Introduction

Species of the red seaweed genus Asparagopsis (A. armata and A. taxiformis) have the ability to produce and accumulate a high amount of diverse bioactive halogenated compounds in their cells (Paul et al. 2006). These compounds provide Asparagopsis biomass with anti-microbial, anti-parasitic and antitumor activities, with numerous biotechnological and industrial applications (Paul et al. 2006; Hutson et al. 2012; Mata et al. 2013; Greff et al. 2014; Pinteus et al. 2018). Recently, it was discovered that bromoform (the main halogenated compound found in Asparagopsis) also has anti-methanogenic activity. Supplementing ruminant diets with Asparagopsis at low inclusions rates (0.2–2% of the animal's daily ration weight) significantly reduced enteric methane emissions by up to 98% (Kinley et al. 2016, 2020; Machado et al. 2016; Roque et al. 2019, 2021). Methane released from ruminant digestion is the food system’s single largest negative planetary impact responsible for approximately 80 million metric tons of CO2-equivalent/year (FAO 2020). Realizing global scale climate benefits from Asparagopsis anti-methanogenic properties requires the development of an entirely new seaweed farming sector.

Asparagopsis is a versatile genus for aquaculture due to the heteromorphic nature of its life cycle, which allows a range of different propagation strategies (see Zanolla et al. (2022) for details on cultivation strategies for the different life cycle stages). In this work, we focused on the cultivation of the tetrasporophyte phase, suitable for on-shore cultivation using vertical bioreactors. The algae grow vegetatively (clonally) through apical growth and, when filaments break down, fragments develop into new clumps, allowing a continuous propagation and production of biomass, assuming all growth requirements are met. Growing the sporophyte can be a standalone production method of Asparagopsis biomass (Schuenhoff et al. 2006; Mata et al. 2010), as well as a source of biomass (broodstock) to induce sporogenesis and produce the spores for gametophyte cultivation in the ocean. Therefore, mastering the cultivation of the sporophyte phase is of paramount importance to produce Asparagopsis biomass, regardless of the chosen culture method (in- or off-shore).

Light management is a particularly valuable tool in seaweed cultivation due to its direct influence on photosynthetic activity, biomass growth, and biochemical composition (Godínez-Ortega et al. 2008; Borlongan et al. 2020; Zepeda et al. 2020; Ghedifa et al. 2021; Liang et al. 2022). The sporophyte of Asparagopsis can be produced both in outdoor (using natural sunlight) or indoor systems (using artificial light) (Schuenhoff et al. 2006; Mata et al. 2010; Nilsson and Martin 2022). In both cases, light can be managed by controlling the spectral composition (using filters or LED lights) and the intensity (using neutral density filters or dimmable lights), as well as by manipulating the density of the algae inside the culture vessels. In the only study that focused on the effects of light quality and intensity on Asparagopsis growth and morphology, Monro and Poore (2005) demonstrated that spectra with medium to high ratios of Blue:Red light promoted compact phenotypes of A. armata sporophyte (higher number of meristems correlated with second-order branch production) and consequently optimized growth, particularly using higher irradiance levels (40 and 60 μmol photons m−2 s−1). Studies on the effects of light intensity on Asparagopsis sporophyte physiology have so far only evaluated the photosynthetic response through photosynthesis-irradiance curves (PI-curves) (Mata et al. 2006; Zanolla et al. 2015). The two studies suggested different photoinhibiting irradiances, with Mata et al. (2006) reporting photoinhibition at light intensities above 100 µmol photons m−2 s−1, but no photoinhibition was reported in Zanolla et al. (2015) at intensities as high as 600 µmol photons m−2 s−1. While PI-curves provide valuable information, the irradiance values used as a reference may not be directly used to optimise seaweed culture, particularly because the experiments from the above-mentioned studies were carried out in static conditions, which do not correspond to the reality of cultivation, where tetrasporophytes are cultivated in a dynamic light environment (biomass in tumbling suspension at varying densities), with variable self-shading patterns that affect both the quality and intensity of light, regardless of the light source settings.

