Effects of Selenite on Growth and Selenium Accumulation in Three Terrestrial Microalgae with Application Potential in Food and Agriculture

Abstract

Selenium (Se)-enriched microalgae are emerging as new food or agricultural resources. Thus far, the microalgae tested for Se accumulation or biofortification are still very limited. Here, we investigated the effects of selenite on the growth of three terrestrial microalgae (Vischeria sp. WL1, Chlorella sp. WL1, and Nostoc flagelliforme) and their total Se accumulation levels. The three microalgae were previously collected at the same location and show great application potential but differ in classification and growth speed. Three concentrations (10, 20, and 40 mg/L) of selenite were used for treatment. The former two species could resist 40 mg/L selenite but the last one could not. At the relatively optimal concentration (20 mg/L selenite), their specific growth rates were 0.09, 0.10, and 0.05 μ day⁻¹, respectively. Vischeria sp. WL1 could accumulate 3.3 mg/g dry weight (DW) Se after 16 days of cultivation; N. flagelliforme could accumulate 5.6 mg/g DW Se after 24 days of cultivation. Chlorella sp. WL1 accumulated a relatively lower amount of Se, being 0.74 mg/L DW after 18 days of cultivation, but its more rapid growth would be beneficial to saving production costs. These results indicate that three microalgae are capable of functioning as excellent carriers for Se nutrients. This study will contribute to enriching the microalgal resource pool for Se biofortification in food and agricultural areas.

1. Introduction

Selenium (Se) is a natural trace element that exhibits close physical and chemical properties with sulfur and tellurium in the Periodic Table of Elements. It serves as an essential micronutrient for humans and various other organisms including bacteria, archaea, and microalgae [1]. It plays a crucial role in the body’s antioxidant function, immune regulation, and cancer/disease prevention [2,3]. However, Se at a high dose may give rise to toxic effects by inducing oxidative stress and generating abnormal selenoproteins [4,5]. Currently, there is a growing focus on the application of Se as a nutrient enhancer in the fields of food and agriculture [6].

The concentration of Se in soils exhibits high variability and is site-specific. The typical Se concentration in soil generally falls within the range of 0.01 to 2.00 mg/kg, and soil Se concentrations ranging from 1 to 5000 mg/kg are regarded as seleniferous [7]. Low soil Se concentration leads to a deficiency of the Se element in crop grains, and insufficient Se intake has emerged as a global health concern [8]. Se-enriched new food resources, particularly microalgae, have been rapidly developed to deal with this situation [1,9,10]. Inorganic Se can be converted to organic Se compounds in plants and microalgae, which are more biocompatible and more readily absorbed [11]. Microalgae-based biofertilizers can enhance crop production and soil fertility and are emerging as an alternative solution to sustainable agriculture [12]. Thus, Se-enriched microalgae also serve as an ideal Se-biofertilizer to improve crop quality [13]. In addition, Se-enriched microalgae can be used as functional feed supplements for livestock and poultry production [9,14]. Therefore, microalgae have become a very important carrier for Se nutrient supplementation.

The uptake, accumulation, and metabolism of Se as well as its physiological effects in microalgae have been summarized in review papers [1,10,15,16]. Microalgae can take up Se in the form of either selenite or selenate, and the absorbed Se is further assimilated into amino acids (selenocysteine, methylselenocysteine, and selenomethionine) and proteins (selenoproteins) through the sulfur metabolic pathway as part of the detoxification mechanism. Most importantly, microalgae can perform Se methylation to produce less toxic volatile compounds such as dimethyldiselenide (DMDSe) and/or dimethylselenide (DMSe) [17,18,19]. Thus, the final level of Se accumulation in microalgae is basically dependent on the form and concentration of the given Se and the algal species (tolerance level and metabolism capability) [1]. Thus far, Se bioaccumulation has been assessed in a limited number of microalgal species, including Chlamydomonas reinhardtii, Scenedesmus quadricauda, Dunaliella salina, Nannochloropsis oceanica, Haematococcus pluvialis, Chlorella pyrenoidosa, and Tribonema minus [15,18,20,21,22,23]. These species are derived from aquatic environments. Almost no relevant reports are available on terrestrial microalgae.

