Biochemical composition, β-glucan and phenolic content of a marine diatom Chaetoceros muelleri cultivated in Guillard’s modified medium

Cultural medium preparation and modification

The standard Guillard’s medium was prepared following the method described by Chiovitti et al. (2004) (see Supplemental Material for general composition). The sodium carbonate concentration was set at 0.5 g L⁻¹, selected based on the results of preliminary tests evaluating concentrations of 0.05, 0.5, and 5 g L⁻¹ (Pimolrat et al., 2010). For nitrogen levels, preliminary screening was conducted at 20%, 50%, and 75% of the standard concentration. The optimal nitrogen level was chosen based on its promotion of the highest β-glucan synthesis and will be used in subsequent experiments.

Diatom cultivation

A diatom, C. muelleri, was cultured in general laboratory conditions. The initial volume of diatom was transferred to 200 mL (0.5 × 10⁶ cellsmL⁻¹) of culture medium. Three cultural media consist of standard Guillard F/2 medium (T1), modified Guillard F/2 medium supplemented with 0.05 L⁻¹ sodium bicarbonate (T2), and modified Guillard F/2 medium with reduction in nitrogen (T3, based on the previously screening) were used in this experiment. The experimented diatom was cultivated at 25 °C under white fluorescent light (36 W G13 cool white linear fluorescent; Pilips, Gujarata, India) at an intensity of 115 µmol/s/m² for 12 h a day until harvesting. The cell density was measured daily using a Neubauer Hemocytometer (Thermo Fisher Scientific, Waltham, MA, USA). The cells were harvested after 5 days of cultivation period, once they reached the stationary phase of growth. At this stage, the cell densities were approximately 3.90 ± 0.03 × 10⁶, 4.03 ± 0.50 × 10⁶, and 4.20 ± 0.04 × 10⁶ cells mL⁻¹, starting from an initial density of 0.5 × 10⁶ cells mL⁻¹. The diatoms were harvested using polyaluminum chloride (PAC; Q RëC™, Auckland, New Zealand) and centrifuged at 2,688 relative centrifugal force (RCF) for 3 min. After washing with distilled water, the wet diatoms were centrifuged, weighed, and preserved at −20 °C for further experiments.

Biochemical composition analysis

Total phenolic content

The total phenolic content (TPC) of the diatom was analyzed via a modified Folin–Ciocalteu reagent method, previously described by Karthikeyan et al. (2013) and Hemalatha et al. (2015). Briefly, 20 µL of the extracts were combined with 100 µL of 1:10 diluted Folin–Ciocalteu reagent, followed by the addition of 80 µL of 7.5% Na₂CO₃. After 2 h of incubation in the dark at room temperature, the absorbance was measured via a spectrophotometer at 600 nanometers (nm). Gallic acid served as the standard reference, and the TPC results was express as milligrams of gallic acid equivalents (GAE) per gram of dried extract (mg GAE g⁻¹).

Antioxidant activity assessment

β-glucan content

β-glucan extraction was performed via a modified warm water method as described by (Chiovitti et al., 2004). Briefly, diatom pellets frozen in 30 mL of Milli-Q water were thawed and heated at 30 °C for 2 h, with stirring every 10 min. The extracted cells were centrifuged at 10,000 CRF, and the supernatant was collected and filtered through 0.8 and 0.2 µm filters to remove impurities. The solution was then freeze-dried via an EYELA FDU-2100 freeze dryer (Tokyo, Japan).

Statistical analysis

Statistical analysis was performed via SPSS (version 29.0). One-way analysis of variance (ANOVA) was performed, and the mean values were compared via Duncan’s multiple range test at a 95% confidence interval (p < 0.05).

Guillard’s medium modification

Preliminary tests were conducted by supplementing the culture medium with nitrogen concentrations of 25%, 50%, and 75% to assess β-glucan accumulation in diatom cells. The highest β-glucan accumulation was observed at the 50% nitrogen level as shown in Table S1 of supplementary data. This N concentration was further selected for the next experiment.

