Rare earth elements (REEs) are a group of 17 chemical elements that comprise yttrium (Y), scandium (Sc), and a series of 15 lanthanides. REEs have practically identical physical and chemical properties although they have rather unique magnetic and catalytic properties (Cheisson and Schelter, 2019; Čížková et al., 2021). These properties make them desirable in a wide range of industries such as electrical, electronics, laser, glass, magnetic materials, energy technology, aquaculture, and agriculture (Hu et al., 2004; Chakhmouradian and Wall, 2012; Balaram, 2019). In recent decades, the use of fluorescent lamps (FLs), involving both tubes and compact fluorescent lamps (CFLs), has increased globally to improve energy efficiency. However, due to the presence of mercury and REEs as essential components, their use can be environmentally harmful (Balaram, 2019; Pagano et al., 2019). The shift from incandescent lights and CFLs to light-emitting diodes has generated a considerable amount of waste electrical equipment (WEE) (Baldé et al., 2017; Gwenzi et al., 2018; Pagano et al., 2019). Among the most common elements present in WEE are the REEs yttrium (Y), europium (Eu), and terbium (Tb). These economically important REEs are usually discarded into our environment as wastes, having a consequential effect on the linear flow of goods throughout the economy (Balaram, 2019). Increased demands for REEs, has exerted pressure on industrialized countries to look for alternatives to fulfill their demand in an eco-friendly manner. Bio-removal of WEE containing REEs, and their recycling can have a positive impact, balancing both environmental and economic benefits.

The role of plants, microbes (i.e., bacteria, cyanobacteria, and fungi), and algae as REE accumulators, bio-removers, and potential bio-miners have been studied extensively (Dubey and Dubey, 2011; Qu and Lian, 2013; Pinto et al., 2020; Jalali and Lebeau, 2021). Among these, algae-based bioaccumulation/bio-removal is considered to be one of the most promising methods due to its high efficiency, wide applicability and the low-cost recovery. However, mostly contaminated water, soil or red mud were used as secondary sources of REEs (Pinto et al., 2020; Lima and Ottosen, 2021). Regarding WEE (e.g., CFL lights) as secondary source of REEs, only a few studies have demonstrated the use of microalgae as potential REE accumulators/sorbents or removers (Čížková et al., 2021). However, several studies have been conducted that demonstrate the growth of green and red algae in the presence of a single REE, or waste containing a mixture of REEs (Čížková et al., 2021; Pinto et al., 2021). The growth of green algae Chlamydomonas reinhardtii, Chlorella sp. or Arthrospira sp. improved in the presence of REEs (Liu and Shizong, 1999) whereas, another study showed that due to the presence of multiple REEs in CFL powder, Galdieria growth and dry matter accumulation slowed compared to the control (Čížková et al., 2021).

In addition, extensive research has been conducted to investigate heavy metal (HM) bioaccumulation/bioremediation by algae employing different chemical agents and phytohormones. However, the underlying mechanisms controlling the effect of phytohormones on bioremediation are elusive (Piotrowska-Niczyporuk et al., 2012, 2018). Unlike HM bioremediation, fewer studies have been conducted to understand REE bio-removal/bioaccumulation (Pinto et al., 2020; Čížková et al., 2021; Pinto et al., 2021). Literature describing the use of chemical agents commonly employed in bioremediation, or phytohormones that affect the biosorption/bio-removal of REEs are also scarce. This study is the first showing the use of two phytohormones, 6-Benzylaminopurine (BAP - Cytokinin family, known to affect cell division) and 1-Naphthaleneacetic acid (NAA - Auxin family, generally known as growth stimulators) on REE accumulation by the red algae G. sulphuraria.

