Algae explosive growth mechanism enabling weather-like forecast of harmful algal blooms

As a global problem in coastal environments, harmful algal blooms (HABs) have seriously affected the health of coastal ecosystems and regional economies. Here we report an aerosol-trigger mechanism for the occurrence of HABs based on long-term field data and laboratory experiments. The occurrence times of HABs and aerosol events had a significant correlation from 2005 to 2013 in the East China Sea, indicating that aerosol transport was probably an alternative trigger of HABs. HABs mostly occur in the transition time between winter and summer, during which northwest monsoon transport substantial aerosol (rich in phosphate, iron and other trace metals) to coastal waters, as revealed by chemical measurements, transmission electron microscope and electron microprober results. Such nutrients can stimulate algal growth in our incubation experiments, suggesting that such aerosol transport can be important nutrient sources for the East China Sea where phytoplankton growth is relatively phosphate limited. Air-borne nutrients are available for algal growth by rapid downward air flow, which additional results a clear weather condition, and thus adequate light intensity for algal growth. At last, the transition from northwest monsoon to warm southwest monsoon establishes favorable seawater temperature for algal blooms. Such weather-related aerosol-trigger mechanism suggests possibly forecast of HABs.

(e and f) Fe and P concentrations of aerosol particles, respectively; (g) relationship between HABs occurrence and aerosol event; (h) TEM of aerosol particles (TEM images revealed that large grains are surrounded by colloidal materials. The mineralogical composition of the large grains is dominated by silicate (I, EMP result in j) and calcium-containing minerals (III, EMP result in l), while iron and other micronutrients are the major chemical composition in the colloidal parts (II, EMP result in k)); (i) TEM images of aerosol particles after contacting with sea water. In the left top of g, monthly variations of larger-scale HABs events (>300 km 2 ) were also shown.
Scientific RepoRTS | (2018) 8:9923 | DOI: 10.1038/s41598-018-28104-7 Atmospheric iron and phosphate transport for algal blooms. To interrogate the potential causal relationship between HABs and aerosol events, atmospheric particles were collected on the route of aerosol migration from inland China to the East China Sea (suburban of Hangzhou and Tiantai cities). Total suspended particles (TSP) concentration varied significantly with time during HABs months (Fig. 1d). The collected particles were rich in iron (Fe), phosphate (P), and other micronutrients Co, Cr, Cu, Mn, and Zn (Figs 1e,f and S2). Previous study have indicated that trace metal concentrations during aerosol events are larger than that during non-aerosol events 22 . Furthermore, transmission electron microscopy (TEM) and electron microprobe (EMP) analysis of the aerosol particles revealed that Fe, P and other micronutrients primarily existed as coating or cementing materials that connected Si or carbonate mineral particles together (Fig. 1h,j,k,l). When these particles contacted with sea water, the coating and cementing materials dissolved, making nutrient elements exist in aqueous phase (Fig. 1i). Fe transported by aerosol in the China coastal waters was also confirmed by TEM results in the latest research 27 .
Vertical air-velocity analysis indicated that downward air flows prevailed during HABs events in the East China Sea (Fig. S3). Such downward flow suggested nutrients in the aerosol particles would probably precipitate and be available for algal. The atmospheric Fe flux to East China Sea could be 0.01-0.1 Mt a −1 , larger than the riverine Fe input (~0.02 Mt a −1 ) 25 . In addition, Kim et al. 28 showed that atmospheric precipitation increased N concentration in the East China Sea and northwestern Pacific Ocean. Utilizing Climate Nested Air Quality Prediction Modeling System (NAQPMS) 29 , the average concentration of N carried by the downdraft air reached 3296.03 nmol m −2 two days before and first four days of the HABs event (May 20 to 27,2006). Although riverine material (Fe, P, N and Si) were important nutrient sources for coastal waters 30,31 , most of them may be consumed completely during long-distance transport due to intense phytoplankton consumption in the Changjiang River plume 16,32 and nutrient deposition with particles 33 . Consequently, riverine nutrients may not be able to meet the continuous nutrient requirement of phytoplankton bloom, particularly for those large-scale HAB events that expanded several thousand square kilometers. On the other hand, there is no transport limitation for the air-borne Fe and P.
Fe and P are two essential nutrients for phytoplankton metabolism, which are required for photosynthetic carbon acquisition and the nitrate reductase synthesis 34,35 . Cryptomonas sp and Prorocentrum micans Ehrenberg are important algae species in the formation of HABs in the East China Sea. Results showed that when Fe < 0.1 μmol L −1 , the measured chlorophyll concentration in incubated Cryptomonas sp and Prorocentrum micans Ehrenberg was relatively low, suggesting that algal growth rate was probably limited 36 . As a comparison, it increased significantly when Fe > 0.5 μmol L −1 (Fig. 2a), which is consistent with previous observations 37,38 . Under rich iron condition (1 μmol L −1 ) but relatively low P concentration (0.5-1.0 μmol L −1 ), the cell growth was significantly inhibited. Similarly, under condition of rich phosphorus (10 μmol L −1 ) but relatively low iron (0.01-0.1 μmol L −1 ), the algal growth ( Fig. 2c) was slow, and the maximum cell number was much lower than the case without Fe and P limitations. The results indicated that explosive algal growth will probably be limited if Fe and P in the East China Sea cannot be supplied continuously. When Fe (1 μmol L −1 ) and P (5-50 μmol L −1 ) were both not limiting, phytoplankton cells multiplied exponentially after three days of incubation. Also, the P and Fe concentrations of the phytoplankton cells show an excellent correlation (r = 0.9979, p < 0.001) (Fig. 2d) with a molar ratio of 356:1 for P to Fe, indicating that both P and Fe are needed for cell construction and metabolic processes of algae 35 . It should be noted that our experimental conditions were not exactly the same as those in real oceanic environments. Consequently the limiting nutrient concentrations we showed here may be different from the limiting threshold for algae growth in ocean. What can be inferred from the experiments is that the high nutrient concentrations benefit the algae growth, and decreasing nutrient concentration will probably decrease algae growth.
Light condition for algal blooms. Light intensity is another important condition for algal bloom, especially for the turbid coastal water in the East China Sea 39,40 . The downward air flow not only makes air-borne nutrients available, but will also lead to a clear weather condition 41 , and will probably increase euphotic zone depth in turbid coastal waters. In our experiment, the specific growth rates of Prorocentrum micans Ehrenberg, Cryptomonas sp., and diatom cells increased with increasing light intensity until reaching to a plateau (saturation) (Fig. 3a). The saturation light intensity was different for different algal species. An ideal saturation light intensity of 200 μE m −2 s −1 was observed for Prorocentrum micans Ehrenberg, and 150 μE m −2 s −1 for Cryptomonas sp. and diatom cells. Figure 3b,c showed the effect of light intensity on the uptakes of Co, Mn, Mo, Zn, and Fe, P by Cryptomonas sp, respectively. Increase uptakes of Fe, P, and microelements were observed under lower light intensity than under saturated light. For example, under light intensity of 10 μE m −2 s −1 , the uptakes of Fe, P, Zn, Mn, Co and Mo were 2.5, 1.9, 1.8, 2.0, 2.8 and 3.5 times as large as those under 100 μE m −2 s −1 , respectively. Under low-light conditions, algae cells absorb more Fe, P, Zn, Mn, Co and Mo than under strong illumination. The results were consistent with the findings that under low light, algae will improve the light absorption efficiency by increasing the surface area of the thylakoid membranes and the number of pigment protein complex 42,43 . Consequently, lots of Fe and P were stored in individual cells. On the other hand, under intense light condition, assimilated Fe, P and essential trace elements were used for organism growth and split, which subsequently decreased Fe and P content per cell. Our results indicated that the algae growth was less limited by Fe and P availability under stronger light condition and explained why HABs always occurred on sunny days.