Light can also be used as a management tool to enhance the concentration of valuable compounds in seaweed aquaculture. To the best of our knowledge, no studies analysed the effects of light quality or intensity on the concentration of bromoform in Asparagopsis. There is substantial evidence that bromoform and other halogenated molecules are produced as a part of a reactive oxygen species production and manipulation cascade, which converts hydrogen peroxide to halogenated chemicals via specific halogenated enzymes (Thapa et al. 2020). Consequently, light intensity may affect the production of bromoform indirectly, as increasing irradiances may lead to oxidative stress as a result of increasing production of hydrogen peroxide and other reactive oxygen species (Dummermuth et al. 2003). On the other hand, directly adding hydrogen peroxide in A. taxiformis cultures resulted in physiological damage to the seaweed, inhibition of the activity of halogenated enzymes, and ultimately in a decrease of bromoform concentration in the tissue (Mata et al. 2011).

The objectives of this study were to evaluate how light quality and intensity affect biomass growth, photosynthetic performance, and bromoform content of Asparagopsis taxiformis tetrasporophyte, cultured in indoor tumbling conditions at two different biomass densities.

Materials & methods

Algal material, culture conditions and species identification

Samples of Asparagopsis tetrasporophytes were collected by snorkel from a depth range of 1–3 m at La Herradura bay, South Spain (36°43′23.4"N, 3°43′36.5"W), in October 2018. Samples were taken to the laboratory at the Centre of Marine Sciences (CCMAR, Portugal) and cleaned from visible epiphytes under a dissecting microscope. Seaweeds were cultured in transparent culture vessels with bottom aeration to keep the biomass in constant movement and maintained in a walk-in culture chamber at constant temperature and light (22 °C, 12:12 light/dark cycle, 30 μmol photons m−2 s−1), using UV-sterilized natural seawater (salinity of 33 ppt and pH of 8.2), enriched with one-half (f/2) of medium f (Guillard 1975), refreshed weekly. The biomass density was maintained between 1–2 g fresh weight (FW) L−1 for at least 30 months prior to the start of the experiments.

Due to morphological similarities between tetrasporophytes of the two species of the Asparagopsis genus (A. armata and A. taxiformis), the mitochondrial marker cytochrome oxidase subunit 2—subunit 3 (cox2-3) spacer (Zuccarello et al. 1999) was chosen to obtain phylogenetic information and properly identify the species used in this study. DNA extraction and PCR-amplification were performed following procedures described in Torres et al. (2021). PCR products were sequenced in an automated capillary sequencer (Applied Biosystems, CCMAR, Portugal). Newly generated and downloaded sequences of the cox2-3 spacer from Asparagopsis spp. (Andreakis et al. 2016) from GenBank were proofread, edited, and aligned in GENEIOUS v.2019.2.1 (http://www.geneious.com). The cox2-3 sequence of the tetrasporophyte used in this study was similar to the Italy (AY589530) and South Africa (JF820073) specimens of Asparagopsis taxiformis Lineage 2 and, therefore, identified as A. taxiformis.

