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All issues Volume 56 (2020) Ann. Limnol. - Int. J. Lim., 56 (2020) 28 Full HTML
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Ann. Limnol. - Int. J. Lim.
Volume 56, 2020
Article Number 28
Number of page(s) 8
DOI https://doi.org/10.1051/limn/2020026
Published online 13 November 2020

© EDP Sciences, 2020

1 Introduction

Microcystis bloom outbreaks often occur in eutrophic lakes in summer and have been found in many regions of the world (Otten et al., 2012; Park et al., 1993). The toxin microcystin produced by Microcystis has caused great harm to the survival of animals and plants in water (Nishiwaki et al., 1992). Microcystis overwinters at the lake bottom in winter after their growth season (Brunberg and Blomqvist, 2002; Preston et al., 1980). In the spring, Microcystis slowly rises to the water column with the increase of temperature and light intensity. During the summer, Microcystis floats to the water surface and multiplies. Microcystis gradually sinks to the bottom of the water as the temperature and light intensity decrease in autumn. The movement of Microcystis in lakes is reciprocating in water as an annual cycle (Sigee, 2005; Walsby, 1994). Two factors of Microcystis buoyancy have been proposed based on the previous studies; gas vesicle and extracellular polysaccharides (EPS). Microcystis has a particular structure called gas vesicle and can obtain buoyancy by synthesizing gas vesicles (Walsby, 1994). Under laboratory conditions, Microcystis aeruginosa NIES-843 synthesized gas vesicles and floated in a test tube, while none of the gas vesicles were detected at the bottom of the test tube (Wei et al., 2018).

It was reported that the buoyancy of Microcystis is also strictly related to colony formation (Wei et al., 2019). Wild Microcystis strain presents strong buoyancy, and the large size of the colony can be observed with a microscope (Liu et al., 2018; Wei et al., 2019). On the surface of Microcystis cells, there is a carbohydrate called extracellular polysaccharides (EPS) that play a significant role in the colony formation of Microcystis (Sato et al., 2017; Zhao et al., 2011). EPS contains polysaccharides, proteins, and DNA (De Philippis and Vincenzini, 1998; Flemming and Wingender, 2010). Carboxyl groups in polysaccharides can combine with divalent metal ions by the ionic attractive force, which induces M. aeruginosa colony formation in culture medium (Hahn et al., 2004; Sato et al., 2017; Wang et al., 2011). M. aeruginosa could combine with calcium or cadmium ions to form a colony (Bi et al., 2016; Pradhan and Rai, 2001; Wang et al., 2011; Zhao et al., 2011). With the EPS supplement, M. aeruginosa (strain: PCC7005) obtain buoyancy and form a bloom (Dervaux et al., 2015). The combination of cationic ions and EPS addition could make M. aeruginosa NIES-843 form a similar colony as wild M. aeruginosa and possessed buoyancy in a measuring cylinder (Wei et al., 2019).

EPS consists of bound EPS (bEPS) and soluble EPS (sEPS) (Wingender et al., 1999). bEPS can be divided into loosely cell-bound EPS (LB-EPS) and tightly cell-bound EPS (TB-EPS) (Basuvaraj et al., 2015; Qu et al., 2012). TB-EPS is one of the main components of M. aeruginosa EPS. Chemical analysis shows that the protein content of TB-EPS exceeds that of EPS, and more enormous amounts of carboxy groups were observed in TB-EPS (Qu et al., 2012). Colony formation induced by TB-EPS is more efficient than EPS in wild Microcystis (Omori et al., 2019; Sakurai et al., 2019). The relationship between two types of bound EPS and M. aeruginosa colony formation has been investigated, and TB-EPS could influence the formation of M. aeruginosa aggregates, and LB-EPS contributed to the development from aggregates to mucilaginous colonies (Tan et al., 2019). However, environmental factors such as light and temperature on the effect of EPS and TB-EPS are unknown.

In this study, from the perspective of colony size, M. aeruginosa NIES-843 and wild Microcystis were cultivated to examine the relationship between colony formation and buoyancy. Based on the experimental data, the efficiency of EPS and TB-EPS to colony formation was investigated. Furthermore, the illumination and temperature conditions on the M. aeruginosa NIES-843 colony formation and buoyancy were discussed.

