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Isolation and identification of four airborne microalgal strains belonging to three genera from Dongsha Island in the South China Sea

Four microalgal strains belonging to three genera were successfully isolated in the collection trip in December 2013 and all the four could not grow in 2f seawater medium10 (in both 33 and 22‰ salt levels), indicating they were freshwater species. As shown in Fig. 1, based on the morphology revealed by Scanning Electron Microscopy and the phylogenetic analysis, the four strains were named Scenedesmus sp. DSA1 (stands for Dongsha airborne #1), Coelastrella sp. DSA2, Coelastrella sp. DSA3, and Desmodesmus sp. DSA6 (hereafter referred to as DSA1, DSA2, DSA3 and DSA6, respectively). All four strains are members of the family Scenedesmaceae in Chlorophyta. Sequences of the 18S rDNA and ITS1-5.8S-ITS2 fragments of the four airborne microalgal strains were deposited into GenBank under the accession numbers KX818834–KX818841.

Figure 1
figure1

A Scanning electron microscopy (SEM) micrographs of the four airborne microalgae in the early stationary phase. Upper panel, left and right: Scenedesmus sp. DSA1 and Coelastrella sp. DSA2. Lower panel, left and right: Coelastrella sp. DSA3 and Desmodesmus sp. DSA6. B Phylogenetic analysis of the four airborne microalgal strains (underlined) and their related species based on the fusion sequences of ITS1 and ITS2 of each species. The numbers in the parentheses are accession numbers of each sequence in GenBank. The numbers at the nodes indicate bootstrap values (expressed as percentage) with 500 replicates.

The airborne green microalgae had better UV tolerance than the waterborne green microalgae

When traveling in the air, airborne microalgae are inevitably exposed to much higher levels of UV radiation compared to microalgae in the aquatic environments. In order to survive aerial travel, these cells must have a mechanism(s) to protect themselves against the damaging effects of the radiation, a mechanism that is of less concern to waterborne microalgal populations. To verify whether the four airborne microalgae resisted UV radiation better than waterborne microalgae, these microorganisms were spread onto agar plates using the top agar method11, and exposed to UV-B radiation to determine their survival rates. The survival rates were compared to those of the two waterborne microalgae, Desmodesmus sp. F512 and Neodesmus sp. UTEX 2219-413 (hereafter referred to as F5 and 2219-4, respectively), both are members of Scenedesmaceae as well. As shown in Fig. 2A, the survival rates of the two waterborne microalgae were about 68 and 46%, respectively, after 1 min of the UV-B exposure, compared to more than 90% for all four airborne microalgae. After 3 min of the exposure, however, all of the waterborne cells died but the survival rates of the airborne cells were about 98, 73, 26, and 11% for DSA3, DSA2, DSA1 and DSA6, respectively. Significant differences could still be observed among the survival rates of DSA3, DSA2, and the other four species after 5 min of the UV-B exposure.

Figure 2
figure2

A UV-B stress tolerance of the four airborne microalgae and the two waterborne microalgae used as controls. About 500 cells in the early stationary phase were spread onto each agar plate and irradiated with 302 nm UV for the specified durations. The survival rate of each strain was defined as the colony numbers on the UV-treated plates compared to those on the non-treated plates (n = 6, mean ± SE). F5, Desmodesmus sp. F5; 2219-4, Neodesmus sp. UTEX 2219-4. B Different autofluorescence intensities from the cell wall of the six microalgal strains.

For algal cells, cell wall is the first barrier to defend the cells against UV attack. It is well known that the cell walls of terrestrial plants are autofluorescent when excited by UV14. In this context, the four airborne microalgal strains would have better UV tolerance if their cell walls could absorb UV, and therefore are autofluorescent as well. To examine this possibility, the pigment-free cells were examined under an epifluorescence microscope. Indeed, as shown in Fig. 2B, the cell walls of the four airborne and the two waterborne green microalgae were able to emit autofluorescence when excited by UV under the microscope. Furthermore, the autofluorescence intensities emitted from the six microalgal strains varied, with DSA3 and DSA2 being the strongest, DSA1 and DSA6 in the middle, and F5 and 2219-4 the weakest as detected using the same parameters. The varied intensities suggested that these microalgae had different levels of UV tolerance because their cell walls absorbed different amounts of UV energy.

