Control of the buoyancy of Microcystis aeruginosa via colony formation induced by regulating extracellular polysaccharides and cationic ions
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It is well recognized that gas vesicles influence the buoyancy of Microcystis and control the migration in the water column. Some of Microcystis strains do not have gas vesicles, so these strains cannot obtain buoyancy from them. In this study, we used two types of Microcystis strains such as the non-gas vesicle synthesizing strain M. aeruginosa UTEX-2061 and the gas vesicle synthesizing strain M. aeruginosa NIES-843 in the buoyancy control experiments of M. aeruginosa. By inducing colony formation and calculating the relative buoyancy rate of each strain, it was proven that the colony formation was strictly related to the buoyancy of both Microcystis strains. M. aeruginosa NIES-843 possessing gas vesicles could obtain buoyancy under the lower concentration of calcium and magnesium than M. aeruginosa UTEX-2061 possessing no gas vesicles. With the addition of the extracellular polysaccharides (EPS) extracted from Microcystis blooms into the culture medium, M. aeruginosa NIES-843 and UTEX-2061 could present higher buoyancy at the lowest concentrations of calcium and magnesium. It was suggested that EPS combined with the divalent metal cations such as calcium and magnesium made Microcystis form colony effectively. Calcium and magnesium ions enhanced the ability of colony formation of Microcystis and had a significant influence on buoyancy.
KeywordsBuoyancy Colony formation Extracellular polysaccharides Cationic ions Microcystis
In recent years, with deepening lake eutrophication and the excessive discharge of industrial effluent, cyanobacterial blooms mainly composed of the genus Microcystis (Microcystis blooms) in eutrophic lakes have become more frequent . The outbreak of Microcystis blooms causes a series of environmental problems such as the production of cyanotoxins (e.g., microcystin), which could affect the survival of animals and plants in water, and people who drink the affected water could suffer from serious diseases such as liver cancer .
It is well known that Microcystis blooms appear in eutrophic lakes and accumulate at the water surface during the summer, and as the water temperature drops, they sink to the bottom of the lake and survive as “seeds” until the next year . Then, they recruit from sediment and accumulate again at the surface water, repeating floating and sinking as an annual cycle . There are two main reasons that Microcystis can float and settle in water. The first reason is that Microcystis has a particular structure called gas vesicle and can obtain buoyancy and float to the water surface by synthesizing gas vesicles . The second reason is that photosynthetic production such as carbohydrates causes an increase in cellular weight . As a result, Microcystis sinks into the bottom of the water. When the respiration reduces the carbohydrate content at night, the weight is lost leading Microcystis to float to the water surface.
It was reported that the cyanobacterium Nostoc muscorum could synthesize gas vesicles in the cell  and the gas vesicle structure was found in the cyanobacterium Microcystis aeruginosa . It is also indicated from  that the gas vesicle affected the buoyancy of M. aeruginosa and that the gas vesicle is composed of proteins. The buoyancy of M. aeruginosa is also influenced by solution pH. Microcystis sp. FACHB 928 cultured in a high solution pH (pH 10) was easy to float compared to the solution at pH 8.5, and by using a transmission electron microscope (TEM), it was found that the gas vesicle was more synthesized under high pH condition . In the latest study, the genes from Bacillus megaterium pNL29 and Anabaena flos-aquae pET28a were added to the Escherichia coli gene, and E. coli could synthesize gas vesicles exhibiting buoyancy in a test tube . In a study of , M. aeruginosa NIES-843 floated in a test tube had gas vesicles, and none of the gas vesicles were detected for M. aeruginosa at the bottom of the test tube. These findings indicate that the gas vesicle plays a significant role in the cyanobacterial buoyancy, and the gas vesicle is generally considered to give the motility in Microcystis that obtains buoyancy .