Light quality experiment

To test the effect of light quality on growth and bromoform content of A. taxiformis, cultures were subjected to a combination of four light qualities: High-Blue:Red (High-BR); Medium Blue:Red (BR); High-Blue:Green:Red (High-BGR); and White (W) (Fig. 1), and two biomass densities (0.5 and 2.0 g FW L−1) for 3 weeks. The first week was considered as the acclimatory period so no data from that time was included in the final analysis. The different qualities of light were achieved using light emitting diodes (LEDs) (ELIXA 600W, Heliospectra), programmed using the Heliospectra System Assistant software to produce different relative spectral distributions with equivalent total PAR (Fig. 1). Spectral distribution was measured by using a spectroradiometer (OceanOptics USB2000 + UV–VIS). For this study, blue light was considered as the 400–485 range, red light was 630–700 nm, and the intermediate waveband (486–629 nm) was referred to as green light, although this waveband also includes orange and yellow spectra components. For all treatments, the irradiance (PAR) was maintained constant at 30 μmol photons m−2 s−1, a level within the light-limited portion of the PI curve, as determined by preliminary tests. Experimental vials were installed inside white foamboard cabinets, to create homogenous and discrete light fields for all light sources. For each light quality treatment, five replicates (n = 5) of 0.5 or 2 g FW L−1 (depending on each biomass density treatment) of A. taxiformis were placed in transparent glass flasks whit 1 L of UV-sterilized natural seawater (salinity 33 ppt, pH 8.2) enriched with f/2 medium (Guillard 1975) and maintained under controlled temperature (22 °C), with a light:dark cycle of 12:12 h. Culture flasks with constant bottom aeration provided water and seaweed motion. At the end of each week, algae were weighed and returned to refreshed culture media at the experimental stocking densities. At the end of the experiment, all algae biomass was stored at − 20 °C until chemical analysis. Prior to the analysis, all biomass was freeze-dried (− 50 °C, 80 μbar, Labconco, USA) for 48 h and homogenized.

Fig. 1
figure 1

Relative spectral distributions of the four light qualities used in this study: (a) High Blue: Red, (b) Medium Blue:Red, (c) High Blue:Green:Red and, (d) White

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Light intensity experiment

To test the effect of light intensity on growth and bromoform content, A. taxiformis tetrasporophyte was exposed to a combination of four light intensities (30, 60, 90, and 120 μmol photons m−2 s−1) and two biomass densities (0.5 and 2.0 g FW L−1) for 3 weeks. The first week was considered the acclimatory period, so no data from that time was included in the final analysis. The spectral composition was maintained at the W light quality (white light). Irradiance levels were achieved using LEDs attenuated with neutral-density light filters. White foamboard cabinets were again used to create homogenous and discrete light fields for each light source. For each light intensity treatment, five replicates (n = 5) of 0.5 or 2 g FW L−1 (depending on each biomass density treatment) of A. taxiformis were placed in transparent 1-L glass flasks filled with UV-sterilized natural seawater (salinity 33ppt, pH 8.2) enriched with f/2 medium (Guillard 1975) and maintained under controlled temperature (22 °C), with a light:dark cycle of 12:12 h. Culture flasks were supplied with constant bottom aeration to provide water motion and supply carbon. At the end of each week, algae were weighed and returned to refreshed culture media at the experimental stocking densities. At the end of the experiment, the effect of light intensity on photosynthetic response was evaluated by measuring oxygen evolution rates. The remaining algal biomass was stored at − 80 °C until chemical analysis. Before analysis, all material was freeze-dried (− 50 °C, 80 μbar, Labconco, USA) for 48 h and homogenized.

Daily growth rates

Growth was assessed weekly as the difference in total biomass, over the three weeks of each experiment. Before weighting, algae were centrifuged (30 s, 1690 × g) to remove residual water. The daily growth rate (DGR), expressed as % day−1, was determined using the following formula, as recommended by Yong et al. (2013):

$$DGR=\left[{\left(\frac{{W}_{t}}{{W}_{0}}\right)}^{1/d}-1\right]\times 100$$

where Wt and W0 are the final and initial FW mass, and d is the time in days.

Since the first week of each experiment was considered the acclimation period, DGR was reported as the mean of the second and third weeks.

Photosynthetic response

The photosynthetic response of A. taxiformis to light intensity was assessed through photosynthesis-irradiance (PI) curves, by measuring oxygen evolution rates after the light intensity experiment. For each PI curve (n = 5), 150 or 600 mg FW of A. taxiformis (depending on each biomass density treatment) were transferred to the measuring chamber, filled with 300 mL of refreshed culture medium, where nitrogen was briefly bubbled to lower O2 saturation and thus prevent potential inhibitory effects on photosynthesis caused by oxygen supersaturation. Algae were kept in constant motion inside the vial using a magnetic stirrer. Water temperature was kept constant at 22 ֯C. For each replicate curve (n = 5), 8 light levels were applied sequentially, increasing from 9 to 158 µmol photons m−2 s−1. Different irradiance levels were achieved by setting neutral density filters between the light source (W light quality) and the reaction chamber. Each light level was imposed for 10 min, except at 9 µmol photons m−2 s−1, where exposure times was 15 min.