Discussion
Most large-scale HABs occurred from April to September (Fig. 1a)  carried moist and warm air from southwest, and generated ideal sea temperature conditions, whereas northwest monsoon transported nutrients-rich aerosol from northwest for algae growth. The contribution of various factors to the formation of HABs was summarized in Fig. 4 and can be described by the following equation: where NW a denotes particles and nutrients carried by northwest monsoon, SW h denotes warm and moist air carried by southwest monsoon, SST denotes surface seawater temperature, and Sink denotes downward air. In this model NW a , SW h , SST, Sink can be obtained from meteorological observations, enabling the weather-like forecast of HABs events. Modern monitoring technologies can predict and observe the spatial and temporal distribution of aerosol events, and vertical variation of air flow dynamics. Based on these monitoring data, the described interaction of weather condition and nutrients transport on algal growth, the occurrence place, time and scale of HABs can probably be forecasted. Thus such weather-like forecast of HABs events can be used to minimize the finical loss caused by HABs.

Materials and Methods
Time series data collection. HABs  The concentrations of Fe, P, Zn, Cu, Co, Mn, and Cr in the flask solution were determined by inductively coupled plasma-mass spectrometry (ICP-MS) (Fig. S2). Firstly, a square piece (4 × 4 cm) was cut off from a   particle-bearing membrane and then was placed in a 25 ml Teflon beaker. Subsequently, a 6 ml HNO 3 -HClO 4 mixture with a ratio of 4:2 (v/v) was added to the beaker, which was covered with a glass dish. The acid-treated and dissolved membrane were heated at 170 °C on a temperature-controlled electrothermal plate for about 5 h and then cooled to room temperature. After cooling, the membrane, beaker and the glass dish were rinsed three times with deionized water. The mixture solution and rinsed water were transferred into a 50 ml volumetric flask and diluted to 50 ml with deionized water for analysis.
Selected atmospheric particles were also examined by transmission electron microscope (TEM) to determine particle morphology and aggregation. In addition, the chemical compositions of whole particles on filters were characterized using electron microprober (EMF). A JEOL JXA-8530F Field Emission HyperProbe Electron Probe Microanalyzer equipped with an energy-dispersive X-ray spectrometer, operating in backscattered electron emission mode at 20 keV was used in this study. At least three randomly selected areas on each sample were measured. Conventional standard ZAF (atomic number, mass absorption and fluorescence) correction was carried out automatically for semi-quantitative energy dispersive spectroscopy (EDS) analysis.
Laboratory algal incubation. Independent experiments were performed to determine the growth of HABs-related species Cryptomonas sp., Prorocentrum micans Ehrenberg, and diatom, under different iron and phosphorus concentrations. Algae Prorocentrum micans and Cryptomonas sp. were incubated in 500 mL media in 1000 mL glass bottles, while diatom was incubated in polyethylene bottles to avoid the effect of glass on algae growth. The growth media was the modified artificial sea water with chemical compositions provided in Table S1 45 . The growth experiments were performed in a light incubator with light intensity of 60 μE m −2 s −1 and light(L)/dark(D) time ratio of 12/12 (hours) at temperature of 21 ± 0.5 °C, pH of 8.0, and salinity of 30.
The effects of light intensity on the algal growth were also evaluated. Daily specific growth rate µ was calculated using the following formula 46 : μ = (lnN t2 − lnN t1 )/Δt, where N t1 and N t2 are cell numbers at two different times during experiment and Δt is the time interval (in days) between N t1 and N t2 . The uptake of iron, phosphorus, and micronutrients by Cryptomonas sp. were also investigated under different light intensity.