Lake Taihu, the third largest freshwater lake in China, is located in the Yangtze River Delta region. It’s area is 2338 km², mean depth 2 m, and the maximum depth is no more than 3 m with flat-bottomed bathymetry. There is no persistent stratification during summertime. Its eutrophication began in the 1980s (Qin et al., 2007), and cyanobacteria bloom coverage has gradually extended from Meiliang Bay (Chen et al., 2003) to the entire northwest zone of the lake. The bloom-forming species is high buoyant Microcystis which account for overwhelming majority during summer (Chen et al., 2003). The nutrient enrichment combined with warm spring resulted in the earlier occurrecne of intensive cyanobacterial bloom which caused the drinking water crisis in late May 2007 (Qin et al., 2010). With a series of pollution control measures implemented since 2007 (Stone, 2011), the water quality has not further deteriorated, but the notorious harmful cyanobacterial blooms still persist and the bloom induced anaerobic “black water” recur throughout summer in this lake (Zhang et al., 2016).

To investigate the impact of wind-induced mixing on the cyanobacteria bloom occurrence, multi-parameter monitoring was conducted synchronously during both Typhoon Morakot (August 12–14, 2009) and Typhoon Soulik (July 12–13, 2013). Instruments to record wind velocity and direction, at a frequency of every 10 min, were moored at the pier end in Taihu Laboratory for Lake Ecosystem Research (TLLER), Chinese Academy of Sciences (CAS) nearby the Meiliang Bay in Lake Taihu (Figure 1). We were particularly interested in the vertical migration speed of Microcystis colonies, therefore, an Acoustic Doppler Current Profilers (ADCP) was deployed at the bottom of the water column near Wuguishan Islet to record three-dimensional current velocity (Figure 1). At the same site, multi-function water quality sensors (YSI6600) were deployed near the water surface (ca. 20 cm under surface) and the bottom (50 cm above bottom of lake). The sensors of water quality parameters, including pH, conductivity, turbidity, were recording with a 10 min resolution, while the chlorophyll-a concentration was recorded every 30 min by in vivo fluorescent light detectors which need to be adjusted before deploying.

Additionally, cyanobacteria colonies were sampled during typhoon Soulik at the end of pier in TLLER (Figure 1). Samples were taken at noon of July 12 and 13, 2013, followed by a period during which the sampling interval was 12 h from the midnight of July 13 to the noon of July 15, 2013. Samples were also collected around midnight of July 15 and in the morning of July 16, 2013. The depths of sampling included the bottom layer (0.1 m above sediments), middle layer (1.2 m above sediments) and surface layer (2 m above sediments). The colony size of Microcystis were counted from at least 30 colonies (>100 cells) with microscopy (Nikon E200). Microscopic examination showed that the dominant phytoplankton species were Microcystis aeruginosa, M. flos-aquae, and M. wesenbergii during observations, and the proportion of Microcystis accounted for more than 90% of total phytoplankton biomass. For convenience, we took the concentration of Chl a to represent Microcystis biomass.

In order to examine whether the disturbance by wind is significant to promote the formation of Microcystis colony, an experiment was carried out under the pier in TLLER on September 23, 2017. Some M. aeruginosa and M. flos-aquae colonies separated from Meiliang Bay, Lake Taihu, were cultured with BG11 (TN = 50 mg/L, TP = 2.5 mg/L) for 2 months followed by extended 2 months culturing with modified BG11 (TN = 10 mg/L, TP = 0.5 mg/L); and a samples with single cell, unicells and small colonies of pure Microcystis were obtained. About 500 ml cultured pure M. aeruginosa (1.59 × 10⁵ cells/mL) and M. flos-aquae (1.59 × 10⁵ cells/mL) were transferred to pre-cleaned plastic bottle. Three 500 ml bottles were fixed to iron pile of the nearby as control of no wind induced disturbance; while the other three bottles as treatments, tethered to cement pile by rope, swing with the water movements induced by wind (Supplementary Figure S2). This experiment took 48 h. The wind speeds were measured every 1 h during experiment. Microcystis biomass, Microcystis colony size and EPS concentration in the bottles were measured at-beginning and at-end of experiment. The content of soluble extracellular polysaccharide (sEPS) and bound extracellular polysaccharide (bEPS) were quantified spectrophotometrically by the anthrone method (Herbert et al., 1971). Samples of M. aeruginosa (5 mL) and M. flos-aquae (5 mL) were preserved with Lugol’s iodine solution. After the M. aeruginosa and M. flos-aquae settled for 48 h, samples of M. aeruginosa (5 mL) and M. flos-aquae were concentrated (1 mL). Then M. aeruginosa and M. flos-aquae colony size was measured (400× magnification) with a Nikon E200 microscope and QCapture pro. software. The mean colony size of M. aeruginosa and M. flos-aquae were measured under microscopy with at least 50 colonies.