Changes in dissolved oxygen concentration were measured using a Microx4 fiber optic oxygen meter (PreSens, Germany). Net photosynthesis rates (NP; μmol O2 g FW−1 h−1) were calculated as follows:

$$NP=\frac{\frac{{\left({O}_{2}\right)}_{f}-{\left({O}_{2}\right)}_{i}}{T}\times V}{FW}$$

where \({\left({O}_{2}\right)}_{f}\)= Final O2 concentration (μmol L−1); \({\left({O}_{2}\right)}_{i}\)= Initial O2 concentration (μmol L−1); T = Incubation time (h); V = Volume of the chamber (L); FW = fresh weight (g).

PI curves were fitted with the model equation of Smith and Talling (Smith 1936; Talling 1955):

$$P=P_m\left[\frac{\alpha E}{{\left({P_m}^{2}+{\left(\alpha E\right)}^{2}\right)}^\frac{1}{2}}\right]$$

in which E is the irradiance, α is the ascending slope at limiting irradiances, and Pm is the maximum photosynthetic rate. Curves were fitted iteratively using SigmaPlot 11.0 (Systat Software, Inc.) and the parameters Pm and α as well as their standard error were estimated for a confidence interval of 95%. The minimum saturation irradiance, Ik, was calculated as the ratio between the estimated Pm and α for each treatment, incorporating error propagation.

Bromoform content

The bromoform content was determined by GC–MS analysis according to Romanazzi et al. (2021) and adapted to the amount of biomass available. Briefly, samples (n = 5) from each treatment were split into three analytical replicates of 20 mg of powdered freeze-dried algae. These 20 mg were extracted twice with 10 mL of methanol sonicating at 0 °C for 30 min. Each extract was centrifuged (3000 × g for 10 min at 4 °C) prior to the collection of the supernatant. The two extracts were combined and further diluted (0.5 mL of the mixture to 10 mL with methanol) before GC–MS analysis. Analysis was performed on a Bruker 456 TQ GC–MS gas chromatograph equipped with a triple quadrupole mass analyzer, and GC was fitted with a ZB-waxplus capillary column (30 m × 0.25 mm i.d., 0.25 μm, Phenomenex). The injector was maintained at 180 °C, operated in split-less mode and an injection volume of 1 μL. The GC oven temperature was programmed to be held at 40 °C for 1 min, raised at 16 °C min−1 to 250 °C, and then held at 250 °C for 2 min. Helium was used as the carrier gas, with a flow rate of 1 mL min−1. MS Data Review software 8.2.1 (MS Workstation, Bruker Daltonics) was used for data analysis. A calibration standard curve for quantitation of bromoform was generated by serial dilution of a bromoform commercial standard (Merck Life Science S.L.). The concentration of bromoform was normalised according to the amount of dry biomass.

Data analysis

All results are presented as mean values ± standard deviation of replicate samples (n = 5), except when noted differently. To meet the analysis of variance (ANOVA) criteria of equal variance, data were square root-transformed. Differences in daily growth rates and bromoform concentration were tested using two-way ANOVAs, with light treatments (quality or intensity) and biomass density as the independent factors. Differences in the photosynthetic parameters observed at different light intensity treatments were tested using one-way ANOVA for each biomass density. Post hoc, multiple comparison analyses were done using the Turkey test. Differences were considered significant at a level of p < 0.05. Statistical analyses were performed using SigmaPlot 11.0 (Systat Software, Inc.).

