017) and Haro et al., (2019b), who measured maximal J<sub>PS</sub> of approximately 4 and 16% under immersion at low irradiances in cyanobacteria-dominated microbial mats, and 2% in microbial biofilms associated to corals.

The majority of incident light energy was dissipated as heat (J<sub>H</sub> = J<sub>Hup</sub> + J<sub>Hdown</sub>). In the control samples, the percentage of J<sub>H</sub> decreased with increasing irradiance, varying between 69 and 81%, while in the Lat-A treated samples it varied much less, only between 75 and 77% (Table S2). As a result of the dissipated heat, sediment temperature was higher at the highest irradiance (Lichtenberg et al. 2017; Haro et al. 2019b) (Fig. 6). Temperature values at 600 µm increased progressively over the emersion period and were slightly higher at E800 than E400 (Fig. S5). Maximum temperature values were always measured 2 h after *in-situ* low tides (i.e. ALT), although the highest rate of increase was measured from BLT to LT (between 0.6 and 1.3 °C) for all treatments (Table 1). Note that the temperature profiles were
<figure><img src="image_12.png" /><figcaption>**Fig. 6** Vertical profiles of temperature change relative to air, ΔT (°C), measured in a microphytobenthic biofilm at different emersion time (2 h before lowest tide, BLT; lowest tide hour *in-situ*, LT; and 2 h after lowest tide, ALT) at 400 and 800 µmol photon m⁻² s⁻¹ in samples treated with/without Latrunculin A (Lat). Y=0 indicates the surface of biofilm, while X=0 indicates a 0 °C temperature differences as compared to the air temperature. Values represent mean ± SE (n=3). Lat-A was added between BLT and LT in samples with migration inhibition</figcaption></figure>

measured with a depth resolution of 200 µm using a needle
sensor with a dip of 430 µm, which may have introduced
some errors in the temperature measurements. Additionally,
there were instances where the temperature exhibited nearly

vertical profiles with very weak TBL, primarily during the BLT periods (Fig. 6). Weak TBL have been reported in other studies where temperature profiles were measured under low irradiance conditions (Brodersen et al. 2014; Lichtenberg et al. 2017; Haro et al. 2019b).

Of the energy dissipated as heat, < 16% was dissipated upward (J<sub>Hup</sub>), i.e., the majority of heat was dissipated downwards (J<sub>Hdown</sub>), independently of the treatment (Fig. 5). This is due to the low thermal conductivity of heat in the air overlying the sediment under emersion. In addition, the higher heat conductivity of water in the sediment pore spaces facilitates a higher downward heat flux into the sediment. This was previously described by Haro et al. (2019b), who observed in a cyanobacteria-dominated microbial mat that heat dissipation under immersion was mainly towards the water column, while most of the heat flux is directed downward under emersion. In this work, heat dissipation flux was mainly downwards over the emersion period (> 59%) but decreased at the end of the emersion period independently of treatment. A slightly higher J<sub>Hdown</sub> at lowest tides could be due to NPQ to avoid photodamage when the motile cells are on the surface. This excess energy dissipated as heat may be higher in LatA treated samples at E800 because the J<sub>Hdown</sub> was a 6.7% higher in the Lat-A treated samples. In contrast, J<sub>Hdown</sub> was 9.3% higher in control samples than in Lat-A treated samples at E400. This could be due to the lack of activation of NPQ in Lat-A treated samples at E400.

Light utilization efficiency

Light utilization efficiency (J<sub>PS</sub> (J<sub>ABS</sub>)) was slightly higher at LT for control biofilms especially at E800 (Fig. 7a); J<sub>PS</sub> (J<sub>ABS</sub>) increased by 16% in LT at E800, as compared to BLT period; and decreased 28–33% from LT to ALT, at photon irradiances of 400 and 800 µmol photons m<sup>-2</sup> s<sup>-1</sup>. At LT, the J<sub>PS</sub> (J<sub>ABS</sub>) decreased only 13.5% at E800 in comparison to