In this study, we aimed to investigate Se accumulation in three terrestrial microalgae available in our laboratory. They were isolated from the arid steppe of northwestern China. Among them, Vischeria sp. WL1 (Eustigmatophyceae) shows a remarkable capability to accumulate oils with a high eicosapentaenoic acid content [24,25]. The second one is a filamentous cyanobacterium with edible and medical values, Nostoc flagelliforme (Cyanophyceae) [26,27]. This alga also shows the potential to improve soil quality and combat desertification [28,29]. The remaining one is Chlorella sp. WL1 (Chlorophyceae), which is part of the commercially popular algae [30,31]. This study will contribute to enriching the species resource of microalgae as Se carriers for diversified food and agricultural applications.

2. Materials and Methods

2.1. Algal Strains and Cultivation

Vischeria sp. WL1, Chlorella sp. WL1, and N. flagelliforme were collected on the eastern side (an arid steppe) of Helan Mountain in Yinchuan, Ningxia, China. Vischeria sp. WL1 is an oil-producing strain of Eustigmatophyceae first isolated by our group [24]. Chlorella sp. WL1 was reported for the first time in this study, and its 18S rDNA sequence was deposited into NCBI (Accession no. PP595731). All three strains are kept in our laboratory. Natural N. flagelliforme presents filamentous colonies, and its suspension culture was developed for polysaccharide production and environmental adaptation studies [27]. They were regularly cultivated with 120 mL of BG11 medium in 250 mL Erlenmeyer flasks. The flasks were placed on a shaker (TS-300T, MIULAB, Hangzhou, China) with a rotation rate of 130 rpm. The light intensity of constant LED illumination was 40 μmol photons m⁻²·s⁻¹ and the temperature was 25 °C. The cultures at the exponential phase were collected by centrifugation and subjected to Se treatment experiments.

2.2. Selenite Treatment

Selenate and selenite are the primary forms of Se absorbed by microalgae, but the latter is preferentially ingested [17]. Thus, selenite was adopted for Se treatment in this study. Briefly, 120 mL of microalgal cultures with an initial OD₇₅₀ of 0.15–0.2 were added with different volumes of Na₂SeO₃ stock solution (10 g/L). The final concentrations of selenite were set at 0, 10, 20, and 40 mg/L. The cultures were cultivated under a constant light intensity of 40 μmol photons m⁻²·s⁻¹ at 25 °C for 16, 18, and 24 days, respectively. For each treatment, three replicates were conducted.

2.3. Growth Determination

During cultivation, 2 mL of each algal culture was collected and subjected to growth evaluation using the cell density parameter OD₇₅₀ [24,32]. The value of OD₇₅₀ was determined using a UV–Vis spectrophotometer (Spectrum, SP-1920, Shanghai, China). The specific growth rate was calculated using the equation μ = ln(N₂/N₁)/(t₂ − t₁), where μ denotes the specific growth rate, and N₁ and N₂ are the OD₇₅₀ values at the beginning (t₁) and end (t₂) day of the determination, respectively.

2.4. Biomass Collection

The algal culture of approximately 30 mL was collected and centrifuged at 7140× g for 5 min. The pellet was rinsed with deionized water 3 times to remove the residual selenite. The rinsed sample was frozen at −20 °C for 12 h and then subjected to vacuum freeze-drying to constant weight and weighed.

2.5. Se Content Determination

The Se content in microalgae was analyzed by the Shiyanjia Lab (Xiyuan 8th Road, Xihu District, Hangzhou, China) using inductively coupled plasma mass spectrometry (ICP-MS) [33], based on the national standard (GB5009.268-2016). In brief, dried samples (50 mg) were subjected to microwave digestion with 10 mL of nitric acid. They were pre-digested at 120 °C for 0.5 h, followed by microwave digestion (at 130 °C for 3 min, 150 °C for 10 min, and 180 °C for 30 min). The samples were then cooled to 60 °C and diluted in 1% nitric acid (25 mL) for testing. An Agilent 7800 ICP-MS instrument (Agilent Technologies, Tokyo, Japan) was used, and the standard mode was adopted for element determination. The optimized operational conditions are as follows: generator power, 1300 W; ion lens voltage, 6.00 V; nebulizer flow rate, 0.98 L/min; cooling gas flow, 17.0 L/min; auxiliary gas rate, 1.20 L/min.

2.6. Statistical Analysis

All treatments and controls were performed in triplicate. The experimental data were presented as the mean ± standard deviation. The statistical analyses were carried out using IBM SPSS 26.0 with Tukey’s multiple comparison at p < 0.05.