Results

Effect of light quality

The DGR of A. taxiformis cultivated under different light qualities at two biomass density conditions is shown in Fig. 2. Average DGRs varied from 1.88 ± 0.16 to 8.08 ± 0.49% day−1 during the two weeks following the acclimation period. DGR was significantly affected by both light quality (F3,32 = 138.557, p < 0.001) and by biomass density (F1,32 = 2012.773, p < 0.001), with a significant interaction between both factors (F3,32 = 5.660, p = 0.003). DGRs decreased significantly (up to 2.8-fold) in cultures stocked at high biomass density (2 g FW L−1), regardless of the light quality used. The significantly highest DGRs were found at the High-BGR and W treatments at 0.5 g FW L−1 (8.08 ± 0.49 and 7.99 ± 0.35% day−1, respectively). These two light qualities (High-BGR and W) also promoted the highest rates within density 2 g FW L−1. There were no significant differences observed in DGRs between W and High-BGR, nor between BR and High-BR light qualities, regardless of the culture density (Fig. 2).

Fig. 2
figure 2

Daily growth rates (DGR) of Asparagopsis taxiformis at different light qualities and biomass density conditions. Data are presented as mean ± SD (n = 5). Different letters represent significant differences among the treatments (p < 0.05). High-BGR = High Blue:Green:Red; High-BR = High Blue:Red; BR = Medium Blue:Red; and W = White

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Regarding the concentration of bromoform in A. taxiformis, values ranged from 7.75 ± 2.63 to 18.56 ± 5.11 mg g−1 DW at the different light qualities and biomass densities (Fig. 3). Bromoform concentration was significantly affected by both light quality (F3,32 = 8.312, p < 0.001) and biomass density (F1,32 = 7.720, p = 0.009), and the interaction between these factors was not significant (F1,32 = 1.571, p = 0.216). The highest concentration of bromoform was in the combination of W light and 0.5 g FW L−1 (18.56 ± 5.11 mg g−1 DW). Regardless of culture density, there were no significant differences in the concentration of bromoform among the High-BGR, BR and High-BR groups (Fig. 3).

Fig. 3
figure 3

Bromoform concentration (mg g−1 DW) of Asparagopsis taxiformis cultivated at different light qualities and biomass density conditions. Data are presented as mean ± SD (n = 5). Different letters represent significant differences among the treatments (p < 0.05). High-BGR = High Blue:Green:Red; High-BR = High Blue:Red; BR = Medium Blue:Red; and W = White

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Effect of light intensity

The DGR of A. taxiformis cultivated under different W light intensities and biomass densities varied from 4.04 ± 0.29 to 13.10 ± 2.35% day−1 during the two weeks following the acclimation period (Fig. 4). DGR differed significantly between light intensities (F3,32 = 28.462, p < 0.001) and biomass densities (F1,32 = 1089.073, p < 0.001) and there was no significant interaction between these two factors (F1,32 = 2.885, p = 0.051). Again, DGRs were significantly lower (up to 2.7-fold) for replicates cultivated at higher density (2 g FW L−1), regardless of the light intensity used. The significantly highest DGRs were found at 90 and 120 μmol photons m−2 s−1 treatments at 0.5 g FW L−1 (12.93 ± 2.14 and 13.10 ± 2.35% day−1, respectively) and there was no significant difference between the two rates at these conditions. There were also no significant differences when A. taxiformis was cultivated under 30 or 60 μmol photons m−2 s−1 within each biomass density level (Fig. 4).

Fig. 4
figure 4

Daily growth rates (DGR) of Asparagopsis taxiformis cultivated at different white light intensities and biomass density conditions. Data are presented as mean ± SD (n = 5). Different letters represent significant differences among the treatments (p < 0.05)