<figure><img src="image_13.png" /><figcaption>**Fig. 7** Light utilization efficiency (in % of absorbed light energy) in an intertidal microphytobenthic biofilm at irradiances of 400 and 800 µmol photon m⁻² s⁻¹ in control samples (black bars; panel A)</figcaption></figure>

and Latrunculin A treated samples (red bars; panel B) under different
emersion time: 2 h before lowest tide (BLT), lowest tide hour (LT)
and 2 h after lowest tide (ALT). Mean values ± standard error (n=3)

<figure><img src="image_14.png" /></figure>
E400 (Fig. 7a). Haro et al., (2019b) observed that $J_{PS}(J_{ABS})$ was slightly lower during emersion (0.6–2.1%) compared to immersion (0.9–2.6%) in cyanobacteria-dominated microbial mats, decreasing with increasing irradiance from 100 to 800 µmol photons m⁻² s⁻¹. The reasons for this decrease are unknown.

In our work, applying Lat-A to the biofilms resulted in a decrease of light utilization efficiency in the order of 16–39% in comparison with control samples at LT (Fig. 7b), as consequence of the incapacity of individual MPB cells to move to their optimum depth along the vertical axis, decreasing the photosynthetic efficiency. Light utilization efficiency at ALT was not affected by Lat-A at the lower irradiance but it decreased 27% at higher irradiances. These facts suggest

photoinhibition of the motile cells at the sediment surface after Lat-A addition, being the photoinhibition higher at high irradiance (Perkins et al. 2010; Serôdio et al. 2012). Although small changes were observed in $J_{PS}(J_{ABS})$ for Lat-A treated samples, our statistical analysis does not support an effect of vertical migration on the light efficiency utilization over the emersion cycle (p-values > 0.05; PERMANOVA). Recently, Lichtenberg et al., (2020) made similar observations, and suggested that phototaxis enabling movement in a motile cyanobacteria-dominated microbial mat along a light gradient does not affect the light utilization efficiency, although no explanation for the effect could be provided.

## Photophysiological parameters

An increase in the photosynthetic flux ($J_{PS}(\%J_{IN})$) and in the NPQ, i.e. $\Delta\alpha$, were observed for both studied irradiances (Table 2). This significant linear relationship was slightly stronger at E800 (r=0.58, p < 0.05, n=12) than E400 (r=0.554, p < 0.05, n=11). At E800, a significant positive linear correlation was also observed between NPQ and light utilization efficiency ($J_{PS}(\%J_{ABS})$) (r=0.58, p < 0.05, n=12), suggesting NPQ activation to maximise photosynthetic efficiency (Cartaxana et al. 2013; Lavaud and Goss 2014). In this context, PSII maximum quantum efficiency of light adapted MPB ($F_q/F_m'$) increased by 11.3% from E800 to E400 for control samples (student test; p value < 0.01) (Fig. 8a). However, significant linear regressions were not observed between $F_q/F_m'$ and photosynthetic flux ($J_{PS}(J_{IN})$) (Table 2) or $F_q/F_m'$ and $\Delta\alpha$ (used here as a proxy of NPQ) (Table 2; Fig. 8a,c). The decrease of $F_q/F_m'$ was larger for samples treated with Lat-A (18.4%; student test, p

<table><caption>**Table 2** Significant Pearson correlations, r, (p < 0.05, n=11) between PSII maximum efficiency of light adapted microphytobenthos ($F_q/F_m'$) and variations in $\alpha$ ($\Delta\alpha$), used as a proxy for *in-situ* NPQ, with fluxes calculated from the radiative energy budget (see definitions of abbreviations in Appendix) in samples exposed to photon irradiances of 400 and 800 µmol photons m⁻² s⁻¹</caption><thead><tr><th rowspan="2"></th><th colspan="2">400 µmol photons<br>m<sup>-2</sup> s<sup>-1</sup></th><th colspan="2">800 µmol photons<br>m<sup>-2</sup> s<sup>-1</sup></th></tr><tr><th>F<sub>q</sub>/F<sub>m</sub>'</th><th>Δα</th><th>F<sub>q</sub>/F<sub>m</sub>'</th><th>Δα</th></tr></thead><tbody><tr><td>R (%)</td><td></td><td>-0.79</td><td></td><td></td></tr><tr><td>J<sub>ABS</sub> (%)</td><td></td><td>0.79</td><td></td><td></td></tr><tr><td>J<sub>PS</sub> (%)</td><td></td><td>0.55</td><td></td><td>0.58</td></tr><tr><td>J<sub>H-up</sub> (%)</td><td>0.77</td><td></td><td>-0.73</td><td></td></tr><tr><td>J<sub>H-down</sub> (%)</td><td>-0.76</td><td>0.55</td><td>0.70</td><td></td></tr><tr><td>J<sub>PS</sub> (J<sub>ABS</sub>) (%)</td><td></td><td></td><td></td><td>0.58</td></tr></tbody></table>