3. Results

Depending on its concentration, Se exerts either beneficial or adverse effects on microalgae. At a low concentration, Se is capable of enhancing photosynthesis and algal growth; conversely, an elevated Se concentration might result in cell toxicity and growth inhibition [1]. Microalgae also differ in their capacity to tolerate and accumulate Se. Referring to the published literature [21,22], we chose a concentration of sodium selenite within 40 mg/L to treat three terrestrial microalgae, mainly evaluating the effects on growth (in terms of OD₇₅₀) and total Se accumulation (Figure 1, Figure 2 and Figure 3). In consideration of the disparity in their growth rates, we opted for diverse time points to cultivate them and determine the Se content.

As shown in Figure 1A, 40 mg/L selenite did not inhibit the growth of Vischeria sp. WL1 during 16 days of cultivation. In fact, 20 mg/L selenite slightly stimulated its growth compared to the control (without selenite treatment). The specific growth rate at this treatment concentration was 0.09 μ day⁻¹. The Se contents in samples cultivated for 8 days and 16 days were determined (Figure 1B). The samples treated with 40 mg/L selenite accumulated the highest Se on day 8, reaching 3.33 mg/g dry weight (DW). However, the two lower concentrations (10 and 20 mg/L) of selenite only resulted in 1.55 mg/g DW and 0.92 mg/g DW Se on day 8, respectively. On day 16, the Se contents of all three samples significantly decreased, implying that long-term cultivation is not suitable for Se biofortification in this alga.

Similar to Vischeria sp. WL1, 40 mg/L selenite did not reduce the growth of Chlorella sp. WL1 (Figure 2A). In fact, a very slight promoting effect was observed at 10 mg/L selenite. The specific growth rates at three treatment concentrations were the same (0.10 μ day⁻¹). The Se contents in samples cultivated for 6, 12, and 18 days were determined (Figure 2B). On days 6 and 12, the levels of Se accumulation seemed to be proportional to the selenite concentrations. However, extended cultivation (18 days) under higher concentrations (20 and 40 mg/L) of selenite led to reduced Se contents in the samples. The highest Se content (0.74 mg/g DW) in Chlorella sp. WL1 was achieved at 40 mg/L selenite on day 12, which is only about 22% of that achieved in Vischeria sp. WL1.

N. flagelliforme is a filamentous cyanobacterium with relatively slower growth. It was treated with three concentrations of selenite for 24 days. Its growth was promoted by lower concentrations of selenite (10 and 20 mg/L), while a higher selenite concentration (40 mg/L) initially promoted the growth and then exhibited an inhibitory impact (Figure 3A). The specific growth rates at the two favorable concentrations were 0.05 μ day⁻¹. The changes in Se content along with the cultivation time did not seem particularly significant at 10 and 20 mg/L selenite, while Se accumulation at 40 mg/L selenite was proportional to the extended cultivation time (Figure 3B). The highest Se content in N. flagelliforme was 5.65 mg/g DW, which is about 1.7 times that achieved in Vischeria sp. WL1.

4. Discussion

Se-enriched foods or crops have become an important part of the Se industrial chain. Microalgae can be explored as a potential source of Se fortification in the field of food and agriculture. The mechanisms of Se accumulation, tolerance, and metabolism in microalgae have been well summarized [1,16,19]. It is well-known that inorganic Se can be transformed into organic Se in microalgae and the latter is more bioavailable. Also, the organic Se content is generally tightly associated with the total Se content in cells [18,23]. Therefore, the total Se level is, to an extent, indicative of the organic Se level. However, excess Se will be further detoxified by volatilization, thus causing the loss of intracellular Se. Therefore, the total Se accumulation may serve as a critical criterion for evaluating the application prospect of new microalgal species. By testing the Se accumulation level in three terrestrial microalgae, our study has provided new candidates for food and agricultural applications and has also expanded our understanding of Se biofortification in terrestrial algal species.

The Se accumulation levels in three terrestrial microalgae were compared with those reported in other literature (

Table 1. The total Se accumulation in microalgal strains after selenite treatment. DW, dry weight.