For further investigating the influence of disturbance on the formation of Microcystis colony, a simulated perturbation experiment was carried out in July in TLLER. Similarly, we took six big plastic barrels (three for control and three for treatment) and each barrel has 90 cm in diameter and 80 cm in height (Supplementary Figure S3). All barrels were filled with water containing Microcystis colonies taken from Meiliang Bay, Lake Taihu, to the depth of 60 cm. Total nitrogen (TN) and total phosphorus (TP) concentrations in barrels were adjusted with addition of NaNO₃ and K₂HPO₄ 3H₂O to 5 mg/L for TN and 0.25 mg/L for TP which were almost two times of TN and TP concentration in Meiliang Bay, Lake Taihu. Six barrels were cultured in static condition during the first 5 days. During this period, samples were taken at 10:00 am every morning from two layers at the surface (50 cm above bottom) and the bottom (0 cm above bottom). From the 6th day, the treatments were imposed with wave produced by mini wave action generators (WP-60, Triopo Electric Co. Ltd.) which were deployed at 10 cm below the surface. The wave was produced with a frequency of 1 Hz and the maximum wave height was 5 cm (Supplementary Figure S3). Wave disturbances were lasting for 24 h. During the disturbance period, water samples were taken at 3, 6, 12, and 24 h after the beginning; and during the post-disturbance (wave generator stopped to work but the wave effects still existed), samples were taken at 3, 6, 12, 24, and 48 h. Sample analysis for Microcystis biomass and colony size and the concentrations of EPS were determined as described in Section “In Situ Experiment of Mixing Induced by Wind-Wave on Colony Size of Microcystis in Lake Taihu.” The concentrations of Chl a, TN and TP were determined by spectrophotometry. Microscopic examination showed that Microcystis was the overwhelming dominant species during experiment.

Single colony of M. aeruginosa and M. flos-aquae were isolated from lake water in Meiliang Bay (dominated by Microcystis bloom) in Lake Taihu and were cultured with BG-11 medium. Two months later, unialgal cultures of M. aeruginosa and M. flos-aquae were transferred to modified BG-11 medium (TN = 50 mg/L, TP = 2.5 mg/L), respectively. Until the beginning of experiment, M. aeruginosa and M. flos-aquae, present as single-cells, paired-cells and small colonies, were cultured in modified BG-11 media for 5 months. At the beginning of the experiment, 150 mL M. aeruginosa (4.83 × 10⁶ cells/mL) and M. flos-aquae (1.78 × 10⁶ cells/mL) were transferred to 500 mL Erlenmeyer flasks containing 200 mL modified BG-11 medium. Considering the velocities (0.005–0.077 m/s) of Lake Taihu (Zhou, 2016), five different mixing intensities were designed as following: 0, 50, 100, 200, and 400 rpm, which were corresponding to velocities of 0, 0.16, 0.32, 0.64, and 1.28 m/s (Camacho et al., 2007; Guadayol et al., 2009; Gallardo Rodríguez et al., 2009), respectively. The control had three 500 ml flasks with shaking intensity of 0 rpm. Each treatment also had three 500 ml flasks (triplicate) placed on shaking incubator. There were four shaking incubators with shaking intensities of 50, 100, 200, and 400 rpm. The shaking experiment lasted for 24 h at 25°C and cool/white fluorescent lights at an intensity of 40.5 mol m^-2 s^-1 with a light-dark cycle of 12:12 h. The total concentration of nitrogen and phosphorus in all treatments and control were maintained about 50 and 2.5 mg/L, respectively. Samples were taken before and after the experiment. The measurements of colony size, biomass of M. aeruginosa, concentrations of Chl a, EPS, TN, and TP were the same as described in Section “In Situ Experiment of Mixing Induced by Wind-Wave on Colony Size of Microcystis in Lake Taihu” above.