2 Materials and methods

2.1 Collection of test algae sample and culture conditions

The wild Microcystis strain was collected from Lake Senba, where Microcystis blooms often occur in summer (36°22′N; 140°27′E), Ibaraki, Japan, on September 2019 (Fig. 1). The collection of the sample was carried out at a depth of 5 cm from the water surface. The samples were brought to the laboratory and kept at 4 °C in the dark condition until they were used for microscopic observation and EPS isolation.

M. aeruginosa NIES-843 obtained from National Institute for Environmental Studies (NIES), Japan. This strain was cultured in 500 mL of modified Wright's Cryptophytes (WC) medium in 1 L Erlenmeyer flasks at 25 °C for about 14 days under 4500 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), Vitamin B12 (0.005 mg), ferric citrate (3 mg), citric acid (3 mg), 500 mg tris-(hydroxymethyl)-aminomethane (Tris buffer) in 1 L of distilled water (Guillard and Lorenzen, 1972). The pH of the medium was adjusted to 8.0 ± 0.1 by using 0.5 M HCl. The 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.

thumbnail Fig. 1

Wild Microcystis floating in the surface of Lake Senba.

2.2 Isolation of EPS and TB-EPS

The EPS extraction protocol was referred to as the previous studies (Amemiya and Nakayama, 1984; Nishikawa and Kuriyama, 1974; Sato et al., 2017). Under the room temperature condition, 0.25 M sodium hydroxide (NaOH) and 2% (w/v) ethylenediaminetetraacetic acid (Na2EDTA · 2H2O) were added to the wild Microcystis sample. After stirring the solution well, the sample was allowed to be left for 1 hour to dissolve the EPS from Microcystis cells. The sample solution was centrifuged at 3000 rpm for 15 minutes, 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 the solution. Then, the mixture was stored at ‑20 °C for 16 hours. 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 TB-EPS extraction protocol was referred to as the previous studies (Sakurai et al., 2019; Xu et al., 2013). Sodium chloride (NaCl) was added to the wild Microcystis sample to concentrate of 0.05% (w/v). The supernatant of the sample was then removed by centrifugation at 3000 rpm for 15 min. After that, distilled water was added into the Microcystis residue and filled to the same original volume. NaCl (0.05% (w/v)) was added into the solution and heated at 60 °C for 30 min. After the sample was cooled to room temperature, it was centrifuged at 3000 rpm for 15 min, and the supernatant was collected. The sample was filtrated by GF/C filter (Whatman, UK) and mixed with 1.5 times the amount of ice-cold ethanol to precipitate EPS. The subsequent procedure was the same as mentioned above, and TB-EPS was obtained. The powdered EPS and TB-EPS sample were stored in a desiccator until use.

2.3 Colony formation and buoyancy experiment of M. aeruginosa NIES-843

M. aeruginosa NIES-843 was precultured for 14 days. The cations (Ca2+ and Mg2+) concentration were analyzed by atomic absorption spectrometer (novAA 300, Analytik Jena AG, Jena, Germany). Then, 50 mL of the sample was poured into a graduated cylinder, and then calcium chloride (CaCl2), magnesium chloride hexahydrate (MgCl2 · 6H2O), EPS, and TB-EPS were added. Four experimental groups were set up in the experiment, “control”, “Ca2+ + Mg2+ group”, “EPS added group”, and “TB-EPS added group”. The concentration of EPS and TB-EPS was 200 mg/L in EPS and TB-EPS added group. The control medium was prepared without any addition of Ca2+, Mg2+, EPS, or TB-EPS. In the previous study, the influences of cationic ions and extracellular polysaccharides (EPS) on colony formation of Microcystis buoyancy were investigated (Sakurai et al., 2019; Wei et al., 2019). With different proportional addition of magnesium and calcium concentration, M. aeruginosa exhibited the strongest buoyancy at the mass ratio of 3 (Wei et al., 2019). Therefore, the mass ratio of Ca2+ and Mg2+ was kept constant at 3 in all experiments. The Ca2+ concentration (w/v) used in the buoyancy experiments was controlled from 0 mg/L to 50 mg/L, while Mg2+ (w/v) was from 0 mg/L to 150 mg/L.

The prepared medium was cultivated at 25 °C for 24 hours at 10,000 lx and 0 lx in the light-controlled experiment. The prepared medium was cultivated at 25 °C and 30 °C for 24 hours at 10,000 lx in the temperature-controlled experiment. The experiment was conducted in triplicate (n = 3), and the results were expressed as the mean value ± standard deviation (SD).