In addition to gas vesicles, the synthesis of carbohydrates by cyanobacteria through photosynthesis is also considered to be one of the driving forces controlling the cyanobacterial buoyancy . The representative carbohydrate in cyanobacteria is called extracellular polysaccharides (EPS), and EPS has been proven to be strongly related to the colony formation of M. aeruginosa . EPS was first discovered in the microbial cells, releasing copious extracellular slime secretions which formed a capsule-like structure around the cell . EPS capsule is thought to be closely related to the cell adhesion and movement in the water column . By ionic attractive force, two carboxyl groups can bind to divalent metal cations to achieve a relatively stable composition [9, 44, 48]. In the recent reports of [28, 29], a mixture of divalent metal ions and EPS was added to the culture medium of unicellular M. aeruginosa, and M. aeruginosa could form a larger colony. M. aeruginosa could also absorb iron and cadmium ion in water to form colonies [1, 4], and this absorption property can be considered as an effective adsorbent for toxic ions in water. The addition of microcystin to the culture medium also caused the colony formation of Microcystis sp. FACHB1027, and with the higher concentration of microcystin, Microcystis could form the larger colony .
Consequently, the colony formation of Microcystis would be related to the secretion of algal toxins . Some zooplankton may prey on cyanobacteria in the water, and in order to avoid predation by predators, cyanobacteria synthesize EPS and form a larger colony [15, 45].
In fact, under natural conditions, some of Microcystis strains do not have gas vesicles, so these strains cannot obtain buoyancy from them . The theory that gas vesicles control the buoyancy of Microcystis cannot explain the movement of all Microcystis strains in water. Thus, in this study, we used the non-gas vesicle synthesizing strain M. aeruginosa UTEX-2061 and the gas vesicle synthesizing strain M. aeruginosa NIES-843 [18, 26, 27] in the buoyancy control experiments. By inducing colony formation using EPS extracted from Microcystis blooms and calculating the relative buoyancy rate of each strain, it was proven that the colony formation was strictly related to the buoyancy of both Microcystis strains.
2 Materials and methods
2.1 Collection of Microcystis blooms
Lake Senba (36°22′N, 140°27′E), located in Ibaraki Prefecture, Japan, has the storage volume and the average depth of 365,000 m3 and 1.0 m, respectively, and was the site for the collection of Microcystis blooms. The water quality such as chemical oxygen demand (COD) in Lake Senba was improved from 28.5 to 10 mg/L (as the mean annual value) due to lake water purification project conducted in 1986. However, because of recent water quality degradation, the mean COD and total phosphorus (TP) concentration values in 2017 were 15.0 and 0.16 mg/L, respectively, which still exceeded the target values of environmental standards related to water pollution (COD: ≤ 8.0 mg/L, TP: ≤ 0.10 mg/L) in Lake Senba .
2.2 M. aeruginosa culture
M. aeruginosa (strain: NIES-843) was obtained from the National Institute for Environmental Studies (NIES), Tsukuba, Japan. M. aeruginosa NIES-843 possesses the ability of colony formation. This strain was cultured in 300 mL of modified MA medium  in 1 L Erlenmeyer flasks at 25 °C for 14 or 28 days under continuous illumination of 4,500 lx. The MA medium consisted of a mixture of bicine (500 mg), Ca(NO3)2·4H2O (50 mg), KNO3 (100 mg), NaNO3 (50 mg), Na2SO4 (40 mg), MgCl2·6H2O (50 mg), β-Na2glycerophosphate (100 mg) and a metal mixed solution (1 mL containing 1 mg of Na2EDTA, 0.1 mg of FeCl3·6H2O, 1 mg of MnCl3·4H2O, 0.1 mg of ZnCl2, 1 mg of CoCl2·6H2O, 0.16 mg of Na2MoO4·2H2O, and 4 mg of H3BO3 in 200 mL of distilled water) in 1 L of distilled water. The pH of the medium was adjusted to 8.6 ± 0.1 by a D-51 pH meter (Horiba, Japan) using 0.5 M NaOH.