<figure><img src="image_15.png" /><figcaption>**Fig. 8** Photosynthetic parameters, (a-b) variations in $\alpha$ ($\Delta\alpha$), and (c-d) PSII maximum efficiency of light adapted microphytobenthos ($F_q/F_m'$), where the parameters $\alpha$ is the initial slope of the curve, used as a proxy for *in-situ* NPQ, plotted for control samples (black bars, panels a, c) and Latrunculin A treated samples (panels b, d) under different emersion time, 2 h before lowest tide (BLT, black bar plots Lat had not been added), *in-situ* lowest tide hour (LT, red bar) and 2 h after lowest tide (ALT, red bar), at 400 and 800 µmol photon m⁻² s⁻¹. Mean values ± standard error (n=3)</figcaption></figure>
value < 0.01) (Fig. 8b). This likely occurred due to an higher irradiance during the continuous exposure of the immobilised Lat-A treated epipelic diatoms at the sediment surface (Fig. 3b,d) (Perkins et al 2010). BLT values were similar between control and before Lat-A addition for both light conditions (Fig. 8a,b). Differences between the BLT, LT and ALT for control conditions at both irradiances (Fig. 8a) and Lat-A treated samples at E400 were not clear (less than 3%). Lat-A treated samples at E800, Fq/Fm' increased from BLT to LT (8.6%) and decreased between LT and ALT (17.9%) (Fig. 8b).

The average Δα was similar for control samples exposed to the two irradiances (Fig. 8c) and no clear differences were found (student test, p value = 0.382). No clear changes were observed during emersion for both light conditions. For Lat-A biofilms, an Δα decrease was observed at E400 between BLT and LT and LT and ALT (respectively 9.0 and 15.2%) (Fig. 8d). A decrease in α, in addition to other parameters (e.g. ETRmax and sediment O2 concentrations), has been previously recorded in MPB biofilm treated with Lat-A under high light exposure (Perkins et al. 2010; Serôdio et al. 2012; Cartaxana et al. 2016). Although a decrease in α at E400 was observed in our work, Δα increased ~23% after adding Lat-A at E800 (23.2% between BLT and LT and 52.0% between LT and ALT) (Fig. 8d). At present we have no explanation for this discrepancy, which was, however, statistically not clear (p-values > 0.05; PERMANOVA).

In cyanobacteria-dominated microbial mats under emersion, Haro et al. (2019b) observed that heat dissipation was mainly directed towards the sediment, i.e. % JHdown > % JHup. The authors suggested that it could be due to the dissipation of the excess energy as heat by means of NPQ activation, as well as a higher thermal conductivity of water in the sediment pore spaces. At E400, we found a significant positive linear correlation (r = 0.55, p < 0.05, n = 11) between the variable Δα and the downward heat dissipation flux (JHdown), as well as an inverse correlation between the heat dissipation fluxes (JHup and JHdown) and the variable Fq/Fm' for both irradiances, as shown in Table 2. Therefore, although clear statistical differences were not found between treatments (p-values > 0.05; PERMANOVA), a small part of heat dissipation energy could be attributed to NPQ. In fact, the higher the PSII effective quantum efficiency, the less thermal energy is generated, because more of the captured light energy is directed into electron transport and less to NPQ thermal dissipation mechanisms (Serôdio et al. 2005a).