2.4 Microscopic observation and measurement of colony size

The microscope (Eclipse E100, Nikon, Japan) was used to observe the colonial morphology of M. aeruginosa. 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). Since the M. aeruginosa NIES-843 colony always presents irregular morphologies, the following method was used to measure and calculate the diameter. The length and width of colonies were measured directly from the longest axis (length, µm) and the shortest axis (width, µm, aligned perpendicular to the longest axis) (Li et al., 2014). The following equation calculated the diameter (µm) of the colony. D i a m e t e r = l e n g t h × w i d t h

The measurement of colony size was conducted in triplicate (n = 3), and the results were expressed as the mean value ± SD.

2.5 Evaluation of the buoyant ability of Microcystis

The ability of buoyancy can be reflected by calculating the buoyancy rate (Wang et al., 2011). Wild Microcystis was cultivated for 24 hours and exhibited strong buoyancy floating to the upper 10 mL of the water surface, as mentioned in detail below (Fig. 2). From these trends, wild Microcystis in the upper 10 mL layer (V1, mL) of the graduated cylinder was considered to possess strong buoyant ability, and the cell density was recorded as C1 (cells/mL). On the other hand, wild Microcystis in the lower 40 mL (V2, mL) of the medium was supposed to present weak buoyant ability, and the density of this layer was recorded as C2 (cells/mL). To evaluate buoyancy of M. aeruginosa, the relative buoyancy (RB20,%) was calculated from the cell density (C1, C2) and the solution volume (V1, V2) by the following equation (Wang et al., 2011). R B 20 ( % ) = C 1 V 1 C 1 V 1 + C 2 V 2 × 100

When M. aeruginosa was uniformly distributed in the graduated cylinder, the RB value was calculated to be 20%.

thumbnail Fig. 2

Wild Microcystis strain cultured in a cylinder for (A) 0 hour and (B) 24 hours.

3 Results and discussion

3.1 Buoyancy of wild Microcystis

The sample of wild Microcystis obtained from Lake Senba was cultivated for 24 h in a cylinder (Fig. 2A). Wild Microcystis floated to the water surface after 24 hours of cultivation and presented strong buoyancy (Fig. 2B). The relative buoyancy value of wild Microcystis was calculated to be nearly 100%. Microscopic observation showed that wild Microcystis formed a large colony, indicating that the colony size was about 100 µm (Fig. 3).

thumbnail Fig. 3

Morphology of Microcystis wild strain.

3.2 Buoyancy regulation of M. aeruginosa NIES-843 under the light-limited condition

Under the light condition, the buoyancy of M. aeruginosa NIES-843 increased in all groups except control. In the TB-EPS added group, M. aeruginosa NIES-843 presented high buoyancy when concentrations of Ca2+ and Mg2+ were more than 10 mg/L and 30 mg/L, respectively. In the EPS added group, the RB value of M. aeruginosa NIES-843 was close to 80%, with the Ca2+ and Mg2+ concentrations of 50 mg/L and 150 mg/L, respectively. At the same concentration of Ca2+ and Mg2+, the buoyancy of EPS added group buoyancy was lower than the TB-EPS added groups (Fig. 4).

Under the dark condition, the buoyancy of M. aeruginosa NIES-843 increased in the EPS added group. In the EPS added group, the RB value of M. aeruginosa NIES-843 was close to 75%, with the Ca2+ and Mg2+ concentrations of 50 mg/L and 150 mg/L, respectively (Fig. 5). In the TB-EPS added group, the buoyancy of M. aeruginosa NIES-843 changed slightly, and RB values were nearly 30% with the Ca2+ and Mg2+ concentrations of 50 mg/L and 150 mg/L, respectively. The buoyancy of M. aeruginosa NIES-843 did not change obviously with the addition of Ca2+ + Mg2+ (Fig. 5). M. aeruginosa NIES-843 presented higher buoyancy in light conditions than dark conditions under the same concentration (Figs. 4 and 5). In the northern rivers of Lake Taihu (China), where Microcystis blooms often occur, the Ca2+ and Mg2+ concentrations were reported to be about 58 mg/L and 15 mg/L, respectively (Ye et al., 2010). The cationic ion concentrations in our study are same level in the field study.

The TB-EPS addition can change the buoyancy more efficiently than EPS addition in the case of lighting conditions. The colony size analysis showed that the colony size in Ca2++ Mg2+ added group was about 35 µm, while that of the EPS added group was about 70 µm. The largest colony size was about 110 µm observed in the TB-EPS added group, which was similar to the wild Microcystis strain is about 100 µm (Fig. 6). With the same concentration of addition, TB-EPS can induce M. aeruginosa NIES-843 to form a more massive colony than EPS. TB-EPS contains more carboxyl groups than EPS (Omori et al., 2019). In conclusion, M. aeruginosa can combine with more cationic ions such as Ca2 and Mg2+ and obtain higher buoyancy with TB-EPS addition.