Recently, algae from the extremophile group of cyanidiophyceae, especially the unicellular red alga Galdieria, has been proposed as suitable models that have the potential to accumulate or adsorb different REEs (Minoda et al., 2015; Čížková et al., 2019, 2021). G. sulphuraria grows under thermo-acidophilic conditions, and inhabits hot sulfur springs, toxic metal-containing and geothermal habitats (Gross et al., 1998; Yoshimura et al., 1999; Reeb and Bhattacharya, 2010; Minoda et al., 2015). The organism is known to thrive over in moderate/high temperatures ranging from 35°C to 56°C (Reeb and Bhattacharya, 2010; Carfagna et al., 2015; Bottone et al., 2019), and pH ranging from 0.2 to 6 (Yoon et al., 2006; Náhlík et al., 2021; Abiusi et al., 2022). Its resistance to toxic metals and REEs is exceptional among other eukaryotic algae, thus it is a suitable organism to achieve bioaccumulation/bio-removal of metals from waste material (Yoshimura et al., 1999; Minoda et al., 2015; Čížková et al., 2021). Unlike other microalgae, G. sulphuraria produces highly branched and low molecular weight glycogen as an energy and carbon reserve instead of starch supplying energy for processes related to cell multiplication such as DNA replication, nuclear division, cytokinesis, daughter cell formation and release of autospores (Vítová et al., 2015; Martinez-Garcia et al., 2017). The accumulation of starch during the cell cycle, and its consumption during the cell division in dark phase has been described in several synchronized cultures of green algae (Brányiková et al., 2011; Vítová et al., 2015). Recently, Náhlík et al. studied the accumulation of glycogen in G. sulphuraria throughout the cell cycle following light and dark phases. Results showed that some accumulated glycogen was consumed during cell division (Náhlík et al., 2021). Like other microalgae, G. sulphuraria divides by multiple fission and releases 2ⁿ autospores from a single mother cell within one cell cycle (Jong et al., 2021).

One of the important fluorescence parameters followed in photosynthetic microalgae is the F_v/F_m ratio, a valuable bio-indicator for overall photosynthetic performance, indicating maximal photochemical quantum efficiency of photosystem II (PSII). The F_v/F_m index also indicates stress conditions such as temperature, light, pH, nutrient concentration, or toxic metals (Schreiber et al., 1986; Iovinella et al., 2020). The ratio of F_v/F_m differs in photosynthetic organisms from plant to cyanobacteria, in land plants F_v/F_m varies between ∼0.75–0.8, and slightly lower values ∼0.7 were reported in green algae. However, red algae and cyanobacteria have significantly lower values of F_v/F_m ratio between ∼0.5–0.6 and ∼0.2–0.4, respectively (Oesterhelt et al., 2007; Iovinella et al., 2020). Like photosynthetic efficiency, photosynthetic pigments are also vulnerable to stress conditions. Until now, the effect of REEs on photosynthetic pigments have not been investigated thoroughly, although research has shown that exposure to Lu³⁺ (lutetium) and Sc³⁺ (scandium) significantly reduced levels of photosynthetic pigments in Parachlorella kessleri (Goecke et al., 2017).

In the present study, we describe the effect of a CFL acid extract on a synchronous culture of G. sulphuraria throughout the cell cycle. Furthermore, accumulated glycogen and the photosynthetic pigment profile of a synchronously growing and dividing culture were analyzed. This study provides insight into the impact of REEs on the cell cycle of G. sulphuraria. The study also determined how efficiently this red alga could accumulate REEs from a CFL extract and the effect of two synthetic plant hormones (BAP and NAA).

Before conducting the experiments and because CFL was extracted into 10% HNO₃, the effect of 10% HNO₃ on a synchronous culture of G. sulphuraria was studied. Results of this preliminary study showed that there was no negative effect of 10% HNO₃ on the cell shape, size, or growth of this organism (Supplementary Figures S1, S2).

The extremophilic red alga G. sulphuraria was selected for the present study due to its ability to grow in a range of diverse habitats including those having thermophilic, acidophilic, halophilic and toxic metal conditions (Oesterhelt et al., 2007; Weber et al., 2007). The growth of cyanobacteria and algae in the presence of REEs has been demonstrated previously. Dubey and Dubey reported on the growth of three cyanobacteria, Phormidium, Oscillatoria and Lyngbya, in presence of red mud (Dubey and Dubey, 2011). Similarly, the growth of 6 living macroalgae species, i.e., Ulva lactuca, Ulva intestinalis, Fucus spiralis, Fucus vesiculosus, Osmundea pinnatifida and Gracilaria sp. in laboratory-prepared seawater solution containing REEs (Y, La, Ce, praseodymium (Pr), neodymium (Nd), Eu, Gd, Tb, dysprosium (Dy)) has been studied (Pinto et al., 2021). However, until now, only a few studies have been conducted to evaluate the growth and accumulation of REEs present in waste luminophores. Interestingly, this is the first study that examines the effect of a CFL acid extract on growth and the bioaccumulation of REEs present in electronic waste of CFL lights. Prior to this study, Čížková et al., used the unicellular red alga Galdieria phlegrea to examine the growth and bioaccumulation of REEs from luminophore powder (of two different sources such as energy saving light bulbs - CFL, and fluorescent lamps - FL) (Čížková et al., 2021). The findings of the present study confirmed that the growth of G. sulphuraria was optimal under the acidic conditions (Supplementary Figures S1, S2). Thus low growth (dry matter) observed in the presence of the CFL acid extract compared to the control culture (Figure 1) could have been due to a negative effect of REEs.