## Conclusions

Vertical migration in epipelic diatom-dominated biofilms has been associated to a diel circadian rhythm (light/dark and immersion/emersion cycles) and to photoprotection mechanisms that optimize photosynthetic efficiency. Our study observed certain trends, but could not conclusively demonstrate a significant effect of vertical migration on photosynthetic flux and light use efficiency during an emersion cycle. However, the following patterns were observed: (1) Downward vertical movements at the end of emersion period were much weaker in Lat-A treated samples, confirming that Lat-A addition successfully inhibited the majority of diatom movements; (2) Reflected light energy remained constant after Lat-A addition (~23%) due to the accumulation of immobilized cells on the sediment surface, which increased absorbed energy and decreased reflected light; and (3) a decrease in the photosynthetic flux and light use efficiency were observed after Lat-A addition, independently of emersion time and irradiance, suggesting a photoinhibition effect. Additionally, a clear effect of NPQ in the REB and heat dissipation fluxes was not observed either. Therefore, the observed increase in heat dissipation flux under emersion was probably mainly due to the amount of incident light energy heating the sediment surface (the low thermal conductivity of air could facilitate heat transfer to the sediment), rather than the excess energy dissipation via NPQ and similar active photoprotection mechanisms. Although determining the amount of heat dissipation fluxes derived from NPQ was not possible, clear relationships were observed between JHdown and Δα. Both physiological mechanisms, i.e. vertical migration and NPQ, contribute to photosynthetic efficiency, affecting directly MPB diel primary production. Gaining further insights on the ecophysiology and energy use efficiency of these productive systems is essential to be able to predict how the variations in temperature and light due to shifting climatic conditions would affect the productivity of these ecosystems. In this context, the development of algorithms for estimating microphytobenthic primary production in tidal flats at global scale becomes an essential preliminary step. Such development is pivotal for scaling up these energy flux models to broader spatial and temporal scales through remote sensing techniques. This includes observing or modelling biomass (using reflectance indices as proxies) and temperature from satellite imagery.