Biomass yield and biochemical compositions

Table 1 presents the results of the biochemical composition analyzed from the diatom extracts. The data revealed that T3 yielded highest biomass, carbohydrate, and lipid levels, with the exception of total protein. The experimental data assumed that diatoms grew best in the T3 medium. The cells were harvested during the stationary phase (at the fourth day, Fig. S1), with a maximum cell density of 5.75 ± 0.06 × 10⁶ cells mL⁻¹ was observed at T3, whereas those of T1 and T2 reached 3.85 ± 0.13 and 4.03 ± 0.08 × 10⁶ cells mL⁻¹, respectively. Statistical calculation showed that T3 significantly different (p < 0.05) from that at T1, but did not differ from that at T2. The carbohydrate content in cultured diatoms was 5.81 ± 0.50%, 6.90 ± 1.14%, and 13.71 ± 1.56% for T1, T2, and T3, respectively. Statistical analysis manifested significant differences when T3 was compared with T1 and T2. The results indicated that the carbohydrate content in T3 was approximately 1.5 times greater (13.71 ± 1.56%) than that in T1 (5.81 ± 0.50%) and T2 (6.90 ± 1.14%). The protein contents in T1, T2, and T3 were 94.84 ± 0.08, 31.39 ± 0.72, and 30.91 ± 0.38 mg g⁻¹, respectively. Statistical analysis displayed significant differences between three treatments, as shown in Table 1. Similarly, the total lipid content in T3 (4.34 ± 0.03%) was significantly higher (p < 0.05) than in T1 (1.1 ± 0.08) and T2 (2.21 ± 0.62).

β-glucan accumulation

Megazyme analysis was conducted on crude diatoms cultured in the modified media has showed in Table 2. The β-glucan concentrations in T1, T2, and T3 were 0.38 ± 0.01, 0.35 ± 0.01, and 0.41 ± 0.01 g L⁻¹, respectively. Statistical analysis revealed significant differences among the three extraction yields. The types of glucans contained in the extracts are shown in Table 2 that expressed as percentages (w/w) for β-glucan, α-glucan, and total glucans. Statistical significance (95% confidence interval) was evaluated for the levels of α-glucan, β-glucan, and total glucans in the diatoms. T3 showed a significantly higher β-glucan content (79.45 ± 1.40) compared to T1 (2.92 ± 0.30) and T2 (11.59 ± 0.18). The β-glucan content in crude diatoms was 2.92 ± 0.30 g 100 g⁻¹ DM, 11.59 ± 0.18, and 79.45 ± 1.40 g 100 g⁻¹ DM for T1, T2, and T3, respectively. The increasing of β-glucan content in T1:T3 was 96.3%, and in T2:T3 was 85.41%.

Total phenolic content and antioxidant activity

The TPC and antioxidation ability of diatom crude extracts were determined.