Large size colony has high buoyancy to overcome the mixing and gravity to float at water surface and form visible bloom. To measure the upward movement velocity of different size Microcystis colonies, the field samples of Microcystis were taken with net filtering (mesh sizes were 425, 100, 64, and 20 μm) to obtain five different size classes of Microcystis colonies (>425 μm, 100–425 μm, 64–100 μm, 20–64 μm, and <20 μm). For each colony size group, the cell numbers and proportion to all colonies were estimated before the experiment. A plexiglass column (1 m in height and 0.1 m in diameter) was used to measure the upward movement velocity of different size colonies. There was an inlet for injection at the column bottom and five sampling outlets distributed along the column every 0.2 m. The column was filled with distilled water. Different size colony samples were injected at the bottom. After 1 (>425 μm), 3 (100–425 μm), and 10 min (<100 μm), samples were taken from 5 sampling outlets simultaneously and the density of algae was counted with microscope. Five different size Microcystis colonies were injected into the columns separately. Each colony size experiment had triplicate.

The floating velocity of different size colonies was calculated as follows:

where B_t (the total biomass) equal to the product of injection density and injection volume, B_i (the biomass of each outlet) was calculated by the cell density of each outlet by 1/6 of the volume of plexiglass column, v_i (floating velocity of each outlet) were calculated by the height of each sampling outlet divided by experimental time.

Remote sensing about the cyanobacterial bloom area were used to investigate the cyanobacterial bloom changes in space. The lowest cyanobacteria biomass at which the bloom could be detected by satellite was equivalent to the Chl a concentration around ∼10¹ mg/m³. All the remote sensing images of cyanobacteria bloom were obtained from NASA¹. The interpretation of MODIS images was according to the re-projection and geometric correction via ERDAS 9.1, and the information of blooms was retrieved by means of the ratio of near-infrared and red spectrum (Ma et al., 2008; Duan et al., 2009).

Lake Taihu basin and delta of Changjiang River (Yangtze River) often experience typhoon during summer. These typhoons normally formed in the tropical or subtropical Pacific Ocean and moved westward or northwestward and influenced the eastern China coastal zone. Typhoon Morakot passed Lake Taihu during August 11–14, 2009. Its effects on the lake started in the early morning of August 11. The dominant wind directions were westerly and northwesterly and the maximum wind speed occurred in the evening of August 11 at 11.8 m s^-1 (Figure 2A), then the wind speed gradually dropped until clam condition in the evening of August 12, while the wind direction shifted to east and northeast. During the typhoon, the near-surface vertical current velocity measured by Acoustic Doppler Current Meter (ADCP), was typically mixing with up and down movement and a maximum velocity was about 0.5 cm/s (Figure 2B) to correspond to the high wind speed. After the typhoon, the vertical direction current turned to be upward uniformly and the maximum velocity reached 1.0 cm/s (Figure 2B). Because of the measured vertical velocity representing the moving speed of particulate matter in the water column (Holdaway et al., 1999). Thus the measured vertical velocity actually indicated the cyanobacteria colonies moving speed in the water column. In situ observation after the typhoon showed that the algal cells/colonies generally ascended to the water surface, instead of sinking, representing that cyanobacteria cells or colonies moving upward was driven by the buoyancy.

Interestingly chlorophyll-a concentrations differed between the layers at near-bottom and near-surface during the post-typhoon. During the typhoon peak period (06:00 h 11 to 06:00 h of August 12), the chlorophyll-a concentrations was almost identical in the water column, suggesting well-mixed conditions during this period (Figure 2C). However, after the typhoon, the chlorophyll-a concentration at near-surface layer was increased and that near the bottom layer declined (Figure 2C), indicating that cyanobacteria cells/colonies in water column started to differentiate and aggregate at the water surface, which probably a precursor of the cyanobacteria bloom occurrence. Remote sensing images in the morning of August 12, 2009 (during Typhoon Morakot) showed the area of the bloom was only 92 km² (Supplementary Figure S4A), which rapidly expanded to 391 km² in the morning of August 13 (Supplementary Figure S4B).

Typhoon Soulik affected Lake Taihu during July 12–17, 2013. The period could be divided into three stages according to the wind speed. The pre-typhoon period (00:00–22:00 of July 12), the wind speed increased gradually and average wind speed was 3.82 m s^-1 (Figure 3A). During the typhoon period (from 22: 00 h, 12–18:00 h, July 15) when a high wind speed persisted with average of 6.63 m s^-1 and a maximum speed of 13.8 m s^-1 (at 18:00 h, July 13) (Figure 3A). The post-typhoon period (18:00 h, 15-00:00 h, July 17) had an average wind speed of 5.26 m s^-1 which gradually reduced to 1 m s^-1 near the end of the typhoon (Figure 3A). In situ measurements revealed that the mean size of Microcystis colonies in the water column increased from 32.8 to 69.4 μm within 48 h (Figure 3B).

