Chlorella vulgaris and Scenedesmus obliquus were purchased from the Freshwater Algae Bank of the Chinese Academy of Sciences. Two dominant algal species were selected based on our previous study of the distribution of dominant algal species in Aha Reservoir, a typical karst lake in Guizhou Province, southwest of China (Ge et al., 2021). Based on our previous sampling results (Zhou et al., 2022), modified BG11 medium (Supplementary Table S1, Supplementary Information) was configured to simulate the karst conditions for algae cultivation. Both algae were cultured in a light incubator (BS-2E; Wampuda, China) at 22˚C ± 1°C with a light intensity of 2000 lux and a light–dark ratio of 12 h: 12 h. After algae were cultured to the logarithmic phase, the algal solution was concentrated using a centrifuge (GT10-1; Beili, China) at 4,500 r·min ⁻¹ for spreading cultivation. Initial inoculum concentrations of Chlorella vulgaris and Scenedesmus obliquus were 2 × 10⁶ cells·mL⁻¹ and 1.65 × 10⁶ cells·mL⁻¹, respectively, and the incubation time was 10 days. The growth cycle of the algal cells was recorded by direct counting under a microscope (UB203i; Aopu, China) using a hemocytometer plate.

EOM was extracted by the following protocol: algal supernatant was obtained by centrifugation at 8,000 r·min⁻¹ for 5 min, then filtered through deionized water (18.2 MΩ cm), and pre-cleaned by filtering through 0.70 μm glass fiber paper (GF/F 47 mm; Whatman, USA) to obtain EOM (Wang et al., 2021). After EOM extraction, remaining algal cells were washed with deionized water and then centrifuged for 1 min (repeated twice), repeatedly grounded and freeze–thawed three times, and filtered via 0.45-µm CA membranes to obtain IOM (Pivokonsky et al., 2014). The organic carbon levels of EOM and IOM were determined using a total organic carbon analyzer (Vario TOC; Elementar, Germany).

Spectral data processing was performed using Origin 8.5 (USA). Analysis of variance at the 0.05 level and Pearson correlation analysis (at the 0.01 and 0.05 levels) were performed using SPSS 20.

The growth of Chlorella vulgaris and Scenedesmus obliquus in simulated karst water is shown in Figure 1. The growth cycle of algae can be divided into four stages: adaptation stage, logarithmic stage, plateau stage, and decline stage. Both Chlorella vulgaris and Scenedesmus obliquus entered the logarithmic stage of rapid growth and reproduction on the third day, but the logarithmic stage lasted for a short time. In addition, the growth of the two algae entered a stable period on days 4–7. In the stable stage, algal cells maintained a relatively stable level, the maximum algal cell concentration reached 5.7×10⁶ cells·mL⁻¹ and 5.9×10⁶ cells·mL⁻¹, and the algae entered a period of decline on the eighth day. Results showed that the maintenance time of the stable period in the karst area was shortened, which led to the overall growth cycle being obviously shortened.

The variations in DOC values of Chlorella vulgaris and Scenedesmus obliquus are shown in Table 1. With the change in the growth cycle of the two algal species of, DOC increased significantly from the adaptation stage to the logarithmic stage. In the stable period, more cell metabolites were released in AOM, and the concentration of DOC increased. The amount of DOC in EOM was higher than that in IOM at each growth stage of Chlorella vulgaris, indicating that EOM would produce more DOC under the simulated condition in the karst lake reservoir. The higher DOC content in stable EOM represents more DOC accumulation with the growth of algae (Zhao et al., 2020; Ni et al., 2021). During the adaptation period, the amount of DIC was abundant and reached the maximum in EOM (20.32 ± 0.12a mg/L and 22.60 ± 0.08a mg/L). Although DIC provided the carbon source for the photosynthesis of algae, it can also be extrapolated that the biological pump effect may provide a promoting effect on the growth of algae (Supplementary Table SI2).

