Component structure and characteristic analysis of cyanobacterial organic matters Open Access

In recent years, with the gradual acceleration of China's industrialization, the phenomenon of water eutrophication is becoming more and more serious (Zhang et al. 2020). Complex water environment and changes in climate variability also play a role in promoting cyanobacteria blooms (O'Neil et al. 2012; Chapra et al. 2017). These bring in severe challenges to the safety of water quality. The rapid propagation of cyanobacteria caused by water eutrophication and climate change will produce a large amount of algae organic matters (AOM) with complex organic components, mainly including polysaccharides, proteins, peptides, amino sugars, hydroxy acids, and also a certain number of toxic and harmful substances, such as typical algal toxins and odor substances (Fang et al. 2010; Li et al. 2012). Part of AOM is stored inside the cells; that is, intracellular organic matters (IOM), which is the main part of the cyanobacteria cell matrix. After the damage or death of algal cells, it can be released to the surrounding water environment in large quantities, causing organic pollution of surrounding water (Fang et al. 2010; Li et al. 2012). Another part of AOM is directly released to the surrounding water or wrapped around the cells to form extracellular organic matters (EOM). The EOM wrapped outside the cells has the characteristics of gelatinous coating, which is composed of micro fibrils and has important physiological and ecological functions. The EOM could promote the aggregation of individual cells into groups and form air filled intercellular spaces. These spaces produced buoyancy for cyanobacteria groups and became one of the main reasons for the formation of cyanobacteria blooms (Zhang et al. 2011; Xu et al. 2014; Qian et al. 2017). During the outbreak of cyanobacteria bloom in summer, the AOM secreted during the rapid growth and metabolism of algal cells brings not only serious organic pollution to the water body, but also great technical challenges to the subsequent water treatment processes (Vandamme et al. 2012; Qian et al. 2017; Xu et al. 2018; Sun et al. 2020a). Therefore, the characteristics of the component structure of AOM deserves special attention.

It was found that the EOM and IOM had different properties under different growth states (Gu et al. 2015; Chen et al. 2017). The surrounding environment of cyanobacteria and the secretion state of AOM are likely to have different effects on the degree of pollution caused by cyanobacteria. In addition, in various researches on cyanobacteria AOM, the purified single cyanobacteria species, is often used as the research object for targeted problem analysis and investigation (Gu et al. 2015; Chen et al. 2017; Xu et al. 2018), while ignoring the impact of the complexity of the actual water environment and climate change on the secretion characteristics of cyanobacteria AOM. Therefore, the conclusion may not be suitable for the characteristic analysis and pollution control of cyanobacteria and their AOM in the actual water environment. On this basis, this study takes both the pure cyanobacteria cultured in the laboratory and the cyanobacteria growing in the actual water body of Taihu Lake as the common research objects, and compares and analyzes the characteristics of AOM secreted by cyanobacteria under different growth environment conditions, so as to ensure the scientific and practical significance of the research.

The cyanobacteria species used in the laboratory culture environment is Microcystis aeruginosa (FACHB-912), which was purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences. Microcystis aeruginosa FACHB-912 is the most common dominant cyanobacteria in water blooms in China and the producer of typical cyanotoxins such as microcystins. Microcystis aeruginosa FACHB-912 was cultured in BG11 medium (NaNO₃ 1.5 g/L, K₂HPO₄·3H₂O 4 × 10⁻² g/L, MgSO₄·7H₂O 7.5 × 10⁻² g/L, CaCl₂·2H₂O 3.6 × 10⁻² g/L, C₆H₈O₇ 6 × 10⁻³ g/L, C₆H₈FeNO₇ 6 × 10⁻³ g/L, EDTA 1 × 10⁻³ g/L, Na₂CO₃ 2 × 10⁻² g/L, H₃BO₃ 2.86 × 10⁻³ g/L, MnCl₂·H₂O 1.81 × 10⁻³ g/L, ZnSO₄·7H₂O 2.22 × 10⁻⁴ g/L, CuSO₄·5H₂O 7.9 × 10⁻⁵ g/L, Na₂MoO₄·2H₂O 3.9 × 10⁻⁴ g/L, Co(NO₃)₂·6H₂O 4.9 × 10⁻⁵ g/L, pH 7.1 ± 0.1, 25 °C) in a light incubator with temperature at 25 °C, light intensity of 1000–2,000 Lux, and the culture cycle of 12 h:12 h (Tsai 2015; Li et al. 2018). Microcystis aeruginosa FACHB-912 species were inoculated in 1 L Erlenmeyer flasks with cotton ball stopper for batch culture. The same amount of mixture was obtained from each culture flask during each sampling. The cyanobacteria density of the sample was counted with a microscope (Nikon Eclipse E100, Japan) at 400× magnification and an Improved Neubauer hemocytometer after washing and dilution with pure water (Tsai 2015; Li et al. 2018). In case of aggregation and adhesion of cyanobacterial cells, vortex treatment shall be carried out in advance to disperse them, and each sample was counted three times.