Biomass yield and biochemical compositions

Our finding indicates that the modified media affected growth, biomass production, and the concentration of biochemical compounds synthesized of diatoms, as reported by Ördög et al. (2012). Shan et al. (2023) reported that Chaetoceros sp. achieved maximum biomass and growth rates under mixotrophic conditions with sodium bicarbonate, indicating its role in enhancing metabolic pathways for growth. Furthermore, the nitrogen source affects the duration of the stationary phase, as observed in Chaetoceros cultures supplemented with ammonia, which maintained a longer stationary phase than did those supplemented with nitrate (Sachindra et al., 2007). This is due to the varying rates of nutrient uptake. Our study recorded that the carbohydrate content in T3 was approximately 1.5 times greater than that in T1 and T2. These findings indicate that nitrogen limitation influences the accumulation of carbohydrates and β-glucan, as proposed by Morales-Plasencia et al. (2023). Nitrogen limitation also impacts carbohydrate and β-glucan accumulation, as well as biomass composition, in Nannochloropsis oculata (López-Elías et al., 2011). In the case of C. muelleri, significant biochemical changes occur in response to nitrogen stress, particularly affecting its chemical composition and fatty acid profile. Under nitrogen limitation, C. muelleri increases lipid and carbohydrate accumulation while maintaining biomass levels (de Jesús-Campos et al., 2020). Our study also revealed greater increases in lipids and β-glucans in T3 (4.34 ± 0.03) than in T1 (1.1 ± 0.08) and T2 (2.21 ± 0.6). Nitrogen limitation triggers extensive cellular physiological changes, resulting in altered biochemical pathways and protein synthesis. Furthermore, different nitrogen sources (nitrate, ammonium, and urea) at the same concentration do not significantly impact growth rates but do influence carbohydrate and protein synthesis (Simionato et al., 2013; López-Elías et al., 2011). Additionally, sodium bicarbonate supplementation has been shown to significantly enhance the growth and biochemical composition of cultures of Tetraselmis suecica and Nannochloropsis salina. A concentration of 1 g L⁻¹ bicarbonate resulted in higher cell abundance and improved photosynthetic efficiency (White et al., 2013). In contrast, in C. gracilis, the lowest bicarbonate concentration (0.05 g L⁻¹) led to the greatest accumulation of carbohydrates and lipids (Pimolrat et al., 2010). Furthermore, 0.15 M bicarbonate supplementation in Dunaliella salina HTBS increased lipid, protein, and carbohydrate synthesis from 34.71% to 43.94%, 22.44% to 26.03%, and 22.62% to 29.18%, respectively (Guo et al., 2022). Hence based on the previously data, carbonate plays an important role as a carbon source in the formation of organic materials and energy for the growth of diatoms (Curcuraci et al., 2022). In contrast, the protein contents in T1, showed maximum level than the others. This is because nitrogen plays a crucial role in amino acid synthesis; therefore, nitrogen deficiency decreases protein production, inhibits the citric acid cycle, and reduces cell division (Msanne et al., 2012). According to this report, a low nitrogen concentration (nitrate concentration <21.66 mg/L) leads to increased lipid content (up to 31.33%) but decreases biomass productivity and protein levels (Liu et al., 2022).

β-glucan accumulation

Polysaccharides produced by C. muelleri contain β-(1 → 3) glucan with β-(1 → 6) branching. The molecular structure does not change with nutrient fluctuations, but it varies in terms of the number of glucose units (degree of polymerization, DP) and degree of β (1 → 6) branching (DB); therefore, the size or molecular weight varies in different organisms (Størseth et al., 2005). Fourier transform infrared (FTIR) spectroscopy was performed to analyze the presence of β-glucan in the diatom extract. The FTIR spectrum of standard β-glucan (Fig. 1) revealed the characteristics at 1,065 and 1,038 cm⁻¹. In addition, The FTIR spectrum of diatom extract (Fig. 2) revealed the sugar region vibrations at 1,200–900 cm⁻¹, the OH functional group at 3,600–2,600, the glycosidic bond at 1,041, and the CH signal at 3,000–2,800 cm⁻¹ were observed. β-glucans are polydisperse, meaning that they exist as mixtures of molecules with different chain lengths and molecular weights, making it difficult to pinpoint a specific molecular weight. The difficulty in determining the exact molecular weight of β-glucan stems from its structural diversity, source-related variability, and challenges associated with its extraction and analysis. Instead of a single molecular weight, β-glucans are often described by a molecular weight range. However, we determined the LC-MS/MS spectral data of diatom extract (T3) for primary analysis of the β-glucan contained in the C. muelleri extract. LC-MS/MS chromatogram (Fig. 3) revealed the loss of two proton patterns of gluconic acid at m/z 198, 196, and 194 (the typical molecular weight of gluconic acid is 196 Da). This evidence also tentatively confirmed the presence of the glucose unit of the β-glucan contained in the diatom extract. Glucans levels in diatoms are low during growth but increase during the stationary phase, particularly under limited nitrogen conditions; this indicated a high level of β-glucan accumulation in N-depleted media (Myklestad, 1989). In general, nitrogen limitation can induce glucan synthesis in diatom cells, similar to other metabolites, such as fatty acids and triacylglycerol, because carbon fixation for protein synthesis is interrupted (Frick et al., 2023). This phenomenon was observed in several cultured diatoms, such as Nanochlopsis oculata, C. pseudocurvisetus, Contricriba weissflogii, C. pseudocuvisetus, Thalassiosira pseudonana, and Scenedesmus obtusiusculus, in which beta-glucan increasingly accumulated in the cell under N-source depletion (Myklestad, 1989), which occurred during the stationary phase, similar to the findings of this study (Frick et al., 2023; Teunis, 2013). A few examples confirmed this finding, including nitrate starvation increasing the β-glucan content of S. obtusiusculus A189 from 16 to 23% and that of S. ovalternus SAG 52.80 from 23.3 to 36.7% (Schulze et al., 2016). In Rodomanas marina culture, media containing low available N induced beta-glucan starvation in their cells (Fernandes et al., 2017). In the case of Nanochloropsis oculata cultures, reducing the N level by 50% in culture media results in a 75% increase in β-glucan accumulation in the cells (Morales-Plasencia et al., 2023). These data suggest that the function of uridine diphosphatase glucose pyrophosphate, a key beta-glucan (chrysolaminarin) synthesis enzyme, is activated when diatoms grow in N starvation media (Guerrini et al., 2000).