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The photosynthetic activity of A. taxiformis responded to increasing light intensity stimulation with a typical P-I hyperbolic-shaped response (Fig. 5). The parameter estimates, that describe the significant features of each P-I curve, are shown in Table 1. At the 0.5 g FW L−1 treatments, the maximum photosynthetic rates (Pm) were not affected by light intensity levels (F3,16 = 1.324, p = 0.301), while the photosynthetic quantum efficiency (α) decreased, and minimum saturation irradiance (Ik) increased with the increased light intensity levels (F3,16 = 11.312, p < 0.001 and F3,16 = 10.362, p < 0.001, respectively). On the other hand, the 2 g FW L−1 treatments, Pm rates were affected by light intensity levels (F3,16 = 3.312, p = 0.0471), presenting a trend of increasing at 60 μmol photons m−2 s−1 and then decreasing when the light intensity levels increased. These Pm were always lower (threefold or more) than those in treatments of 0.5 g FW L−1. No significant difference was observed among α or Ik (F3,16 = 0.937, p = 0.446 and F3,16 = 2.844, p = 0.071, respectively).

Fig. 5
figure 5

Light response curves of Asparagopsis taxiformis cultivated at different white light intensities (30 (a); 60 (b); 90 (c); and 120 (d) 120 μmol photons m−2 s−1) and two biomass density conditions. The model equation of Smith and Talling (Smith 1936; Talling 1955) was adjusted to the observed points

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Table 1 Photosynthetic parameters of Asparagopsis taxiformis obtained after fitting the data with the Smith and Talling (Smith 1936; Talling 1955) P-I model
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Concerning the concentration of bromoform in A. taxiformis, values varied between 18.34 ± 3.32 and 29.26 ± 3.45 mg g−1 DW at the different light intensities and biomass densities (Fig. 6). Bromoform concentration was significantly affected by light intensity (F3,32 = 24.648, p < 0.001) and not by biomass density levels tested (F1,32 = 0.311, p = 0.581), with a significant interaction between both factors (F3,32 = 3.404, p = 0.029). The significantly highest concentration of bromoform was in the combination of 90 µmol photons m2 s−1 irradiance and 2 g FW L−1 (34.18 ± 2.00 mg g−1 DW). No significant differences were observed in the bromoform concentration between 30 and 60 μmol photons m−2 s−1 treatments, regardless of biomass density, and bromoform concentration significantly decreased when A. taxiformis was cultivated under 120 μmol photons m−2 s−1 at 0.5 g FW L−1 (Fig. 6).

Fig. 6
figure 6

Bromoform concentration (mg g−1 DW) in Asparagopsis taxiformis cultivated under different white light intensities and biomass density conditions. Data are presented as mean ± SD (n = 5). Different letters represent significant differences among the treatments (p < 0.05)

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Discussion

Our results show that the light spectra containing intermediate wavelengths promoted the highest growth rates of A. taxiformis, compared to the other spectral conditions tested. Increases in the growth rate of up to 50% were observed in the W and High-BGR quality treatments at both biomass densities. The W and High-BGR treatments consisted of continuous wavebands containing a broad range of intermediate wavelengths (485–630 nm), whereas the lower performing BR and High-BR qualities consisted mostly of restricted blue and red lights. In this regard, it is known that Rhodophytes contain high phycoerythrin levels, a pigment that absorbs efficiently in the green region of the spectrum (495–570 nm) (Hurd et al. 2014). Therefore, the positive effect found on growth under intermediate wavelengths may be associated with a higher quantum efficiency under green-including light. The use of green and white lights also shown to promote the growth of other red seaweed species, such as Halymenia floresii (Godínez-Ortega et al. 2008) and Pyropia haitanensis (Wang et al. 2020). Moreover, green light was able to enhance the photosynthetic activity of Meristotheca papulosa (Borlongan et al. 2020), while in Gracilaria gracilis the electron transport rate significantly increased under green light when compared to the control group (Ghedifa et al. 2021).