The cyanobacteria growing in the actual water body was also studied to ensure the scientificity and practical significance of this research. Considering the frequency and normalization of cyanobacteria blooms at the sampling point, the cyanobacteria-containing water samples in the actual water environment were obtained by the mixed sampling method of surface distribution and vertical distribution at the algae water separation station in Yangwan, Taihu Lake, Wuxi City, Jiangsu Province (31°32′50″ N, 120°11′43″ E). The sampling work was carried out from June to August, 2020, three times a month. The obtained samples were taken back to the laboratory immediately for water quality analysis and AOM extraction. The characteristic parameters of the water samples were: middle eutrophic water body, pH of 7.6–8.7, turbidity of 204–278 NTU, temperature of 27 ± 3 °C, DO of 8.7–9.5 mg/L, conductivity of 510–640 μS/cm, chlorophyll-a concentration of 0.856 × 10⁻³–1.055 × 10⁻³ g/L, and the dominant algae species of Microcystis aeruginosa (accounted for 98% by microscopic counting). The cyanobacteria density was recorded as the average value of each sampling test. The extracted AOM was preserved at −20 °C and mixed in equal quantities for analysis.

The cyanobacteria EOM was extracted by centrifugation at 5000 g for 15 min with the CR21GIII ultra-high speed centrifuge (Beckman Coulter, USA). The supernatant was extracted with 0.45 μm mixed fiber filter membrane to obtain the cyanobacteria EOM (Qu et al. 2012; Xu et al. 2013; Zhao et al. 2015). The cyanobacteria IOM was extracted by repeated freeze-thaw method. The cyanobacteria cell sediment in the centrifuge tube after extracting EOM was washed with pure water 3 times, and then re-suspended to the original volume. The cell suspension was then treated by freezing and thawing for 3 cycles. After this treatment, the supernatant was centrifuged at 5000 g for 15 min and filtrated with 0.45 μm mixed fiber filter membrane to obtain the cyanobacteria IOM (Li et al. 2012; Zhou et al. 2014; Chen et al. 2017).

The AOM samples were taken for quantitative and qualitative analyses. All tests were performed in triplicate and the mean value was calculated.

DOC was quantified by a total organic carbon analyzer (model TOC4200, Shimadzu, Japan) (Cheng et al. 2005; Leloup et al. 2013; Koistinen et al. 2017).

The UV absorbance at 254 nm was measured by a UV-Vis spectrophotometer (model UV759S, Shanghai Jingke, China) (Cheng et al. 2005; Leloup et al. 2013).

The SUVA is defined as the ratio of the UV₂₅₄ absorbance to the DOC value of the sample and reported in units of L/(m·mg) (Cheng et al. 2005; Leloup et al. 2013).

The water sample was separated using the ultrafiltration tubes (Amicon^® Ultra-15, Millipore, USA) with different molecular weights to obtain the different sample fractions with molecular weight of <100 kda, <50 kDa, <30kda, <10 kDa, and <3 kDa, respectively (Qu et al. 2012; Ye et al. 2021). The DOC value of the sample fractions was measured and calculated to analyze the molecular weight distribution.

Excitation-emission matrices of cyanobacteria EOM and IOM were measured with a fluorescence spectrophotometer (model F-7000, Hitachi, Japan). The measured parameters were set as follows: the excitation wavelength was 220–400 nm, the emission wavelength was 250–550 nm, the excitation slit width was 5 nm, the emission slit width was 1 nm, the voltage of the photomultiplier tube was 725 V, and the scanning speed was 12,000 nm/min. The obtained data was drawn using Origin software. According to the distribution position and intensity of fluorescence peaks in different regions, the fluorescence spectra were divided into five regions for different organic compounds (Chen et al. 2003, 2017; Gu et al. 2015; Sun et al. 2020b).