M. aeruginosa (strain: UTEX-2061) was obtained from the Culture Collection of Algae, the University of Texas, USA. This strain was unicellular and never forms a colony. M. aeruginosa UTEX-2061 was cultured in 500 mL of modified Wright’s Cryptophytes (WC) medium  in 1 L Erlenmeyer flasks at 25 °C about 28 days under 4,500 lx continuous illumination. The WC medium consisted of a mixture of CaCl2 (36.76 mg), MgSO4·7H2O (36.97 mg), NaHCO3 (12.60 mg), K2HPO4 (8.71 mg), NaNO3 (85.01 mg), Na2·EDTA (4.36 mg), FeCl3·6H2O (3.15 mg), CuSO4·5H2O (0.01 mg), ZnSO4·7H2O (0.022 mg), CoCl2·6H2O (0.01 mg), MnCl2·4H2O (0.18 mg), Na2MoO4·2H2O (0.006 mg), H3BO3 (1.0 mg), thiamin·HCl (0.1 mg), biotin (0.005 mg), B12 (0.005 mg), 500 mg tris-(hydroxymethyl)-aminomethane (Tris buffer) in 1 L of distilled water. The pH of the medium was adjusted to 8.0 ± 0.1 by using 0.5 M HCl. The MA and WC media used in the experiment were sterilized by autoclaving at 115 kPa for 20 min at 121 °C. Inoculation and sampling of the culture medium were conducted in a clean bench to minimize bacterial contamination.
2.3 Isolation of EPS and protocol of colony formation and buoyancy experiment of M. aeruginosa
The EPS extraction protocol was referred to the study of [2, 22, 28]. Under the room temperature condition, 0.25 M sodium hydroxide (NaOH) and 2% (w/v) ethylenediaminetetraacetic acid (EDTA) were added to the Microcystis blooms sample collected in Lake Senba. After stirring the solution well, the sample was allowed to leave for 1 h to dissolve the EPS from Microcystis cells. The sample solution was centrifuged at 3000 rpm for 15 min, and the supernatant was collected. The solution was further filtered with GF/C filter (Whatman, UK) to remove impurities, and ethanol was added into the filtrate to give a final concentration of 60% (v/v), and it was allowed to precipitate the EPS in solution. Then, the mixture was stored in − 20 °C for 16 h. After that, the solution was centrifuged at 3000 rpm for 15 min, and the precipitate was collected. The collected sample was freeze-dried at -0.1 MPa, and the dried sample was ground with a mortar and a pestle. The powdered EPS sample was stored in a desiccator until use.
M. aeruginosa NIES-843 and UTEX-2061 were precultured for 14 or 28 days. Although the cell density of M. aeruginosa remains stable after 14 days of culture [30, 47], the long-term cultivation for 28 days was also conducted because M. aeruginosa would synthesize large amounts of EPS. The use of these different aged M. aeruginosa leads to a deep understanding of the relationship between EPS and colony formation. After both strains were cultivated in each duration, 50 mL of the sample was poured into a graduated cylinder, and then calcium chloride (CaCl2), magnesium chloride hexahydrate (MgCl2·6H2O) and EPS were added to the graduated cylinder.
In the previous study, the influences of Ca2+ and Mg2+ concentrations on extracellular polysaccharides (EPS) content and colony formation of Microcystis were investigated . It was also revealed that the addition of EPS and Ca2+ into the medium could lead M. aeruginosa to form colonies effectively when the EPS and Ca2+ concentrations were adjusted to 200 and 1000 mg/L, respectively . In the present study, the Ca2+ concentration (w/v) used in the buoyancy control experiments was controlled from 0 to 5000 mg/L, while that of Mg2+ and EPS (w/v) was from 0–1500 mg/L to 14,000 mg/L, respectively. Each concentration was gradually increased until the colony was formed and M. aeruginosa floated to the water surface. The control medium was also prepared without any additions of Ca2+, Mg2+ and EPS. Each prepared medium was cultivated at 25 °C for 24 h at 4500 lx. Each prepared medium was represented with components added. For example, the medium with the addition of Ca2+, Mg2+ and EPS was designated as “Ca2++Mg2++EPS”.