Asparagopsis taxiformis growth rates were lower under BR and High-BR supplemented light, and it should be noted that they were not affected by the different ratios of blue and red light. These results contrast those reported by Monro and Poore (2005), who found that blue light-enriched treatments induced faster growth rates in the congeneric species A. armata, and that a low ratio of blue to red light was correlated with lower growth rates. Two factors may have contributed to this discrepancy; firstly, a potential species-specific physiological difference between the two taxa (A. armata and A. taxiformis), and secondly the drastic difference between the dynamic light environment used in this study (tumble culture) compared to the stable light environment used in Monro and Poore’s study (static culture). In this sense, the seaweed in the tumble culture may act as a non-neutral density filter, adding a degree of self-shading and preventing the wavelengths reflected or absorbed by the algae from penetrating deeper into the culture unit. As a result, the conditions of light quantity and quality that the algae are exposed to as they move up and down in the tumble culture vary significantly with their distance to the light source and may also vary with increasing biomass density, as higher densities increase the self-shading phenomenon. However, our findings suggest a weak interaction between light quality and biomass density on growth rates, as both biomass densities tested showed the same effects of light quality on growth. Hence, within the tested range of biomass densities, the growth-stimulating effect of intermediate wavelengths on A. taxiformis does not seem to be affected by the increasingly dynamic light environment associated with increased biomass density.

The highest bromoform concentration was obtained in the W light treatment, but only at density of 0.5 g FW L−1. In seaweeds, bromoform, and other halogenated molecules are produced along with several reactive oxygen species that convert hydrogen peroxide into halogenated chemicals (Thapa et al. 2020). Our results suggest that for A. taxiformis, W light with low shelf-shading (lower biomass density) may induce some level of oxidative state inside the cells, leading to increasing bromoform production, compared to other light qualities. Zepeda et al. (2020) found an increase in antioxidant activity (an indicator of reactive oxygen species increases and eventual oxidative stress) under white light and white + blue light in Solieria filiformis and Gracilaria cornea, respectively. On the other hand, at the higher density of 2 g FW L−1, the bromoform concentration in A. taxiformis was not sensitive to the different light qualities, suggesting that with this higher level of self-shading the W light does not induce enough of an oxidative state to result in higher bromoform levels. Thus, unlike growth, the stimulatory effect of W light on bromoform production appears to be counterbalanced by biomass increase.

Considering the above-mentioned results, the W light spectrum was selected to investigate the effects of light intensity on the growth rates and bromoform content of A. taxiformis. Growth responded positively to increasing light intensity regardless of the stocking biomass density used, with a plateau above 90 μmol photons m−2 s−1, suggesting that, beyond this irradiance, increased light intensity does not promote increased growth. The most notable differences in growth were observed between the 2 stocking densities, regardless of the light intensity. For example, the seaweed stocked at 0.5 g FW L−1 under the lowest light intensity of 30 μmol photons m−2 s−1 had a twofold higher growth than those cultures with the highest light intensity of 120 μmol photons m−2 s−1 but stocked at 2 g FW L−1 (higher self-shading). These results highlight the importance of adjusting biomass density (self-shading) in A. taxiformis tetrasporophyte cultures in order to manage biomass growth rates.

The parameters estimated of the P-I curves performed at the end of the experiment also highlight the power of the biomass density effect, where the maximum photosynthetic rates (Pm) and α values in the low biomass density were more than 3.5-fold higher than in the 2 g FW L−1 cultures, possibly due to the decrease in the available light associated with self-shading, whereas changing light intensity within the same biomass density had mild effects on these two parameters. At 0.5 g FW L−1, increasing light intensities above 30 μmol photons m−2 s−1 did not induce significant alterations in the Pm, suggesting that A. taxiformis is capable of optimal photosynthetic efficiency at low light intensities. This is corroborated by the significantly higher initial slope (α) of the P-I curve at 30 μmol photons m−2 s−1. These photosynthetic parameters suggests that A. taxiformis is a shade-type macroalgae (Reiskind et al. 1989), as also concluded previously by Mata et al. (2006) for the tetrasporophyte of A. armata. At the culture density of 2 g FW L−1, Pm increased from 30 to 60 μmol photons m−2 s−1 and then tended to decrease at the uppermost irradiance levels, and no changes were observed in α. The saturating irradiance (Ik) parameter provides valuable information to set the optimal irradiance for the cultures. Seaweed should not be continuously exposed to the saturating irradiance levels (Ik) as it may cause damage to the photosynthetic apparatus. In this sense, our results show that the cultures at the highest light intensity treatments (90 and 120 μmol photons m−2 s−1) were already above the saturation irradiance, for both culture densities (for example, at 90 μmol photons m−2 s−1, Ik was 65.2 and 53.9 μmol photons m−2 s−1 at densities of 0.5 and 2 g FW L−1, respectively). This result suggests that long-term cultures of A. taxiformis should be maintained at light intensities no higher than 60 μmol photons m−2 s−1. It should be noted that all the cultures at light intensities above 60 μmol photons m−2 s−1 had filamentous cyanobacteria developing amidst the branches of A. taxiformis after 21 days of cultivation, while at 30 and 60 μmol photons m−2 s−1 no contamination was noticeable. Contamination control in cultures of Asparagopsis is a significant challenge (Dishon et al. 2023), due to the size and filamentous nature of Asparagopsis tetrasporophytes. As such, maintaining A. taxiformis cultures at irradiances between 30 and 60 μmol photons m−2 s−1 is advisable in order to prevent the development of contamination, with the caveat of a sub-optimal growth rates.