The growth curve of Microcystis aeruginosa is shown in Figure 1. The growth phase was divided into four stages: (1) in the adaptation phase (0–5 d), the change of cyanobacteria density was not obvious, and it was basically maintained in the content state of initial cultured Microcystis aeruginosa; (2) in the exponential growth phase (6–40 d), the growth rate of cells was greater than the death rate, and the Microcystis aeruginosa began to reproduce rapidly with the cyanobacteria density continuing to rise; (3) in the stationary growth phase (41–81 d), the cyanobacteria density remained at a relatively stable level; (4) in the decline phase (>82 d), the death rate of Microcystis aeruginosa cells was greater than the growth rate, resulting in an obvious downward trend of the overall cyanobacteria density.

In the growth period of Microcystis aeruginosa cells, the secretion and release of AOM also showed a certain regularity, which was basically distributed in 4–6 μg COD/10⁶ cells. By measuring the DOC content of EOM and IOM respectively, we found that the content of IOM was higher than that of EOM in the adaptation phase and exponential growth phase, and the content ratio was about EOM:IOM = 0.83. It indicated that most of the organic matters secreted by Microcystis aeruginosa was still stored inside the cells, and the released parts to the extracellular environment was relatively small. This phenomenon directly explains that the damage of algal cells should be avoided during cyanobacteria treatment to lighten the continuous increase of various organic pollution indicators in the water (Zuo et al. 2021). With the extension of growth period, the content ratio of EOM and IOM changed significantly in the stationary growth phase and decline phase, and gradually increased to 3.33. That was probably due to the direct consumption of energy substances inside Microcystis aeruginosa and the decline of intracellular metabolic and secretory functions, or the gradual infiltration and loss of IOM with the damage of cell membrane.

The data in Figure 1 also showed that the organic content of AOM in the actual water body of Taihu Lake was 4.3 μg COD/10⁶ cells with EOM:IOM = 1.82. It was similar to the secretion state of AOM in the stationary growth phase of the laboratory culture. In order to facilitate the comparative analysis of cyanobacteria AOM properties based on similar EOM and IOM yields, the cyanobacteria in the stationary growth phase of the laboratory culture environment and in the actual Taihu Lake environment were chosen for the follow-up study.

Cyanobacteria AOM is a complex heterogeneous mixture with different size, structure and functions. Therefore, the molecular weight distribution analysis of organic matters can be used to identify the characteristics of cyanobacteria AOM (Fang et al. 2010; Gao et al. 2010; Li et al. 2012; Qu et al. 2012; Ye et al. 2021). In this study, the molecular weight distribution characteristics of cyanobacteria EOM and IOM were investigated respectively in Figure 2. It can be seen that the molecular weight of cyanobacteria EOM was mainly distributed in the small molecular range of <3 kDa, while the molecular weight of cyanobacteria IOM was mainly distributed in the two ranges of <3 kDa and >100 kDa. For the EOM in lab-culture environment, the proportion of substances with <3 kDa molecular weight was 36.2%, and the proportion of substances with >100 kDa molecular weight was 24.08%. For the IOM in lab-culture environment, the proportion of substances with <3 kDa molecular weight was 31.18%, and the proportion of substances with >100 kDa molecular weight was 51.08%. For the EOM in actual Taihu Lake, the proportion of substances with <3 kDa molecular weight was 55.65%, and the proportion of substances with >100 kDa molecular weight was 9.56%. For the IOM in actual Taihu Lake, the proportion of substances with <3 kDa molecular weight was 42.05%, and the proportion of substances with >100 kDa molecular weight was 31.11%. Under the two environmental conditions, the proportion of substances with small molecular weight was less and the proportion of substances with large molecular weight was heavier in IOM compared with in EOM, which indicated that the macromolecular organic substances synthesized by cyanobacterial cells were mainly stored inside the cells, and the organic components released outside the cells were mainly small molecular substances. Relevant studies showed that the organic substances less than 3 kDa in IOM included chlorophyll, algal toxins, odor compounds, amino acids, etc., and the organic substances greater than 100 kDa mainly included phycocyanin and macromolecular carbohydrates (Gao et al. 2010). This also explained the color difference between EOM and IOM extracts of Microcystis aeruginosa. The reason why IOM extracts showed bright blue was mostly due to the large amount of phycocyanin in IOM. The mass of proteins accounted for more than 30% of the total intracellular organic mass (Gao et al. 2010), and there were many nitrogen-containing organics in the proteins. The decomposition of protein would cause the rise of ammonia-nitrogen in water, and it was more likely to produce toxic nitrogen-containing disinfection by-products. Moreover, these organics with high nitrogen content were very easy to provide material basis for bacterial growth and reproduction, and a large number of growing bacteria and their metabolites would produce toxic and harmful transformants again, causing serious pollution to the water environment (Fang et al. 2010; Bond et al. 2012). In addition, most of the known cyanotoxins, such as microcystins, are released extracellularly during cell lysis (Merel et al. 2013), which pushed the pollution threat caused by cell damage and IOM release to urgent.