At the same time, remote sensing satellite images showed that little of the cyanobacteria blooms could be seen in the pelagic area of the lake during the Soulik Typhoon peak period from July 12 to July 14; however, around noon of July 17, after the typhoon, blooms occurred with coverage of 288 km² (Supplementary Figure S5).

During this experiment, the mean wind speed was 3.15 m/s (range 1.33–4.83 m/s) (Figure 4A). In the treatments (the bottles were not fixed), the colony size of M. aeruginosa and M. flos-aquae increased quickly after mixing induced by wind-wave for 48 h. The colony size of M. aeruginosa in controls (the bottles were fixed) and treatments were 23.6 (±1.2) and 59.0 (±1.8) μm, respectively (Figure 4B). The colony size of M. flos-aquae in controls and treatments were 25.5 (±1.1) and 40.8 (±1.7) μm, respectively (Figure 4C). ANOVA analysis showed that the difference of M. aeruginosa and M. flos-aquae colony sizes between control and treatment were significantly (p < 0.01) (Figures 4B,C). Also, ANOVA analysis showed that the EPS concentrations of M. aeruginosa and M. flos-aquae in treatments were significantly higher than that in controls (p < 0.01) (Figures 4D,E).

Before the disturbance, the Microcystis spp. colony size in the control and treatment decreased gradually (Figure 5A). To the 6th day when the disturbance experiment started, the size of Microcystis in the control and treatment had reduced from 46.8 μm and 45.4 μm to 19.8 μm and 20.2 μm, respectively. During the disturbance experimental period, the mean Microcystis colony size decreased continuously in control while it increased rapidly to 68.4 μm after 24 h disturbance (Figure 5A). At the end of experiment (48 h after 24-h disturbance), the average colony size in treatment was 70.1 μm, while the mean colony size in control was 12.6 μm (Figure 5A). The colony size difference between control and treatment was significant (p < 0.01). After 24-h disturbance, the EPS concentration in the control and the treatment were 1.26 and 1.49 ng/cell, respectively, which was significantly different (p < 0.05) (Figure 5B). Before the experiment, the initial averaged Chl a concentration at the surface and the bottom in the experimental barrels were 36.8 and 33.4 μg/L (Figure 5C), respectively. There was no significant difference (p > 0.05). During the disturbance period, the surface concentration of Chl a decreased sharply to 51.2 μg/L while the Chl a concentration at bottom increased rapidly to 42.1 μg/L (Figure 5D), which was no significant difference (p > 0.05). At the end of the experiment (48 h after 24-h disturbance), the cyanobacterial colonies returned to water surface resulted in Chl a concentration of 102.6 μg/L at surface, which was higher than that in control (Figure 5D); while the Chl a near the bottom was only 28.6 μg/L which differed significantly from that at water surface (p < 0.05). The stratification recurred at the end of experiment in treatment (Figure 5D).

In this experiment, the colony size of M. aeruginosa and M. flos-aquae in 50, 100, 200, and 400 rpm experiments were significantly larger than that in control (p < 0.05) (Figures 6A,C) after 24-h continuously mixing. The colony size of M. aeruginosa in control, 50, 100, 200, and 400 rpm experiments were 23.6 (±1.8), 50.1 (±8.6), 92.9 (±4.8), 67.8 (±10. 9), and 37.3 (±3.9) μm, respectively (Figure 6A and Supplementary Figure S6). The colony size of M. flos-aquae in control, 50, 100, 200, and 400 rpm were 10.6 (±0.6), 24.0 (±1.2), 33.0 (±1.2), 19.2 (±1.6), and 14.4 (±1.8) μm, respectively (Figure 6C). Both Microcystis species showed an increase firstly with the intensity of mixing followed by a decrease when the mixing intensity larger than 100 rpm (Figures 6A,C). In addition, ANOVA analysis indicated that the EPS concentration of M. aeruginosa and M. flos-aquae in all treatments were significantly higher than that in controls (p < 0.01) (Figures 6B,D).

Experimental results showed that the large size colony (cell count of each colony > 100) accounted for an overwhelming majority in colony size distribution, which was consistent with our field observations (Figure 7), and floating rates increased with the colony size. The largest cell colony (>425 μm in diameter) had the floating rate of up to 0.77 ± 0.23 cm s^-1 (Supplementary Table S3). The largest proportion of colony size occurred at colony diameters between 100 and 425 μm with a high floating rate of 0.30 ± 0.08 cm s^-1 (Figure 7 and Supplementary Table S3), while colony sizes <20 μm were solitary cells or small colonies with floating rates about 0.0002 cm s^-1 (Supplementary Table S3).