Under dark conditions, the colony induced by the Ca2+ + Mg2+ added group and TB-EPS added group was smaller than under light conditions. The largest colony size was observed about 80 µm in the EPS added group (Fig. 6). M. aeruginosa cannot obtain energy without photosynthesis (Takamura et al., 1985). EPS is mainly composed of polysaccharides, and the carboxyl groups combined with Ca2 and Mg2+ do not require energy, while TB-EPS is mainly composed of protein (Basuvaraj et al., 2015; Liu et al., 2010). Protein hydrolysis requires energy, and some of the carboxyl groups exist as peptide bonds in protein molecules. These carboxyl groups need hydrolysis to combinate with cationic ions (Liu et al., 2008; Mimmack et al., 1989). Therefore, the addition of TB-EPS cannot change the buoyancy of M. aeruginosa NIES-843 in the absence of light. In the previous study, the addition of powdered TB-EPS sample and the adjustment of cationic ion concentrations were promoted effective the colony formation and enlarged the colony size of wild Microcystis (Sakurai et al., 2019). Consequently, Microcystis buoyancy was enhanced by enlarging the colony size, indicating that the control of EPS and cationic ion concentrations would be one of the options for the removal of Microcystis blooms from the viewpoint of cost-effective, low-energy, environmentally-friendly.

M. aeruginosa NIES-843 induces higher buoyancy than dark conditions even at the same concentration of addition, indicating that light affects the buoyancy of M. aeruginosa NIES-843 (Fig. 7). In the present study, M. aeruginosa NIES-843 generated many bubbles in the graduated cylinder with TB-EPS under the light condition (Fig. 7A). Under the dark condition, no bubbles were observed in the graduated cylinder, and the buoyancy of M. aeruginosa NIES-843 was lower than the light condition (Fig. 7B). EPS enhanced the photosynthesis of M. aeruginosa, M. aeruginosa generated many bubbles via photosynthesis after the addition of EPS (Dervaux et al., 2015; Wei et al., 2019). These bubbles have a particular effect on buoyancy reconfirmed in this experiment. In this study, the concentration of calcium and magnesium greatly enhanced the colony formation of M. aeruginosa NIES-843. Compared with the chemical composition in WC (Guillard and Lorenzen, 1972) and BG-11 (Waterbury and Stanier, 1981) medium, their concentrations are not significantly different. Therefore, the same experiment could be carried out in BG-11 medium or other media.

thumbnail Fig. 4

Relative buoyancy of M. aeruginosa NIES-843 under light conditions.
thumbnail Fig. 5

Relative buoyancy of M. aeruginosa NIES-843 under dark conditions.
thumbnail Fig. 6

Colony size of M. aeruginosa NIES-843 under light conditions and dark conditions.
thumbnail Fig. 7

 M. aeruginosa NIES-843 in the surface layers of a cylinder and bubbles can be confirmed: (A) light condition, Mg2+ 90 mg/L, Ca2+ 30 mg/L, and TB-EPS 200 mg/L (B) dark condition, Mg2+ 90 mg/L, Ca2+ 30 mg/L, and TB-EPS 200 mg/L.

3.3 Buoyancy regulation of M. aeruginosa NIES-843 under the temperature controlled condition

The specified concentration conditions (Ca2+ and Mg2+ of 30 mg/L and 90 mg/L, EPS and TB-EPS of 200 mg/L, respectively) were set in the experiment to confirm temperature effect on buoyancy and colony formation. In the control group, the buoyancy of M. aeruginosa NIES-843 did not change significantly under different temperature conditions. With the temperature increased from 20 °C to 30 °C, the buoyancy of the Ca2+ + Mg2+ added group increased from 50% to 53%, the EPS added group increases from 70% to 73%, and the TB-EPS added group increased from 90% to 94% (Fig. 8). The colony size of Ca2+ + Mg2+ added group increased to 51 µm, the colony size of the EPS addition group increased to 94 µm, and the colony size of the TP-EPS added group increased to 134 µm (Fig. 9). Under the same concentration conditions, the increase in temperature is conducive to colony formation and presents higher buoyancy of M. aeruginosa NIES-843.