Compared with the Microcystis aeruginosa cultured in the laboratory environment, the proportions of small molecules in cyanobacteria EOM and IOM in the actual Taihu Lake were larger. This should be due to the complex chemical components and diverse microbial communities in the actual water environment. During the growth of lab-cultured cyanobacteria, the similarity between IOM and EOM synthesized and secreted by Microcystis aeruginosa was higher. Under the conditions of complex water environment in Taihu Lake, the structure of macromolecular organic matters was likely to be chemically oxidized or biodegraded into small molecular states, resulting in small scale and high dispersion of organic components in the water body, for which it was difficult to implement follow-up treatment. Therefore, the targeted analysis and investigation of organic components in the actual water body still faces a highly challenging technical height.

From the appearance of the EOM and IOM extracts, the component structures of cyanobacteria EOM and IOM should be significantly different. Researches found that DOC and UV₂₅₄ values were the visual spectroscopic reflection of dissolved organic pollutants in water (Leloup et al. 2013; Kong et al. 2019). The SUVA value related to these two parameters was also often used to characterize the aromatic characteristics of organic compounds, which was positively correlated with the aromaticity of substances and the number of hydrophobic substances. When the SUVA value of the water is >4 L/(m·mg), it means that the water body contains more aromatic and high molecular weight substances, and the hydrophobicity of the organic matters is relatively high. If the SUVA value of the water is <2–3 L/(m·mg), it means that it contains more non-aromatic and low molecular weight substances, and the hydrophilicity of the organic matters in the water body is high (Kitis et al. 2002; Cheng et al. 2005). In Figure 3, the DOC, UV₂₅₄, and SUVA values for the EOM in lab-culture environment were 35.21 mg/L, 0.232 cm⁻¹, and 0.66 L/(m·mg), respectively. The DOC, UV₂₅₄, and SUVA values for the IOM in lab-culture environment were 27.23 mg/L, 0.259 cm⁻¹, and 0.95 L/(m·mg), respectively. While the DOC, UV₂₅₄, SUVA values for the EOM and IOM in the actual Taihu Lake were 28.11 mg/L, 0.134 cm⁻¹, 0.48 L/(m·mg), and 15.43 mg/L, 0.184 cm⁻¹, 1.192 L/(m·mg), respectively. Whether in the lab-culture environment or in the actual Taihu Lake, the DOC value of EOM was higher, the UV₂₅₄ value was lower, and the SUVA value was lower than that of IOM under the stable cell growth state. In both the two environments, the DOC yield of EOM was higher than that of IOM, but the SUVA value of EOM was lower than that of IOM, indicating that the EOM had relatively low aromaticity and high hydrophilicity due to the low content of hydrophobic organic matters with high molecular weight. Therefore, it is very difficult to completely remove the EOM with high content of small molecular organic matters by coagulation and other conventional water treatment processes, so that an effective control before the outbreak of cyanobacteria bloom is necessary. Compared with those in the lab-culture environment, the DOC and UV₂₅₄ values of EOM and IOM in the actual Taihu Lake were lower, which might be because the nutrient supply in the actual Taihu Lake for the growth and metabolism of cyanobacterial cells were not sufficient and targeted as that in the lab-culture environment, or be due to the fact that the complex actual water was rich in more chemical components and biological organisms that could degrade some organic matters produced by cyanobacteria. However, the SUVA value of EOM was lower and the SUVA value of IOM was higher in the actual Taihu Lake than those in the lab-culture environment. This indicated that the EOM was more prone to degradation and transformation in the actual Taihu Lake, further increasing its solubility in water, while the secretion of hydrophobic substances with high molecular weight in IOM was relatively higher under the stimulation of complex Taihu Lake environment. It also clarified that the EOM and IOM produced by cyanobacteria had more convergence characteristics in the comfortable environment of lab-culture, but had more obvious heterogeneity under the stimulation of actual complex environmental factors in Taihu Lake.