The accumulated β-glucan in marine diatoms is significant mainly on biological and ecological manners. β-glucans particularly chrysolaminarin (a β-1,3-glucan with β-1,6 branches) play the biological function as storage energy, stress responsible and life cycle controlling (Frick et al., 2023). In ecological significant, diatoms fix large amounts of CO₂ via photosynthesis process that β-glucans are stored as a portion of this fixed carbon resulting carbon sequestration and food web dynamics (Schulze et al., 2016).

Total phenolic content and antioxidant activity

Phenolic compounds in diatom cells act as a defense against oxidative stress caused by abiotic factors, such as nutrient deficiency. Variations in phenolic compounds concentrations among diatom species are well studied as strategies for adapting to nutrient stress (Goiris et al., 2012). Diatoms produce phenolic compounds that serve various biological functions, particularly as antioxidants that protect cells from oxidative stress caused by UV radiation and other environmental stressors (Re et al., 1999). In the marine environment, these phenolic compounds also play a defensive role by deterring grazing by zooplankton. The production of phenolic compounds in marine diatoms under laboratory conditions is influenced by the composition of the culture medium (Frleta Matas et al., 2024). According to a study by Curcuraci et al. (2022), a diatom Phaeodactylum tricornutum produced a greater phenolic content when cultured in N-enriched media (3.07 ± 0.17 mg GAE g⁻¹ dry weight) than cultured under N limitation (1.12 ± 0.00 mg GAE g⁻¹ DW). Hemalatha et al. (2015) reported that the methanolic extract of the marine diatom Odontella aurita had a greater total phenolic content and antioxidant properties than did C. curvisetus and Thalassiosira subtilis. The study reported a TPC of 0.55 ± 0.031 mg ⁻¹g GAE, total antioxidant activity of 0.97 ± 0.044 mg g⁻¹ ascorbic acid equivalent, DPPH radical scavenging activity of 15.25%, and ferric reducing power of 1.032 ± 0.031 mg g⁻¹ ascorbic acid equivalent. Therefore, crude extracts from diatoms cultured in N-enriched media presented increased antioxidant activity (Frleta Matas et al., 2024). In contrast, our study recorded that the extract from C. muelleri had the highest activity, whereas T1 had a higher total phenolic content of 14.91 ± 0.97 mg g⁻¹ GAE. Additionally, T3 exhibited a DPPH radical scavenging activity of 65.33 ± 2.90% at 10 mg mL⁻¹ extract and a ferric reducing power of 53.23 ± 6.61 mg g⁻¹ ascorbic acid equivalent, with no significant difference (p < 0.005) (Table 3). Despite the lowest TPC observed in T3, it exhibited the highest antioxidant potential. Typically, higher antioxidant values would be expected with increased phenolic content, suggesting that β-glucan may play a crucial role in the observed antioxidant activity. Various metabolites, including carotenoids, quinones, and other compounds contained hydroxyl groups or chromophores, contribute to antioxidant activity. For instance, fucoxanthin extracted from Odontella aurita demonstrated strong ABTS radical scavenging activity, with an EC50 value of 0.03 mg mL⁻¹ (Suttisuwan et al., 2019). This suggests that the antioxidation potential observed might be played by the hydroxyl groups present in β-glucan, however no evidences of minor constituents in crud extract may also influenced.