The biomass loss recorded in the dark phase of both control and treated cultures (Figure 1, dark phase) was due to the phenomenon of night biomass loss or respiration loss. This phenomenon is an essential property of photosynthetic algae, and acts as a tax on day biomass gains; approximately 30% of the algal biomass produced during the day can be lost at night (Guterman et al., 1989; Hu et al., 1998; Edmundson and Huesemann, 2015). Edmundson and Huesemann, studied night biomass loss in three potential commercial biomass-producing algal strains, Chlorella sorokiniana, Nannochloropsis salina and Picochlorum sp. They reported that specific night biomass loss rates were highly variable, and varied between −0.006 and −0.59 μ_dark day⁻¹ (Edmundson and Huesemann, 2015). The result also showed that night biomass loss was species-specific and influenced by environmental conditions such as culture temperature and light intensity prior to and during the dark phase. The night biomass loss was positively corelated with increasing cultivation temperature (Torzillo et al., 1991; Edmundson and Huesemann, 2015). Given the high optimal cultivation temperature (40°C) of G. sulphuraria, loss of biomass during the dark phase was not surprising.

All three photosynthetic pigments increased with progression of the cell cycle in both control and CFL–treated cultures (Figure 2). However, the CFL–treated culture showed reduced levels of Chl a as compared to the control, probably due to adverse effects of REEs present in the CFL acid extract (Figure 2A). A negative effect of abiotic stresses on photosynthetic pigments of algae and cyanobacteria has also been reported (Sun et al., 2014; Fu et al., 2020). In the present experiment, the phycocyanin was least affected pigment by CFL acid stress (Figure 2C). The reason could be a high stability of phycocyanin of extremophilic G. sulphuraria which was significantly more stable compared to phycocyanin of common cyanobacteria Spirulina platensis (Wan et al., 2021). All the members of cyanidiophyceae were considered as best organisms for the production of stable phycobiliproteins (Carfagna et al., 2018; Ferraro et al., 2020). Regarding carotenoids, their function also comprises a defense mechanism to mitigate the damaging effects of stress in photosynthetic organisms (Shi et al., 2020; Potijun et al., 2021). Microalgae respond to an increased exposure to dissolved metals by accumulating carotenoids as antioxidant compounds (Gauthier et al., 2020). In the present study, carotenoid content increased during the initial 4 h of the cell cycle (Figure 2B), probably to mitigate the stress caused by prime accumulation of REEs (Figure 6; 2 h) in the CFL–treated culture. Nonetheless, due to increased accumulation of certain REEs (e.g., Y, Eu) with the progression of the cell cycle (Figure 6; 10 h, 24 h) and due to their toxicity, carotenoids were not able to cope with this stress and levels began to decrease (Figure 2B). It is known that under acute metal stress the antioxidant capacity of microalgae can be depleted (Pinto et al., 2003). Similarly to this study, a selenium (Se) concentration of up to 75 mg L⁻¹ increased both the chlorophyll and carotenoid contents in Chlorella vulgaris; a higher concentration (>75 mg L⁻¹) of Se caused a significant decline in the overall content of carotene and chlorophyll a (Sun et al., 2014). This result showed that if the level of stress was higher, then no defense mechanism of the cell could protect it. Similarly, Cheng et al., showed that an increasing concentration and exposure time to cadmium (Cd) can cause a decline in Chl a, Chl b and carotenoids in C. vulgaris (Cheng et al., 2016). Like other metal stresses, photosynthetic pigments of Trachydiscus minutus and Parachlorella kessleri were evaluated after exposure to several single REEs and monazite (Goecke et al., 2017). Results showed that pigment content was variable according to the element and algal species used.