2.4 Microscopic observation and relative buoyancy
Microscope (Eclipse E100, Nikon, Japan) was used to observe the colony of M. aeruginosa. Because of long-term cultivation in the laboratory condition, M. aeruginosa UTEX-2061 was always observed as a single cell or twin cells. In this experiment, more than three cells in the aggregation were regarded as a colony. Optical microscopic images were taken with a digital camera system (AM-4023X, AnMo Electronics Corp, Taiwan).
When M. aeruginosa was uniformly distributed in the graduated cylinder, the RB value was calculated to be 20% (RB20).
3.1 Buoyancy regulation of wild Microcystis strain
3.2 Buoyancy regulation of M. aeruginosa NIES-843
When Mg2+ was added to M. aeruginosa NIES-843 cultured for 14 days, the RB value increased from 20 to 40% at the Mg2+ concentration of 700 mg/L (Fig. 4B), whereas when M. aeruginosa NIES-843 was cultivated for 28 days, the RB value of M. aeruginosa NIES-843 was close to 80% and presented strong buoyancy at 250 mg/L or more.
The EPS addition into M. aeruginosa NIES-843 cultured for 14 days caused changes in the buoyancy. When the amount of added reached 16 g/L, the RB value of M. aeruginosa NIES-843 showed 80% and presented strong buoyancy (Fig. 4C). M. aeruginosa NIES-843 cultured for 28 days showed the same buoyancy when the addition of EPS reached 12 g/L. Although the addition of EPS alone could also make M. aeruginosa obtain buoyancy, a large amount of EPS addition would cause an increase in solution pH up to 12 (data not shown).
Ca2+ and Mg2+ ions were simultaneously added into the M. aeruginosa NIES-843 medium cultured for 14 days and 28 days with different proportions, and the magnesium and calcium mass ratio (Mg2 + Ca2+ ratio) of 3 exhibited the strongest buoyancy (Fig. 4d). From these results, the Mg2+ + Ca2+ ratio was fixed to 3 in the experiments hereafter. M. aeruginosa NIES-843 cultivated for 14 days with the Ca2+ and Mg2+ concentrations of 70 and 210 mg/L, respectively, indicated the strong buoyancy (RB = ca. 95%), while as for 28 days cultivation, the same high buoyancy (RB = 90%) was obtained when the Ca2+ and Mg2+ concentrations reached 60 and 180 mg/L, respectively (Fig. 4e).
Microcystis aeruginosa NIES-843 cultured for 14 days and 28 days was added to a mixture of Ca2+, Mg2+ and EPS. Since the addition of EPS into the culture medium caused the rise of pH, the amount of EPS in this experiment was controlled to 200 mg/L. This condition gave the pH value in the solution to be about 9.2. M. aeruginosa cultivated for 14 days exhibited the strong buoyancy, indicating that the RB value was close to 90% at the Ca2+ and Mg2+ concentrations of 10 and 30 mg/L under constant EPS concentration (Fig. 4f). The 28-day cultivation resulted in the further increase in the buoyancy at the same concentrations (Ca2+ = 10 mg/L, Mg2+ = 30 mg/L, EPS = 200 mg/L), indicating almost 100% of the RB value. The medium with Ca2+, Mg2+ and EPS additions could make M. aeruginosa achieve much higher buoyancy than the combined addition of Ca2+ and Mg2+ (Fig. 4d, e).