Three dimensional fluorescence spectroscopy can further realize structure classification and identification in cyanobacteria AOM. It can be seen from the comparison between Figure 4(a) and 4(b), the characteristics of fluorescent substances in IOM were more obvious than those in EOM. There were two main types of obvious fluorescence regions in the spectrum of EOM, which were mainly concentrated in D and E regions. This means that the EOM of Microcystis aeruginosa mainly contains humic acids, tryptophan-like proteins and dissolved microbial metabolites. The dissolved microbial metabolites are mainly composed of tyrosine, tryptophan, proteins and other substances rich in organic nitrogen. Therefore, organic nitrogen substances might account for a large proportion in EOM, which was consistent with the previous relevant reports (Li et al. 2012; Gu et al. 2015). There were mainly five kinds of fluorescence regions in the spectrum of IOM, indicating that there were more abundant biochemical organics inside cells. Among them, the peak areas of A, B (tyrosine and protein like substances with aromatic structure) and D (tryptophan-like proteins and soluble microbial metabolites) were wider and stronger, while the fluorescence response values of region C (fulvic acid-like substances) and E (humic acid-like substances) were relatively lower, which was similar to the reports of Gu et al. (2015) and Wang et al. (2012). Therefore, the species of fluorescent substances in IOM were richer than EOM, and the content level of corresponding substances was higher. Combined with the analysis of the research results in Figures 2 and 3, the proportion of hydrophobic aromatic proteins in IOM was higher, while the proportion of hydrophilic humic acids, fulvic acids and dissolved microbial metabolites in EOM was higher.

Comparing the cyanobacteria EOM and IOM in actual Taihu Lake with those in lab-culture environment (Figure 4(a)–4(d)), it could be clearly seen that different growth environments were not the main reason for the difference in the classification of IOM substances. However, the EOM would have some component changes due to their release and dissolving in different water quality conditions, which showed two more peak signal changes of A and B than that in lab-culture. This indicated that cyanobacteria were likely to be affected by the complex actual water environment and show more diverse types of released substances in EOM, or the EOM was transformed under the influence of complex chemical components and diverse microbial communities in the actual water environment after being released. Combined with the above results in this study, the stimulation of the actual water environment in Taihu Lake on EOM should tend to the transformation of amino acids, fulvic acids and other substances with smaller molecular and higher hydrophilicity. For the secretion of IOM, although the actual water environmental factors in Taihu Lake had little effect on its component species, they still had a certain effect on the secretion amount of the components. Figure 4(d) showed stronger signal responses at C and E regions than Figure 4(b), which indicated that the growth and secretion of humic acids and fulvic acids of IOM in the actual environment of Taihu Lake were more obvious than those in the lab-culture environment. Therefore, the complex actual water environment of Taihu Lake not only leads to the diversity transformation of component types of EOM, but also leads to the secretion stimulation of some components of IOM. The targeted organic pollution control of cyanobacteria AOM may face higher technical difficulties.

During the growth period of Microcystis aeruginosa, the secretion and release of AOM was about 4–6 μg COD/10⁶ cells. From the adaptation phase to the exponential growth phase, due to the rich nutrients in the culture system and the strong metabolic activities of cyanobacterial cells, the formation and secretion of IOM increased steadily and was higher than the release of EOM with the content ratio being about EOM:IOM = 0.83. From the stationary growth phase to the decline phase, the lack of nutrients and the decline of cyanobacterial cells lead to the decrease of IOM synthesis and the gradual infiltration outside of the cell membrane, decreasing the content ratio to EOM:IOM = 3.33. The components of EOM had a high proportion of hydrophilic humic acids, fulvic acids and small molecular amino acids of <3 kDa, showing low SUVA value. While the molecular weight of IOM was mainly distributed in the macromolecules of >100 kDa and the micromolecules of <3 kDa. Compared with EOM, the components of small molecular weight of IOM accounted for less, and the aromaticity and hydrophobicity were higher. EOM and IOM produced by Microcystis aeruginosa had more convergence characteristics in the comfortable environment of lab-culture, but had more obvious heterogeneity under the stimulation of actual complex water environmental factors in Taihu Lake. Cyanobacteria EOM was more prone to be degraded and transformed in Taihu Lake, resulting in lower molecular level, lower SUVA value and richer component types, which was likely to make the organic pollution control for cyanobacteria EOM more difficult. The growth and secretion of humic acids and fulvic acids in cyanobacteria IOM stimulated by the complex Taihu Lake environment were more obvious, and its organic pollution in water could not be underestimated.

This work was supported by the National Natural Science Foundation of China [grant number 51708480]; the Qing Lan Project of Yangzhou University; the Natural Science Foundation of Jiangsu Province [grant number BK20150456]; the Water Environmental Protection Engineering Laboratory of Jiangsu Province [grant number W1801]; and the Water Conservancy Science and Technology Project of Jiangsu Province [grant number 2020033].

All relevant data are included in the paper or its Supplementary Information.