The F_v/F_m ratio signifies the maximum potential quantum yield of photosystem II, once all the reaction centers are exposed. It is used as an indicator of stress on PSII. Under stress conditions, the photosynthetic efficiency of PSII is reduced as the cell activates all photo-protective mechanisms. The determined starting values of F_v/F_m ratio (0.58; 0.55) are typical for red algae (Oesterhelt et al., 2007; Iovinella et al., 2020), and are not comparable with green algae or plants due to a fluorescence interference of phycocyanin present in red algae. Our results showed that both the control and CFL–treated cultures expressed a fall in the F_v/F_m ratio at the initial hour of the experiment, which could be due to sudden light exposure after a long (8 h) dark phase (Figure 3). Comparable F_v/F_m values and pattern were determined during the cell cycle of G. sulphuraria treated with red mud extract (Náhlík et al., 2022). Fu et al. also observed a similar pattern in Galdieria partita, where the F_v/F_m value started to decrease rapidly within 1 h of high light exposure (Fu et al., 2020). In our study, after the 1^st h of incubation, the control culture began to adapt to the new condition and eventually recovered significantly during the dark phase of the cell cycle. Nevertheless, CFL–treated cells were unable to recover significantly during the light phase of the experiment due to simultaneous light and CFL stress (Figure 3). Likewise, the maximum photochemical quantum yield (F_v/F_m) of the PSII reaction center decreased under La exposure in D. quadricauda (Ashraf et al., 2021). However, in G. sulphuraria, high light conditions stimulated cell growth and dry matter in both control and CFL–treated cultures. Recently, Kselíková et al., studied the effect of two different concentrations of deuterated water and three different light intensities on the F_v/F_m ratio in C. reinhardtii and D. quadricauda. They observed reduced F_v/F_m ratios with increasing light intensity and the concentration of deuterated water (Kselíková et al., 2022). The studies by Kselíková et al., and Ashraf et al., support results observed in the present study showing that increasing stress can reduce the photosynthetic efficiency of PSII (Ashraf et al., 2021; Kselíková et al., 2022).

Glycogen synthesis exactly followed the pattern of dry matter accumulation throughout the cell cycle in both control and CFL–treated cultures. Around 20–30% of the glycogen content produced in the light phase was consumed during the dark phase (Figure 4). Náhlík et al. observed a similar pattern of glycogen synthesis in synchronized cultures of G. sulphuraria (Náhlík et al., 2021). So far, the accumulation of glycogen during the cell cycle in Galdieria has not been studied. However, the consumption of glycogen during cell division was comparable to consumption of starch during the cell cycle in green algae (Vítová et al., 2015).

The algal cell cycle is associated with the nature of algal species and growth conditions. In the present study, the control culture of G. sulphuraria attained its 1^st division at 12 h and 2^nd at 16 h of the cell cycle (Figure 5A). Under the same growth conditions, G. sulphuraria attained the first cell division into two cells at 12 h and the second division into four cells at the 16th h of the cell cycle (Náhlík et al., 2021). Although, CFL–treated cells showed a 1–2 h delay in cell division which could be a toxicity adaptation time, eventually they completed their second division within the time course of the cell cycle (Figure 5B). At the end of the cell cycle (24 h of experiment) all cells had divided into four daughter cells in both control and CFL–treated cultures, but not released from the mother cell wall (Figures 5A,B). The 2 h shift was also confirmed by the calculation of the mass doubling time being about 10 h in the control culture and 12 h in the CFL treated culture. Although the cells finished the division to four in the CFL treated culture, the biomass gain was lower implying the daughter cell size had to be smaller (compare Figures 1, 5, 24 h). In contrast to Cyanidioschyzon, in Galdieria and Cyanidium, the divided cells (autospores) were surrounded by the mother cell wall before hatching (Jong et al., 2021). There was also a correlation between the number of following cell divisions and commitment cell size in cyanidialean red algae, including G. sulphuraria. The delayed cell division of the CFL–treated culture could be due to slower growth for cells to reach commitment size, as compared to the control culture (Jong et al., 2021). However, CFL–treated cells also finished the 2^nd division during the dark phase of the 24 h cell cycle. Similarly, Pseudokirchneriella subcapitata, a freshwater alga was exposed to different metals, i.e., cadmium (Cd), chromium (Cr) and copper (Cu), and their growth, cell volumes, and cell divisions were investigated over a period of 72 h (Machado and Soares, 2014). Results showed that the highest metal concentrations of Cr(VI) and Cu(II) arrested cell growth before the first nuclear division whereas Cd(II) arrested the cell after the second nuclear division but before the release of autospores from the mother cell wall (Machado and Soares, 2014). This variable impact of metals on the cell cycle of algae suggests that different metals trigger different toxicity mechanisms.