The changes in SUVA₂₅₄, SUVA₂₈₀, and URI values in different growth stages of the two algae are shown in Figure 2. The SUVA₂₅₄ of logarithmic IOM for Chlorella vulgaris and Scenedesmus obliquus was 1.898 ± 0.043 L mg⁻¹·m⁻¹ and 2.619 ± 0.007 L mg⁻¹·m⁻¹ (Figure 2A), respectively, suggesting more aromatic substances were produced in IOM during this period (Dong et al., 2019; Sun F. et al., 2022). SUVA₂₅₄ in AOM was generally less than 1.5 L mg⁻¹·m⁻¹ (Zhou et al., 2014), showing that the aromaticity of AOM was low—AOM seemed to contain less protein-like substances and organic nitrogen (Hua et al., 2018). The DOC content in EOM of both algae during all periods was higher than that in IOM, except for the stationary phase of Scenedesmus obliquus, but SUVA₂₅₄ in EOM was lower than that in IOM in all growth periods, showing a stronger aromatic expression in IOM under the influence of complex karst water condition (Li et al., 2012). SUVA₂₈₀ and SUVA₂₅₄ share the same trend, suggesting a potent correlation between aromaticity and protein levels (Wang et al., 2013; Liu W. et al., 2018). The SUVA₂₅₄ and SUVA₂₈₀ values in Figures 2A, B were the highest in the logarithmic phase of IOM, indicating high accumulation of aromatic compounds and proteins in the algal cells during rapid growth. However, the decrease in SUVA₂₈₀ in IOM during the decline phase may be due to the release of more organic matter outside the cell during decay of the algae (Hua et al., 2017) and higher accumulation of polysaccharides than proteins (Henderson et al., 2008). In addition, the trends of SUVA₂₅₄ and SUVA₂₈₀ in the two algae at different growth periods were completely opposite in EOM and IOM, with a gradual decrease in IOM after the logarithmic phase, indicating higher release of aromatic protein substances from cells. This was different from the results of many studies in non-karst areas (Gough et al., 2015; Chen et al., 2017). After the logarithmic phase, higher accumulation of aromatic substances in EOM than consumption was observed as SUVA₂₅₄ in EOM gradually increased.

URI values in EOM reached their highest during the adaptation period (43.877 ± 0.442a and 35.235 ± 1.059a) and gradually decreased with algal growth (Figure 2C). IOM was found to have higher URI values in the study of AOM in karst areas (Ma et al., 2022), which showed that the concentration of amino groups was higher than that of aromatic groups in EOM, and there were more proteins in EOM at the early growth stage of algae in karst lakes. The concentration of amino groups in IOM in the decline stage increased compared with that in the stable stage, which may result in the formation of unsaturated aromatic substances during the decline stage (Her et al., 2004).

The FT-IR spectra of EOM and IOM of the two algae at different growth periods are shown in Figure 3. The spectral peaks of EOM at 3,500–3,300 cm⁻¹ indicated O-H and N-H stretching vibrations (Zhang et al., 2014), and both algae exhibited N-H stretching at 3,200–3,100 cm⁻¹ during the decline phase (Ma et al., 2022). The peaks at 1700–1,600 cm⁻¹ can be explained by C=O vibrations of amide I (Fawzy, 2016; Zambrano et al., 2021), while those at 1,500–1,400 cm⁻¹ correspond to CH₂/CH₃ vibrations (Yang et al., 2018). Wavelength ranges of 1,200–1,100 cm⁻¹ and 875 cm⁻¹ show C-O-C stretching vibrations. The peak of Chlorella vulgaris IOM at 3,200–3,100 cm⁻¹ was not observed (Figure 3A), whereas a significant N-H stretching during the adaptation and decline phases of Scenedesmus obliquus was noted. In addition, C-H stretching at 3,000–2,800 cm⁻¹ was observed in IOM in each growth period (Dias et al., 2021; Fawzy and Alharthi, 2021). Vibrations of amide II were also observed at 1,580–1,500 cm⁻¹ in the latter two periods of Scenedesmus obliquus (Figure 3B; Singh et al., 2018); these peaks caused by amide groups proved the presence of proteins (Zhou et al., 2014).

The main components of both algae showed different patterns: both Chlorella vulgaris and Scenedesmus obliquus had more than 50% protein content, with lipids and polysaccharides forming 7%–24% of the main compounds (Becker, 2007; Wang et al., 2013). The obvious functional groups of proteins and polysaccharides were found at 3,500–3,300 cm⁻¹ during the adaptation phase of both algal species, and the concentration of these biochemical constituents gradually decreased during the growth period. Protein peaks corresponding to 1,700–1,400 cm⁻¹ similarly weakened in peak intensity after the adaptation phase, and polysaccharide peaks corresponding to 1,200–1,000/875 cm⁻¹ showed a similar trend. However, the functional groups in the 3,200–3,100 cm⁻¹ band were observed much frequently during the decline phase, suggesting that algal growth requires consumption of organic substances such as proteins and polysaccharides by the algal cells after the stationary phase. The peak at 3,000–2,800 cm⁻¹ was more strongly expressed in IOM and can be used to explain the presence of lipids (Figure 3B). The FT-IR results revealed that both algae contain proteins and polysaccharides in EOM and IOM and that DIC promotes the production of more protein and polysaccharide material during the adaptation phase to provide energy for algal growth. The functional groups of proteins were more abundant, and as the growth of algae increased, more lipids were retained in the IOM.