The UV tolerance of the microalgae was positively correlated with their cell wall thickness

There appeared to be a correlation between the autofluorescence intensities emitted by the cell walls and the survival rates of the six microalgal strains exposed to the UV-B radiation for 3 min. It was intriguing to investigate whether the cell wall thickness of these microalgae played a role in the survival rates. To measure the cell wall thickness of the six microalgal strains, these cells were fixed and sectioned, and then observed using Transmission Electron Microscopy (TEM). As shown in Fig. 3A, the cell wall thickness of the six strains varied, with DSA3 and DSA2 being the thickest, DSA1 and DSA6 intermediate, and F5 and 2219-4 the thinnest. This relation well-correlated with that of the autofluorescence intensities from their cell walls. When the survival rates under 3 min of the UV-B exposure were plotted against the cell wall thickness of the six strains, a good correlation (r = 0.99, p < 0.0001, Correlation Coefficient method in StatView) was revealed (Fig. 3B). This finding suggests that a thicker cell wall, which absorbs more UV energy and emits stronger autofluorescence, provides better protection against UV radiation in the case of green microalgae.

Figure 3
figure3

A Transmission electron microscopy (TEM) micrographs of the four airborne microalgae and the two waterborne microalgae used as controls. These cells were sampled in the early stationary phase. Note the differences in the cell wall thickness among these strains. B The correlation between the UV-B stress survival rates and the cell wall thickness of the six microalgal strains (r = 0.99, p < 0.0001, analyzed using the StatView Correlation Coefficient method). The cell wall thickness of each strain was measured based on the TEM images (n = 10, mean ± SE).

The four airborne green microalgae survived the freeze–desiccation stress treatment

In addition to UV radiation, airborne microalgae are also subjected to desiccation and extreme low temperature stresses especially dispersed in winter. To examine whether the four airborne strains had better tolerance to the two stresses than the waterborne microalgae, the six strains were grown in the B3N liquid medium until early stationary phase. These cells were collected, transferred onto nylon membranes and air-dried on the bench. The samples were then placed at − 20 °C in the dark for 1, 2 and 4 weeks and at the end spread onto agar plates to examine their vitalities. As shown in Fig. 4A, the cell lawns of the four airborne microalgae grew denser in 7 days in the incubation compared to those on day 0. On the other hand, the cells of the two waterborne species died after the double stress treatment. In the less challenging conditions, the air-dried cells of the six strains were placed at room temperature in the dark for 14 and 21 days, and spread onto agar plates (Fig. 4B). Again, the two waterborne species could not survive the desiccation stress while the four airborne strains grew well.

Figure 4
figure4

A Vitalities of the six microalgal strains after freeze–desiccation stress treatments. Early stationary phase cells of the six microalgal species were air-dried on the bench and placed at − 20 °C for 1, 2 and 4 weeks before spread onto the agar plates. All the four airborne microalgae DSA1, DSA2, DSA3, and DSA6 grew into dense cell lawns in 7 days but the two waterborne species died after these stress treatments. B Vitalities of the six microalgal strains after desiccation stress treatments. Early stationary phase cells of the six strains were air-dried on the bench and placed at room temperature in the dark for 14 or 21 days before spread onto the agar plates. The four airborne microalgae DSA1, DSA2, DSA3, and DSA6 grew into dense cell lawns in 7 days but the two waterborne species died after the stress treatments. C Vitalities of the six microalgal strains after lyophilization treatment. Early stationary phase cells of the six microalgal strains were air-dried on the bench and lyophilized for 16 h. One hundred mg of the treated cells of each strain was inoculated into a bottle and allowed to grow for 7 days. The three airborne microalgae DSA1, DSA2, DSA3 grew into dense cultures but the airborne species DSA6 and the two waterborne species died after the stress treatments.