Microalgal Strains	Na2SeO3	Total Se Accumulation	References
Dunaliella salina	50 mg/L	65 μg/g DW	[22]
Dunaliella salina	25 mg/L	52 μg/g DW	[22]
Haematococcus pluvialis	13 mg/L	646 μg/g DW	[18]
Chlorella pyrenoidosa	50 mg/L	435 μg/g DW	[21]
Chlorella sorokiniana	50 mg/L	964.7 μg/g DW	[36]
Chlorella vulgaris R117	8 mg/g DW	10,000 μg/g DW	[34]
Tribonema minus	8 mg/L	964.7 μg/g DW	[23]
Nannochloropsis oceanica	30 mg/L	131 μg/g DW	[20]
Scenedesmus quadricauda	50 mg/L	1430 μg/g DW (in presence of 4 mM SO42−)	[38]
Chlamydomonas reinhardtii	3.16 mg/L	1960 μg/g DW	[35]
Spirulina platensis	40 mg/L	<50 μg/g DW	[39]
Spirulina platensis	400 mg/L	219 μg/g DW	[39]
Vischeria sp. WL1	40 mg/L	3331 μg/g DW	This study
Chlorella sp. WL1	40 mg/L	740 μg/g DW	This study
Nostoc flagelliforme	40 mg/L	5648 μg/g DW	This study

). The previously reported two highest contents of Se accumulation were ~10 mg/g DW in Chlorella vulgaris R117 [34] and 1.95 mg/g DW in C. reinhardtii [35]. However, it should be noted that the former was realized under periodic selenite treatment (twice a day). Vischeria sp. WL1 (3.33 mg/g DW) and N. flagelliforme (5.65 mg/g DW) accumulated approximately 1.7 and 2.9 times as much Se as C. reinhardtii. This should benefit its exploitation as Se nutrient carriers. Chlorella sp. WL1 shows relatively weak Se accumulation capability but is still comparable to other Chlorella species such as C. pyrenoidosa and C. sorokiniana [21,36]. The cyanobacterium S. platensis has long been recognized as a valuable food source [37]. It can only accumulate no more than 50 μg/g DW Se (Table 1), which is far less than N. flagelliforme, another popular edible cyanobacterium in Southeast Asia [26]. In addition, it is noteworthy that compared to the other two species, Chlorella sp. WL1 has more rapid growth under the same selinite condition (Figure 1A, Figure 2A and Figure 3A), which is beneficial for saving production costs. Thus, the three microalgae can be recognized as new promising Se-enriched species.

Among the tested Se concentrations, 40 mg/L selenite did not inhibit the growth of Vischeria sp. WL1 and Chlorella sp. WL1 but impaired the growth of N. flagelliforme. Given its relatively low Se resistance, it is suggested to use 20 mg/L selenite for cultivating N. flagelliforme, which is still effective in promoting a high Se accumulation (3.04 mg/g DW). Furthermore, it has been noted that long-term cultivation is not invariably advantageous for Se accumulation, especially when the Se concentration surpasses a particular threshold. For Vischeria sp. WL1, the threshold is 10 mg/L (Figure 1A), while for Chlorella sp. WL1, the threshold is 20 mg/L (Figure 2A). Similar dynamic changes in Se accumulation, either related to long-term cultivation or declining with high-dose treatment, were also observed in other microalgae such as H. pluvialis and C. pyrenoidosa [17,18,21]. This might be attributed to Se methylation and subsequent volatilization upon excessive absorption of Se [17,19]. In N. flagelliforme, such potential Se volatilization was not observed at 40 mg/L selenite after 24 days of cultivation (Figure 3B). N. flagelliforme cells show a remarkable capability to synthesize and secrete exopolysaccharides [27]. Se and polysaccharides can form Se-polysaccharides, which have better biological activities compared to polysaccharides or inorganic Se [40]. It has been reported that S. platensis polysaccharides can improve the stability and reduce the toxicity of Se nanoparticles [41]. Also, S. platensis Se-polysaccharides can provide significant protection against Cd-induced toxicity [42]. Thus, it may be assumed that N. flagelliforme exopolysaccharides have chelated Se and thus exhibit a high level of Se accumulation (Figure 3). Together, the three microalgae exhibit distinct dynamics and capabilities in Se accumulation.

In summary, the Se accumulation in three terrestrial microalgae was investigated, which provides new food or agricultural resources for application. Vischeria sp. WL1 and Chlorella sp. WL1 could resist 40 mg/L selenite, while N. flagelliforme could not. The optimal selenite treatment for them is suggested to be 20 mg/L. Vischeria sp. WL1 and N. flagelliforme could accumulate more than 3.33 mg/g DW Se, much higher than most of the tested microalgae thus far. The Se accumulation capability of Chlorella sp. WL1 is similar to that of other Chlorella species. Of course, as mentioned for C. vulgaris R117, periodic selenite treatment (or domestication) might be a good strategy to further enhance their Se accumulation potential. Although there is still much exploration to be performed, such as evaluating the compositions of selenium components, this study has enriched the Se-enriched resource pool by providing terrestrial microalgae.