The 3D-EEM-PARAFAC results of EOM and IOM in Chlorella vulgaris and Scenedesmus obliquus are shown in Figure 4. All analyses yielded two fluorescence fractions: the C1 fraction had two excitation peaks and one emission peak, representing aromatic protein-like organic matter (AP) and soluble metabolites (SMP), respectively, and the C2 fraction for humic acid-like organic matter (HA). It was similarly found that AP and SMP were the dominant fluorescent components in AOM (Wang et al., 2021).

Although both algae showed the same fluorescence components, the fluorescence intensity differed in different periods, and the fluorescence intensity of two fluorescence components of Chlorella vulgaris (113.02 and 13.54) and EOM (102.89 and 17.61) increased significantly after entering the stationary phase (Figure 4A). Tryptophan-like aromatic substances, biochemical organic substances with abundant activity in algal cells, were found at 230/350 nm. This means that Chlorella vulgaris accumulates more protein-like substances with aromatic compounds in the AOM after entering the stationary phase (Li et al., 2012). SMP is mainly composed of tryptophan, tyrosine, and protein-like substances, and production of these substances shows that AOM is enriched in organic nitrogen and promotes the production of compounds rich in organic nitrogen (Li et al., 2012; Zhao et al., 2020). Humic acid-like substances were mainly derived from dead algal cells, and damage of algal cells was caused during grinding or freeze–thaw extraction of IOM (Zhao et al., 2020). C2 in EOM had stronger fluorescence intensity during the decline phase, and post-death, algal cells exhibited obvious humic signals. The low intensity of C2 fluorescence might be caused by powerful cell activity in the algal solution (Rochelle-Newall and Fisher, 2002).

Unlike Chlorella vulgaris, the C1 fraction of Scenedesmus obliquus had the greatest fluorescence intensity during the IOM stationary phase (112.73) (Figure 4B) and higher protein enrichment during the stationary phase, which might be driven by intracellular AOM release (Chen et al., 2017). During the decline phase, due to the death of a large quantity of algae, protein materials also decreased. Although IOM has been proven to be mainly organic nitrogen compounds with more protein aromatic substances, the soluble protein generated would be gradually broken down by the activities of algal cells (Hua et al., 2017). The highest fluorescence intensity of humic substances in the C2 fraction of Scenedesmus obliquus EOM was found during the decline phase (10.94), associated with the death of algae.

The spectral parameters of both algae were correlated to the chemical characteristics of AOM. In Chlorella vulgaris (Figures 5A, B), the correlation between DIC and TN was high (0.87 and 0.99), indicating that there was a good correlation between the accumulation of N production in algae and the change in DIC during the growth period. The correlation between DOC, DIC, and TN of Chlorella vulgaris and SUVA₂₅₄ and SUVA₂₈₀ showed the opposite trend in EOM and IOM, proving differences in the chemical properties of AOM, and the growth change in AOM could be better judged based on the changes in DOC, DIC, and TN. There was a high correlation between TN and URI in IOM (0.97) (Figure 5B), showing that changes in the amino-structured organic matter in IOM of Chlorella vulgaris can be analyzed in conjunction with the changes in TN. The correlation between DOC and C1 and C2 indicated a positive effect of organic carbon on the production of aromatic, soluble biometabolites and humic substances in EOM.

The correlations of the indices of Scenedesmus obliquus were slightly different from those of Chlorella vulgaris (Figures 5C, D). As shown in Figure 5C, DIC showed negative correlations with SUVA₂₅₄ and SUVA₂₈₀ in EOM; the differences arising between the two algae may be related to the transformation of DIC and organic matter production. The negative correlation between DIC and TN further reflected that the chemical composition and changes in EOM in Scenedesmus obliquus were more complex. The relationship between DOC and C1 and C2 was significantly different from that in Chlorella vulgaris. It is recommended to avoid using a single DOC trend for interpretation of the derivation of compositional changes in AOM (Hestir et al., 2015; Zeng et al., 2019). SUVA₂₅₄ and SUVA₂₈₀ showed an intense correlation in AOM of both algae, confirming that it is possible to discuss the analysis of aromaticity and protein content in organic matter by combining two UV coefficients (Figure 5). In addition, URI and C1 fractions were negatively correlated in both algae, and the negative correlation was more significant in EOM (Figures 5A, C), indicating that the content of amino-structured substances in EOM gradually decreased with the growth of algae. High levels of aromatic proteins (tryptophan-like substances) were produced in the algal cells, suggesting lower aromaticity in EOM. FT-IR of the adaptation phase also reflected weakening of the peak intensity of the functional groups containing amino groups, and more aromatic structure-related functional groups were generated during subsequent growth periods (Figure 3).