Microcystis wesenbergii and Microcystis ichthyoblabe cultivated under strong light intensity and warmer temperatures might enhance the growth of surface Microcystis directly through increasing the colony size (Duan et al., 2018). Under high-temperature conditions, M. aeruginosa NIES-843 failed to form a colony in the control group, indicating the temperature has different effects on different types of Microcystis strains (Fig. 8). The buoyancy of M. aeruginosa AK1 increased with high temperature, but the gas vesicle volume showed no significant change during the transient state. The high temperature helps M. aeruginosa consume carbohydrates in cells and lead to the changes in polysaccharide ballast (Kromkamp et al., 1988). M. aeruginosa NIES-843 can obtain buoyancy by synthesizing gas vesicles. The buoyancy of M. aeruginosa NIES-843 did not change obviously when the temperature rises without addition, suggesting that the changes in the number of gas vesicles do not affect the buoyancy of M. aeruginosa NIES-843 (Fig. 8). The number of Microcystis colonies decrease as a decrease in water temperature under the natural environment. Wild Microcystis strain isolated from Lake Nieuwe Meer in Holland, the percentage of total colonies that were sinking increased a few days to 100% after the temperature shifted from 20 °C to 10.5 °C (Visser et al., 1995). Lake Taihu is the third-largest freshwater lake in China, and seasonal Microcystis blooms have regularly formed in the lake for the past three decades. With global warming, Microcystis growing season has advanced by approximately 20 days over the last two decades (Deng et al., 2014; Otten and Paerl, 2011). Therefore, monitoring the lakes' temperature where Microcystis bloom often occurs is incredibly essential to protect the water environment.

thumbnail Fig. 8

Relative buoyancy of M. aeruginosa NIES-843 under the condition of 25 °C and 30 °C.
thumbnail Fig. 9

Colony size of M. aeruginosa NIES-843 under the condition of 25 °C and 30 °C.

4 Conclusions

In this study, we induced M. aeruginosa NIES-843 to form colony by using EPS and TB-EPS extracted from Microcystis blooms. The change of the M. aeruginosa buoyancy was examined, and principal conclusions were summarized as follows:

  • The light influenced the colony formation and buoyancy of M. aeruginosa NIES-843. Under light conditions, the addition of TB-EPS colud induce M. aeruginosa to obtain high buoyancy at low Ca2+ and Mg2+ concentration.

  • The colony size analysis showed that at the same cationic concentration, the addition of TB-EPS could induce M. aeruginosa to form the largest colony and present strong buoyancy.

  • The increase in temperature is conducive to the colony formation of M. aeruginosa and presents higher buoyancy.

Funding

The present work was supported in part by JFE 21st Century Foundation and JSPS KAKENHI Grant Number JP18K04404.

Acknowledgments

The authors would like to extend deep gratitude to Prof. Dr. Fumio Imazeki, Safety and Health Organization of Chiba University, for his fruitful and helpful discussion.

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Cite this article as: Wei K, Amano Y, Machida M. 2020. Impacts of different extracellular polysaccharides on colony formation and buoyancy of Microcystis aeruginosa. Ann. Limnol. - Int. J. Lim. 56: 28

All Figures

thumbnail Fig. 1

Wild Microcystis floating in the surface of Lake Senba.
In the text
thumbnail Fig. 2

Wild Microcystis strain cultured in a cylinder for (A) 0 hour and (B) 24 hours.
In the text
thumbnail Fig. 3

Morphology of Microcystis wild strain.
In the text
thumbnail Fig. 4

Relative buoyancy of M. aeruginosa NIES-843 under light conditions.
In the text
thumbnail Fig. 5

Relative buoyancy of M. aeruginosa NIES-843 under dark conditions.
In the text
thumbnail Fig. 6

Colony size of M. aeruginosa NIES-843 under light conditions and dark conditions.
In the text
thumbnail Fig. 7

 M. aeruginosa NIES-843 in the surface layers of a cylinder and bubbles can be confirmed: (A) light condition, Mg2+ 90 mg/L, Ca2+ 30 mg/L, and TB-EPS 200 mg/L (B) dark condition, Mg2+ 90 mg/L, Ca2+ 30 mg/L, and TB-EPS 200 mg/L.
In the text
thumbnail Fig. 8

Relative buoyancy of M. aeruginosa NIES-843 under the condition of 25 °C and 30 °C.
In the text
thumbnail Fig. 9

Colony size of M. aeruginosa NIES-843 under the condition of 25 °C and 30 °C.
In the text

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