3.3 Microscopic observation of M. aeruginosa NIES-843
3.4 Buoyancy regulation of M. aeruginosa UTEX-2061
When M. aeruginosa UTEX-2061 was cultivated in the Mg2+ added medium for 14 days the RB value increased to 60% with the addition of 1.1 g/L. In the case of 28-day cultivation, the strong buoyancy with the RB value of 90% was observed at the Mg2+ concentration of 0.5 g/L (Fig. 6b). Contrary to the cases of Ca2+ and Mg2+ ions, the single addition of EPS could not significantly change the buoyancy of M. aeruginosa UTEX-2061 (Fig. 6c).
Different proportions of Ca2+ and Mg2+ were added to the M. aeruginosa UTEX-2061 medium cultured for 14 days and 28 days, and it was found that M. aeruginosa presented the strongest buoyancy (Fig. 6d) at the Mg2/Ca2+ ratio of 3 as similar to the case of M. aeruginosa NIES-843. Based on the result, the ratio of Mg2+ and Ca2+ in the medium was maintained at 3. M. aeruginosa UTEX-2061 cultured for 14 days was grown in the medium at Ca2+ of 200 mg/L and Mg2+ of 600 mg/L and represented the relative buoyancy of 80% (Fig. 6e). M. aeruginosa UTEX-2061 cultured for 28 days showed much higher buoyancy, indicating that the RB value was ca. 95% at Ca2+ = 160 mg/L and Mg2+ = 480 mg/L.
Microcystis aeruginosa UTEX-2061 cultured for 14 and 28 days was added into the Ca2++Mg2++EPS medium. The high buoyancy (RB = 90%) of M. aeruginosa UTEX-2061 was observed at Ca2+ = 100 mg/L and Mg2+ = 300 mg/L for 14 days cultivation, and Ca2+ = 80 mg/L and Mg2+ = 240 mg/L for 28 days cultivation (Fig. 6F).
3.5 Microscopic observation of M. aeruginosa UTEX-2061
M. aeruginosa UTEX-2061 cultivated for 14 days exhibited the strong buoyancy, indicating that the relative buoyancy (RB) value was close to 90% at the Ca2+ and Mg2+ concentrations of 100 and 300 mg/L under EPS concentration of 200 mg/L (Fig. 6f). However, M. aeruginosa UTEX-2061 did not present strong buoyancy under the high concentration of Ca2+ ions (1500 mg/L) or Mg2+ ions (700 mg/L). Consequently, it is inferred that physical influence like a density rise of solution would not affect the buoyancy of M. aeruginosa UTEX-2061. The long-term cultivation of M. aeruginosa could synthesize large amounts of EPS in the medium . In the present experiment, we found that 28 days cultivated M. aeruginosa was able to obtain buoyancy at lower concentrations of Ca2+ and Mg2+ than that cultivated for 14 days because older M. aeruginosa would synthesize large amounts of EPS in the medium.
Because of synthesizing EPS and gas vesicle conducive to the maintenance of buoyancy, M. aeruginosa can exhibit strong buoyancy and remain stably on the water surface. It was reported that M. aeruginosa PCC6803 secreted less amount of EPS, which affected the buoyancy in the medium . EPS can be divided into dissolved EPS and bound EPS. The bound EPS can be further fractionated into loosely bound (LB-EPS) and tightly bound EPS (TB-EPS) because it inherently has a dynamic double-layered structure . Omori et al.  reported that TB-EPS induced colony formation more efficiently than EPS (not fractionated EPS, i.e., a mixture of dissolved, LB- and TB-EPS), and more enormous amounts of carboxy groups were observed in the surface of TB-EPS. The carboxy groups can attract cationic ions via ionic attractive force and make cross-linking reaction between the carboxy groups and cationic ions. Therefore, the carboxy groups on TB-EPS would be contributed to colony formation, which causes buoyancy of M. aeruginosa. Xavier and Foster examined the relationship between oxygen concentration in water and EPS synthesis of M. aeruginosa, and revealed that under the low oxygen concentration, carbohydrate was mainly used to synthesize EPS. On the other hand, under the high oxygen concentration, M. aeruginosa primarily produces biomass and less amount of EPS via photosynthesis . Therefore, M. aeruginosa presented at the bottom of the lake, where the oxygen concentration is low, can produce EPS to increase buoyancy. After floating from the bottom (low oxygen) to the water surface (high oxygen), photosynthesis mainly produces biomass and maintains the stability of the colony. The present study could further explain the phenomenon that wild Microcystis annually floats and sinks in water column. In summer, M. aeruginosa can synthesize a large amount of EPS by photosynthesis. In the winter when the photosynthesis is weakened, M. aeruginosa synthesizes little amount of EPS to maintain the stability of the colony and sinks into the bottom of water until the next summer.