There was no evident pattern between bromoform content, irradiance levels and biomass density. Notably, the biomass subjected to the highest light exposure (higher irradiance of 120 μmol photon m−2 s−1 and lower self-shading at 0.5 g FW L−1) had the lowest bromoform content. This goes against the initial expectations, that increasing irradiances that lead to hydrogen peroxide production and oxidative stress (Dummermuth et al. 2003) result in increasing bromoform synthesis (Ohsawa et al. 2001). However, it is in accordance with the results obtained by Mata et al. (2011) for A. taxiformis, where exposure to oxidative stress, through the addition of hydrogen peroxide in the culture water, inhibited the activity of iodoperoxidase (used as a proxy for bromoperoxidase, the enzyme involved in the production of bromoform) and reduced the bromoform concentration over exposure time. After 8 days of exposure to hydrogen peroxide the bromoform levels were 70% lower than initial values (Mata et al. 2011). In the current study, A. taxiformis was exposed to potentially oxidative stress inducing irradiances at 120 μmol photons m−2 s−1 (above the Ik) for 21 days, which may have inhibited the synthesis of bromoform. This pattern was similar in the marine diatom Ditylum brightwelli (Abe et al. 2022), where exposure to irradiances of 120 μmol photons m−2 s−1 tented to reduce the relative production rates of bromoform, but stimulated the production of other halogenated compounds, such as chloroform and dibromomethane. A possible explanation for the decrease of bromoform content with increasing light (and hydrogen peroxide) could reside in the prosthetic group of the haloperoxidase. Two distinct types of marine haloperoxidases are identified in algae: those that contain vanadium as a cofactor and those containing Fe-heme (Butler and Walker 1993). Heme-containing peroxidises are inhibited at much lower concentration of hydrogen peroxide than vanadium-enzyme activity (Zuurbier et al. 1990). Thapa et al (2020), recently demonstrated that vanadium-dependent haloperoxidases are involved in bromoform synthesis but also detected multiple highly transcribed heme-dependent peroxidases in the Asparagopsis transcriptomes. The heme type haloperoxidase of the unicellular rhodophyte Porphyridium purpureum, was inhibited at relatively low concentrations of hydrogen peroxide concentrations (Murphy et al. 2000), and when exposed to high irradiance did not result in increased production of halogenated compounds (Scarratt and Moore 1999).

Our study shows that light containing intermediate wavelengths supports fast-growing A. taxiformis tetrasporophytes in indoor cultures, with white light being the preferred option, as it also promotes higher bromoform content. Additionally, it is suggested that cultures stocked with up to 2 g FW L−1 of biomass should be maintained below the irradiance of 60 μmol photons m−2 s−1 to prevent photosynthetic stress and to avoid the development of biological contamination over time in culture, even if higher growth rates could be achieved with higher irradiances. These results provide valuable information to optimise the production of biomass and bioactive compounds in indoor cultures of the genus Asparagopsis.