## Appendix

See below Table 3

<figure><img src="image_16.png" /></figure>
<table><caption>**Table 3** Definition of abbreviations and parameters</caption><thead><tr><th>Abbreviation</th><th>Definition</th><th>Unit</th></tr></thead><tbody><tr><td>MPB</td><td>Microphytobenthos</td><td></td></tr><tr><td>REB</td><td>Radiative energy budget</td><td></td></tr><tr><td>Lat-A</td><td>Latrunculin A</td><td></td></tr><tr><td>BLT</td><td>2 h before low tide</td><td></td></tr><tr><td>LT</td><td>At low tide hour</td><td></td></tr><tr><td>ALT</td><td>2 h after low tide</td><td></td></tr><tr><td>C_E400</td><td>Control at 400 µmol photon m<sup>-2</sup> s<sup>-1</sup></td><td></td></tr><tr><td>C_E800</td><td>Control at 800 µmol photon m<sup>-2</sup> s<sup>-1</sup></td><td></td></tr><tr><td>Lat_E400</td><td>Lat-A at 400 µmol photon m<sup>-2</sup> s<sup>-1</sup></td><td></td></tr><tr><td>Lat_E800</td><td>Lat-A at 800 µmol photon m<sup>-2</sup> s<sup>-1</sup></td><td></td></tr><tr><td>P<sub>G</sub></td><td>Areal rate of gross photosynthesis</td><td>nmol O<sub>2</sub> cm<sup>-2</sup> s<sup>-1</sup></td></tr><tr><td>P<sub>G</sub>(z)</td><td>Volumetric rates of gross photosynthesis</td><td>nmol O<sub>2</sub> cm<sup>-3</sup> s<sup>-1</sup></td></tr><tr><td>PAR</td><td>Photosynthetic active radiation (400–700 nm)</td><td></td></tr><tr><td>R</td><td>Reflectance</td><td>%</td></tr><tr><td>R<sub>mat</sub></td><td>Reflectance of the biofilm</td><td>nm<sup>-1</sup></td></tr><tr><td>R<sub>ref</sub></td><td>Reflectance standard</td><td>nm<sup>-1</sup></td></tr><tr><td>E<sub>ABS</sub>(λ)</td><td>Absolute downwelling irradiance or spectral irradiance availability</td><td>W m<sup>-2</sup> nm<sup>-1</sup></td></tr><tr><td>R570/R635</td><td>Green–Red reflectance ratio</td><td></td></tr><tr><td>RLC</td><td>PAM fluorescence rapid light-response curves</td><td></td></tr><tr><td>F<sub>o</sub></td><td>Minimum fluorescence yield after dark incubation</td><td></td></tr><tr><td>F<sub>s</sub></td><td>Minimum fluorescence yield under illumination</td><td></td></tr><tr><td>F<sub>m</sub></td><td>Maximum fluorescence yield after dark incubation</td><td></td></tr><tr><td>F<sub>m</sub>'</td><td>Maximum fluorescence yield under illumination</td><td></td></tr><tr><td>F<sub>q</sub></td><td>F<sub>m</sub>'-F<sub>s</sub></td><td></td></tr><tr><td>F<sub>q</sub>/F<sub>m</sub>'</td><td>PSII effective quantum yield</td><td></td></tr><tr><td>rETR</td><td>Relative electron transport rates</td><td></td></tr><tr><td>rETR<sub>max</sub></td><td>Maximum relative electron transport rate</td><td></td></tr><tr><td>E</td><td>Photon irradiance level</td><td></td></tr><tr><td>E<sub>k</sub></td><td>Photon irradiance at onset of light saturation coefficient</td><td></td></tr><tr><td>α</td><td>Initial RLC slope under light limitation</td><td></td></tr><tr><td>Δα</td><td>Proxy for in-situ non-photochemical quenching</td><td></td></tr><tr><td>NPQ</td><td>Non-photochemical quenching</td><td></td></tr><tr><td>J<sub>IN</sub></td><td>Total incoming light energy</td><td>J m<sup>-2</sup> s<sup>-1</sup></td></tr><tr><td>J<sub>ABS</sub></td><td>Absorbed light energy in the MPB biofilm</td><td>J m<sup>-2</sup> s<sup>-1</sup></td></tr><tr><td>J<sub>PS</sub></td><td>Areal rate of gross photosynthesis in energy terms</td><td>J m<sup>-2</sup> s<sup>-1</sup></td></tr><tr><td>J<sub>Hup</sub></td><td>Upward heat flux in energy terms</td><td>J m<sup>-2</sup> s<sup>-1</sup></td></tr><tr><td>J<sub>Hdown</sub></td><td>Downward heat flux in energy terms</td><td>J m<sup>-2</sup> s<sup>-1</sup></td></tr><tr><td>J<sub>H</sub></td><td>Heat dissipation flux (J<sub>H</sub>↑ – J<sub>H↓</sub>) in energy terms</td><td>J m<sup>-2</sup> s<sup>-1</sup></td></tr><tr><td>TBL</td><td>Temperature bounding layer</td><td></td></tr><tr><td>J<sub>PS</sub> (J<sub>IN</sub>)</td><td>Incident light energy conserved by photosynthesis</td><td>%</td></tr><tr><td>J<sub>PS</sub> (J<sub>ABS</sub>)</td><td>Light utilization efficiency (in % of absorbed light energy)</td><td>%</td></tr></tbody></table>

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s00227-024-04518-y.

**Author contributions** S. Haro: conceptualization, investigation, writing – original draft, visualization, formal analysis, funding acquisition; A. Barnett: investigation, writing – review & editing; S. Papaspyrou: formal analysis, visualization, writing – review & editing, funding acquisition; M. Kühl: writing – review & editing; A. Corzo: writing – review & editing, funding acquisition; B. Jesus: conceptualization,

supervision, writing – original draft, writing – review & editing, funding acquisition.

**Funding** Funding for open access publishing: Universidad de Cádiz/ CBUA. This project was funded by the Spanish Ministry of Economy and Business (MINECO) through the project EXTREME-FUN (PID2020-112488RB- I00) and MICROBAHIA II (CTM2017-82274), awarded to SP and AC. SH was funded by a postdoctoral fellowship from the University of Cádiz, Spain (2018–011/PU/AY.PUENTE/CD;
2019–004/PU/PP-EST/MV). BJ and MK acknowledge support by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement N° 860125.

**Data availability** The datasets are available on request. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

## Declarations

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

**Ethical approval** No ethical approval was required for this study.

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