The strong tolerance of the four airborne microalgae prompted the question whether these cells could survive the harsh conditions in the lyophilization chamber that had extreme low temperature, air pressure and moisture that were harsher than the conditions they could encounter during traveling at high altitudes in the air? To answer this question, the air-dried cells of each species were lyophilized for 16 h before inoculated to bottles containing the B3N medium. As shown in Fig. 4C, DSA1, DSA2 and DSA3 grew well in the cultivation but DSA6 and the two waterborne microalgae failed to grow after the treatment.

The thick cell walls did not confer water retention in the airborne microalgal cells

Since the cell wall of airborne DSA3 and DSA2 is much thicker (average 713 and 477 nm, respectively) than that of the two waterborne species (average 85 and 59 nm for F5 and 2219-4, respectively) and their desiccation tolerance is also much better, the question whether the thick cell wall played a role in water retention in the airborne microalgae arose. To determine the water content of these air-dried and the lyophilized cells, the weight of each sample was measured before and after heating at 105 °C for 24 h using a high accuracy analytical balance. As shown in Fig. 5, the water contents of the air-dried cells of the six strains were about 12%, which did not explain their differences in the desiccation tolerance. The water content of the lyophilized cells was below 1% with the exception for 2219-4. This did not explain their survival rates after the lyophilization treatment either.

Figure 5
figure5

Water content of the lyophilized and the air-dried cells of the six microalgae (n = 3, mean ± SE).

Total antioxidation capacities of the airborne microalgae did not confer their freezing and desiccation tolerance

Intracellular oxidative stress is common in photosynthetic cells under environmental stress. Eukaryotic cells in general are equipped with various mechanisms to ameliorate oxidative stress. One mechanism to prevent damage caused by reactive oxygen species (ROS) such as superoxide, hydrogen peroxide and hydroxyl radicals in photosynthetic cells is production of antioxidants in the cells to detoxify these harmful molecules. To examine whether the levels of antioxidants in the airborne microalgae contributed to their better freezing and desiccation tolerance, the total antioxidation capacities of the six strains in the early stationary phase were compared. As shown in Fig. 6A, the total antioxidation capacity of DSA3, the strain with strong tolerance to the stresses tested, was at the same level as that of F5, the waterborne species with weak tolerance. The antioxidant levels of the other strains did not show dramatic difference either. Therefore, their differences in freezing and desiccation tolerance demonstrated in this study did not appear to be conferred by their total antioxidation capacities.

Figure 6
figure6

A Antioxidation capacity of the six microalgal extracts from the early stationary phase cells. One mg lyophilized microalgal cells of each species was analyzed (n = 3, mean ± SE). Antioxidation capacity is expressed as the Trolox equivalent. B Production of carotenoids by the four airborne microalgae before and after the stress treatment. Upper panel, cells in the early stationary phase; lower panel, cells after nitrogen deprivation and 1.5% NaCl stress under high light intensity (800 μmole photo/m2/s) treatment for 7 days.

The airborne microalgae produced carotenoids under prolonged stressful conditions

Colonies of the airborne microalgae became reddish on agar plates after prolonged incubation (more than 4 weeks) under continuous light. This phenomenon is reminiscent of Haematococcus pluvialis15, Coelastrella striolata16 and Coelastrella sp. F5017, which accumulate high levels of carotenoids, which are strong antioxidants, under stressful conditions. Two of the four airborne microalgae are members of Coelastrella, and they form a clade with Coelastrella sp. F50 (Fig. 1), suggesting they are able to produce carotenoids as well. To verify this view, the four airborne microalgae were cultivated in the liquid medium under normal conditions until reaching stationary phase. These cells were then subjected to nitrogen starvation and salt stress under high light intensity. As shown in Fig. 6B, these cells changed color from green to red or yellow–brown in seven days of this stress treatment. HPLC analysis of these pigments of DSA2 and DSA3 demonstrated that the pigments contained carotenoids including astaxanthin, canthaxanthin and β-carotene similar to the pigments identified in Coelastrella sp. F5017.

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