Researchers focusing on the growth of M. aeruginosa do not attract attention to the concentration of Ca2+ and Mg2+ ions in lakes. In Lake Taihu, China, Microcystis blooms often occur in summer. M. aeruginosa in Lake Taihu can form a colony [20, 25, 42]. M. aeruginosa cultured under laboratory conditions is different from natural lakes. Without the addition of cationic ions and EPS, it is difficult to form M. aeruginosa colonies from unicellular and laboratory-cultured M. aeruginosa like wild colonies [3, 37]. In recent years, the local government has taken measures to control the concentration of nitrogen and phosphorus, but the inhibition effect on M. aeruginosa is not satisfactory. It was reported that the addition of Ca2+ into the culture medium of M. aeruginosa FACHB 905 could promote the synthesis of EPS within 48 h . Ca2+ promoted the growth rate of M. aeruginosa, and the buoyancy increased with the high Ca2+ concentration . Ye et al.  reported that the Ca2+ concentration was about 58 mg/L, and Mg2+ concentration was about 15 mg/L in the northern rivers of the Lake Taihu, where Microcystis blooms often occurred. The concentration of cationic ions in MA (Ca2+ = 8.5 mg/L, Mg2+ = 6 mg/L) and WC (Ca2+ = 10 mg/L, Mg2+ = 3.65 mg/L) medium is lower than those in Lake Taihu, China. It is reported that a high amount of calcium was concentrated in EPS, and approximately 200 mg/L Ca2+ concentration was detected in colonies of wild strain M. aeruginosa . In the present study, M. aeruginosa cultured under laboratory conditions cannot synthesize high amount of EPS and also Ca2+ and Mg2+ were not concentrated in EPS. This would require high Ca2+ and Mg2+ concentration in the medium to form M. aeruginosa colonies. Under the Ca2+, Mg2+ and EPS concentrations of 10, 30 and 200 mg/L, respectively, M. aeruginosa NIES-843 cultured for 28 days formed colonies and presented strong buoyancy, showing the RB value was close to 100%. Therefore, the addition of Ca2+, Mg2+ ions and EPS into the medium promoted the ability of colony formation of Microcystis and had a significant influence on buoyancy. It suggests that the monitoring of the concentrations of these ions and substance in lakes, where Microcystis blooms occur frequently, is particularly essential.
The colony formation of both M. aeruginosa UTEX-2061 and NIES-843 had a significant influence on their buoyancy.
With the addition of EPS into medium, M. aeruginosa presented higher buoyancy at the lowest concentrations of Ca2+ and Mg2+, suggesting that EPS combined with the divalent metals can make Microcystis form colony effectively.
The combination of Ca2+ and Mg2+ addition into medium could enhance the buoyancy of M. aeruginosa effectively compared with that with the addition of them separately.
The authors would like to extend deep gratitude to Prof. Dr. Masu Hyuma, Center for Analytical Instrumentation Chiba University, for his fruitful and helpful discussions about the freeze-dried method.
The present work was supported in part by JFE 21st Century Foundation and by JSPS KAKENHI Grant Number 18K04404.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
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