In the present study, REE accumulation was concentration-independent and highly selective with the progression of the cell cycle (Figure 6; Table 1). In G. sulphuraria, the accumulation of single REEs was not concentration dependent, e.g., the concentration of La and Ce was very high in the CFL acid extract, however the accumulation of Y and Eu was greater than the accumulation of La and Ce (Figures 6, 7). The increased or decreased metal concentration in cells at different phases of the cell cycle could be due to the selective behavior of G. sulphuraria towards individual REEs. If the cells were not able to tolerate toxicity of several REEs such as Ce and La, they excluded these metals from cells in a later phase of the cell cycle. This phenomenon could be a base for a future selective “extraction” of lanthanides from medium by continuous sampling during the cultivation. Similar findings were observed in Galdieria phlegrea, where the ratio of accumulated REEs differed significantly from their ratio in the growth medium (Čížková et al., 2021). Several studies show that the accumulation of light lanthanides, i.e., La, Ce, Pr, Nd, promethium (Pm), samarium (Sm) were preferred by organisms over heavy lanthanides (González et al., 2015; Yang and Wilkinson, 2018). Our results clearly showed that G. sulphuraria has tremendous capacity for lanthanide bio-mining/bio-removal, but further investigation is required to understand the detailed mechanisms behind the bioaccumulation/biosorption of REEs from e-waste (CFL lights) containing environments.

Interestingly, almost no research to date has been conducted on the effect of hormones on the bioaccumulation of REEs by microalgae. This is the first study that shows the accumulation of Y and Eu can be doubled in the presence of the plant hormones NAA and BAP (Figures 7A,B). However, the mechanism behind increased REE accumulation following NAA exposure remains elusive. A possible explanation could be that the application of exogenous cytokinins and auxins mitigated the toxicity of REEs and promoted cell growth, development, and regulated their adsorption similar to HM adsorption in green algae C. vulgaris and Acutodesmus obliquus (Piotrowska-Niczyporuk et al., 2012, 2018).

Being acidophilic in nature, the red alga G. sulphuraria can grow in the presence of a CFL acid extract containing 10% HNO_3, although growth was slightly slower than the control culture. Photosynthetic pigments such as chlorophyll a, carotenoids and phycocyanin were also decreased under CFL treatment, although carotenoid synthesis minimized the deleterious effects of CFL up to the initial 4^th h of the cell cycle. During the light phase of the cell cycle, photosynthetic performance, expressed as the ratio F_v/F_m, was negatively affected by CFL treatment, but later in the dark phase, recovered significantly. In the initial hours of the cell cycle, cell division was delayed by about 2 h with CFL treatment, although later cells were able to complete their 2nd division within 24 h of the cell cycle. The produced daughter cells were of smaller size. Accumulation of REEs by G. sulphuraria was concentration-independent and selective. The REEs most accumulated by G. sulphuraria were Y and Eu, followed by La, Ce, Gd, and Tb, respectively. The plant hormone NAA stimulated the accumulation of REEs and almost doubled Y accumulation. BAP also had a pronounced effect on Y and Eu accumulation, although it did not increase the accumulation of other REEs. Waste luminophores like CFL bulbs could be a great secondary source of REEs, and using the red alga G. sulphuraria may be promising in REE bio-removal/accumulation technology. Recovery of REEs from algal biomass could lead to an economic and eco-friendly solution for the removal of hazardous waste from CFL lights and for the generation of REEs for future use.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

MV, DS, and DM: conceptualization and funding acquisition. AS, MČ, and VN: methodology, investigation, and writing—original draft preparation. DS and DM: validation. MR: formal analysis. AS and MČ: resources. MV and DM: data curation, writing—review and editing, and project administration. MV and MR: visualization. MV: supervision. All authors contributed to the article and approved the submitted version.

This research was funded by the European fund for regional development, the program Interreg V-A Austria – Czech Republic, the Project ATCZ172 REEgain, the COST Action 19116 – PLANTMETALS and by the institutional support RVO 61388971 and RVO 67985939 of the Czech Academy of Sciences.

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

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.